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Sample records for calcium-independent phospholipase a2

  1. Calcium-independent phospholipase A2 in rat tissue cytosols

    NARCIS (Netherlands)

    Pierik, A.J.; Nijssen, J.G.; Aarsman, A.J.; Bosch, H. van den

    1988-01-01

    Cytosols (105000 X g supernatant) from seven rat tissues were assayed for Ca²⁺-independent phospholipase A₂ activity with either 1-acyl-2-[1-¹⁴C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[l-¹⁴C]linoleoyl-snglycero- 3-phosphoethanohunine or 1-0-hexadecyl-2-[9,10-³H₂]oleoyl-sn-glycero-3-phosphoc

  2. Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A(2).

    Science.gov (United States)

    Barbour, S E; Marciano-Cabral, F

    2001-02-26

    Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A(2) (PLA(2)) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA(2)s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA(2) activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA(2), cPLA(2). However, unlike the cPLA(2) enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA(2) activity is sensitive to inhibitors that block the activities of cPLA(2)-alpha and the 80 kDa calcium-independent PLA(2), iPLA(2), that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [(3)H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA(2) that may play a role in the basal phospholipid metabolism of these cells.

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

    OpenAIRE

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

    2010-01-01

    Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA2 inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA2 (XI(50) 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA2 than for GIVA cPLA2 and...

  4. Interaction between VEGF and calcium-independent phospholipase A2 in proliferation and migration of retinal pigment epithelium

    DEFF Research Database (Denmark)

    Kehler, Anne Katrine; Andersen, Emelie Cammilla; Andreasen, Jens Rovelt;

    2012-01-01

    Inhibition of VEGF in the eye is an important treatment modality for reducing proliferation and migration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). Additionally, previous studies suggest calcium-independent phospholipase A(2) group VIA (iPLA(2)-VIA) to be a po...

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

    Science.gov (United States)

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

    2010-05-13

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

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

    OpenAIRE

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

    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 hom...

  7. Calcium-independent phospholipase A2 regulates retinal pigment epithelium proliferation and may be important in the pathogenesis of retinal diseases

    DEFF Research Database (Denmark)

    Kolko, M; Kiilgaard, J F; Wang, J;

    2009-01-01

    -VIA decreased the rate of proliferation, whereas over expression of iPLA2-VIA increased the rate of proliferation. Using an experimental porcine model of RPE proliferation we demonstrated significant nuclear upregulation of iPLA2-VIA in proliferating RPE cells in vivo. We furthermore evaluated......Calcium-independent phospholipase A2, group VIA (iPLA2-VIA) is involved in cell proliferation. This study aimed to evaluate the role of iPLA2-VIA in retinal pigment epithelium (RPE) cell proliferation and in retinal diseases involving RPE proliferation. A human RPE cell line (ARPE-19) was used...... to explore this role in vitro. Proliferating ARPE-19 cells had increased expression and activity of iPLA2-VIA. iPLA2-VIA was found in the nuclei of proliferating ARPE-19 cells, whereas in confluent ARPE-19 cells, with limited proliferation, iPLA2-VIA was primarily found in the cytosol. Inhibition of iPLA2...

  8. Regulation of Calcium-Independent Phospholipase A2 Expression by Adrenoceptors and Sterol Regulatory Element Binding Protein-Potential Crosstalk Between Sterol and Glycerophospholipid Mediators.

    Science.gov (United States)

    Chew, Wee-Siong; Ong, Wei-Yi

    2016-01-01

    Calcium-independent phospholipase A2 (iPLA2) is an 85-kDa enzyme that releases docosahexaenoic acid (DHA) from glycerophospholipids. DHA can be metabolized to resolvins and neuroprotectins that have anti-inflammatory properties and effects on neural plasticity. Recent studies show an important role of prefrontal cortical iPLA2 in hippocampo-prefrontal cortical LTP and antidepressant-like effect of the norepinephrine reuptake inhibitor (NRI) antidepressant, maprotiline. In this study, we elucidated the cellular mechanisms through which stimulation of adrenergic receptors could lead to increased iPLA2 expression. Treatment of SH-SY5Y neuroblastoma cells with maprotiline, another tricyclic antidepressant with noradrenaline reuptake inhibiting properties, nortriptyline, and the adrenergic receptor agonist, phenylephrine, resulted in increased iPLA2β mRNA expression. This increase was blocked by inhibitors to alpha-1 adrenergic receptor, mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) 1/2, and sterol regulatory element-binding protein (SREBP). Maprotiline and phenylephrine induced binding of SREBP-2 to sterol regulatory element (SRE) region on the iPLA2 promoter, as determined by electrophoretic mobility shift assay (EMSA). Together, results indicate that stimulation of adrenoreceptors causes increased iPLA2 expression via MAP kinase/ERK 1/2 and SREBP, and suggest a possible mechanism for effect of CNS noradrenaline on neural plasticity and crosstalk between sterol and glycerophospholipid mediators, that may play a role in physiological or pathophysiological processes in the brain and other organs.

  9. Group VIB calcium-independent phospholipase A2 (iPLA2γ regulates platelet activation, hemostasis and thrombosis in mice.

    Directory of Open Access Journals (Sweden)

    Emiko Yoda

    Full Text Available In platelets, group IVA cytosolic phospholipase A2 (cPLA2α has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A2 and anti-thrombotic 12-(S-hydroxyeicosatetranoic acid production. However, studies using cPLA2α-deficient mice have indicated that other PLA2(s may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca2+-independent PLA2 (iPLA2γ-deficient platelets showed decreases in adenosine diphosphate (ADP-dependent aggregation and ADP- or collagen-dependent thromboxane A2 production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA2γ-null mice. Furthermore, mice lacking iPLA2γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA2γ is an additional, long-sought-after PLA2 that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.

  10. Role of prefrontal cortical calcium independent phospholipase A₂ in antidepressant-like effect of maprotiline.

    Science.gov (United States)

    Lee, Lynette Hui-Wen; Tan, Chay-Hoon; Shui, Guanghou; Wenk, Markus R; Ong, Wei-Yi

    2012-09-01

    There is increasing interest in the pathophysiology and neurochemistry of the prefrontal cortex (PFC) in depression. Blood flow and metabolism are decreased in the PFC of patients with depression compared to controls. Changes in long-chain polyunsaturated fatty acids (PUFAs) are also associated with depression. This study was conducted to elucidate a possible role of PFC activity of an enzyme involved in the release of docosahexaenoic acid (DHA), i.e. calcium-independent phospholipase A2 (iPLA₂), in the effects of the norepinephrine reuptake inhibitor (NRI) antidepressant, maprotiline, in mice. Treatment of Balb/C mice with maprotiline for 4 wk resulted in reduction in the level of behavioural despair, as determined by decreased immobility and increased climbing during the forced swim test. In contrast, mice treated with maprotiline plus bilateral prefrontal cortical injections of antisense oligonucleotide to iPLA₂, showed significantly increased immobility and decreased climbing, to levels comparable to saline-treated controls, indicating abolishment of the antidepressant-like effect of maprotiline. Lipidomic analyses showed significant decreases in phosphatidylcholine species containing long-chain PUFAs and increases in lysophosphatidylcholine after maprotiline treatment, indicating increased PLA₂ activity and endogenous release of eicosapentaenoic acid (EPA) or DHA after maprotiline treatment. These changes in lipid profiles were absent in mice that received maprotiline and PFC injections of antisense oligonucleotide to iPLA₂. Together, the results indicate that PFC iPLA₂ activity plays an important role in the antidepressant-like effect of maprotiline, possibly through endogenous release of long-chain PUFAs.

  11. Calcium-independent phospholipase A₂, group VIA, is critical for RPE cell survival

    DEFF Research Database (Denmark)

    Kolko, Miriam; Vohra, Rupali; Westlund, Barbro S.;

    2014-01-01

    PURPOSE: To investigate the significance of calcium-independent phospholipase A₂, group VIA (iPLA2-VIA), in RPE cell survival following responses to sodium iodate (SI) in cell cultures. METHODS: The human retinal pigment epithelium (RPE) cell line (ARPE-19) cells and primary mouse-RPE cultures were...... treated with SI to induce cell death. Cells were transfected with an iPLA₂-VIA promoter-luciferase construct to evaluate the regulation of iPLA-VIA after exposure to SI. PCR analysis, western blot analysis, and activity assays were performed to evaluate the mRNA level, protein level, and activity levels...... of iPLA₂-VIA after SI exposure. Inhibitors of iPLA₂-VIA were used to explore a potential protective role in cells exposed to SI. Primary RPE cell cultures were grown from iPLA₂-VIA knockout mice and wild-type mice. The cultures were exposed to SI to investigate a possible increased protection against...

  12. Diverse Functions of Secretory Phospholipases A2

    OpenAIRE

    Preetha Shridas; Webb, Nancy R.

    2014-01-01

    Phospholipase A2 enzymes (PLA2s) catalyze the hydrolysis of glycerophospholipids at their sn-2 position releasing free fatty acids and lysophospholipids. Mammalian PLA2s are classified into several categories of which important groups include secreted PLA2s (sPLA2s) and cytosolic PLA2s (cPLA2s) that are calcium-dependent for their catalytic activity and calcium-independent cytosolic PLA2s (iPLA2s). Platelet-activating factor acetylhydrolases (PAF-AHs), lysosomal PLA2s, and adipose-specific ...

  13. Phospholipase A2 Biochemistry

    OpenAIRE

    Burke, John E.; Dennis, Edward A.

    2008-01-01

    The phospholipase A2 (PLA2) superfamily consists of many different groups of enzymes that catalyze the hydrolysis of the sn-2 ester bond in a variety of different phospholipids. The products of this reaction, a free fatty acid, and lysophospholipid have many different important physiological roles. There are five main types of PLA2: the secreted sPLA2’s, the cytosolic cPLA2’s, the Ca2+ independent iPLA2’s, the PAF acetylhydrolases, and the lysosomal PLA2’s. This review focuses on the superfam...

  14. Neuronal damage by secretory phospholipase A2

    DEFF Research Database (Denmark)

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

    2003-01-01

    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 neura...

  15. Action of pure phospholipase A2 and phospholipase C on human erythrocytes and ghosts

    NARCIS (Netherlands)

    Roelofsen, B.; Zwaal, R.F.A.; Comfurius, P.; Woodward, C.B.; Deenen, L.L.M. van

    1971-01-01

    1. 1.|Pancreatic phospholipase A2 (phosphatide acyl-hydrolase, EC 3.1.1.4) and phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus appeared not to be lytic for human erythrocytes, either before or after treatment of the cells with trypsin, pronase or neuram

  16. Secretory Phospholipase A2-IIA and Cardiovascular Disease

    DEFF Research Database (Denmark)

    Holmes, Michael V; Simon, Tabassome; Exeter, Holly J;

    2013-01-01

    This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease.......This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease....

  17. Secretory Phospholipase A(2) Activity toward Diverse Substrates

    DEFF Research Database (Denmark)

    Madsen, Jesper Jonasson; Linderoth, Lars; Subramanian, Arun Kumar;

    2011-01-01

    We have studied secretory phospholipase A(2)-IIA (sPLA(2)) activity toward different phospholipid analogues by performing biophysical 1 characterizations and molecular dynamics simulations. The phospholipids were natural substrates, triple alkyl phospholipids, a prodrug anticancer etherlipid, and...... charged residues, but relatively large fluctuations are observed, suggesting that these interactions are not necessarily important for stabilizing substrate binding to the enzyme....

  18. Phospholipases A2 in ocular homeostasis and diseases

    DEFF Research Database (Denmark)

    Wang, Jinmei; Kolko, Miriam

    2010-01-01

    Phospholipases A(2) (PLA(2)s) and its generation of second messengers play an important role in signal transduction, cell proliferation, cell survival and gene expression. At low concentrations mediators of PLA(2) activity have a variety of physiological effects whereas high levels of PLA(2) and ...

  19. Secretory Phospholipase A(2)-IIA and Cardiovascular Disease

    NARCIS (Netherlands)

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

    2013-01-01

    Objectives This study sought to investigate the role of secretory phospholipase A(2) (sPLA(2))-IIA in cardiovascular disease. Background Higher circulating levels of sPLA(2)-IIA mass or sPLA(2) enzyme activity have been associated with increased risk of cardiovascular events. However, it is not clea

  20. Identification of intracellular phospholipases A2 in the human eye: involvement in phagocytosis of photoreceptor outer segments

    DEFF Research Database (Denmark)

    Kolko, Miriam; Wang, Jinmei; Zhan, Chen;

    2007-01-01

    PURPOSE: To identify intracellular phospholipases A(2) (PLA(2)) in the human retina and to explore the role of these enzymes in human retinal pigment epithelium (RPE) phagocytosis of photoreceptor outer segments (POS). METHODS: PCR amplification and Western blot analysis were used to identify m......RNA and protein expression of intracellular PLA(2) subtypes in the retinal pigment epithelial cell line ARPE-19. Immunohistochemical staining of normal human eye sections was performed to reveal the cellular location of the enzymes. A model of RPE phagocytosis of POS was used to explore the role of...... intracellular PLA(2) in phagocytosis. An activity assay was used to evaluate PLA(2) activity, and inhibitors of specific PLA(2) were applied to evaluate the role of PLA(2) in RPE phagocytosis. RESULTS: Genes encoding calcium-independent (i)PLA(2), group VIA; calcium-dependent cytosolic (c)PLA(2), groups IVA...

  1. Activation of phospholipase A2 by Hsp70 in vitro

    DEFF Research Database (Denmark)

    Mahalka, Ajay K; Code, Christian; Rezaijahromi, Behnam;

    2011-01-01

    We recently suggested a novel mechanism for the activation of phospholipase A2 (PLA2), with a (catalytically) highly active oligomeric state, which subsequently becomes inactivated by conversion into amyloid. This process can be activated by lysophosphatidylcholine which promotes both oligomeriza......We recently suggested a novel mechanism for the activation of phospholipase A2 (PLA2), with a (catalytically) highly active oligomeric state, which subsequently becomes inactivated by conversion into amyloid. This process can be activated by lysophosphatidylcholine which promotes both...... oligomerization and amyloid activation/inactivation. The heat shock protein 70 (Hsp70), has been demonstrated to be able to revert the conversion of a-synuclein and Alzheimer ß-peptide to amyloid fibrils in vitro. Accordingly, we would expect Hsp70 to sustain the lifetime of the active state of the enzyme...

  2. Secretory phospholipase A2 in patients with coronary artery disease.

    Science.gov (United States)

    Lima, Luciana Moreira; Carvalho, Maria das Graças; da Fonseca Neto, Cirilo Pereira; Garcia, José Carlos Faria; Sousa, Marinez Oliveira

    2010-04-01

    This study investigated the correlation of sPLA2 (secretory phospholipase A2) activity with the atheromatosis extent in subjects with coronary artery disease (CAD) undergoing coronary angiography. We analyzed 123 patients, including 35 subjects with angiographically normal coronary arteries (controls), 31 with mild/moderate atheromatosis (stenosis of 30-70% of the luminal diameter in one or more coronary arteries) and 57 with severe atheromatosis (>70% stenosis). Plasma sPLA2 activity was significantly higher in subjects with severe [127.7 U/ml (102.3-162.7); p tabagism, hypertension, sedentarism, family history for coronary artery disease, diabetes mellitus, total cholesterol, HDLc, LDLc, triglycerides, high sensitivity C-reactive protein and phospholipase A2, only sPLA2 was observed to be independently associated with severe CAD (>70% of stenosis) (p < 0.0001). PMID:19449149

  3. Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry

    OpenAIRE

    Yuan-Hao Hsu; Dumlao, Darren S.; Jian Cao; Dennis, Edward A.

    2013-01-01

    Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A2 (PLA2) in vivo. We have developed mass spectrometric methodology for analyzing PLA2 activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA2, cPLA2 and...

  4. Cytosolic phospholipase A2 in hypoxic pulmonary vasoconstriction

    OpenAIRE

    Ichinose, Fumito; Ullrich, Roman; Sapirstein, Adam; Jones, Rosemary C; Bonventre, Joseph V.; Serhan, Charles N.; Bloch, Kenneth D.; Zapol, Warren M.

    2002-01-01

    Cytosolic phospholipase A2 (cPLA2) releases arachidonic acid (AA) from phospholipids in cell membranes. To assess the role of cPLA2 in hypoxic pulmonary vasoconstriction (HPV), we measured the increase in left lung pulmonary vascular resistance (LPVR) before and during hypoxia produced by left main stem bronchus occlusion (LMBO) in mice with and without a targeted deletion of the PLA2g4a gene that encodes cPLA2α. LMBO increased LPVR in cPLA2α+/+ mice but not in cPLA2α–/– mice. cPLA2α+/+ mice ...

  5. Discrete Role for Cytosolic Phospholipase A2α in Platelets

    OpenAIRE

    Wong, Dennis A.; Kita, Yoshihiro; Uozumi, Naonori; Shimizu, Takao

    2002-01-01

    Among several different types of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2)α and group IIA (IIA) secretory PLA2 (sPLA2) have been studied intensively. To determine the discrete roles of cPLA2α in platelets, we generated two sets of genetically engineered mice (cPLA2α−/−/sPLA2-IIA−/− and cPLA2α−/−/sPLA2-IIA+/+) and compared their platelet function with their respective wild-type C57BL/6J mice (cPLA2α+/+/sPLA2-IIA−/−) and C3H/HeN (cPLA2α+/+/sPLA2-IIA+/+). We found that cPLA2α is needed for...

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

    Science.gov (United States)

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

    2014-07-01

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

  7. Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2.

    Directory of Open Access Journals (Sweden)

    Dan Zhang

    Full Text Available BACKGROUND: Antimicrobial peptides derived from the natural processing of chromogranin A (CgA are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. METHODOLOGY/PRINCIPAL FINDINGS: PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR or Catestatin (CAT induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate, a store operated channels (SOCs blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. CONCLUSIONS/SIGNIFICANCE: For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.

  8. Anti-phospholipase A2 receptor antibody in membranous nephropathy.

    Science.gov (United States)

    Qin, Weisong; Beck, Laurence H; Zeng, Caihong; Chen, Zhaohong; Li, Shijun; Zuo, Ke; Salant, David J; Liu, Zhihong

    2011-06-01

    The M-type phospholipase A2 receptor (PLA2R) is a target autoantigen in adult idiopathic membranous nephropathy (MN), but the prevalence of autoantibodies against PLA2R is unknown among Chinese patients with MN. Here, we measured anti-PLA2R antibody in the serum of 60 patients with idiopathic MN, 20 with lupus-associated MN, 16 with hepatitis B (HBV)-associated MN, and 10 with tumor-associated MN. Among patients with idiopathic MN, 49 (82%) had detectable anti-PLA2R autoantibodies using a Western blot assay; an assay with greater sensitivity detected very low titers of anti-PLA2R in 10 of the remaining 11 patients. Using the standard assay, we detected anti-PLA2R antibody in only 1 patient with lupus, 1 with HBV, and 3 with cancer, producing an overall specificity of 89% in this cohort limited to patients with secondary MN. The enhanced assay detected low titers of anti-PLA2R in only 2 additional samples of HBV-associated MN. In summary, these results suggest that PLA2R is a major target antigen in Chinese idiopathic MN and that detection of anti-PLA2R is a sensitive test for idiopathic MN.

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

    Science.gov (United States)

    Lee, Gihyun; Bae, Hyunsu

    2016-02-01

    Bee venom therapy has been used to treat immune-related diseases such as arthritis for a long time. Recently, it has revealed that group III secretory phospholipase A2 from bee venom (bee venom group III sPLA2) has in vitro and in vivo immunomodulatory effects. A growing number of reports have demonstrated the therapeutic effects of bee venom group III sPLA2. Notably, new experimental data have shown protective immune responses of bee venom group III sPLA2 against a wide range of diseases including asthma, Parkinson's disease, and drug-induced organ inflammation. It is critical to evaluate the beneficial and adverse effects of bee venom group III sPLA2 because this enzyme is known to be the major allergen of bee venom that can cause anaphylactic shock. For many decades, efforts have been made to avoid its adverse effects. At high concentrations, exposure to bee venom group III sPLA2 can result in damage to cellular membranes and necrotic cell death. In this review, we summarized the current knowledge about the therapeutic effects of bee venom group III sPLA2 on several immunological diseases and described the detailed mechanisms of bee venom group III sPLA2 in regulating various immune responses and physiopathological changes. PMID:26907347

  10. CONTROL OF ANGIOGENESIS BY INHIBITOR OF PHOSPHOLIPASE A2

    Institute of Scientific and Technical Information of China (English)

    Chen Wenming(陈文明); Li Lihong(李利红); Zhu Jiazhi(朱嘉芷); Liu Jinwei(刘晋玮); Soria Jeannette; Soria Claudine; Yedgar Saul

    2004-01-01

    Objective To investigate the potential effects of angiogenic process by secretory phospholipase A2(sPLA2) inhibitor-HyPE (linking N-derivatized phosphatidyl-ethanolamine to hyaluronic acid) on human bone marrow endothelial cell line (HBME-1). Methods In order to examine the suppressing effects of HyPE on HBME-1 proliferation, migration, and capillary-like tube formation, HBME-1 were activated by angiogenic factor, specifically by basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), and oncostatin M (OSM) (at a final concentration of 25, 20, and 2.5 ng/mL, respectively), then HBME-1 proliferation, migration, and tube formation were studied in the absence or presence of HyPE. HBME-1 tube formation was specially analyzed in fibrin gel. Results HyPE effectively inhibited HBME-1 proliferation and migration as a dose-dependent manner,whatever HBME-1 were grown in the control culture medium or stimulated with b-FGF, VEGF, or OSM.In fibrin, the formations of HBME-1 derived tube-like structures were enhanced by all angiogenic factors,but these were strongly suppressed by HyPE. Conclusions The results support the involvement of sPLA2 in angiogenesis. It is proposed that sPLA2inhibitor introduces a novel approach in the control of cancer development.

  11. Inhibitory effects of Swietenia macrophylla on myotoxic phospholipases A2

    Directory of Open Access Journals (Sweden)

    Jaime A. Pereañez

    2013-12-01

    Full Text Available Activity-guided fractionation of an ethanol-soluble extract of the leaves of Swietenia macrophylla King, Meliaceae, led to several fractions. As a result, sample Sm13-16, 23 had the most promising activity against phospholipases A2 (PLA2, Asp49 and Lys49 types. This fraction inhibited PLA2 activity of the Asp49 PLA2, when aggregated substrate was used. On the other hand, this activity was weakly neutralized when monodispersed substrate was used. In addition, Sm13-16, 23 inhibited, in a dose dependent manner, the cytotoxicity, myotoxicity and edema induced by PLA2s, as well as the anticoagulant activity of Asp49 PLA2. Overall, this fraction exhibited a better inhibition of the toxic activities induced by the Lys49 PLA2than those caused by the Asp49 PLA2. The spectral data of Sm13-16, 23 suggested the presence of aromatic compounds (UV λ max (nm 655, 266, and 219; IR λ max KBr (cm-1: ~ 3600-3000 (OH, 2923.07 and 1438.90 (C-H, 1656.69 (C = O, 1618.63 and 1607.67 (C-O, 1285.47772.60. We suggest that phenolic compounds could interact and inhibit the toxins by several mechanisms. Further analysis of the compounds present in the active fraction could be a relevant contribution in the treatment of accidents caused by snake envenomation.

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

    Science.gov (United States)

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

    1989-02-01

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

  13. Identification and Functional Characterization of Adipose-specific Phospholipase A2 (AdPLA)*S⃞

    OpenAIRE

    Duncan, Robin E.; Sarkadi-Nagy, Eszter; Jaworski, Kathy; Ahmadian, Maryam; Sul, Hei Sook

    2008-01-01

    Phospholipases A2 (PLA2s) catalyze hydrolysis of fatty acids from the sn-2 position of phospholipids. Here we report the identification and characterization of a membrane-associated intracellular calcium-dependent, adipose-specific PLA2 that we named AdPLA (adipose-specific phospholipase A2). We found that AdPLA was highly expressed specifically in white adipose tissue and was induced during preadipocyte differentiation into adipocytes. Clearance of AdPLA by immuno...

  14. A neurotoxic phospholipase A2 impairs yeast amphiphysin activity and reduces endocytosis.

    Directory of Open Access Journals (Sweden)

    Mojca Mattiazzi

    Full Text Available BACKGROUND: Presynaptically neurotoxic phospholipases A(2 inhibit synaptic vesicle recycling through endocytosis. PRINCIPAL FINDINGS: Here we provide insight into the action of a presynaptically neurotoxic phospholipase A(2 ammodytoxin A (AtxA on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin. CONCLUSIONS: We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein-protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A(2 work can open new ways to regulate endocytosis.

  15. Structural comparison of phospholipase-A2-binding regions in phospholipase-A2 receptors from various mammals.

    Science.gov (United States)

    Higashino, K; Ishizaki, J; Kishino, J; Ohara, O; Arita, H

    1994-10-01

    We determined the nucleotide sequence of a mouse cDNA encoding the receptor for pancreatic group I phospholipase A2 (PLA2-I). Interspecies structural comparison of the mouse receptor with bovine PLA2-I receptor, whose structure had been clarified, revealed that the fourth carbohydrate-recognition domain (CRD)-like domain (CRD-like 4) was the most conserved among the domains in the PLA2-I receptor, suggesting the functional importance of CRD-like 4. A transient expression experiment with a truncated form of the receptor consisting of three CRD-like domains, from the third to the fifth, demonstrated that the PLA2-I-binding site of the receptor is constituted from these three CRD-like domains, supporting the functional indispensability of CRD-like 4 in the receptor. Since the PLA2-I-binding region was thus assigned to be CRD-like domains 3-5, we further analyzed the structures of the PLA2-I-binding regions in the PLA2-I receptors from the rat, rabbit and human. Furthermore, the obtained PLA2-I receptor cDNA fragments from these animals made it possible to examine the tissue expression patterns of this receptor in various mammals. The results, together with the results of the genomic structural analysis of this gene, indicated that a PLA2 receptor recently characterized by Lambeau et al. [Lambeau, G., Ancian, P., Barhanin, J. & Lazdunski, M. (1994) J. Biol. Chem. 269, 1575-1578] is a rabbit counterpart of the PLA2-I receptor although these two PLA2 receptors have distinctive PLA2-binding specificities.

  16. Secretory Phospholipase A2-IIA and Cardiovascular Disease

    Science.gov (United States)

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

    2013-01-01

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

  17. DMPD: Regulation of arachidonic acid release and cytosolic phospholipase A2activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10080535 Regulation of arachidonic acid release and cytosolic phospholipase A2activ...on of arachidonic acid release and cytosolic phospholipase A2activation. PubmedID 10080535 Title Regulation of arachidonic acid relea...ation. Gijon MA, Leslie CC. J Leukoc Biol. 1999 Mar;65(3):330-6. (.png) (.svg) (.html) (.csml) Show Regulati...se and cytosolic phospholipase A2activation. Authors Gij

  18. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Steffen Nyegaard

    Full Text Available Secretory phospholipase A2 (sPLA2 is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2's do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50-60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2.

  19. Tenidap sodium inhibits secretory non-pancreatic phospholipase A2 synthesis by foetal rat calvarial osteoblasts

    OpenAIRE

    Pruzanski, W; Kennedy, B P; van den Bosch, H; Stefanski, E.; M. Wloch; Vadas, P.

    1995-01-01

    Tenidap (TD) was initially defined as a dual inhibitor of cyclooxygenase and lipoxygenase. This study was designed to assess its inhibitory activity against proinflammatory phospholipase A2. This study shows that TD inhibits the synthesis of pro-inflammatory secretory non-pancreatic phospholipase A2 (sPLA2). Concentrations as low as 0.25 μg/ml (0.725 μM) reduced the release of sPLA2 by 40% from foetal rat calvarial osteoblasts stimulated with IL-1β and TNFα, whereas a concentration of 2.5 μg/...

  20. Plasma Lipoprotein-associated Phospholipase A(2) Is Inversely Correlated with Proprotein Convertase Subtilisin-kexin Type 9

    NARCIS (Netherlands)

    Constantinides, Alexander; Kappelle, Paul J. W. H.; Lambert, Gilles; Dullaart, Robin P. F.

    2012-01-01

    Background and Aims. Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a proatherogenic phospholipase A(2), which is predominantly complexed to low-density lipoprotein (LDL) particles. Proprotein convertase subtilisin-kexin type 9 (PCSK9) provides a key step in LDL metabolism by stimulating L

  1. Lipocortin inhibition of extracellular and intracellular phospholipases A2 is substrate concentration dependent

    NARCIS (Netherlands)

    Aarsman, A.J.; Mynbeek, G.; Bosch, H. van den; Rothhut, B.; Prieur, B.; Comera, C.; Jordan, J.; Russo-Marie, F.

    1987-01-01

    Hydrolysis of Escherichia coli membrane phospholipids by pancreatic phospholipase A2 was inhibited by lipocortin from human monocytes in a substrate dependent manner. Inhibition was completely overcome at substrate concentrations above 250 μM. Lipocortin also inhibited partially purified preparation

  2. Secretory phospholipase A2 potentiates glutamate-induced rat striatal neuronal cell death in vivo

    DEFF Research Database (Denmark)

    Kolko, M; Bruhn, T; Christensen, Thomas;

    1999-01-01

    The secretory phospholipases A2 (sPLA2) OS2 (10, 20 and 50 pmol) or OS1, (50 pmol) purified from taipan snake Oxyuranus scutellatus scutellatus venom, and the excitatory amino acid glutamate (Glu) (2.5 and 5.0 micromol) were injected into the right striatum of male Wistar rats. Injection of 10 an...

  3. Moderate alcohol consumption and lipoprotein-associated phospholipase A2 activity

    NARCIS (Netherlands)

    Beulens, J.W.J.; Berg, van den R.; Kok, F.J.; Helander, A.; Vermunt, S.H.F.; Hendriks, H.F.J.

    2008-01-01

    Background and aims To investigate the effect of moderate alcohol consumption on lipoprotein-associated phospholipase A2 activity, markers of inflammation and oxidative stress and whether these effects are modified by BMI. Methods and results Eleven lean (BMI: 18.5¿25 kg/m2) and 9 overweight (BMI &g

  4. Lipoprotein-associated phospholipase A2 and coronary calcification - The Rotterdam coronary calcification study

    NARCIS (Netherlands)

    Kardys, Isabella; Oei, Hok-Hay S.; Hofman, Albert; Oudkerk, Matthijs; Witteman, Jacqueline C. M.

    2007-01-01

    Objectives: Although several studies have recently suggested that lipoprotein-associated phospholipase A2 (Lp-PLA2) is an independent predictor of coronary events, only one study has examined the association between Lp-PLA2 and coronary calcification, using young adults. We investigated the associat

  5. Expression and induction of secretory phospholipase A2 group IB in brain

    DEFF Research Database (Denmark)

    Kolko, Miriam; Christoffersen, Nanna R; Varoqui, Hélène;

    2005-01-01

    Secretory phospholipases A2 (sPLA2) form a diverse family of enzymes involved in physiologic and pathologic processes. Common among all sPLA2 is the ability to cleave acyl groups of phospholipids at C2 of the glycerol backbone, thereby releasing fatty acid and a lysophospholipid. Several sPLA2 ha...

  6. Bactericidal properties of group IIA and group V phospholipases A2

    NARCIS (Netherlands)

    Grönroos, J.O.; Laine, V.J.O.; Janssen, M.J.W.; Egmond, M.R.; Nevalainen, T.J.

    2010-01-01

    Group V phospholipase A2 (PLA2) is a recently characterized 14-kDa secretory PLA2 of mammalian heart and macrophage-derived cells. Group IIA PLA2, which is structurally close to group V PLA2, has been shown to kill Gram-positive bacteria in vitro and to prevent symptoms of Gram-positive infection in

  7. X-ray structure of bovine pancreatic phospholipase A(2) at atomic resolution

    NARCIS (Netherlands)

    Steiner, RA; Rozeboom, HJ; Kalk, KH; Murshudov, GN; Wilson, KS; Dijkstra, BW

    2001-01-01

    Using synchrotron radiation and a CCD camera, X-ray data have been collected from wild-type bovine pancreatic phospholipase A(2) at 100 K to 0.97 Angstrom resolution allowing full anisotropic refinement. The final model has a conventional R factor of 9.44% for all reflections, with a mean standard u

  8. Regulation of cytosolic Phospholipase A2 activity plays a central role in cell responses

    NARCIS (Netherlands)

    Rossum, Gerarda Sophia Agnes Theodora van

    2002-01-01

    Phospholipases A2 are enzymes that hydrolyse fatty acids from the sn-2 position of phospholipids, resulting in the release of free fatty acids and lysophospholipids. The sn-2 position of phospholipids in mammalian cells is enriched with arachidonic acid, which is a substrate for cyclooxygenases, lip

  9. Moderate alcohol consumption and lipoprotein-associated phospholipase A2 activity

    NARCIS (Netherlands)

    Beulens, J.W.J.; Berg, R. van den; Kok, F.J.; Helander, A.; Vermunt, S.H.F.; Hendriks, H.F.J.

    2008-01-01

    Background and aims: To investigate the effect of moderate alcohol consumption on lipoprotein-associated phospholipase A2 activity, markers of inflammation and oxidative stress and whether these effects are modified by BMI. Methods and results: Eleven lean (BMI: 18.5-25 kg/m2) and 9 overweight (BMI

  10. Darapladib, a lipoprotein-associated phospholipase A2 inhibitor, in diabetic macular edema

    DEFF Research Database (Denmark)

    Staurenghi, Giovanni; Ye, Li; Magee, Mindy H;

    2015-01-01

    PURPOSE: To investigate the potential of lipoprotein-associated phospholipase A2 inhibition as a novel mechanism to reduce edema and improve vision in center-involved diabetic macular edema (DME). DESIGN: Prospective, multicenter, randomized, double-masked, placebo-controlled phase IIa study. PAR...

  11. Activation of phospholipase A2 by carbon tetrachloride in isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Freshly isolated rat hepatocytes were incubated with 3H-arachidonic acid or 14C-ethanolamine for 1 hour in order to label cellular lipids. Thin-layer chromatographic analysis indicated that of the 3H-arachidonate incorporated into lipid nearly 50% was found in phosphatidylcholine and 15% in phosphatidylethanolamine. 14C-Ethanolamine was incorporated mainly into phosphatidylethanolamine. Hepatocytes labeled as such were exposed to carbon tetrachloride (CCl4) for periods up to 4 hours. Phospholipase A2 of these preparations was determined by measuring either the release of 4H-arachidonic acid from cellular phospholipids prelabeled with 3H-arachidonic acid or measuring the formation of 14C-lysophosphatidylethanolamine from cellular lipids prelabeled with 14C-ethanolamine. Through the use of hexane-partition extraction and thin-layer chromatographic analysis of hepatocyte lipid extracts it was found that CCl, stimulated phospholipase A2 activity in a dose- an time-dependent manner. Carbon tetrachloride at concentrations of 0.23 to 1.3 mM produced a 1.4- to 5.3-fold increase in phospholipase activity which was initiated within 30 to 60 minutes of incubation at 370. A role for phospholipase activation as a secondary mechanism of CCl4-induced hepatocyte injury is proposed

  12. Cross-reactivity and phospholipase A2 neutralization of anti-irradiated Bothrops jararaca venom antibodies

    International Nuclear Information System (INIS)

    The detoxified Bothrops jararaca venom, immunized rabbits with the toxoid obtained and investigated cross-reactivity of the antibodies obtained against autologous and heterelogous venoms was presented. It was also investigated the ability of the IgGs, purified by affinity chromatography, from those sera to neutralize phospholipase. A2, an ubiquous enzyme in animal venoms. Results indicate that venom irradiation leads to an attenuation of toxicity of 84%. Cross-reactivity was investigated by ELISA and Western blot and all venoms were reactive to the antibodies. On what refers to phospholipase A2 activity neutralization, the antibodies neutralized autologous venoms efficiently and, curiously, other venoms from the same genus were not neutralized, while Lachesis muta venom, a remote related specier, was neutralized by this serum. These data suggest that irradiation preserve important epitopes for induction of neutralizing antibodies and that these epitopes are not shared by all venoms assayed. (author). 8 refs, 2 figs, 3 tabs

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

    OpenAIRE

    Wilensky, Robert L.; Shi, Yi; Mohler, Emile R; Hamamdzic, Damir; Burgert, Mark E.; Li, Jun; Postle, Anthony; Fenning, Robert S.; Bollinger, James G.; Hoffman, Bryan E; Pelchovitz, Daniel J.; Yang, Jisheng; Mirabile, Rosanna C; Webb, Christine L; Zhang, LeFeng

    2008-01-01

    Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA2 is a causative agent. Here we show that selective inhibition of Lp-PLA2 with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA2 activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene express...

  14. Elevated serum secretory type Ⅱ phospholipase A2 in patients with coronary heart disease

    Institute of Scientific and Technical Information of China (English)

    于路

    2006-01-01

    Objective To measure the serum level of secretory type H phospholipase A2 (sPLA2) in patients with coronary heart disease and investigate the possible relationship with IL-8 and LPA. Methods A total of 110 patients with acute coronary syndrome (ACS) , 63 patients with stable coronary heart disease (SCHD) group and 89 non-CHD control patients were studied. Serum levels of sPLA2, IL-8, LPA and hs-CRP were measured and the

  15. Phospholipase A(2) - An enzyme that is sensitive to the physics of its substrate

    DEFF Research Database (Denmark)

    Høyrup, Lise Pernille Kristine; Jørgensen, Kent; Mouritsen, O.G.

    2002-01-01

    A simple statistical mechanical model of lipid bilayers is proposed to account for the non-equilibrium action of the enzyme phospholipase A(2). The enzyme hydrolyses lipid-bilayer substrates and produces product molecules that lead to local variations in the bilayer interfacial pressure. Computer...... simulation of the model shows, in quantitative agreement with experimental data, that the enzyme activity is modulated by nano-scale lipid-domain formation in the lipid bilayer leading to a characteristic lag-burst behavior....

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

    Directory of Open Access Journals (Sweden)

    Gargouri Youssef

    2011-02-01

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

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

    OpenAIRE

    Miki, Yoshimi; Yamamoto, Kei(Department of Physics, Niigata University, Niigata 950-2181, Japan); Taketomi, Yoshitaka; Sato, Hiroyasu; Shimo, Kanako; Kobayashi, Tetsuyuki; Ishikawa, Yukio; Ishii, Toshiharu; NAKANISHI, Hiroki; Ikeda, Kazutaka; Taguchi, Ryo; Kabashima, Kenji; Arita, Makoto; Arai, Hiroyuki; Lambeau, Gérard

    2013-01-01

    Resolution of inflammation is an active process that is mediated in part by antiinflammatory lipid mediators. Although phospholipase A2 (PLA2) enzymes have been implicated in the promotion of inflammation through mobilizing lipid mediators, the molecular entity of PLA2 subtypes acting upstream of antiinflammatory lipid mediators remains unknown. Herein, we show that secreted PLA2 group IID (PLA2G2D) is preferentially expressed in CD11c+ dendritic cells (DCs) and macrophages and displays a pro...

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

    OpenAIRE

    Mark Merchant; Kate Juneau; Jared Gemillion; Rodolfo Falconi; Aaron Doucet; 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 cr...

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

    OpenAIRE

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

    2011-01-01

    Abstract Background Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food. Results The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used...

  20. A Tentative Mechanism of Solubilization of Neuropathy Target Esterase from Chicken Embryo Brain by Phospholipase A2

    Directory of Open Access Journals (Sweden)

    Josef Seifert

    2008-01-01

    Full Text Available The neuropathy target esterase is a membrane-bound enzyme linked to organophosphate-induced distal neuropathy. Here we report a tentative mechanism of its solubilization from chicken embryo brains by using phospholipase A2. The enzyme was released from brain membranes after degradation of their structural phospholipids initiated by phospholipase A2. L-α-lysophosphatidylcholine, tested as a representative product of phospholipid hydrolysis, was identified as a new efficient detergent for solubilization of the neuropathy target esterase.

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

    Science.gov (United States)

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

    2010-10-20

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

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

    Science.gov (United States)

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

    1989-01-01

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

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

    Science.gov (United States)

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

    1994-09-01

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

  4. Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A2-induced degranulation in mast cells

    International Nuclear Information System (INIS)

    Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of β-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (GM1), di-sialoganglioside (GD1a) and tri-sialoganglioside (GT1b). In contrast, honeybee venom-derived phospholipase A2 induced the net degranulation directly without cytotoxicity, which was not inhibited by GM1, GD1a and GT1b. For analysis of distribution of Gαq and Gαi protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of Gαq and Gαi at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A2-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A2-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.

  5. Implication of lipoprotein associated phospholipase A2 activity in oxLDL uptake by macrophages

    OpenAIRE

    Markakis, Konstantinos P.; Koropouli, Maria K.; Grammenou-Savvoglou, Stavroula; van Winden, Ewoud C.; Dimitriou, Andromaxi A.; Demopoulos, Constantinos A.; Tselepis, Alexandros D; Kotsifaki, Eleni E.

    2010-01-01

    Recognition and uptake of oxidized LDL (oxLDL) by scavenger receptors of macrophages and foam cell formation are mediated by the oxidatively modified apolipoprotein B (ApoB) and lipid moiety of oxLDL. A great amount of oxidized phosphatidylcholine (oxPC) of oxLDL is hydrolyzed at the sn-2 position by lipoprotein associated phospholipase A2 (Lp-PLA2) to lysophosphatidylcholine and small oxidation products. This study examines the involvement of Lp-PLA2 in the uptake of oxLDL by mouse peritonea...

  6. Group X secretory phospholipase A2 negatively regulates adipogenesis in murine models

    OpenAIRE

    Li, Xia; Shridas, Preetha; Forrest, Kathy; Bailey, William; Webb, Nancy R.

    2010-01-01

    Studies in vitro indicate that group X secretory phospholipase A2 (GX sPLA2) potently releases arachidonic acid (AA) and lysophosphatidylcholine from mammalian cell membranes. To define the function of GX sPLA2 in vivo, our laboratory recently generated C57BL/6 mice with targeted deletion of GX sPLA2 (GX−/− mice). When fed a normal rodent diet, GX−/− mice gained significantly more weight and had increased adiposity compared to GX+/+ mice, which was not attributable to alterations in food cons...

  7. Group X Secretory Phospholipase A2 Enhances Toll-Like Receptor 4 Signaling in Macrophages

    OpenAIRE

    Shridas, Preetha; Bailey, William M.; Talbott, Kayla R; Oslund, Rob C.; Gelb, Michael H; Webb, Nancy R.

    2011-01-01

    Secretory phospholipase A2’s (sPLA2) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although Group X (GX) sPLA2 is recognized as the most potent mammalian sPLA2 in vitro, its precise physiological function(s) remain unclear. We recently reported that GX sPLA2 suppresses activation of the Liver X receptor (LXR) in macrophages, resulting in reduced expression of LXR-responsive genes including ABCA1 and ABCG1, and a consequent decrease in cellular cholesterol ...

  8. Bee Venom Phospholipase A2: Yesterday’s Enemy Becomes Today’s Friend

    OpenAIRE

    Gihyun Lee; Hyunsu Bae

    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 inc...

  9. Biophysical mechanisms of phospholipase A2 activation and their use in liposome-based drug delivery

    DEFF Research Database (Denmark)

    Jørgensen, Kaj; Davidsen, Jesper; Mouritsen, Ole G.

    2002-01-01

    Secretory phospholipase A(2) (PLA(2)) is a ubiquitous water-soluble enzyme found in venom, pancreatic, and cancerous fluid. It is also known to play a role in membrane remodeling processes as well as in cellular signaling cascades. PLA(2) is interfacially active and functions mainly on organized...... types of substrate, e.g. micelles and lipid bilayers. Hence the activity of the enzyme is modulated by the lateral organization and the physical properties of the substrate, in particular the structure in the nanometer range. The evidence for nano-scale structure and lipid domains in bilayers is briefly...

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

    OpenAIRE

    Liao, Hui-Ling; Burns, Jacqueline K.

    2010-01-01

    The low-molecular weight secretory phospholipase A2α (CssPLA2α) and β (CsPLA2β) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2α displayed distinct diurnal patterns in fruit tissues. CssPLA2α and CsPLA2β diurnal expression exhibited periods of approximately 24 h; CssPLA2α amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2β amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2α and CsPLA2β gene expressi...

  11. Secretory Phospholipase A2 Enzymes as Pharmacological Targets for Treatment of Disease

    OpenAIRE

    Nhat D Quach; Arnold, Robert D.; Cummings, Brian S.

    2014-01-01

    Phospholipase A2 (PLA2) cleave phospholipids preferentially at the sn-2 position, liberating free fatty acids and lysophospholipids. They are classified into six main groups based on size, location, function, substrate specificity and calcium requirement. These classes include secretory PLA2 (sPLA2), cytosolic (cPLA2), Ca2+-independent (iPLA2), platelet activating factor acetylhydrolases (PAF-AH), lysosomal PLA2 (LyPLA2) and adipose specific PLA2 (AdPLA2). It is hypothesized that PLA2 can ser...

  12. Phospholipase A2 gene expression and activity in histologically normal ileal mucosa and in Crohn's ileitis.

    OpenAIRE

    Lilja, I; Smedh, K; Olaison, G; Sjödahl, R.; Tagesson, C; Gustafson-Svärd, C

    1995-01-01

    Increased activity of phospholipase A2 (PLA2) in the ileal mucosa may contribute to the inflammation in Crohn's disease. The results of this study showed that (a) three months after ileocolonic resection for Crohn's disease the neoterminal ileal mucosa showed endoscopically new inflammation and had higher PLA2 activity than at the time of the operation (n = 8); no such findings were seen in controls (n = 7), (b) histologically normal ileal mucosa (n = 3) contained mRNA for three isoforms of P...

  13. Production of phosphatidylcholine containing conjugated linoleic acid mediated by phospholipase A2

    OpenAIRE

    Yamamoto, Yukihiro; Hosokawa, Masashi; Miyashita, Kazuo

    2006-01-01

    Esterification of lysophosphatidylcholine (LPC) with conjugated linoleic acid (CLA) was carried out using porcine pancreatic phospholipase A2 (PLA2). PLA2 only slightly synthesized phosphatidylcholine containing CLA (CLA-PC) at 2.6% by the addition of water. Addition of formamide in place of water markedly increased the yield of CLA-PC. In addition, synthesis of CLA-PC by PLA2 was affected by the amount of substrate CLA and PLA2 in the reaction system. Under optimal reaction conditions using ...

  14. Characteristics of phospholipase A2 mutant of the starfish Asterina pectinifera

    OpenAIRE

    Kishimura, Hideki; ANDO, Seiichi

    2007-01-01

    Site-directed mutagenesis study of phospholipase A2 (PLA2) from the pyloric ceca of starfish Asterina pectinifera was used to probe the relationship between polar-group specificity and structure of the pancreatic loop region. The sequence of the cDNA encoding the starfish PLA2 was exchanged by the oligonucleotide-directed dual amber-long and accurate polymerase chain reaction method to insert Lys residue between Cys-62 and Gly-63. The modified cDNA was inserted into the expression plasmid pET...

  15. Purification and properties of phospholipase A2 isozymes from pyloric ceca of the starfish (asterina pectinifera)

    OpenAIRE

    Kishimura, Hideki; Hayashi, Kenji

    2004-01-01

    Phospholipase A2 isozyme II (PLA2 II) which showed different mobility on native PAGE from that of the PLA2 isozyme I (PLA2 I) isolated previously was purified from pyloric ceca of the starfish (Asterina pectinifera). The PLA2 II was mainly released oleic acid from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. N-terminal amino acid sequence of the PLA2 II was SVYQF. Temperature and pH optimums of the PLA2 II were at around 50C and pH 9.0, respectively, and the enzyme activity was enhanced ...

  16. [Manoalide: a new phospholipase A2 inhibitor of marine origin with potential immunoregulatory effect].

    Science.gov (United States)

    Mayer, A M

    1989-01-01

    Manoalide, a non-steroidal sesterterpenoid isolated from a marine sponge, is a potent analgesic and antiinflammatory compound. Manoalide inhibits phospholipase A2 from extracellular sources (snake venoms, bee, etc.), the release of arachidonic acid from rabbit polymorphonuclear leukocytes as well as calcium mobilization. This suggests that the anti-inflamatory effect might be caused by the regulation of eicosanoid biosynthesis. The macrophage plays a major role in the immune response and the inflammatory process, it has the capacity to synthesize and secrete arachidonic acid oxygenation products derived from both cyclooxygenase and lipoxygenase catalyzed pathways, and has been used extensively to study the effect of inhibitors of phospholipases, cyclooxygenase and lipoxygenase enzymes. Our results demonstrate that Manoalide modified the release of arachidonic acid and its further metabolism into prostaglandins and leukotrienes in mouse cultured peritoneal macrophages stimulated by phorbol myristate acetate, calcium ionophore A23187 and zymosan. Since eicosanoids have been shown to cause pain, we studied the possibility that the analgesic effect of Manoalide might be correlated with a decrease of eicosanoid release in vivo. The fact that Manoalide reduced both zymosan-induced peritoneal writhing in the mouse and the synthesis of both 6-keto-prostaglandin F1 alfa and leukotriene C4 suggests that the analgesic effect of Manoalide is at least in part linked to the inhibition of eicosanoid production in vivo. Since it has been shown that eicosanoids have immunoregulatory functions, a future possibility is that a phospholipase A2 inhibitor such as Manoalide may prove useful to investigate the biological role of eicosanoid metabolites on the immune function.

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

    Institute of Scientific and Technical Information of China (English)

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

    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 LpPLA2 inhibitor darapladib, which seem to challenge the pathogenic role of Lp-PLA2, will also be discussed.

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

    Science.gov (United States)

    Shit, Sanjoy; Atreja, S K

    2004-05-01

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

  19. Role of phospholipase A2 in the induction of drip loss in porcine muscle

    DEFF Research Database (Denmark)

    Poulsen, Kristian A; Young, Jette F; Theil, Peter;

    2007-01-01

    The role of phospholipase A2 in the induction of drip loss from pig muscle has been investigated. In samples from porcine M. longissimus dorsi, total PLA2 activity as well as mRNA and protein levels of the group VIA iPLA2 (iPLA2-VIA) increased during the initial 4 h post-mortem period. Morphologi......The role of phospholipase A2 in the induction of drip loss from pig muscle has been investigated. In samples from porcine M. longissimus dorsi, total PLA2 activity as well as mRNA and protein levels of the group VIA iPLA2 (iPLA2-VIA) increased during the initial 4 h post-mortem period...... different breeds of pigs with variations in drip loss revealed no clear correlation between drip loss level and iPLA2-VIA expression. Together, these data indicate that during the post-mortem period, iPLA2-VIA expression and activity is increased at the muscle fiber membranes. PLA2 activity may affect...

  20. Quercetin modulates activities of Taiwan cobra phospholipase A2 via its effects on membrane structure and membrane-bound mode of phospholipase A2

    Indian Academy of Sciences (India)

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

    2012-06-01

    The goal of the present study is to elucidate the mechanism of quercetin on modulating Naja naja atra phospholipase A2 (PLA2) activities. Sphingomyelin inhibited PLA2 enzymatic activity and membrane-damaging activity against egg yolk phosphatidylcholine (EYPC), while cholesterol and quercetin abrogated the sphingomeyelin inhibitory effect. Quercetin incorporation led to a reduction in PLA2 enzymatic activity and membrane-damaging activity toward EYPC/sphingomyelin/cholesterol vesicles. Both cholesterol and quercetin increased detergent resistance and reduced membrane fluidity of EYPC/sphingomyelin vesicles. Quercetin reduced detergent insolubility but increased ordered lipid packing of EYPC/sphingomyelin/cholesterol vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that quercetin altered the membrane-bound mode of PLA2 differently upon absorption onto the membrane bilayers of different lipid compositions. However, 8-anilinonaphthalene sulphonate-binding assay revealed that quercetin marginally affected the interaction between active site of PLA2 with phospholipid vesicles. Collectively, our data indicate that membrane-inserted quercetin modulates PLA2 interfacial activity and membrane-damaging activity via its effects on membrane structure and membrane-bound mode of PLA2.

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Possible involvement of phospholipase A2 and cyclooxygenase in stimulatory action of L-histidine on protein synthesis in L6 myotubes

    OpenAIRE

    Yagasaki, Kazumi; Hatano, Naoko; Fujii, Motoki; Miura, Yutaka; Funabiki, Ryuhei

    2002-01-01

    Effects of L-histidine and related compounds on protein synthesiswere studied in cultured L6 myotubes. L-Histidine specifically stimulated protein synthesis, whereas D-histidine, histamine, L-arginine and L-lysine did not. Inhibitors of phospholipase A2, phospholipase C and cyclooxygenase intercepted the stimulatory action of L-histidine on protein synthesis, while inhibitors of protein kinase C and 5-lipoxygenase did not. These results suggest an involvement of phospholipase A2 and cyclooxyg...

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

    OpenAIRE

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

    2014-01-01

    The M-type phospholipase A2 receptor (PLA2R) is expressed in podocytes in human glomeruli. Group IB secretory phospholipase A2 (sPLA2 IB), which is one of the ligands of the PLA2R, is more highly expressed in chronic renal failure patients than in controls. However, the roles of the PLA2R and sPLA2 IB in the pathogenesis of glomerular diseases are unknown. In the present study, we found that more podocyte apoptosis occurs in the kidneys of patients with higher PLA2R and serum sPLA2 IB levels....

  6. Comparative immunohistochemical study of group II (synovial-type) and group IV (cytosolic) phospholipases A2 in disc prolapse tissue

    OpenAIRE

    Habtemariam, A.; Grönblad, M; Virri, J.; Airaksinen, O.; Luukkonen, M; Turunen, V.; Savolainen, S; Karaharju, E.

    1998-01-01

    Phospholipase A2 (PLA2) has been suggested to be present in herniated disc tissue and it could possibly be involved in sciatica/ discogenic back pain mechanisms. In the present study the occurrence of two different phospholipase A2 enzymes, (1) low molecular weight (14 kDa) group II synovial-type (sPLA2) and (2) high molecular weight (85 kDa) group IV cytosolic (cPLA2), were compared. Fifty-three disc prolapses obtained at disc operations were analyzed by immunohistochemistry, using anti-huma...

  7. Comparison of the activities of wild type and mutant enhancing factor/mouse secretory phospholipase A2 proteins

    Indian Academy of Sciences (India)

    Bhakti M Kirtane; Rita Mulherkar

    2002-09-01

    Enhancing factor (EF) protein, an isoform of secretory phospholipase A2 (PLA2), was purified as a modulator of epidermal growth factor from the small intestine of the Balb/c mouse. It was for the first time that a growth modulatory property of sPLA2 was demonstrated. Deletion mutation analysis of EF cDNA carried out in our laboratory showed that enhancing activity and phospholipase activity are two separate activities that reside in the same molecule. In order to study the specific amino acids involved in each of these activities, two site-directed mutants of EF were made and expressed in vitro. Comparison of enhancing activity as well as phospholipase A2 activity of these mutant proteins with that of wild type protein helped in identification of some of the residues important for both the activities.

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

    Science.gov (United States)

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

    2016-06-01

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

  9. Antibodies to m-type phospholipase A2 receptor in children with idiopathic membranous nephropathy.

    Science.gov (United States)

    Kumar, Vinod; Ramachandran, Raja; Kumar, Ashwani; Nada, Ritambhra; Suri, Deepti; Gupta, Anju; Kohli, Harbir Singh; Gupta, Krishan Lal; Jha, Vivekanand

    2015-08-01

    Idiopathic membranous nephropathy (IMN), the commonest cause of adult nephrotic syndrome (NS), accounts for only a minority of paediatric NS. Antibodies to m-type phospholipase A2 receptor (PLA2R) are seen in two-thirds of adult IMN cases. PLA2R staining in glomerular deposits is observed in 74% and 45% of adult and paediatric IMN cases, respectively. However, there are no reports of anti-PLA2R in paediatric IMN. We evaluated anti-PLA2R levels and PLA2R in gloemrular deposits in paediatric IMN seen at our center. Five cases were enrolled, all the cases stained for PLA2R in glomeruli and three (60%) had antibodies to PLA2R antigen. There was a parellel reduction in proteinuria and anti-PLA2R titer. The present report suggests that PLA2R has a contributory role in the pathogenesis of paediatric IMN.

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve cell

    International Nuclear Information System (INIS)

    Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A2 acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment

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

    DEFF Research Database (Denmark)

    Leidy, Chad; Ocampo, Jackson; Duelund, Lars;

    2011-01-01

    . Differential scanning calorimetry results show that this preferential hydrolysis in the presence of lipid domains leads to a membrane system with a higher-temperature melting profile due to enrichment in DSPC. Together, these results show that the presence of lipid domains can induce specificity......Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (Lα/Lβ) lipid domains. In this work, we explore whether lipid domains can also...... direct the activity of the enzyme by inducing hydrolysis of certain lipid components due to preferential activity of the enzyme toward lipid domains susceptible to sPLA(2). Specifically, we show that the presence of Lα/Lβ and Lα/Lβ, phase coexistence in a 1,2-dimyristoyl-sn-glycero-3-PhosPhocholine (DMPC...

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

    Science.gov (United States)

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

    2015-03-01

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

  14. Gene cloning, expression, purification and characterization of lipoprotein- associated phospholipase A2 in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Fu-junZHANG; Yi-pingWANG

    2005-01-01

    AIM To express and purify Lipoprotein -associated phospholipase A2 (Lp-PLA2), and to establish a screening model for Lp-PLA2 inhibitors through the recombinant Lp-PLA2. METHODS The full-length gene of Lp-PLA2 was cloned from the differentiated THP-1 cells by RT-PCR and PCR. The Lp-PLA2 gene was subcloned into the Pichia expression vector pPIC9 and introduced a sequence encoding a C-terminal stretch of six histidine residues at the same time. The recombinant plasmid was transformed into Pichia pastoris GS115 by spheroplasting and the gene was then integrated into the GS115 genome. Lp- PLA2 was expressed in the yeast strain GS115 by inducing with 0.5% methanol.

  15. Role of the Phospholipase A2 Receptor in Liposome Drug Delivery in Prostate Cancer Cells

    Science.gov (United States)

    2015-01-01

    The M-type phospholipase A2 receptor (PLA2R1) is a member of the C-type lectin superfamily and can internalize secreted phospholipase A2 (sPLA2) via endocytosis in non-cancer cells. sPLA2 itself was recently shown to be overexpressed in prostate tumors and to be a possible mediator of metastasis; however, little is known about the expression of PLA2R1 or its function in prostate cancers. Thus, we examined PLA2R1 expression in primary prostate cells (PCS-440-010) and human prostate cancer cells (LNCaP, DU-145, and PC-3), and we determined the effect of PLA2R1 knockdown on cytotoxicity induced by free or liposome-encapsulated chemotherapeutics. Immunoblot analysis demonstrated that the expression of PLA2R1 was higher in prostate cancer cells compared to that in primary prostate cells. Knockdown of PLA2R1 expression in PC-3 cells using shRNA increased cell proliferation and did not affect the toxicity of cisplatin, doxorubicin (Dox), and docetaxel. In contrast, PLA2R1 knockdown increased the in vitro toxicity of Dox encapsulated in sPLA2 responsive liposomes (SPRL) and correlated with increased Dox and SPRL uptake. Knockdown of PLA2R1 also increased the expression of Group IIA and X sPLA2. These data show the novel findings that PLA2R1 is expressed in prostate cancer cells, that PLA2R1 expression alters cell proliferation, and that PLA2R1 modulates the behavior of liposome-based nanoparticles. Furthermore, these studies suggest that PLA2R1 may represent a novel molecular target for controlling tumor growth or modulating delivery of lipid-based nanomedicines. PMID:25189995

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

    Science.gov (United States)

    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

  17. Purification and biochemical characterization of pancreatic phospholipase A2 from the common stingray Dasyatis pastinaca

    Directory of Open Access Journals (Sweden)

    Gargouri Youssef

    2011-02-01

    Full Text Available Abstract Background Mammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. Results A marine stingray phospholipase A2 (SPLA2 was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C in the presence of 4 mM NaTDC and 8 mM CaCl2 using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry. Conclusions SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.

  18. Characterization of phospholipase A2 from the pyloric ceca of two species of starfish, Coscinasterias acutispina and Plazaster borealis

    OpenAIRE

    Kishimura, Hideki; Hayashi, Kenji

    2005-01-01

    Phospholipase A (PLA) activities in the pyloric ceca and viscera from seven species of marine invertebrates (four starfish, one sea urchin, and two shellfish) were determined. Relatively high PLA specific activities were found in the pyloric ceca of two species of starfish (Coscinasterias acutispina and Plazaster borealis). Phospholipase A2s (PLA2s) were partially purified from the pyloric ceca of the starfish, C. acutispina PLA2 (C-PLA2) and P. borealis PLA2 (P-PLA2). The C-PLA2 and P-PLA2 m...

  19. Sensitization to autoimmune hepatitis in group VIA calcium-independent phospholipase A2-null mice led to duodenal villous atrophy with apoptosis, goblet cell hyperplasia and leaked bile acids.

    Science.gov (United States)

    Jiao, Li; Gan-Schreier, Hongying; Tuma-Kellner, Sabine; Stremmel, Wolfgang; Chamulitrat, Walee

    2015-08-01

    Chronic bowel disease can co-exist with severe autoimmune hepatitis (AIH) in an absence of primary sclerosing cholangitis. Genetic background may contribute to this overlap syndrome. We previously have shown that the deficiency of iPLA2β causes an accumulation of hepatocyte apoptosis, and renders susceptibility for acute liver injury. We here tested whether AIH induction in iPLA2β-null mice could result in intestinal injury, and whether bile acid metabolism was altered. Control wild-type (WT) and female iPLA2β-null (iPLA2β(-/-)) mice were intravenously injected with 10mg/kg concanavalinA (ConA) or saline for 24h. ConA treatment of iPLA2β(-/-) mice caused massive liver injury with increased liver enzymes, fibrosis, and necrosis. While not affecting WT mice, ConA treatment of iPLA2β(-/-) mice caused severe duodenal villous atrophy concomitant with increased apoptosis, cell proliferation, globlet cell hyperplasia, and endotoxin leakage into portal vein indicating a disruption of intestinal barrier. With the greater extent than in WT mice, ConA treatment of iPLA2β(-/-) mice increased jejunal expression of innate response cytokines CD14, TNF-α, IL-6, and SOCS3 as well as chemokines CCL2 and the CCL3 receptor CCR5. iPLA2β deficiency in response to ConA-induced AIH caused a significant decrease in hepatic and biliary bile acids, and this was associated with suppression of hepatic Cyp7A1, Ntcp and ABCB11/Bsep and upregulation of intestinal FXR/FGF15 mRNA expression. The suppression of hepatic Ntcp expression together with the loss of intestinal barrier could account for the observed bile acid leakage into peripheral blood. Thus, enteropathy may result from acute AIH in a susceptible host such as iPLA2β deficiency. PMID:25957555

  20. Expression and location of mRNAs encoding multiple forms of secretory phospholipase A2 in the rat retina

    DEFF Research Database (Denmark)

    Christoffersen, Nanna R; Barreiro, Sebastian G; Bazan, Nicolas G;

    2004-01-01

    Low-molecular-weight secretory phospholipases A(2) (sPLA(2)s) are a subgroup of PLA(2)s, which are secreted, bind to receptors, and may act as intercellular signaling modulators. At least 10 different groups have been characterized in mammals, and there is expanding evidence of the significance o...

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

    NARCIS (Netherlands)

    Code, Christian; Domanov, Y.A.; Killian, J.A.; Kinnunen, P.K.J.

    2009-01-01

    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-dip

  2. Direct Imaging by Cryo-TEM Shows Membrane Break-up by Phospholipase A2 Enzymatic Activity

    DEFF Research Database (Denmark)

    Callisen, Thomas Hønger; Talmon, Y.

    1998-01-01

    Phospholipid hydrolysis to free fatty acid and l-lyso-phospholipid by water-soluble phospholipase A(2) (PLA(2)) at the surface of lipid membranes exhibits a poorly understood transition from a low-activity lag phase to a burst regime of rapid hydrolysis. Understanding this kinetic phenomenon may ...

  3. Increased type IIA secretory phospholipase A(2) expression contributes to oxidative stress in end-stage renal disease

    NARCIS (Netherlands)

    van der Giet, Markus; Toelle, Markus; Pratico, Domenico; Lufft, Volkmar; Schuchardt, Mirjam; Hoerl, Matthias P.; Zidek, Walter; Tietge, Uwe J. F.

    2010-01-01

    End-stage renal disease (ESRD) patients exhibit increased in vivo oxidative stress conceivably contributing to cardiovascular mortality. The type IIA secretory phospholipase A(2) (sPLA(2)) has proatherogenic activity. We explored the hypothesis that sPLA(2) contributes to oxidative stress generation

  4. Characterization and location of secretory phospholipase A2 groups IIE, V, and X in the rat brain

    DEFF Research Database (Denmark)

    Kolko, M.; Christoffersen, Nanna Rønbjerg; Barreiro, S.G.;

    2006-01-01

    Secretory phospholipases A2 (sPLA2) form a diverse family of enzymes involved in a variety of physiologic and pathologic processes. Common among all sPLA2 is the ability to cleave the second position of phospholipids, thereby releasing fatty acid and a lysophospholipid. Several sPLA2 have been cl...

  5. Expression of Type IIA Secretory Phospholipase A 2 Inhibits Cholesteryl Ester Transfer Protein Activity in Transgenic Mice

    NARCIS (Netherlands)

    Hurt-Camejo, Eva; Gautier, Thomas; Rosengren, Birgitta; Dikkers, Arne; Behrendt, Margareta; Grass, David S.; Rader, Daniel J.; Tietge, Uwe J. F.

    2013-01-01

    Objective High circulating levels of group IIA secretory phospholipase A(2) (sPLA(2)-IIA) activity and mass are independent cardiovascular risk factors. Therefore, inhibition of sPLA(2)-IIA may be a target for the treatment of atherosclerotic cardiovascular disease. The present study evaluated the e

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

    Directory of Open Access Journals (Sweden)

    Juan Carlos Alarcón Pérez

    2012-12-01

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

  7. Phospholipase A2 as a point of care alternative to serum amylase and pancreatic lipase

    Science.gov (United States)

    Liu, Nathan J.; Chapman, Robert; Lin, Yiyang; Bentham, Andrew; Tyreman, Matthew; Philips, Natalie; Khan, Shahid A.; Stevens, Molly M.

    2016-06-01

    Acute pancreatitis is a relatively common and potentially fatal condition, but the presenting symptoms are non-specific and diagnosis relies largely on the measurement of amylase activity by the hospital clinical laboratory. In this work we develop a point of care test for pancreatitis measuring concentration of secretory phospholipase A2 group IB (sPLA2-IB). Novel antibodies for sPLA2-IB were raised and used to design an ELISA and a lateral flow device (LFD) for the point of care measurement of sPLA2-IB concentration, which was compared to pancreatic amylase activity, lipase activity, and sPLA2-IB activity in 153 serum samples. 98 of these samples were obtained from the pathology unit of a major hospital and classified retrospectively according to presence or absence of pancreatitis, and the remaining 55 were obtained from commercial sources to serve as high lipase (n = 20), CA19-9 positive (n = 15), and healthy (n = 20) controls. sPLA2-IB concentration correlated well with the serum activity of both amylase and lipase, and performed at least as well as either markers in the differentiation of pancreatitis from controls.Acute pancreatitis is a relatively common and potentially fatal condition, but the presenting symptoms are non-specific and diagnosis relies largely on the measurement of amylase activity by the hospital clinical laboratory. In this work we develop a point of care test for pancreatitis measuring concentration of secretory phospholipase A2 group IB (sPLA2-IB). Novel antibodies for sPLA2-IB were raised and used to design an ELISA and a lateral flow device (LFD) for the point of care measurement of sPLA2-IB concentration, which was compared to pancreatic amylase activity, lipase activity, and sPLA2-IB activity in 153 serum samples. 98 of these samples were obtained from the pathology unit of a major hospital and classified retrospectively according to presence or absence of pancreatitis, and the remaining 55 were obtained from commercial sources to

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

    OpenAIRE

    Huang, Qi; Wu, Yuan; Qin, Chao; HE, WENWU; Wei, Xing

    2014-01-01

    The phospholipase A (PLA)2 family is the most complex gene family of phospholipases and plays a crucial role in a number of physiological activities. However, the phylogenetic background of the PLA2 gene family and the amino acid residues of the PLA2G7 gene following positive selection gene remain undetermined. In this study, we downloaded 49 genomic data sets of PLA from different species, including the human, house mouse, Norway rat, pig, dog, chicken, cattle, African clawed frog, Sumatran ...

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

    Science.gov (United States)

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

    2015-10-19

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

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

    Directory of Open Access Journals (Sweden)

    Rebai Tarek

    2011-06-01

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

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

    Science.gov (United States)

    Truong, Luan D; Seshan, Surya V

    2015-12-15

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

  12. Progress on the M-type phospholipase A2 receptor in idiopathic membranous nephropathy

    Institute of Scientific and Technical Information of China (English)

    Wang Chao; Lu Huan; Yang Cui; Luo Yuezhong

    2014-01-01

    Objective To highlight current knowledge about M-type phospholipase A2 receptor (PLA2R) which is the first human autoantigen discovered in adult idiopathic membranous nephropathy.Data sources Relevant articles published in English from 2000 to present were selected from PubMed.Searches were made using the terms "idiopathic membranous nephropathy,M-type PLA2R and podocyte." Study selection Articles studying the role of M-type PLA2R in idiopathic membranous nephropathy were reviewed.Articles focusing on the discovery,detection and clinical observation of anti-PLA2R antibodies were selected.Results M-type PLA2R is a member of the mannose receptor family of proteins,locating on normal human glomeruli as a transmembrane receptor.The anti-PLA2R in serum samples from MN were primarily IgG4 subclass.Technologies applied to detect anti-PLA2R autoantibody are mainly WB,lIFT,ELISA and so on.Studies from domestic and overseas have identified a strongly relationship between circulating anti-PLA2R levels and disease activity.Conclusion Recent discoveries corresponding to PLA2R facilitate a better understanding on IMN pathogenesis and may provide a new tool to its diagnosis,differential diagnosis,risk evaluation,response monitoring and patient-specific treatment.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2009-10-01

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

  16. Cellular volume regulation by anoctamin 6: Ca²⁺, phospholipase A2 and osmosensing.

    Science.gov (United States)

    Sirianant, Lalida; Ousingsawat, Jiraporn; Wanitchakool, Podchanart; Schreiber, Rainer; Kunzelmann, Karl

    2016-02-01

    During cell swelling, Cl(-) channels are activated to lower intracellular Cl(-) concentrations and to reduce cell volume, a process termed regulatory volume decrease (RVD). We show that anoctamin 6 (ANO6; TMEM16F) produces volume-regulated anion currents and controls cell volume in four unrelated cell types. Volume regulation is compromised in freshly isolated intestinal epithelial cells from Ano6-/- mice and also in lymphocytes from a patient lacking expression of ANO6. Ca(2+) influx is activated and thus ANO6 is stimulated during cell swelling by local Ca(2+) increase probably in functional nanodomains near the plasma membrane. This leads to stimulation of phospholipase A2 (PLA2) and generation of plasma membrane lysophospholipids, which activates ANO6. Direct application of lysophospholipids also activates an anion current that is inhibited by typical ANO6 blocker. An increase in intracellular Ca(2+) supports activation of ANO6, but is not required when PLA2 is fully activated, while re-addition of arachidonic acid completely blocked ANO6. Moreover, ANO6 is activated by low intracellular Cl(-) concentrations and may therefore operate as a cellular osmosensor. High intracellular Cl(-) concentration inhibits ANO6 and activation by PLA2. Taken together, ANO6 supports volume regulation and volume activation of anion currents by action as a Cl(-) channel or by scrambling membrane phospholipids. Thereby, it may support the function of LRRC8 proteins.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Daishi Yui

    Full Text Available 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.

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

    Science.gov (United States)

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

    2016-07-22

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Zhao, Hongxia; Kinnunen, Paavo K. J.

    2003-01-01

    The antimicrobial peptides magainin 2, indolicidin, and temporins B and L were found to modulate the hydrolytic activity of secretory phospholipase A2 (sPLA2) from bee venom and in human lacrimal fluid. More specifically, hydrolysis of phosphatidylcholine (PC) liposomes by bee venom sPLA2 at 10 μM Ca2+ was attenuated by these peptides while augmented product formation was observed in the presence of 5 mM Ca2+. The activity of sPLA2 towards anionic liposomes was significantly enhanced by the antimicrobial peptides at low [Ca2+] and was further enhanced in the presence of 5 mM Ca2+. Similarly, with 5 mM Ca2+ the hydrolysis of anionic liposomes was enhanced significantly by human lacrimal fluid sPLA2, while that of PC liposomes was attenuated. These results indicate that concerted action of antimicrobial peptides and sPLA2 could improve the efficiency of the innate response to infections. Interestingly, inclusion of a cationic gemini surfactant in the vesicles showed an essentially similar pattern on sPLA2 activity, suggesting that the modulation of the enzyme activity by the antimicrobial peptides may involve also charge properties of the substrate surface. PMID:12604528

  2. Assay of phospholipases A2 and their inhibitors by kinetic analysis in the scooting mode

    Directory of Open Access Journals (Sweden)

    Mahendra Kumar Jain

    1992-01-01

    Full Text Available Several cellular processes are regulated by interfacial catalysis on biomembrane surfaces. Phospholipases A2 (PLA2 are interesting not only as prototypes for interfacial catalysis, but also because they mobilize precursors for the biosynthesis of eicosanoids and platelet activating factor, and these agents ultimately control a wide range of secretory and inflammatory processes. Since PLA2 carry out their catalytic function at membrane surfaces, the kinetics of these enzymes depends on what the enzyme ‘sees’ at the interface, and thus the observed rate is profoundly influenced by the organization and dynamics of the lipidwater interface (‘quality of the interface’. In this review we elaborate the advantages of monitoring interfacial catalysis in the scooting mode, that is, under the conditions where the enzyme remains bound to vesicles for several thousand catalytic turnover cycles. Such a highly processive catalytic turnover in the scooting mode is useful for a rigorous and quantitative characterization of the kinetics of interfacial catalysis. This analysis is now extended to provide insights into designing strategy for PLA2 assays and screens for their inhibitors.

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

    Science.gov (United States)

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

    2005-06-01

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

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

    Science.gov (United States)

    Schröter, Jenny; Süß, Rosmarie; Schiller, Jürgen

    2016-07-15

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

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

    Directory of Open Access Journals (Sweden)

    Mark Merchant

    2011-01-01

    Full Text Available 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.

  6. Snake venom phospholipase A2 inhibitors: medicinal chemistry and therapeutic potential.

    Science.gov (United States)

    Marcussi, Silvana; Sant'Ana, Carolina D; Oliveira, Clayton Z; Rueda, Aristides Quintero; Menaldo, Danilo L; Beleboni, Rene O; Stabeli, Rodrigo G; Giglio, José R; Fontes, Marcos R M; Soares, Andreimar M

    2007-01-01

    Phospholipases A2 (PLA2s) are commonly found in snake venoms from Viperidae, Hydrophidae and Elaphidae families and have been extensively studied due to their pharmacological and physiopathological effects in living organisms. This article reports a review on natural and artificial inhibitors of enzymatic, toxic and pharmacological effects induced by snake venom PLA2s. These inhibitors act on PLA2s through different mechanisms, most of them still not completely understood, including binding to specific domains, denaturation, modification of specific amino acid residues and others. Several substances have been evaluated regarding their effects against snake venoms and isolated toxins, including plant extracts and compounds from marine animals, mammals and snakes serum plasma, in addition to poly or monoclonal antibodies and several synthetic molecules. Research involving these inhibitors may be useful to understand the mechanism of action of PLA2s and their role in envenomations caused by snake bite. Furthermore, the biotechnological potential of PLA2 inhibitors may provide therapeutic molecular models with antiophidian activity to supplement the conventional serum therapy against these multifunctional enzymes. PMID:17456038

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

    Science.gov (United States)

    Hains, P G; Broady, K W

    2000-02-15

    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

  8. Phospholipase A2 changes and its significance on brain tissue of rat in severe acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Yao Xuan; Chen Xi; Ji Zongzheng

    2007-01-01

    Objective To survey changes and the significance of phospholipase A2(PLA2) on brain tissue of SD rat in acute pancreatitis. Methods With retrograde injection of 3% taurocholate sodium into pancreatic and biliary duct, rat model of severe acute pancreatitis (SAP) was made,and it included four groups: the control group, the sham-operation group, the SAP group and the PLA2 inhibitor-treated group of SAP. Serum amylases, PLA2 and PLA2 in brain tissue were measured and the brain tissue changes were observed. Results There were no significant difference in serum amylases, PLA2 and PLA2 in brain tissue between the sham-operation and the control groups; the levels of serum amylases, PLA2 and PLA2 in brain tissue in the SAP group were higher than those in the control. In the SAP group expansion and hemorrhage of meninges, intracephalic arteriolar hyperemia, in meninges and cephalic-parenchyma infiltration of inflammatory cells and interval broaden were observed, significant differences were found between two groups.Compared with the SAP group, the level of serum amylase, PLA2 and PLA2 in brain tissue were reduced significantly in the treatment group of SAP. Pathological damages in the treatment group were significantly reduced when compared with the SAP group. Conclusion PLA2 might play an important role in brain tissue damages in severe acute pancreatitis.

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

    Science.gov (United States)

    Schröter, Jenny; Süß, Rosmarie; Schiller, Jürgen

    2016-07-15

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

  10. EXPRESSION OF A BEE-VENOM PHOSPHOLIPASE A2 FROM APIS CERANA CERANA IN E,.qCHERICHIA COLI

    Institute of Scientific and Technical Information of China (English)

    Li-rongShen; Jia-anCheng; Chuan-xiZhang

    2004-01-01

    The venomous phospholipase A2 (AcPLA2) coding reading region of the Chinese honeybee (Apis cerana cerana), which is composed of 405 bp encoding a mature glycosylated peptide with 134 amino residues was transformed into the expression vector pETblue-1. Then the recombinant vector was introduced into Escherichia coli Tuner (DE3) plac I for expression. Analysis result of SDS-PAGE showed that the expression products had a protein band of about 15 kD. Detection of western blot using ant-European honeybee (Apis mellifera) phospholipase A2 (AmPLA2) polyclonal serum as the first antibody showed that the expression products appeared a special blot same as the native AmPLA2.The result demonstrated that the AcPLA2 peptide had been expressed in E. coli and the AcPLA2 has the similar antigenicity as the AmPLA2.

  11. Synergistic Effects of Secretory Phospholipase A2 from the Venom of Agkistrodon piscivorus piscivorus with Cancer Chemotherapeutic Agents

    OpenAIRE

    Jennifer Nelson; Kristen Barlow; D. Olin Beck; Amanda Berbert; Nathan Eshenroder; Lyndee Francom; Mark Pruitt; Kina Thompson; Kyle Thompson; Brian Thurber; Celestine H.-Y. Yeung; Allan M. Judd; Bell, John D.

    2013-01-01

    Healthy cells typically resist hydrolysis catalyzed by snake venom secretory phospholipase A2. However, during various forms of programmed cell death, they become vulnerable to attack by the enzyme. This observation raises the question of whether the specificity of the enzyme for dying cells could be used as a strategy to eliminate tumor cells that have been intoxicated but not directly killed by chemotherapeutic agents. This idea was tested with S49 lymphoma cells and a broad range of antine...

  12. Evaluation of different glycoforms of honeybee venom major allergen phospholipase A2 (Api m 1) produced in insect cells

    DEFF Research Database (Denmark)

    Blank, Simon; Seismann, Henning; Plum, Melanie;

    2011-01-01

    Allergic reactions to hymenoptera stings are one of the major reasons for IgE-mediated anaphylaxis. However, proper diagnosis using venom extracts is severely affected by molecular cross-reactivity. In this study recombinant honeybee venom major allergen phospholipase A2 (Api m 1) was produced......-derived recombinant Api m 1 with defined CCD phenotypes might provide further insights into hymenoptera venom IgE reactivities and contribute to an improved diagnosis of hymenoptera venom allergy....

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

    OpenAIRE

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

    2016-01-01

    Oxaliplatin, a chemotherapy drug used to treat colorectal cancer, induces specific sensory neurotoxicity signs that are aggravated by cold and mechanical stimuli. Here we examined the preventive effects of Bee Venom (BV) derived phospholipase A2 (bvPLA2) on oxaliplatin-induced neuropathic pain in mice and its immunological mechanism. The cold and mechanical allodynia signs were evaluated by acetone and von Frey hair test on the hind paw, respectively. The most significant allodynia signs were...

  14. Hydrolysis of human milk fat globules by pancreatic lipase: role of colipase, phospholipase A2, and bile salts.

    OpenAIRE

    Bläckberg, L; Hernell, O.; Olivecrona, T

    1981-01-01

    Human milk fat globules were used to explore how dietary triglycerides are hydrolyzed by pancreatic lipase. These triglycerides were hydrolyzed very slowly by lipase alone as if the surface layer of proteins and phospholipids impeded the action of the enzyme. The inhibition of lipase activity could be overcome by addition either of colipase or of pancreatic phospholipase A2. Colipase enhanced triglyceride hydrolysis in a dose-dependent manner whether bile salts were present or not. Bile salts...

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

    OpenAIRE

    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 pancreat...

  16. Pathophysiological role of secretory type I and II phospholipase A2 in acute pancreatitis: an experimental study in rats.

    OpenAIRE

    Uhl, W.; Schrag, H J; Schmitter, N; Nevalainen, T J; AUFENANGER, J; Wheatley, A. M.; Büchler, M W

    1997-01-01

    BACKGROUND: In human acute pancreatitis two different types of secretory phospholipase A2 (PLA2) have been found. AIM: To analyse the specific pattern of distribution of these PLA2 activities and their pathophysiological role in experimental acute pancreatitis. SUBJECTS AND METHODS: Catalytic activities of secretory type I (pancreatic) and type II (non-pancreatic) PLA2 and the protein concentration of immunoreactive pancreatic PLA2 (IR-PLA2) in serum and pancreatic tissue of rats with cerulei...

  17. Phospholipid Synthesis by Staphylococcus aureus during (Sub)Lethal Attack by Mammalian 14-Kilodalton Group IIA Phospholipase A2

    OpenAIRE

    Foreman-Wykert, Amy K.; Weiss, Jerrold; Elsbach, Peter

    2000-01-01

    Killing of gram-positive bacteria by mammalian group IIA phospholipases A2 (PLA2) requires the catalytic activity of the enzyme. However, nearly complete degradation of the phospholipids can occur with little effect on bacterial viability, suggesting that PLA2-treated bacteria can biosynthetically replace phospholipids that are lost due to PLA2 action. In the presence of albumin, phospholipid degradation products are quantitatively sequestered extracellularly. In the absence of albumin, the b...

  18. Increased Serum Phospholipase A2 Activity in Advanced Chronic Liver Disease as an Expression of the Acute Phase Response

    OpenAIRE

    Mario Pirisi; Carlo Fabris; Maria Piera Panozzo; Giorgio Soardo; Pierluigi Toniutto; Ettore Bartou

    1993-01-01

    Phospholipase A2 (PLA2) modifications were investigated in patients with acute and chronic liver diseases, PLA2 variations were related to indices of liver function as well as to parameters of the acute phase response. Serum PLA2 activity modifications were f1uorimetrically measured in 105 patients affected by acute and chronic liver diseases or extra-hepatic diseases. One-way ANOV A demonstrated a significant difference among groups (F= 4.53, P

  19. Bleeding diathesis and gastro-duodenal ulcers in inherited cytosolic phospholipase-A2 alpha deficiency.

    Science.gov (United States)

    Faioni, E M; Razzari, C; Zulueta, A; Femia, E A; Fenu, L; Trinchera, M; Podda, G M; Pugliano, M; Marongiu, F; Cattaneo, M

    2014-12-01

    Arachidonic acid (AA), when cleaved from phospholipids by cytosolic phospholipase A2 alpha (cPLA2a), generates eicosanoids, with pro-hemostatic, pro-inflammatory, vasoactive and gastro-protective functions. We describe a patient (27-year-old man) and his twin-sister with early-onset bleeding diathesis and recurrent gastro-intestinal (GI) ulcers. Platelet aggregation/δ-granules secretion by collagen was impaired, but normal by AA; serum levels of thromboxane (Tx) B2 and 12-hydroxyeicosatetraenoic acid, and urinary levels of 11-dehydro-TxB2 were extremely low. Patients were homozygous for 1723G>C transition in PLA2G4A gene, which changed the codon for Asp575 to His. GI ulcers affected 5/14 heterozygous ( 60 years) family members; none had bleeding diathesis. The proband, his sister and mother also had mildly reduced factor XI levels. Platelet messenger RNA expression did not differ among subjects with different PLA2G4A genotypes. Conversely, platelet cPLA2a was undetectable by Western Blotting in the proband and his sister, and decreased in 1723G>C heterozygous subjects, suggesting that the variant is transcribed, but not translated or translated into an unstable protein. We described a syndromic form of deficiency of cPLA2a , characterised by recurrent GI ulcers and bleeding diathesis, associated with mild inherited deficiency of factor XI. Unlike other reported patients with cPLA2a deficiency, these patients had extremely low levels of platelet TxA2 biosynthesis.

  20. Expression and purification of lipoprotein-associated phospholipase A2, a key enzyme involved in atherosclerosis

    Institute of Scientific and Technical Information of China (English)

    Fu-jun ZHANG; Mao-jun CAI; Jing-kang SHEN; Yi-ping WANG

    2006-01-01

    Aim: To express and purify lipoprotein-associated phospholipase A2 (Lp-PLA2), and to establish a screening model for Lp-PLA2 inhibitors using the expressed Lp-PLA2. Methods: We cloned the full-length cDNA of Lp-PLA2 from differentiated THP-1 cells, and subcloned the cDNA into the baculovirus transfer vector pFastBacl. In addition, we introduced an N-terminal Kozak sequence for highlevel translation initiation and a C-terminal sequence of 6 histidine residues for purification. The fusion enzyme was expressed in Sf9 insect cells and purified by Ni2+ affinity chromatography. Recombinant Lp-PLA2 activity was measured using [3H]PAF as a substrate, and we examined the enzyme activity of recombinant Lp-PLA2 pretreated with the known Lp-PLA2 inhibitor SB435495. Results: We successfully cloned the full-length Lp-PLA2 gene from differentiated THP-1 cells. The fusion enzyme was expressed in Sf9 insect cells at a high level and purified efficiently through a 2-step procedure. The recombinant Lp-PLA2 activity was measured using [3H]PAF as a substrate, and proved to be enzymatically active. Lp-PLA2 inhibitor SB435495 produced a good inhibition curve for inhibition of recombinant Lp-PLA2 with an IC50 of 57±1 μmol/L. Conclusion: We expressed and purified Lp-PLA2 at a high level in insect cell-baculovirus expression system. The yield ratio was much greater than that obtained from human plasma and we established a screening model for Lp-PLA2 inhibitors using the expressed Lp-PLA2.

  1. Trypanocidal efficacy of two indigeneous ethanolic plant extracts (Mimosa pigra and Ipomoea asarifolia) against Trypanosoma evansi phospholipase A2 activity

    Institute of Scientific and Technical Information of China (English)

    Yusuf Alkali; A K Gana; Abdulkadir A; Nzelibe C Humphrey

    2015-01-01

    Objective:To study the inhibitory activity of ethanolic extract from Mimosa pigra and Ipomoea asarifolia against Trypanosoma evansi (T. evansi) calcium dependent phospholipase A2. Methods: The calcium dependent phospholipase A2 (E C 3.1.1.4) enzyme was isolated from T. evansi and purified to electrophoretic homogeneity under non denaturing conditions. It was solubilized from T. evansi cells recovered from white albino rats which were previously inoculated by intraperitoneal injection of infected camel blood. Two indigeneous ethanolic plant extracts used locally for treatment of trypanosomiasis were tested for the inhibition of phospholipases A2. Results: Double reciprocal plots of the initial velocity data of the inhibition by the indigenous plant extracts revealed a noncompetitive pattern of inhibition for the Ipomoea asarifolia and a competitive inhibition for Mimosa pigra in a dose dependent fashion. The extrapolated inhibition binding constant (Ki) of these extracts were found to be 2.0í102μg/mL and 1.12í102μg/mL respectively. Conclusions:The low Ki values obtained for these extracts towards this enzyme are an indication of high affinity of the extract or the active components (present in the plants) are for these enzyme and therefore, could be explored to serve as a cheap source of T. evansi PLA2 antidote and as well help in designing a novel drug with high efficiency.

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

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    Luís F. Leandro

    2015-03-01

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

  3. The first report on coagulation and phospholipase A2 activities of Persian Gulf lionfish, Pterois russelli, an Iranian venomous fish.

    Science.gov (United States)

    Memar, Bahareh; Jamili, Shahla; Shahbazzadeh, Delavar; Bagheri, Kamran Pooshang

    2016-04-01

    Pterois russelli is a venomous fish belonging to scorpionidae family. Regarding to high significance value for tracing potential therapeutic molecules and special agents from venomous marine creatures, the present study was aimed to characterization of the Persian Gulf lionfish venom. Proteolytic, phospholipase, hemolytic, coagulation, edematogenic and dermonecrotic activities were determined for extracted venom. The LD50 of P. russelli venom was determined by intravenous injection in white Balb/c mice. Phospholipase A2 activity was recorded at 20 μg of total venom. Coagulation activity on human plasma was shown by Prothrombin Time (PT) and activated Partial Thromboplastin Time (APTT) assays and coagulation visualized after 7 and 14 s respectively for 60 μg of crude venom. LD50 was calculated as 10.5 mg/kg. SDS-PAGE revealed the presence of major and minor protein bands between 6 and 205 kDa. Different amounts of crude venom ranged from 1.87 to 30 μg showed proteolytic activity on casein. The highest edematic activity was detected at 20 μg. Our findings showed that the edematic activity was dose dependent and persisted for 48 h after injection. The crude venom did not induce dermonecrotic activity on rabbit skin and showed no hemolytic activity on human, mouse and rabbit erythrocytes. This is the first report for phospholipase A2 and coagulation activity in venomous fish and venomous marine animals respectively. Proteolytic activity of P. russelli venom is in accordance with the other genara of scorpionidae family. According to venom activity on intrinsic and extrinsic coagulation pathways, lionfish venom would be contained an interesting pharmaceutical agent. This study is pending to further characterization of phospholipase A2, coagulation, and protease activities and also in vivo activity on animal model of surface and internal bleeding. PMID:26853495

  4. Relation of phospholipase A2-V and indoxam to hippocampal neuronal death

    Institute of Scientific and Technical Information of China (English)

    Fang Liu; Shi Wang; Yan Lin; Runhui Li; Li Ma; Yanjun Li; Qing Jin; Xiao Gong; Yuhua Chen

    2006-01-01

    BACKGROUND: V secretory phospholipase A2 (sPLA2- V ) is abundant in many mammal tissues. However, it remains unknown whether sPLA2-V causes biological or pathological response in central nervous system.OBJECTIVE: To observe the effect of phospholipase A2- V (PLA2- V ) and its inhibitor (indoxam) on hippocampal neuron survival.DESIGN: A repetitive measurement.SETTING: The Animal Center of South Carolina University.MATERIALS: Sprague-Dawley pregnancy day-7, 14, 21 female rats were selected; Reagents: sPLA2- V and indoxam were obtained from the Dennis Research Laboratories METHODS: The experiment was finished at the animal center in South Carolina University from January to December, 2004. 0, 12.5, 25, 50 and 100 μg/L sPLA2-V were added to neuron with none-MgCl2 Eagle's medium at 37 ℃, then changed to normal neuron culture medium after 3 hours. 1, 2.5, 5 and 10 μmol/L indoxam was added at 6 hours after 100 μg/L sPLA2-V was put to Day-21 SD rat hippocampal embryonic neurons with none-MgCl2 Eagle's medium at 37 ℃. After 3 hours in the inhibition experiment, it was changed to normal neuron culture medium. The embryonic hippocampal neurons were primarily cultured, and the neuron survival ratio was detected with morphological method.MAIN OUTCOME MEASURES: Survival ratio of hippocampal neurons.RESULTS: ① Effects of sPLA2-V on neuron survival: When sPLA2-V was 0, 12.5, 25, 50 and 100 μg/L, the neuron survival ratios in embryonic neurons of day-7 SD rats were (95.3±1.1)%, (81.4±3.1)%, (74.2±2.2)%,(62.4±1.7)% and (48.9±1.6)%, those in embryonic neurons of day-14 rats were (93.2±1.4)%, (74.3±1.9)%,(68.1± 1.7)%, (56.1± 1.4)% and (42.5± 1.1)%, and those in embryonic neurons of day-21 rats were (91.2±1.2)%,(69.4±2.1)%, (60.3±2.2)%, (49.1 ± 1.2)% and (35.5± 1.9)%. There were significant differences among difierent concentrations (P< 0.05). ② Effects of indoxam on neuron survival: In case of sPLA2-V 100 μg/L, the neuron survival ratios were (58.65±1

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

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    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.

  6. Fluctuation of lysosomal phospholipase A2 in experimental autoimmune uveitis in rats.

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    Ohkawa, Ei; Hiraoka, Miki; Abe, Akira; Murata, Masaki; Ohguro, Hiroshi

    2016-08-01

    Intraocular inflammation leads to oxidative stress and may generate lipid oxidation products. The present study was conducted to elucidate the pathophysiological roles of the lysosomal phospholipase A2 (LPLA2), a phospholipid-degrading enzyme, and the production of oxidized phospholipids (oxPLs) in autoimmune uveitis using a rat model. Lewis rats were immunized with a bovine interphotoreceptor retinoid-binding protein (bIRBP) peptide with complete Freund's adjuvant (CFA) to induce experimental autoimmune uveitis (EAU). The aqueous humor (AH) and serum were collected every week for 4 weeks from the immunized rats. The LPLA2 activity of the AH and serum was detected using liposomes consisting of 1,2-dioleoylphosphatidylglycerol/N-acetylsphingosine as the substrate under acidic conditions. Immunohistochemical analysis was performed using antibodies against LPLA2 and oxPLs. The ocular inflammation was exacerbated at 2 weeks after immunization. The LPLA2 activity in the rat AH was increased by EAU induction, and was concomitant with the extent of inflammation in the anterior chamber (AC). In contrast, the LPLA2 activity in the rat serum was not influenced by EAU induction. At 2 weeks after immunization, immunoreactivity of LPLA2 was observed in infiltrated macrophages in the AC and vitreous cavity of the EAU rats. Furthermore, immunoreactivity of oxPLs was observed in the infiltrated macrophages of EAU rat eyes. These results demonstrated that the LPLA2 activity of the AH is augmented with the inflammation in the AC. The high expression of LPLA2 and production of oxPLs are found in the infiltrated macrophages in the acute inflammation of EAU rats. The present findings suggest the connection between LPLA2 activity and oxPL metabolism in the inflammation sites in the eye. PMID:27344956

  7. Anti-Phospholipase A2 Receptor Antibodies in Recurrent Membranous Nephropathy

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    Kattah, Andrea; Ayalon, Rivka; Beck, Laurence H.; Sandor, Dana G.; Cosio, Fernando G.; Gandhi, Manish J.; Sethi, Sanjeev; Lorenz, Elizabeth C.; Salant, David J.; Fervenza, Fernando C.

    2015-01-01

    About 70% of patients with primary membranous nephropathy (MN) have circulating anti-phospholipase A2 receptor (PLA2R) antibodies that correlate with disease activity, but their predictive value in post-transplant (Tx) recurrent MN is uncertain. We evaluated 26 patients, 18 with recurrent MN and 8 without recurrence, with serial post-Tx serum samples and renal biopsies to determine if patients with pre-Tx anti-PLA2R are at increased risk of recurrence as compared to seronegative patients and to determine if post-Tx changes in anti-PLA2R correspond to the clinical course. In the recurrent group, 10/17 patients had anti-PLA2R at the time of Tx vs. 2/7 patients in the non-recurrent group. The positive predictive value of pre-Tx anti-PLA2R for recurrence was 83%, while the negative predictive value was 42%. Persistence or reappearance of post-Tx anti-PLA2R was associated with increasing proteinuria and resistant disease in many cases; little or no proteinuria occurred in cases with pre-Tx anti-PLA2R and biopsy evidence of recurrence in which the antibodies resolved with standard immunosuppression. Some cases with positive pre-Tx anti-PLA2R were seronegative at the time of recurrence. In conclusion, patients with positive pre-Tx anti-PLA2R should be monitored closely for recurrent MN. Persistence or reappearance of antibody post-Tx may indicate a more resistant disease. PMID:25766759

  8. Deficiency of phospholipase A2 receptor exacerbates ovalbumin-induced lung inflammation.

    Science.gov (United States)

    Tamaru, Shun; Mishina, Hideto; Watanabe, Yosuke; Watanabe, Kazuhiro; Fujioka, Daisuke; Takahashi, Soichiro; Suzuki, Koji; Nakamura, Takamitsu; Obata, Jun-Ei; Kawabata, Kenichi; Yokota, Yasunori; Murakami, Makoto; Hanasaki, Kohji; Kugiyama, Kiyotaka

    2013-08-01

    Secretory phospholipase A2 (sPLA2) plays a critical role in the genesis of lung inflammation through proinflammatory eicosanoids. A previous in vitro experiment showed a possible role of cell surface receptor for sPLA2 (PLA2R) in the clearance of extracellular sPLA2. PLA2R and groups IB and X sPLA2 are expressed in the lung. This study examined a pathogenic role of PLA2R in airway inflammation using PLA2R-deficient (PLA2R(-/-)) mice. Airway inflammation was induced by immunosensitization with OVA. Compared with wild-type (PLA2R(+/+)) mice, PLA2R(-/-) mice had a significantly greater infiltration of inflammatory cells around the airways, higher levels of groups IB and X sPLA2, eicosanoids, and Th2 cytokines, and higher numbers of eosinophils and neutrophils in bronchoalveolar lavage fluid after OVA treatment. In PLA2R(-/-) mice, intratracheally instilled [(125)I]-labeled sPLA2-IB was cleared much more slowly from bronchoalveolar lavage fluid compared with PLA2R(+/+) mice. The degradation of the instilled [(125)I]-labeled sPLA2-IB, as assessed by trichloroacetic acid-soluble radioactivity in bronchoalveolar lavage fluid after instillation, was lower in PLA2R(-/-) mice than in PLA2R(+/+) mice. In conclusion, PLA2R deficiency increased sPLA2-IB and -X levels in the lung through their impaired clearance from the lung, leading to exaggeration of lung inflammation induced by OVA treatment in a murine model.

  9. Autoantibodies against phospholipase A2 receptor in Korean patients with membranous nephropathy.

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    Oh, Yun Jung; Yang, Seung Hee; Kim, Dong Ki; Kang, Shin-Wook; Kim, Yon Su

    2013-01-01

    The data were presented in abstract form at the 45(th) meeting of the American Society of Nephrology, October 30-November 04 2012, San Diego, CA, USA. Circulating autoantibodies against M-type phospholipase A2 receptor (PLA2R) are important pathogenic antibodies of idiopathic membranous nephropathy (MN) in adults. However, previous studies on the clinical impact of anti-PLA2R antibodies demonstrated several limitations, including insufficient numbers of study subjects and different time points and methods for anti-PLA2R antibody measurement. To verify the clinical significance of anti-PLA2R antibodies in Korean patients with MN, we measured autoantibodies in serum samples obtained at the time of biopsy from a total of 100 patients with idiopathic MN who had not yet received immunosuppressive treatment. We detected anti-PLA2R antibody in 69 patients, and we observed that autoantibody reactivity reflected the severity of disease activity. Proteinuria and hypoalbuminemia were more severe in patients with anti-PLA2R than in those without the autoantibodies (2.95 g/g vs. 6.85 g/g, P = 0.003; 3.1 g/dL vs. 2.5 g/dL, P = 0.004, respectively). Additionally, the clinical severities worsened proportionally as the levels of anti-PLA2R antibodies increased (P = 0.015 and P for trend PLA2R antibody showed a significant correlation with clinical outcomes, such as remission rate and time to remission. In conclusion, we observed that anti-PLA2R antibodies are highly prevalent in Korean patients with idiopathic MN and that they reflect the clinical disease activity before the administration of immunosuppressive treatment. However, the levels of anti-PLA2R antibody at the time of kidney biopsy may not predict the clinical outcomes in current clinical practice.

  10. Phospholipase A2 receptor autoantibodies and clinical outcome in patients with primary membranous nephropathy.

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    Hoxha, Elion; Thiele, Ina; Zahner, Gunther; Panzer, Ulf; Harendza, Sigrid; Stahl, Rolf A K

    2014-06-01

    Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, with an uncertain clinical outcome. The characterization of the phospholipase A2 receptor (PLA2R) as the major target antigen in primary MN and the detection of circulating autoantibodies in these patients is a major advance in understanding this disease. To test whether PLA2R antibody levels reflect disease activity or clinical outcome, we performed a prospective multicenter study of 133 adult patients with primary MN and detectable serum PLA2R antibodies who had not received immunosuppressive therapy. Patients were followed ≤24 months. PLA2R antibody levels associated with clinical disease activity (proteinuria) in patients with immunosuppressive therapy (n=101) or supportive care (n=32). Within 3 months, immunosuppressive therapy led to a sustained 81% reduction in PLA2R antibody levels paralleled by a 39% reduction in proteinuria. Patients who experienced remission of proteinuria after 12 months had significantly lower PLA2R antibody levels at the time of study inclusion compared with patients with no remission. Patients with high PLA2R antibody levels achieved remission of proteinuria significantly later than patients with low PLA2R antibody levels. PLA2R antibody levels fell over time in patients with spontaneous remission but remained elevated in patients who did not show a reduction in proteinuria. Multivariable Cox regression analysis confirmed PLA2R antibody level as an independent risk factor for not achieving remission of proteinuria. We conclude that a decrease in PLA2R antibody level is associated with a decrease of proteinuria in patients with primary MN.

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

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

    2015-10-01

    Rituximab induces nephrotic syndrome (NS) remission in two-thirds of patients with primary membranous nephropathy (MN), even after other treatments have failed. To assess the relationships among treatment effect, circulating nephritogenic anti-phospholipase A2 receptor (anti-PLA2R) autoantibodies and genetic polymorphisms predisposing to antibody production we serially monitored 24-hour proteinuria and antibody titer in patients with primary MN and long-lasting NS consenting to rituximab (375 mg/m(2)) therapy and genetic analyses. Over a median (range) follow-up of 30.8 (6.0-145.4) months, 84 of 132 rituximab-treated patients achieved complete or partial NS remission (primary end point), and 25 relapsed after remission. Outcomes of patients with or without detectable anti-PLA2R antibodies at baseline were similar. Among the 81 patients with antibodies, lower anti-PLA2R antibody titer at baseline (P=0.001) and full antibody depletion 6 months post-rituximab (hazard ratio [HR], 7.90; 95% confidence interval [95% CI], 2.54 to 24.60; PPLA2R antibody depletion. On average, 50% anti-PLA2R titer reduction preceded equivalent proteinuria reduction by 10 months. Re-emergence of circulating antibodies predicted disease relapse (HR, 6.54; 95% CI, 1.57 to 27.40; P=0.01), whereas initial complete remission protected from the event (HR, 6.63; 95% CI, 2.37 to 18.53; PPLA2R1 and HLA-DQA1 polymorphisms and of previous immunosuppressive treatment. Therefore, assessing circulating anti-PLA2R autoantibodies and proteinuria may help in monitoring disease activity and guiding personalized rituximab therapy in nephrotic patients with primary MN.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

    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

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

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    Go Alan S

    2011-06-01

    Full Text Available Abstract Background Lipoprotein-associated phospholipase A2 (Lp-PLA2 is a predictor of cardiovascular events that has been shown to vary with race. The objective of this study was to examine factors associated with this racial variation. Methods We measured Lp-PLA2 mass and activity in 714 healthy older adults with no clinical coronary heart disease and not taking dyslipidemia medication. We evaluated the association between race and Lp-PLA2 mass and activity levels after adjustment for various covariates using multivariable linear regression. These covariates included age, sex, diabetes, hypertension, body mass index, lipid measurements, C-reactive protein, smoking status, physical activity, diet, income, and education level. We further examined genetic covariates that included three single nucleotide polymorphisms shown to be associated with Lp-PLA2 activity levels. Results The mean age was 66 years. Whites had the highest Lp-PLA2 mass and activity levels, followed by Hispanics and Asians, and then African-Americans; in age and sex adjusted analyses, these differences were significant for each non-White race as compared to Whites (p 2 mass and 24.7 nmol/ml-min lower activity, compared with Whites, independent of age and sex (p 2 mass and activity levels (p 2 mass and 17.3 nmol/ml-min lower activity compared with Whites (p Conclusion Biological, lifestyle, demographic, and select genetic factors do not appear to explain variations in Lp-PLA2 mass and activity levels between Whites and non-Whites, suggesting that Lp-PLA2 mass and activity levels may need to be interpreted differently for various races.

  15. Epigenetic Regulation of Cytosolic Phospholipase A2 in SH-SY5Y Human Neuroblastoma Cells.

    Science.gov (United States)

    Tan, Charlene Siew-Hon; Ng, Yee-Kong; Ong, Wei-Yi

    2016-08-01

    Group IVA cytosolic phospholipase A2 (cPLA2 or PLA2G4A) is a key enzyme that contributes to inflammation via the generation of arachidonic acid and eicosanoids. While much is known about regulation of cPLA2 by posttranslational modification such as phosphorylation, little is known about its epigenetic regulation. In this study, treatment with histone deacetylase (HDAC) inhibitors, trichostatin A (TSA), valproic acid, tubacin and the class I HDAC inhibitor, MS-275, were found to increase cPLA2α messenger RNA (mRNA) expression in SH-SY5Y human neuroblastoma cells. Co-treatment of the histone acetyltransferase (HAT) inhibitor, anacardic acid, modulated upregulation of cPLA2α induced by TSA. Specific involvement of class I HDACs and HAT in cPLA2α regulation was further shown, and a Tip60-specific HAT inhibitor, NU9056, modulated the upregulation of cPLA2α induced by MS-275. In addition, co-treatment of with histone methyltransferase (HMT) inhibitor, 5'-deoxy-5'-methylthioadenosine (MTA) suppressed TSA-induced cPLA2α upregulation. The above changes in cPLA2 mRNA expression were reflected at the protein level by Western blots and immunocytochemistry. Chromatin immunoprecipitation (ChIP) showed TSA increased binding of trimethylated H3K4 to the proximal promoter region of the cPLA2α gene. Cell injury after TSA treatment as indicated by lactate dehydrogenase (LDH) release was modulated by anacardic acid, and a role of cPLA2 in mediating TSA-induced injury shown, after co-incubation with the cPLA2 selective inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3). Together, results indicate epigenetic regulation of cPLA2 and the potential of such regulation for treatment of chronic inflammation. PMID:26162318

  16. Botanical Polyphenols Mitigate Microglial Activation and Microglia-Induced Neurotoxicity: Role of Cytosolic Phospholipase A2.

    Science.gov (United States)

    Chuang, Dennis Y; Simonyi, Agnes; Cui, Jiankun; Lubahn, Dennis B; Gu, Zezong; Sun, Grace Y

    2016-09-01

    Microglia play a significant role in the generation and propagation of oxidative/nitrosative stress, and are the basis of neuroinflammatory responses in the central nervous system. Upon stimulation by endotoxins such as lipopolysaccharides (LPS), these cells release pro-inflammatory factors which can exert harmful effects on surrounding neurons, leading to secondary neuronal damage and cell death. Our previous studies demonstrated the effects of botanical polyphenols to mitigate inflammatory responses induced by LPS, and highlighted an important role for cytosolic phospholipase A2 (cPLA2) upstream of the pro-inflammatory pathways (Chuang et al. in J Neuroinflammation 12(1):199, 2015. doi: 10.1186/s12974-015-0419-0 ). In this study, we investigate the action of botanical compounds and assess whether suppression of cPLA2 in microglia is involved in the neurotoxic effects on neurons. Differentiated SH-SY5Y neuroblastoma cells were used to test the neurotoxicity of conditioned medium from stimulated microglial cells, and WST-1 assay was used to assess for the cell viability of SH-SY5Y cells. Botanicals such as quercetin and honokiol (but not cyanidin-3-O-glucoside, 3CG) were effective in inhibiting LPS-induced nitric oxide (NO) production and phosphorylation of cPLA2. Conditioned medium from BV-2 cells stimulated with LPS or IFNγ caused neurotoxicity to SH-SY5Y cells. Decrease in cell viability could be ameliorated by pharmacological inhibitors for cPLA2 as well as by down-regulating cPLA2 with siRNA. Botanicals effective in inhibition of LPS-induced NO and cPLA2 phosphorylation were also effective in ameliorating microglial-induced neurotoxicity. Results demonstrated cytotoxic factors from activated microglial cells to cause damaging effects to neurons and potential use of botanical polyphenols to ameliorate the neurotoxic effects. PMID:27339657

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

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    Dinesh Thotala

    Full Text Available 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.

  18. Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release.

    Directory of Open Access Journals (Sweden)

    Ahmad Arouri

    Full Text Available The feasibility of exploiting secretory phospholipase A2 (sPLA2 enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA2 enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA2-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA2 releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA2. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA2-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.

  19. Biochemical and biological properties of phospholipases A(2) from Bothrops atrox snake venom.

    Science.gov (United States)

    Kanashiro, Milton M; de Cássia M Escocard, Rita; Petretski, Jorge H; Prates, Maura V; Alves, Elias W; Machado, Olga L T; da Silva, Wilmar Dias; Kipnis, Thereza L

    2002-10-01

    Phospholipases A(2) (PLA(2)s), of molecular mass 13-15kDa, are commonly isolated from snake venom. Two myotoxins with PLA(2) activity, BaPLA(2)I and BaPLA(2)III, with estimated molecular masses of 15kDa were isolated from the venom of Bothrops atrox using Sephacryl S-100-HR and reverse-phase chromatography. BaPLA(2)I was basic, with a pI of 9.1, while BaPLA(2)III was neutral with a pI of 6.9. On a molecular basis, BaPLA(2)III exhibited higher catalytic activity on synthetic substrates than BaPLA(2)I. Comparison of the N-terminal residues of BaPLA(2)I with other PLA(2) proteins from snake venoms showed that it has the highest homology (94%) with B. asper myotoxin II and homology with a PLA(2) Lys(49) from B. atrox (89%). In contrast, BaPLA(2)III demonstrated 75, 72, and 71% homology with PLA(2) from Vipera ammodytes meridionalis, B. jararacussu, and B. jararaca, respectively. BaPLA(2)I and BaPLA(2)III were capable, in vitro, of inducing mast cell degranulation and, in vivo, of causing creatine kinase release, edema, and myonecrosis typical of PLA(2)s from snake venoms, characterized by rapid disruption of the plasma membrane as indicated by clumping of myofilaments and necrosis of affected skeletal muscle cells. BaPLA(2)I- and BaPLA(2)III-specific monoclonal and polyclonal antibodies, although incapable of neutralizing PLA(2) edematogenic activity, blocked myonecrosis efficiently in an in vivo neutralization assay. The results presented herein suggest that the biological active site responsible for edema induction by these two PLA(2) enzymes is distinct from the myonecrosis active site and is not dependent upon the catalytic activity of the PLA(2) enzyme. PMID:12234622

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

    Science.gov (United States)

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

    2016-06-15

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

  1. Crystallization and preliminary crystallographic studies of a phospholipase A2 from the venom of the Brazilian snake Bothrops moojeni.

    Science.gov (United States)

    Nonato, M C; Garratt, R C; Mascarenhas, Y P; Jesus, W D; Assakura, M T; Serrano, S M; Oliva, G

    2001-04-01

    A phospholipase A(2) purified from the venom of the snake Bothrops moojeni has been crystallized by vapour-diffusion techniques in hanging drops at 291 K. The crystals, which were grown in the absence of Ca(2+), belong to the cubic system, space group P432, with unit-cell parameters a = b = c = 91.86 A, and contain one molecule in the asymmetric unit (V(M) = 2.71 A(3) Da(-1)). X-ray diffraction experiments provide data to 2.35 A resolution collected on a rotating-anode home source at cryogenic temperatures. The structure has been solved via molecular-replacement techniques using a single monomer of the crystallographic structure of the phospholipase from the Western diamondback rattlesnake (Crotalus atrox) as a search model. PMID:11264594

  2. Preclinical evaluation of an inhibitor of cytosolic phospholipase A2α for the treatment of asthma.

    Science.gov (United States)

    Hewson, Christopher A; Patel, Sheena; Calzetta, Luigino; Campwala, Hinnah; Havard, Suzanne; Luscombe, Emma; Clarke, Philip A; Peachell, Peter T; Matera, Maria G; Cazzola, Mario; Page, Clive; Abraham, William M; Williams, Cara M; Clark, James D; Liu, Wai L; Clarke, Nicholas P; Yeadon, Michael

    2012-03-01

    Asthma is a chronic inflammatory lung disease with considerable unmet medical needs for new and effective therapies. Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme that is ultimately responsible for the production of eicosanoids implicated in the pathogenesis of asthma. We investigated a novel cPLA(2)α inhibitor, PF-5212372, to establish the potential of this drug as a treatment for asthma. PF-5212372 was a potent inhibitor of cPLA(2)α (7 nM) and was able to inhibit prostaglandin (PG)D(2) and cysteinyl leukotriene release from anti-IgE-stimulated human lung mast cells (0.29 and 0.45 nM, respectively). In a mixed human lung cell population, PF-5212372 was able to inhibit ionomycin-stimulated release of leukotriene B(4), thromboxane A(2), and PGD(2) (2.6, 2.6, and 4.0 nM, respectively) but was significantly less effective against PGE(2) release (>301 nM; p < 0.05). In an in vitro cell retention assay, PF-5212372 retained its potency up to 24 h after being washed off. In a sheep model of allergic inflammation, inhalation of PF-5212372 significantly inhibited late-phase bronchoconstriction (78% inhibition; p < 0.001) and airway hyper-responsiveness (94% inhibition; p < 0.001), and isolated sheep lung mast cell assays confirmed species translation via effective inhibition of PGD(2) release (0.78 nM). Finally, PF-5212372 was assessed for its ability to inhibit the contraction of human bronchi induced by AMP. PF5212372 significantly inhibited AMP-induced contraction of human bronchi (81% inhibition; p < 0.001); this finding, together with the ability of this drug to be effective in a wide range of preclinical asthma models, suggests that inhibition of cPLA(2)α with PF-5212372 may represent a new therapeutic option for the treatment of asthma.

  3. Phospholipase A(2) and Lipids as Potential Modulators of c-Raf-1 Kinase.

    Science.gov (United States)

    Bonventre, Joseph V.; Kyriakis, John M.; Spech, Richard; Witzgall, Ralph; Force, Thomas

    1996-04-01

    c-Raf-1 is a proximal serine/threonine kinase in the signaling cascade of many mitogens. The cellular mechanisms responsible for regulation of this kinase remain ill-defined. Although c-Raf-1-associated proteins have been identified, including Ras, none of these have been found to activate c-Raf-1 kinase in vitro. To evaluate whether arachidonic acid or one of its products is implicated in c-Raf-1 activation, c-Raf-1 activity was measured in LLC-PK(1) kidney epithelial cells overexpressing the 100 kDa phospholipase A(2) (PLA(2)). As compared to control neomycin plasmid transfected cells, the cells overexpressing PLA(2) had a greater activation of c-Raf-1 in response to A23187 and phorbol ester stimulation. To explore the possibility that c-Raf-1 activity may be modulated directly by lipids, the enzymatic characteristics of c-Raf-1 were determined, and the effects of various possible lipid modulators on c-Raf-1 activity were examined. The K(m) of c-Raf-1 for ATP and mitogen-activiated protein kinase kinase (MAPKK), the only known physiologic substrate of c-Raf-1, were 11.6 &mgr;M and 0.8 &mgr;M, respectively. Of 13 lipids or combinations of lipids tested, including arachidonic acid and several eicosanoids, only phosphatidylserine and diacylglycerol in the presence of CA(2+) (2.5 mM) increased c-Raf-1 kinase activity significantly. The increase (1.5-fold) was approximately two orders of magnitude less than the stimulation of protein kinase C by these lipids. c-Raf-1 kinase activity and immunoreactivity eluted on gel filtration at a predicted molecular mass of greater than 150 kDa, suggesting that active c-Raf-1 is part of a multimeric complex. The absence of immunoreactive Ras in the active fractions confirms that the interaction is not necessary to maintain c-Raf-1 in an active state. In conclusion, a product of PLA(2) may play a role, together with Ras and another unidentified cofactor, in activating c-Raf-1. This lipid mediator(s) may directly or indirectly

  4. Autoantibodies against phospholipase A2 receptor in Korean patients with membranous nephropathy.

    Directory of Open Access Journals (Sweden)

    Yun Jung Oh

    Full Text Available The data were presented in abstract form at the 45(th meeting of the American Society of Nephrology, October 30-November 04 2012, San Diego, CA, USA. Circulating autoantibodies against M-type phospholipase A2 receptor (PLA2R are important pathogenic antibodies of idiopathic membranous nephropathy (MN in adults. However, previous studies on the clinical impact of anti-PLA2R antibodies demonstrated several limitations, including insufficient numbers of study subjects and different time points and methods for anti-PLA2R antibody measurement. To verify the clinical significance of anti-PLA2R antibodies in Korean patients with MN, we measured autoantibodies in serum samples obtained at the time of biopsy from a total of 100 patients with idiopathic MN who had not yet received immunosuppressive treatment. We detected anti-PLA2R antibody in 69 patients, and we observed that autoantibody reactivity reflected the severity of disease activity. Proteinuria and hypoalbuminemia were more severe in patients with anti-PLA2R than in those without the autoantibodies (2.95 g/g vs. 6.85 g/g, P = 0.003; 3.1 g/dL vs. 2.5 g/dL, P = 0.004, respectively. Additionally, the clinical severities worsened proportionally as the levels of anti-PLA2R antibodies increased (P = 0.015 and P for trend <0.001 for proteinuria and hypoalbuminemia, respectively. However, neither the levels nor the presence or absence of anti-PLA2R antibody showed a significant correlation with clinical outcomes, such as remission rate and time to remission. In conclusion, we observed that anti-PLA2R antibodies are highly prevalent in Korean patients with idiopathic MN and that they reflect the clinical disease activity before the administration of immunosuppressive treatment. However, the levels of anti-PLA2R antibody at the time of kidney biopsy may not predict the clinical outcomes in current clinical practice.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Chenggang Li

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

  7. Inhibition of secreted phospholipase A2 by neuron survival and anti-inflammatory peptide CHEC-9

    Directory of Open Access Journals (Sweden)

    Yao Lihua

    2006-09-01

    Full Text Available Abstract Background The nonapeptide CHEC-9 (CHEASAAQC, a putative inhibitor of secreted phospholipase A2 (sPLA2, has been shown previously to inhibit neuron death and aspects of the inflammatory response following systemic treatment of rats with cerebral cortex lesions. In this study, the properties of CHEC-9 inhibition of sPLA2 enzymes were investigated, using a venom-derived sPLA2 group I and the plasma of rats and humans as the sources of enzyme activity. The results highlight the advantages of inhibitors with uncompetitive properties for inflammatory disorders including those resulting in degeneration of neurons. Methods Samples of enzyme and plasma were reacted with 1-Palmitoyl-2-Pyrenedecanoyl Phosphatidylcholine, a sPLA2 substrate that forms phospholipid vesicles in aqueous solutions. Some of the plasma samples were collected from restrained peptide-treated rats in order to confirm the validity of the in vitro assays for extrapolation to in vivo effects of the peptide. The enzyme reactions were analyzed in terms of well-studied relationships between the degree of inhibition and the concentrations of different reactants. We also examined interactions between different components of the reaction mixture on native polyacrylamide gels. Results In all cases, the peptide showed the properties of an uncompetitive (or anti-competitive enzyme inhibitor with Ki values less than 100 nanomolar. The electrophoresis experiments suggested CHEC-9 modifies the binding properties of the enzyme only in the presence of substrate, consistent with its classification as an uncompetitive inhibitor. Both the in vitro observations and the analysis of plasma samples from restrained rats injected with peptide suggest the efficacy of the peptide increases under conditions of high enzyme activity. Conclusion Modeling studies by others have shown that uncompetitive inhibitors may be optimal for enzyme inhibition therapy because, unlike competitive inhibitors, they are

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mao Yong-jun

    2011-01-01

    Full Text Available Abstract 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 2 was obtained between patients with three and four disorders of metabolic syndrome (P 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.

  10. Cell Swelling Activates Phospholipase A2 in Ehrlich Ascites Tumor Cells

    DEFF Research Database (Denmark)

    Thoroed, S.M.; Lauritzen, L.; Lambert, I.H.;

    1997-01-01

    Ehrlich ascites tumor cells! loaded with H-labeled arachidonic acid and C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo......-osmotic exposure the rate of H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A is activated by cell swelling in the Ehrlich...... cells. Within the same time frame there is no swelling-induced increase in C-labeled stearic acid release nor in the synthesis of phosphatidyl C-butanol in the presence of C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of C...

  11. 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

    OpenAIRE

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

    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. Th...

  12. Cytosolic phospholipase A2 activation correlates with HER2 overexpression and mediates estrogen-dependent breast cancer cell growth.

    OpenAIRE

    Caiazza, Francesco; Harvey, Brian J; Thomas, Warren

    2010-01-01

    Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) catalyzes the hydrolysis of membrane glycerol-phospholipids to release arachidonic acid as the first step of the eicosanoid signaling pathway. This pathway contributes to proliferation in breast cancer, and numerous studies have demonstrated a crucial role of cyclooxygenase 2 and prostaglandin E(2) release in breast cancer progression. The role of cPLA(2)alpha activation is less clear, and we recently showed that 17beta-estradiol (E2) can rapid...

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

    OpenAIRE

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

    2014-01-01

    The role of Group X secreted phospholipase A2 (GX-sPLA2) during influenza infection has not been previously investigated. We examined the role of (Reviewer 2 Minor Comment 2) GX-sPLA2 during H1N1 pandemic influenza infection in a GX-sPLA2 gene targeted mouse (GX−/−) model and found that survival after infection was significantly greater in GX−/− mice than in GX+/+ mice. Downstream products of GX-sPLA2 activity, PGD2, PGE2, LTB4, cysteinyl leukotrienes and Lipoxin A4 were significantly lower i...

  14. Function, Activity, and Membrane Targeting of Cytosolic Phospholipase A2ζ in Mouse Lung Fibroblasts*S

    OpenAIRE

    Ghosh, Moumita; Loper, Robyn; Ghomashchi, Farideh; Tucker, Dawn E.; Bonventre, Joseph V.; Gelb, Michael H; Leslie, Christina C.

    2007-01-01

    Group IVA cytosolic phospholipase A2 (cPLA2α) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA2α−/− lung fibroblasts stimulated with A23187 or serum. cPLA2α+/+ fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA2α−/− fibroblasts non-specifically released multiple fatty acids. Arachidonic acid release from cPLA2α−/− fibroblasts was inhibited by the cPLA2α inhibitors pyrrolidine-2 (IC50, 0.03 μM) and Wyeth-1 (IC50, 0.1 μM), im...

  15. Group II and IV phospholipase A2 are produced in human pancreatic cancer cells and influence prognosis

    OpenAIRE

    Kashiwagi, M; Friess, H; Uhl, W.; Berberat, P; Abou-Shady, M; MARTIGNONI, M.; Anghelacopoulos, S; Zimmermann, A.; Buchler, M.

    1999-01-01

    BACKGROUND—Phospholipase A2 (PLA2) is involved in regulating biosynthesis of arachidonic acid and its metabolites. There are three major structurally different forms of PLA2: group I, also called pancreatic PLA2 (PLA2-I); group II, referred to as secretory non-pancreatic or synovial or platelet PLA2 (PLA2-II); group IV, referred to as cytosolic PLA2 (PLA2-IV).
AIMS—To examine PLA2-I, PLA2-II, and PLA2-IV in normal and pancreatic cancer tissues.
 Patients—PLA2 was studied in 58 pancreatic aden...

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

    OpenAIRE

    Clive Bate; Alun Williams

    2015-01-01

    A key event in Alzheimer’s disease (AD) is the production of amyloid-β (Aβ) peptides and the loss of synapses. In cultured neurons Aβ triggered synapse damage as measured by the loss of synaptic proteins. α-synuclein (αSN), aggregates of which accumulate in Parkinson’s disease, also caused synapse damage. Synapse damage was associated with activation of cytoplasmic phospholipase A2 (cPLA2), an enzyme that regulates synapse function and structure, and the production of prostaglandin (PG) E2. I...

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

    OpenAIRE

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

    2010-01-01

    Bee venom phospholipase A2 (BvPLA2) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids. In this work, a new BvPLA2 (AccPLA2) gene from the Chinese honeybee (Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector. Tn-5B-4 (Tn) cells were transfected with the recombinant bacmid DNA for expression. Sodium dodecylsulfate-polyacrylamide gel electrophoresis...

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

    DEFF Research Database (Denmark)

    Nielsen, Lars Kildemark; Balashev, K.; Callisen, Thomas Hønger;

    2002-01-01

    concentrations, made by Langmuir-Blodgett deposition, we show that small depressions enriched in products are efficiently promoting enzyme degradation of the bilayer. These small depressions, which are indicative of phase separation, are initially present in samples with 75% products. The kinetics of...... phospholipase A(2) exhibit under certain conditions an initial phase of slow hydrolysis, termed the latency phase, followed by a marked increase in the hydrolysis rate. The appearance of the phase-separated bilayer is strikingly similar to that of bilayers; at the end of the latency phase. By analysis of...

  19. Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A(2) and Humicola lanuginosa lipase

    DEFF Research Database (Denmark)

    Balashev, Konstantin; DiNardo, N. John; Callisen, Thomas H.;

    2007-01-01

    at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A(2) (PLA(2)) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution....... Lipid bilayers were prepared by the Langmuir-Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme...

  20. Time-resolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum.

    Science.gov (United States)

    Nevalainen, Timo J; Eerola, Leena I; Rintala, Esa; Laine, V Jukka O; Lambeau, Gérard; Gelb, Michael H

    2005-04-15

    Time-resolved fluoroimmunoassays (TR-FIA) were developed for all human secreted phospholipases A(2) (PLA(2)), viz. group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein. Antibodies were raised in rabbits against recombinant human PLA(2) proteins and used in sandwich-type TR-FIAs as both catching and detecting antibodies, the latter after labeling with Europium. The antibodies were non-cross-reactive. The analytical sensitivities were 1 microg/L for the TR-FIA for GIB PLA(2), 1 microg/L (GIIA), 35 microg/L (GIID), 3 microg/L (GIIE), 4 microg/L (GIIF), 14 microg/L (GIII), 11 microg/L (GV), 2 microg/L (GX), 92 microg/L (GXIIA) and 242 microg/L (GXIIB). All secreted PLA(2)s were assayed by these TR-FIAs in serum samples from 34 patients (23 men and 11 women, mean age 53.2 years) treated in an intensive care unit for septic infections, and in control samples from 28 volunteer blood donors (14 men and 14 women, mean age 57.0 years). Five serum samples (3 in the sepsis group and 2 in the blood donor group) gave high TR-FIA signals that were reduced to background (blank) levels by the addition of non-immune rabbit IgG to the sera. This reactivity was assumed to be due to the presence of heterophilic antibodies in these subjects. In all other subjects, including septic patients and healthy blood donors, the TR-FIA signals for GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were at background (blank) levels. Four patients in the sepsis group had pancreatic involvement and elevated concentration of GIB PLA(2) in serum (median 19.0 microg/L, range 13.1-33.7 microg/L, n = 4) as compared to the healthy blood donors (median 1.8 microg/L, range 0.8-3.4 microg/L, n = 28, P < 0.0001). The concentration of GIIA PLA(2) in the sera of septic patients (median 315.7 microg/L, range 15.9-979.6 microg/L, n = 34) was highly elevated as compared to that of the blood donors (median 1.8 microg/L, range 0

  1. Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A(2) Action at the Single Molecule Level

    DEFF Research Database (Denmark)

    Gudmand, Martin Jesper; Rocha, Susana; Hatzakis, Nikos;

    2010-01-01

    We monitored the action of phospholipase A(2) (PLA(2)) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffus......We monitored the action of phospholipase A(2) (PLA(2)) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity...... and diffusion behavior of single PLA(2) molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from...... interface were between 3.2 microm(2)/s on the L-DPPC and 4.9 microm(2)/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to approximately 0.2 microm(2)/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries...

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

    Institute of Scientific and Technical Information of China (English)

    Jacqueline Leβig; Uta Reibetanz; Jürgen Arnhold; Hans-Jürgen Glander

    2008-01-01

    Aim: To determine the cellular distribution of secretory phospholipase A2 (sPLA2) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes. Methods: Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate in flammatory conditions. To visualize the distribution of sPLA2 and to determine the acrosomal state, immunofluorescence tech-niques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used. Results: Although sPLA2 was detected at the acrosome and tail regions in intact spermatozoa, it disappearedfrom the head region after triggering the acrosome reaction. This release of sPLA2 was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer L and bnding of FITC-iabelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA2, disturbance of acrosome structure, and loss of vitality. Conclusion:The ability of spermatozoa to release secretory phospholipase A2 is related to the acrosomal state. Premature destabi-lization of the acrosome and loss of sPLA2 can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA2 in intact spermatozoa might be an additional parameter for evaluating sperm quality.

  3. Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A2.

    Science.gov (United States)

    Casais-E-Silva, Luciana L; Teixeira, Catarina F P; Lebrun, Ivo; Lomonte, Bruno; Alape-Girón, Alberto; Gutiérrez, José María

    2016-08-22

    The venom of Micrurus lemniscatus, a coral snake of wide geographical distribution in South America, was fractionated by reverse-phase HPLC and the fractions screened for phospholipase A2 (PLA2) activity. The major component of the venom, a PLA2, here referred to as 'Lemnitoxin', was isolated and characterized biochemically and toxicologically. It induces myotoxicity upon intramuscular or intravenous injection into mice. The amino acid residues Arg15, Ala100, Asn108, and a hydrophobic residue at position 109, which are characteristic of myotoxic class I phospholipases A2, are present in Lemnitoxin. This PLA2 is antigenically related to M. nigrocinctus nigroxin, Notechis scutatus notexin, Pseudechis australis mulgotoxin, and Pseudonaja textilis textilotoxin, as demonstrated with monoclonal and polyclonal antibodies. Lemnitoxin is highly selective in its targeting of cells, being cytotoxic for differentiated myotubes in vitro and muscle fibers in vivo, but not for undifferentiated myoblasts or endothelial cells. Lemnitoxin is not lethal after intravenous injection at doses up to 2μg/g in mice, evidencing its lack of significant neurotoxicity. Lemnitoxin displays anticoagulant effect on human plasma and proinflammatory activity also, as it induces paw edema and mast cell degranulation. Thus, the results of this work demonstrate that Lemnitoxin is a potent myotoxic and proinflammatory class I PLA2. PMID:27282409

  4. Darapladib Binds to Lipoprotein-Associated Phospholipase A2 with Meaningful Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Do, Kyungrok; Chang, Byungha; Shin, Jae Min; No, Kyoung Tai; Lee, Jeeyoung [Bioinformatics and Molecular Design Research Center, Seoul (Korea, Republic of); Kim, Chul; Yea, Sangjun; Song, Miyoung [Korea Institute of Oriental Medicine, Daejeon (Korea, Republic of)

    2014-01-15

    Lipoprotein-associated phospholipase A{sub 2} (Lp-PLA{sub 2}) is a crucial enzyme in atherosclerosis as a potential drug target. The most remarkable Lp-PLA{sub 2} inhibitory drug is Darapladib. We determined the binding pose of Darapladib to Lp-PLA{sub 2} through docking study. Darapladib formed two hydrogen bonding interactions with the side chain of Tyr160 and Gln352 and several pi-pi interactions with aromatic and aliphatic hydrophobic residues of Lp-PLA{sub 2}. It is known that the dietylpropan-amine moiety of Darapladib has influence on the improvement of its oral bioavailability and we supposed this in our docking results.

  5. Analyses of Group III Secreted Phospholipase A2 Transgenic Mice Reveal Potential Participation of This Enzyme in Plasma Lipoprotein Modification, Macrophage Foam Cell Formation, and Atherosclerosis*S⃞

    OpenAIRE

    Sato, Hiroyasu; Kato, Rina; Isogai, Yuki; Saka, Go-ichi; Ohtsuki, Mitsuhiro; Taketomi, Yoshitaka; Yamamoto, Kei(Department of Physics, Niigata University, Niigata 950-2181, Japan); Tsutsumi, Kae; Yamada, Joe; Masuda, Seiko; Ishikawa, Yukio; Ishii, Toshiharu; Kobayashi, Tetsuyuki; Ikeda, Kazutaka; Taguchi, Ryo

    2008-01-01

    Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA...

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

    Directory of Open Access Journals (Sweden)

    Laušević Željko

    2010-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  8. Increased expression and activity of group IIA and X secretory phospholipase A2 in peritumoral versus central colon carcinoma tissue

    DEFF Research Database (Denmark)

    Tribler, Line; Jensen, Lotte T; Jørgensen, Kent;

    2007-01-01

    Secretory phospholipase A2 (sPLA2) type IIA and X was analyzed in tumors from 22 patients with colon adenocarcinomas in order to determine the involvement and activity of sPLA2 in colon cancer. Evaluation of immunoreactive sPLA2 IIA by Western blotting showed a significantly higher level in the...... periphery of the tumors, compared to central tumor regions. Increased levels of sPLA2 IIA protein correlated with a two-fold increase in sPLA2 enzymatic activity in the peripheral regions compared to central regions. Nineteen out of 22 tumors showed high levels of sPLA2 IIA, whereas 7 out of the 22 tumors...... showed sPLA2 type X. These data demonstrate that both sPLA2 type IIA and X are present in human colon cancer and suggest a role for sPLA2 in colon cancer tumor immunology and tumorigenesis....

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

    Science.gov (United States)

    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.

  10. Secreted phospholipase A(2) as a new enzymatic trigger mechanism for localised liposomal drug release and absorption in diseased tissue

    DEFF Research Database (Denmark)

    Davidsen, Jesper; Jørgensen, K.; Andresen, Thomas Lars;

    2003-01-01

    Polymer-coated liposomes can act as versatile drug-delivery systems due to long vascular circulation time and passive targeting by leaky blood vessels in diseased tissue. We present an experimental model system illustrating a new principle for improved and programmable drug-delivery, which takes...... advantage of an elevated activity of secretory phospholipase A(2) (PLA(2)) at the diseased target tissue. The secretory PLA2 hydrolyses a lipid-based proenhancer in the carrier liposome, producing lyso-phospholipids and free fatty acids, which are shown in a synergistic way to lead to enhanced liposome......-based proenhancers or prodestabilisers into the liposome carrier, which automatically becomes activated by PLA2 only at the diseased target sites, such as inflamed or cancerous tissue....

  11. Atomic resolution structure of the double mutant (K53,56M) of bovine pancreatic phospholipase A2

    International Nuclear Information System (INIS)

    The atomic resolution crystal structure of the double mutant (K53,56M) of bovine pancreatic phospholipase A2 is reported. The structure of the double mutant K53,56M has previously been refined at 1.9 Å resolution using room-temperature data. The present paper reports the crystal structure of the same mutant K53,56M refined against 1.1 Å data collected using synchrotron radiation. A total of 116 main-chain atoms from 29 residues and 44 side chains are modelled in alternate conformations. Most of the interfacial binding residues are found to be disordered and alternate conformations could be recognized. The second calcium ion-binding site residue Glu92 adopts two alternate conformations. The minor and major conformations of Glu92 correspond to the second calcium ion bound and unbound states

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    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.

  15. Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    González-Méndez Ricardo

    2009-05-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways. Results In this work we describe a new G protein α subunit gene in S. schenckii, ssg-2. The cDNA sequence of ssg-2 revealed a predicted open reading frame of 1,065 nucleotides encoding a 355 amino acids protein with a molecular weight of 40.9 kDa. When used as bait in a yeast two-hybrid assay, a cytoplasmic phospholipase A2 catalytic subunit was identified as interacting with SSG-2. The sspla2 gene, revealed an open reading frame of 2538 bp and encoded an 846 amino acid protein with a calculated molecular weight of 92.62 kDa. The principal features that characterize cPLA2 were identified in this enzyme such as a phospholipase catalytic domain and the characteristic invariable arginine and serine residues. A role for SSPLA2 in the control of dimorphism in S. schenckii is suggested by observing the effects of inhibitors of the enzyme on the yeast cell cycle and the yeast to mycelium transition in this fungus. Phospholipase A2 inhibitors such as AACOCF3 (an analogue of archidonic acid and isotetrandrine (an inhibitor of G protein

  16. Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy

    NARCIS (Netherlands)

    Dahnrich, C.; Komorowski, L.; Probst, C.; Seitz-Polski, B.; Esnault, V.; Wetzels, J.F.M.; Hofstra, J.M.; Hoxha, E.; Stahl, R.A.K.; Lambeau, G.; Stocker, W.; Schlumberger, W.

    2013-01-01

    BACKGROUND: Autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) are specific markers for primary membranous nephropathy (pMN) and anti-PLA2R1 serum levels may be useful to monitor disease activity. So far, a recombinant cell-based indirect immunofluorescence assay (RC-IFA) using rec

  17. Carotid intima media thickness is associated with plasma lipoprotein-associated phospholipase A(2) mass in nondiabetic subjects but not in patients with type 2 diabetes

    NARCIS (Netherlands)

    Constantinides, Alexander; van Pelt, L. Joost; van Leeuwen, Jeroen J. J.; de Vries, Rindert; Tio, Rene A.; van der Horst, Iwan C. C.; Sluiter, Wim J.; Dullaart, Robin P. F.

    2011-01-01

    Background A recent meta-analysis showed that both plasma lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) mass and activity independently predict cardiovascular events. Notably, Lp-PLA(2) activity but not mass was found to be a determinant of cardiovascular outcome in type 2 diabetes mellitus.

  18. Phospholipases A2: enzymatic assay for snake venom (Naja naja karachiensis) with their neutralization by medicinal plants of Pakistan.

    Science.gov (United States)

    Asad, Muhammad H H B; Durr-E-Sabih; Yaqab, Tahir; Murtaza, Ghulam; Hussain, Muhammad S; Hussain, Muhammad S; Nasir, Muhammad T; Azhar, Saira; Khan, Shujaat A; Hussain, Izhar

    2014-01-01

    Phospholipases A2 (PLA2) are the most lethal and noxious component of Naja naja karachiensis venom. They are engaged to induce severe toxicities after their penetration in victims. Present study was designed to highlight hydrolytic actions of PLA. in an egg yolk mixture and to encounter their deleterious effects via medicinal plants of Pakistan. PLA2 were found to produce free fatty acids in a dose dependent manner. Venom at concentration of 0.1 mg was found to liberate 26.6 pmoles of fatty acids with a decline in pH1 of 0.2 owing to the presence of PLA2 (133 Unit/mg). When quantity of venom was increased up to 8 mg, it caused to release 133 pmoles of free fatty acids with a decrease in 1.0 pH due to abundance in PLA, (665 Unit/mg). The rest of other doses of venom (0.3-4.0 mg) was found to liberate fatty acids between these two upper and lower limits. Twenty eight medicinal plants (0.1-0.6 mg) were tried to abort PLA, hydrolytic action, however, all were found useful (50-100%) against PLA,. Bauhinia variegate L., Citrus limon (L.). Burm.f. Enicostemnma hyssopifolium (Willd.) Verdoorn, Ocimum sanctum. Psoralea corylifolia L. and Stenolobium stans (L.) D. Don were found excellent in switching off 100% phospholipases A, at their lowest concentration (0.1 mg). Three plants extract were found useful only at lower concentration (0.1 mg), however, their higher doses were seemed to aggravate venom response. Eight medicinal plants failed to neutralize PLA, rather their higher doses were found effective. Standard antidote and rest of other plants extract were able to show maximum of 50% efficiencies. Therefore, it is necessary to identify and isolate bioactive constituent(s) from above cited six medicinal plants to eradicate the problem of snake bite in the future. PMID:25272888

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

    Directory of Open Access Journals (Sweden)

    Juliana L. Furtado

    2014-01-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2014-01-01

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

  1. Research Progress on Group-XII Secreted Phospholipase A2%分泌型磷脂酶A2-XII的研究进展

    Institute of Scientific and Technical Information of China (English)

    许媛媛; 叶波平

    2011-01-01

    Group-XII secreted phospholipase A2 (sPLA2 - XII) includes two isoforms: sPLA2 - XIIA and sPLA2 - XIIB. They are present only in animals and their primary sequence, distribution and functions are markedly different from other PLA2s. The catalytic activity of sPLA2 - XIIA is very weak, yet it can kill Gram-negative bacteria efficiently. However sPLA2 - IIB is the first mammalian secreted phospholipase A2 that is completely devoid of enzymatic activity. Strong expression was observed in liver, small intestine, and kidney in both human and mouse species. Interestingly, the expression of SPLA2 - XIIBs dramatically decreased in tumors from the same tissues, which suggest that sPLA2 - XIIB probably play an important role in tumorigenesis. The study on sPLA2 - XIIs' structure, distribution and pharmaceutical application, which provide the theoretical basis for further investigations and applications of sPLA2 - XII, was reviewed in this paper.%分泌型磷脂酶A2-XII包括A、B 2个亚型,存在于多种动物的组织和细胞中,在结构、分布与功能上明显异于其他磷脂酶,其中的分泌型磷脂酶A2-XIIA酶活性较低,但具有很强的抗革兰氏阴性菌作用,而分泌型磷脂酶A2-XIIB是首个发现于哺乳动物的完全没有酶活性的分泌型磷脂酶亚型,在人和小鼠的肝脏、小肠和肾脏中含量最高,而在这些组织对应的肿瘤中表达下调,推测其在这些肿瘤发生过程中发挥重要作用.文章综述了分泌型磷脂酶A2-XII的结构、分布以及在药学中的应用,为进一步开发其药用价值提供理论依据.

  2. Correlation of M-type phospholipase A2 receptor genetic polymorphism with idiopathic membranous nephropathy

    Institute of Scientific and Technical Information of China (English)

    周广宇

    2013-01-01

    Objective To investigate the correlation of M-typephos pholipase A2receptor(PLA2R) genetic polymorphism in two single nucleotide polymorphisms(SNPs) with idiopathic membranous nephropathy(IMN) of Chinese

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

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    Anna Södergren

    2015-01-01

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

  4. Synergistic Effects of Secretory Phospholipase A2 from the Venom of Agkistrodon piscivorus piscivorus with Cancer Chemotherapeutic Agents

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    Jennifer Nelson

    2013-01-01

    Full Text Available Healthy cells typically resist hydrolysis catalyzed by snake venom secretory phospholipase A2. However, during various forms of programmed cell death, they become vulnerable to attack by the enzyme. This observation raises the question of whether the specificity of the enzyme for dying cells could be used as a strategy to eliminate tumor cells that have been intoxicated but not directly killed by chemotherapeutic agents. This idea was tested with S49 lymphoma cells and a broad range of antineoplastic drugs: methotrexate, daunorubicin, actinomycin D, and paclitaxel. In each case, a substantial population of treated cells was still alive yet vulnerable to attack by the enzyme. Induction of cell death by these agents also perturbed the biophysical properties of the membrane as detected by merocyanine 540 and trimethylammonium-diphenylhexatriene. These results suggest that exposure of lymphoma cells to these drugs universally causes changes to the cell membrane that render it susceptible to enzymatic attack. The data also argue that the snake venom enzyme is not only capable of clearing cell corpses but can aid in the demise of tumor cells that have initiated but not yet completed the death process.

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

    Science.gov (United States)

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

    2014-01-01

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

  6. Lipoprotein-Associated Phospholipase A2 Mass Level Is Increased in Elderly Subjects with Type 2 Diabetes Mellitus

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    J. Fortunato

    2014-01-01

    Full Text Available Objective. Lipoprotein-associated phospholipase A2 (Lp-PLA2 is extensively expressed by advanced atherosclerotic lesions and may play a role in plaque instability. We selected a group of elderly subjects that underwent transcatheter aortic valve implantation (TAVI or balloon angioplasty (BA and separated them into two groups, diabetic and nondiabetic, to compare the level of Lp-PLA2 mass between them. Methods. 44 patients aged 79.6±5.6 years with symptomatic severe aortic valve stenosis underwent TAVI (n=35 or BA (n=9. 21 subjects had confirmed type 2 diabetes mellitus. Lp-PLA2 mass was measured using an enzyme-linked immunosorbent assay kit (USCN Life Science, China before and 3 days after the procedure. Results. Lp-PLA2 mass was significantly elevated in this population (1296±358 ng/mL before TAVI; 1413±268 ng/mL before BA and further increased after TAVI (1604±437 ng/mL, P<0.01 or BA (1808±303 ng/mL, P<0.01. Lp-PLA2 mass was significantly increased on the diabetic group before these interventions. Conclusion. Lp-PLA2 may be a novel biomarker for the presence of rupture-prone atherosclerotic lesions in elderly patients. Levels of Lp-PLA2 in diabetic patients may accompany the higher amount of small dense LDL particles seen in these subjects.

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

    Science.gov (United States)

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

    2016-01-01

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

  8. Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A2 Action at the Single Molecule Level

    CERN Document Server

    Gudmand, M; Hatzakis, N S; Peneva, K; Muellen, K; Stamou, D; Uji-I, H; Hofkens, J; Bjornholm, T; Heimburg, T

    2009-01-01

    We monitored the action of phospholipase A2 (PLA2) on L- and D-dipalmitoylphosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA2 molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the build-up of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA2. The mobility of individual enzymes on the monolayers was characterized by single particle tracking (SPT). Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3 mu m^2/s on the L-DPPC and 4.6 mu m^/s on the D-DPPC monolayers. In regions enriched with hydrolysis produc...

  9. Mechanism of Cytosolic Phospholipase A(2) Activation in Ghrelin Protection of Salivary Gland Acinar Cells against Ethanol Cytotoxicity.

    Science.gov (United States)

    Slomiany, Bronislaw L; Slomiany, Amalia

    2010-01-01

    Ghrelin, a peptide hormone, newly identified in oral mucosal tissues, has emerged recently as an important mediator of the processes of mucosal defense. Here, we report on the mechanism of ghrelin protection against ethanol cytotoxicity in rat sublingual salivary gland cells. The protective effect of ghrelin was associated with the increase in NO and PGE2, and upregulation in cytosolic phospholipase A(2) (cPLA(2)) activity and arachidonic acid (AA) release. The loss in countering effect of ghrelin occurred with cNOS inhibitor, L-NAME, as well as indomethacin and COX-1 inhibitor, SC-560, while COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced cell capacity for NO production, cPLA(2) activation and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA(2) phosphorylation and S-nitrosylation. Inhibition in ghrelin-induced S-nitrosylation was attained with L-NAME, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA(2) protein phosphorylation as well as S-nitrosylation. Thus, ghrelin protection of salivary gland cells against ethanol involves cNOS-derived NO induction of cPLA(2) activation through S-nitrosylation for the increase in AA release at the site of COX-1 action for PGE2 synthesis.

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

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    Ewan West

    2015-06-01

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

  11. Design of group IIA secreted/synovial phospholipase A(2 inhibitors: an oxadiazolone derivative suppresses chondrocyte prostaglandin E(2 secretion.

    Directory of Open Access Journals (Sweden)

    Jean-Edouard Ombetta

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

  12. Protective Effects of Intratracheally-Administered Bee Venom Phospholipase A2 on Ovalbumin-Induced Allergic Asthma in Mice

    Science.gov (United States)

    Jung, Kyung-Hwa; Baek, Hyunjung; Shin, Dasom; Lee, Gihyun; Park, Sangwon; Lee, Sujin; Choi, Dabin; Kim, Woojin; Bae, Hyunsu

    2016-01-01

    Asthma is a common chronic disease characterized by bronchial inflammation, reversible airway obstruction, and airway hyperresponsiveness (AHR). Current therapeutic options for the management of asthma include inhaled corticosteroids and β2 agonists, which elicit harmful side effects. In the present study, we examined the capacity of phospholipase A2 (PLA2), one of the major components of bee venom (BV), to reduce airway inflammation and improve lung function in an experimental model of asthma. Allergic asthma was induced in female BALB/c mice by intraperitoneal administration of ovalbumin (OVA) on days 0 and 14, followed by intratracheal challenge with 1% OVA six times between days 22 and 30. The infiltration of immune cells, such as Th2 cytokines in the lungs, and the lung histology, were assessed in the OVA-challenged mice in the presence and absence of an intratracheal administration of bvPLA2. We showed that the intratracheal administration of bvPLA2 markedly suppressed the OVA-induced allergic airway inflammation by reducing AHR, overall area of inflammation, and goblet cell hyperplasia. Furthermore, the suppression was associated with a significant decrease in the production of Th2 cytokines, such as IL-4, IL-5, and IL-13, and a reduction in the number of total cells, including eosinophils, macrophages, and neutrophils in the airway. PMID:27669297

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

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    Ryan C Smith

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2012-01-01

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

  16. Translocation of phospholipase A2α to apoplasts is modulated by developmental stages and bacterial infection in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jihye eJung

    2012-06-01

    Full Text Available Phospholipase A2 (PLA2 hydrolyzes phospholipids at the sn-2 position to yield lysophospholipids and free fatty acids. Of the four paralogs expressed in Arabidopsis, the cellular functions of PLA2α in planta are poorly understood. The present study shows that PLA2α possesses unique characteristics in terms of spatiotemporal subcellular localization, as compared with the other paralogs that remain in the ER and/or Golgi apparatus during secretory processes. Only PLA2α is secreted out to extracellular spaces, and its secretion to apoplasts is modulated according to the developmental stages of plant tissues. Observation of PLA2α-RFP transgenic plants suggests that PLA2α localizes mostly at the Golgi bodies in actively growing leaf tissues, but is gradually trans-located to apoplasts as the leaves become mature. When Pseudomonas syringae pv. tomato DC3000 carrying the avirulent factor avrRpm1 infects the apoplasts of host plants, PLA2α rapidly trans-locates to the apoplasts where bacteria attempt to become established. PLA2α promoter::GUS assays show that PLA2α gene expression is controlled in a developmental stage- and tissue-specific manner. It would be interesting to investigate if PLA2α functions in plant defense responses at apoplasts where secreted PLA2α confronts with invading pathogens.

  17. Increased Serum Phospholipase A2 Activity in Advanced Chronic Liver Disease as an Expression of the Acute Phase Response

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    Mario Pirisi

    1993-01-01

    Full Text Available Phospholipase A2 (PLA2 modifications were investigated in patients with acute and chronic liver diseases, PLA2 variations were related to indices of liver function as well as to parameters of the acute phase response. Serum PLA2 activity modifications were f1uorimetrically measured in 105 patients affected by acute and chronic liver diseases or extra-hepatic diseases. One-way ANOV A demonstrated a significant difference among groups (F= 4.53, P<0.001; Bonferroni’s test for pairwise comparisons showed that patients with hepatocellular carcinoma had higher mean values than subjects with benign extra-hepatic diseases (p<0.0 I and mild chronic liver disease (p<0.0S J. Multiple regression analysis, performed choosing PLA2 as the dependent variable and blood urea nitrogen, C-reacti ve protein, alkaline phosphatase and al-fetoprotein as predictor variables was significant (multiple R= 0.7056, multiple R2= 0.4978, F= 15.36, P= <0.0001. The standardized regression coefficients found to be significant were those of Creactive protein, blood urea nitrogen and al-fetoprotein. In conclusion, in patients with chronic liver disease, serum PLA2 activity increases parallel to disease severity and accompanies the expression of proteins of the acute phase response that. like PLA2 activity, increase in serum while liver synthesis declines.

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

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    Carlos A H Fernandes

    Full Text Available 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.

  19. Purification of a basic phospholipase A2 from Indian saw-scaled viper (Echis carinatus) venom: characterization of antigenic, catalytic and pharmacological properties.

    Science.gov (United States)

    Kemparaju, K; Prasad, B N; Gowda, V T

    1994-10-01

    A major basic phospholipase A2 was purified from the Indian saw-scaled viper (Echis carinatus) venom by the combination of column chromatography and electrophoresis. The purified phospholipase A2 (EC-IV-PLA2) has a mol. wt of 14,000 by SDS-PAGE. It is a basic protein with a pI value between 7.2 and 7.6, and has a fluorescence emission maxima at 340 nm. It induces neurotoxicity and oedema in mice with an i.p. LD50 of 5 mg/kg body weight. It is devoid of direct haemolytic, myotoxic, cytotoxic and anticoagulant activities. Rabbit polyclonal antibodies prepared against EC-IV-PLA2 inhibited the in vitro enzymatic activity dose dependently, but did not neutralize the toxic effects of EC-IV-PLA2 in experimental animals. PMID:7846689

  20. An alternative method to isolate protease and phospholipase A2 toxins from snake venoms based on partitioning of aqueous two-phase systems

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    GN Gómez

    2012-01-01

    Full Text Available Snake venoms are rich sources of active proteins that have been employed in the diagnosis and treatment of health disorders and antivenom therapy. Developing countries demand fast economical downstream processes for the purification of this biomolecule type without requiring sophisticated equipment. We developed an alternative, simple and easy to scale-up method, able to purify simultaneously protease and phospholipase A2 toxins from Bothrops alternatus venom. It comprises a multiple-step partition procedure with polyethylene-glycol/phosphate aqueous two-phase systems followed by a gel filtration chromatographic step. Two single bands in SDS-polyacrylamide gel electrophoresis and increased proteolytic and phospholipase A2 specific activities evidence the homogeneity of the isolated proteins.

  1. 蜂毒过敏原磷脂酶A2%Research on Phospholipase A2, the Allergen in Bee Venom

    Institute of Scientific and Technical Information of China (English)

    李英华; 胡福良; 刘艳荷

    2001-01-01

    @@ 蜂毒(bee venom)是由工蜂毒腺和副腺分泌的、具有芳香气味的一种透明液体,贮藏在毒囊中,在蜜蜂蛰刺时由蛰针排出[1].蜂毒具有抗菌、消炎、镇痛、降血压、抗辐射、预防癌症等药理作用,可用于治疗风湿性关节炎、类风湿性关节炎、哮喘、神经痛等多种疑难杂症.目前世界上许多国家都已开展蜂针疗法,并有各种类型的蜂毒软膏和针剂生产.但由于蜂毒易使人产生过敏反应,致使蜂针疗法不能得到广泛推广.鉴于这一点,国内外许多学者对主要引起人类过敏的蜂毒组分--磷脂酶A2(phospholipase A2)进行了研究,并且取得了一定进展.

  2. Inhibition of Group IIA Secretory Phospholipase A2 and its Inflammatory Reactions in Mice by Ethanolic Extract of Andrographis paniculata, a Well-known Medicinal Food

    OpenAIRE

    V Kishore; N S Yarla; Zameer, F.; Nagendra Prasad, M. N.; Santosh, M. S.; S S More; Rao, D.G.; Bhadrapura Lakkappa Dhananjaya

    2016-01-01

    Andrographis paniculata Nees is an important medicinal plant found in the tropical regions of the world, which has been traditionally used in Indian and Chinese medicinal systems. It is also used as medicinal food. A. paniculata is found to exhibit anti-inflammatory activities; however, its inhibitory potential on inflammatory Group IIA phospholipases A2 (PLA2) and its associated inflammatory reactions are not clearly understood. The aim of the present study is to evaluate the inhibitory/neut...

  3. Bee venom phospholipase A2 suppresses allergic airway inflammation in an ovalbumin‐induced asthma model through the induction of regulatory T cells

    OpenAIRE

    Park, Soojin; Baek, Hyunjung; Jung, Kyung‐Hwa; Lee, Gihyun; Lee, Hyeonhoon; Kang, Geun‐Hyung; Lee, Gyeseok; Bae, Hyunsu

    2015-01-01

    Abstract Bee venom (BV) is one of the alternative medicines that have been widely used in the treatment of chronic inflammatory diseases. We previously demonstrated that BV induces immune tolerance by increasing the population of regulatory T cells (Tregs) in immune disorders. However, the major component and how it regulates the immune response have not been elucidated. We investigated whether bee venom phospholipase A2 (bvPLA2) exerts protective effects that are mediated via Tregs in OVA‐in...

  4. Cytosolic phospholipase A2-α expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours.

    LENUS (Irish Health Repository)

    Caiazza, F

    2011-01-18

    The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A(2)α (cPLA(2)α) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA(2)α expression correlated with EGFR\\/HER2 over-expression in a small number of breast cancer cell lines.

  5. Inhibition of toxic actions of phospholipase A2 isolated & characterized from the Indian Banded Krait (Bungarus fasciatus) venom by synthetic herbal compounds

    OpenAIRE

    Gomes, Antony; Bhattacharya, Shamik; Mukherjee, Sanghamitra; Inn-ho-Tsai,; Gomes, Aparna

    2012-01-01

    Background & objectives: Phospholipase A2 (PLA2) is one of the major constituents of krait venom associated with several pathophysiological actions like myotoxicity, cardiotoxicity, neurotoxicity, etc. As there was no specific antiserum available against Bungarus fasciatus venom, this study was done with synthetic herbal compounds, anti PLA2 rabbit antiserum and commercial polyvalent snake venom antiserum to neutralize the PLA2 induced toxicities in experimental models. Methods: B. fasciatus ...

  6. Possible role of mammalian secretory group II phospholipase A2 in T-lymphocyte activation: implication in propagation of inflammatory reaction.

    OpenAIRE

    Asaoka, Y; Yoshida, K.; Sasaki, Y.; Nishizuka, Y; M. Murakami; Kudo, I; Inoue, K.

    1993-01-01

    Both 2-lysophosphatidylcholine and cis-unsaturated fatty acids were previously shown to intensify agonist-induced cellular responses by enhancing the diacylglycerol-dependent activation of protein kinase C. Consistent with these observations, extracellular, secretory group II phospholipase A2, when added directly to human resting T lymphocytes, greatly potentiates their activation that was induced by a membrane-permeant diacylglycerol and ionomycin, as determined by the expression of the alph...

  7. Investigation into the Role of Phosphatidylserine in Modifying the Susceptibility of Human Lymphocytes to Secretory Phospholipase A2 using Cells Deficient in the Expression of Scramblase

    OpenAIRE

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

    2012-01-01

    Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt’s lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells ...

  8. Two phospholipase A2 inhibitors from the plasma of Cerrophidion (Bothrops) godmani which selectively inhibit two different group-II phospholipase A2 myotoxins from its own venom: isolation, molecular cloning and biological properties.

    Science.gov (United States)

    Lizano, S; Angulo, Y; Lomonte, B; Fox, J W; Lambeau, G; Lazdunski, M; Gutiérrez, J M

    2000-01-01

    Myotoxic phospholipases A(2) (PLA(2)s; group II) account for most of the muscle-tissue damage that results from envenomation by viperid snakes. In the venom of the Godman's viper (Cerrophidion godmani, formerly Bothrops godmani), an enzymically active PLA(2) (myotoxin I) and an inactive, Lys-49 variant (myotoxin II) induce extensive muscle damage and oedema. In this study, two distinct myotoxin inhibitor proteins of C. godmani, CgMIP-I and CgMIP-II, were purified directly from blood plasma by selective binding to affinity columns containing either myotoxin I or myotoxin II, respectively. Both proteins are glycosylated, acidic (pI=4) and composed of 20-25-kDa subunits that form oligomers of 110 kDa (CgMIP-I) or 180 kDa (CgMIP-II). In inhibition studies, CgMIP-I specifically neutralized the PLA(2) and the myotoxic, oedema-forming and cytolytic activities of myotoxins I, whereas CgMIP-II selectively inhibited the toxic properties of myotoxin II. N-terminal amino acid sequence analysis and sequencing of cDNAs encoding the two inhibitors revealed that CgMIP-I is similar to gamma-type inhibitors, which share a pattern of cysteine residues present in the Ly-6 superfamily of proteins, whereas CgMIP-II shares sequence identity with alpha-type inhibitors that contain carbohydrate-recognition-like domains, also found in C-type lectins and mammalian PLA(2) receptors. N-terminal sequencing of myotoxin I revealed a different primary structure from myotoxin II [De Sousa, Morhy, Arni, Ward, Díaz and Gutiérrez (1998) Biochim. Biophys. Acta 1384, 204-208], which provides insight into the nature of such pharmacological specificity. PMID:10698689

  9. 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.

    Science.gov (United States)

    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

  10. 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

    Directory of Open Access Journals (Sweden)

    Kazuaki Yamaguchi

    2014-12-01

    Full Text Available 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.

  11. Atomic resolution (0.97 Å) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2

    International Nuclear Information System (INIS)

    The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 has been solved at atomic resolution (0.97 Å) and the refined model features the presence of a second calcium ion and a chloride ion. The enzyme phospholipase A2 catalyzes the hydrolysis of the sn-2 acyl chain of phospholipids, forming fatty acids and lysophospholipids. The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 in which the lysine residues at positions 53, 56 and 121 are replaced recombinantly by methionines has been determined at atomic resolution (0.97 Å). The crystal is monoclinic (space group P2), with unit-cell parameters a = 36.934, b = 23.863, c = 65.931 Å, β = 101.47°. The structure was solved by molecular replacement and has been refined to a final R factor of 10.6% (Rfree = 13.4%) using 63 926 unique reflections. The final protein model consists of 123 amino-acid residues, two calcium ions, one chloride ion, 243 water molecules and six 2-methyl-2,4-pentanediol molecules. The surface-loop residues 60–70 are ordered and have clear electron density

  12. Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica.

    Science.gov (United States)

    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).

  13. Identification of the immunodominant epitope region in phospholipase A2 receptor-mediating autoantibody binding in idiopathic membranous nephropathy.

    Science.gov (United States)

    Kao, Liyo; Lam, Vinson; Waldman, Meryl; Glassock, Richard J; Zhu, Quansheng

    2015-02-01

    Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Recent clinical studies established that >70% of patients with idiopathic (also called primary) MN (IMN) possess circulating autoantibodies targeting the M-type phospholipase A2 receptor-1 (PLA2R) on the surface of glomerular visceral epithelial cells (podocytes). In situ, these autoantibodies trigger the formation of immune complexes, which are hypothesized to cause enhanced glomerular permeability to plasma proteins. Indeed, the level of autoantibody in circulation correlates with the severity of proteinuria in patients. The autoantibody only recognizes the nonreduced form of PLA2R, suggesting that disulfide bonds determine the antigenic epitope conformation. Here, we identified the immunodominant epitope region in PLA2R by probing isolated truncated PLA2R extracellular domains with sera from patients with IMN that contain anti-PLA2R autoantibodies. Patient sera specifically recognized a protein complex consisting of the cysteine-rich (CysR), fibronectin-like type II (FnII), and C-type lectin-like domain 1 (CTLD1) domains of PLA2R only under nonreducing conditions. Moreover, absence of either the CysR or CTLD1 domain prevented autoantibody recognition of the remaining domains. Additional analysis suggested that this three-domain complex contains at least one disulfide bond required for conformational configuration and autoantibody binding. Notably, the three-domain complex completely blocked the reactivity of autoantibodies from patient sera with the full-length PLA2R, and the reactivity of patient sera with the three-domain complex on immunoblots equaled the reactivity with full-length PLA2R. These results indicate that the immunodominant epitope in PLA2R is exclusively located in the CysR-FnII-CTLD1 region.

  14. IgG4-Related Disease Is Not Associated with Antibody to the Phospholipase A2 Receptor

    Directory of Open Access Journals (Sweden)

    Arezou Khosroshahi

    2012-01-01

    Full Text Available Patients with IgG4-related disease (IgG4-RD share histopathological characteristics that are similar across affected organs. The finding of infiltration with IgG4+ plasma cells in the proper clinical and histopathological contexts connects a large number of clinical entities that were viewed previously as separate conditions. The renal involvement in IgG4-RD is usually characterized by tubulointerstitial nephritis, but membranous nephropathy has also been reported to be one of the renal complications of IgG4-RD. The recent discovery that a high proportion of patients with idiopathic membranous nephropathy (IMN have IgG4 autoantibodies to the M-type phospholipase A2 receptor (PLA2R in the circulation and glomerular immune deposits, together with the profound IgG4 hypergammaglobulinemia and occasional reports of membranous nephropathy in IgG4-RD, raised the question of a common antigen. To assess the presence of anti-PLA2R antibody in patients with IgG4-RD, we screened sera from 28 IgG4-RD patients by immunoblot. None of the patients in this cohort had detectable circulating anti-PLA2R antibodies. This study suggests that despite some clinical and serological overlaps between IgG4-RD and IMN,anti-PLA2R antibodies do not play a role in the pathogenesis of IgG4-RD. Additional studies of IgG4-RD with evidence of membranous nephropathy are important to exclude any definite relationship.

  15. Analysis of expression of secreted phospholipases A2 in mouse tissues at protein and mRNA levels.

    Science.gov (United States)

    Eerola, Leena I; Surrel, Fanny; Nevalainen, Timo J; Gelb, Michael H; Lambeau, Gérard; Laine, V Jukka O

    2006-07-01

    Secreted phospholipases A(2) (sPLA(2)) form a group of low-molecular weight enzymes that catalyze the hydrolysis of phospholipids. Some sPLA(2)s are likely to play a role in inflammation, cancer, and as antibacterial enzymes in innate immunity. We developed specific and sensitive time-resolved fluroimmunoassays (TR-FIA) for mouse group (G) IB, GIIA, GIID, GIIE, GIIF, GV and GX sPLA(2)s and measured their concentrations in mouse serum and tissues obtained from both Balb/c and C57BL/6J mice. We also analyzed the mRNA expression of the sPLA(2)s by quantitative real-time reverse transcriptase PCR (qPCR). In most tissues, the concentrations of sPLA(2) proteins corresponded to the expression of sPLA(2)s at the mRNA level. With a few exceptions, the sPLA(2) proteins were found in the gastrointestinal tract. The qPCR results showed that GIB sPLA(2) is synthesized widely in the gastrointestinal tract, including esophagus and colon, in addition to stomach and pancreas. Our results also suggest that the loss of GIIA sPLA(2) in the intestine of GIIA sPLA(2)-deficient C57BL/6J mice is not compensated by other sPLA(2)s under normal conditions. Outside the gastrointestinal tract, sPLA(2)s were expressed occasionally in a number of tissues. The TR-FIAs developed in the current study may serve as useful tools to measure the levels of sPLA(2) proteins in mouse serum and tissues in various experimental settings.

  16. Inhibition of ACh release at an Aplysia synapse by neurotoxic phospholipases A2: specific receptors and mechanisms of action.

    Science.gov (United States)

    Fossier, P; Lambeau, G; Lazdunski, M; Baux, G

    1995-01-01

    1. Monochain (OS2) and multichain (taipoxin) neurotoxic phospholipases A2 (PLA2), purified from taipan snake venom, both inhibited ACh release at a concentration of 20 nM (90% inhibition in 2 h) at an identified synapse from buccal ganglion of Aplysia californica. 2. The Na+ current was unchanged upon application of either OS2 or taipoxin. Conversely, presynaptic K+ currents (IA and IK) were increased by taipoxin but not by OS2. In addition, OS2 induced a significant decrease of the presynaptic Ca2+ current (30%) while taipoxin increased this latter current by 20-30%. 3. Bee venom PLA2, another monochain neurotoxic PLA2, also inhibited ACh release while non-toxic enzymatically active PLA2s like OS1 (also purified from taipan snake venom) or porcine pancreatic PLA2 elicited a much weaker inhibition of ACh release, suggesting a specific action of neurotoxic PLA2s versus non-toxic PLA2s on ACh release. 4. Using iodinated OS2, specific high affinity binding sites with molecular masses of 140 and 18 kDa have been identified on Aplysia ganglia. The maximal binding capacities were 55 and 300-400 fmol (mg protein)-1 for membrane preparations from whole and buccal ganglia, respectively. These binding sites are of high affinity for neurotoxic PLA2s (Kd values, 100-800 pM) and of very low affinity for non-toxic PLA2s (Kd values in the micromolar range), thus indicating that these binding sites are presumably involved in the blockade of ACh release by neurotoxic PLA2s. Images Figure 8 Figure 9 PMID:8583413

  17. Atherosclerotic Plaque Inflammation Varies Between Vascular Sites and Correlates With Response to Inhibition of Lipoprotein‐Associated Phospholipase A2

    Science.gov (United States)

    Fenning, Robert S.; Burgert, Mark E.; Hamamdzic, Damir; Peyster, Eliot G.; Mohler, Emile R.; Kangovi, Shreya; Jucker, Beat M.; Lenhard, Stephen C.; Macphee, Colin H.; Wilensky, Robert L.

    2015-01-01

    Background Despite systemic exposure to risk factors, the circulatory system develops varying patterns of atherosclerosis for unclear reasons. In a porcine model, we investigated the relationship between site‐specific lesion development and inflammatory pathways involved in the coronary arteries (CORs) and distal abdominal aortas (AAs). Methods and Results Diabetes mellitus (DM) and hypercholesterolemia (HC) were induced in 37 pigs with 3 healthy controls. Site‐specific plaque development was studied by comparing plaque severity, macrophage infiltration, and inflammatory gene expression between CORs and AAs of 17 DM/HC pigs. To assess the role of lipoprotein‐associated phospholipase A2 (Lp‐PLA2) in plaque development, 20 DM/HC pigs were treated with the Lp‐PLA2 inhibitor darapladib and compared with the 17 DM/HC untreated pigs. DM/HC caused site‐specific differences in plaque severity. In the AAs, normalized plaque area was 4.4‐fold higher (P<0.001) and there were more fibroatheromas (9 of the 17 animals had a fibroatheroma in the AA and not the COR, P=0.004), while normalized macrophage staining area was 1.5‐fold higher (P=0.011) compared with CORs. DM/HC caused differential expression of 8 of 87 atherosclerotic genes studied, including 3 important in inflammation with higher expression in the CORs. Darapladib‐induced attenuation of normalized plaque area was site‐specific, as CORs responded 2.9‐fold more than AAs (P=0.045). Conclusions While plaque severity was worse in the AAs, inflammatory genes and inflammatory pathways that use Lp‐PLA2 were more important in the CORs. Our results suggest fundamental differences in inflammation between vascular sites, an important finding for the development of novel anti‐inflammatory therapeutics. PMID:25672369

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

    Directory of Open Access Journals (Sweden)

    G L Crawford

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Jantan I

    2014-09-01

    Full Text Available Ibrahim Jantan,1,* Syed Nasir Abbas Bukhari,1,* Olayiwola A Adekoya,2 Ingebrigt Sylte3 1Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia; 2Department of Pharmacy, Faculty of Health Science, UiT The Arctic University of Norway, Tromsø, Norway; 3Department of Medical Biology, Faculty of Health Science, UiT The Arctic University of Norway, Tromsø, Norway *These authors contributed equally to this work Abstract: Arachidonic acid metabolism leads to the generation of key lipid mediators which play a fundamental role during inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as a synergistic anti-inflammatory effect with enhanced spectrum of activity. A series of 1,3-diphenyl-2-propen-1-one derivatives were investigated for anti-inflammatory related activities involving inhibition of secretory phospholipase A2, cyclooxygenases, soybean lipoxygenase, and lipopolysaccharides-induced secretion of interleukin-6 and tumor necrosis factor-alpha in mouse RAW264.7 macrophages. The results from the above mentioned assays exhibited that the synthesized compounds were effective inhibitors of pro-inflammatory enzymes and cytokines. The results also revealed that the chalcone derivatives with 4-methlyamino ethanol substitution seem to be significant for inhibition of enzymes and cytokines. Molecular docking experiments were carried out to elucidate the molecular aspects of the observed inhibitory activities of the investigated compounds. Present findings increase the possibility that these chalcone derivatives might serve as a beneficial starting point for the design and development of improved anti-inflammatory agents. Keywords: anti-inflammatory, tumor necrosis factor-alpha, lipopolysaccharides, molecular docking

  20. Cytosolic phospholipase A2 shows burst kinetics consistent with the slow, reversible formation of a dead-end complex.

    Science.gov (United States)

    Guenther, Matthew G; Witmer, Mark R; Burke, James R

    2002-02-01

    Cytosolic phospholipase A2 catalyzes the hydrolysis of the sn-2 ester of arachidonate-containing phospholipids. In the present research, a "burst" of arachidonate which precedes a somewhat slower, linear rate (upsilon) of product formation was observed and characterized using covesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) containing <10 mol% 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate. The magnitude of the burst (pi) was enzyme dependent, in both the presence and absence of glycerol. Upon subsequent addition of enzyme after the primary burst was complete, a second burst of arachidonate production was observed. This is consistent with the effect resulting from an enzyme effect and not from changes in the substrate. The use of 1,2-dioleoyl-sn-glycero-3-phosphomethanol as the carrier phospholipid instead of DMPM greatly reduced the rate of hydrolysis without a large effect on the pi/upsilon ratio, consistent with the burst not being the result of limitations in the lateral diffusion rate of phospholipids within the covesicles. When the assay is performed in the presence of glycerol, the burst phenomenon was also observed with the monoarachidonoyl glycerol transacylase product which shows that the effect occurs through a common mechanism. The burst and subsequent linear rate of hydrolysis are highly temperature dependent, with a pronounced increase in the pi/upsilon ratio as the temperature is increased from 35 to 45 degrees C. A mechanism in which a slow equilibrium between an active and less active (inactive) state of substrate-bound enzyme is proposed. This may provide a means by which the enzyme is switched off after a few hundred turnovers in order to prevent unabated phospholipid hydrolysis in cells which may be deleterious to membrane integrity. PMID:11811954

  1. Mechanism of Cytosolic Phospholipase A2 Activation in Ghrelin Protection of Salivary Gland Acinar Cells against Ethanol Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Bronislaw L. Slomiany

    2010-01-01

    Full Text Available Ghrelin, a peptide hormone, newly identified in oral mucosal tissues, has emerged recently as an important mediator of the processes of mucosal defense. Here, we report on the mechanism of ghrelin protection against ethanol cytotoxicity in rat sublingual salivary gland cells. The protective effect of ghrelin was associated with the increase in NO and PGE2, and upregulation in cytosolic phospholipase A2 (cPLA2 activity and arachidonic acid (AA release. The loss in countering effect of ghrelin occurred with cNOS inhibitor, L-NAME, as well as indomethacin and COX-1 inhibitor, SC-560, while COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced cell capacity for NO production, cPLA2 activation and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA2 phosphorylation and S-nitrosylation. Inhibition in ghrelin-induced S-nitrosylation was attained with L-NAME, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA2 protein phosphorylation as well as S-nitrosylation. Thus, ghrelin protection of salivary gland cells against ethanol involves cNOS-derived NO induction of cPLA2 activation through S-nitrosylation for the increase in AA release at the site of COX-1 action for PGE2 synthesis.

  2. Secretory phospholipase A2 in dromedary tears: a host defense against staphylococci and other gram-positive bacteria.

    Science.gov (United States)

    Ben Bacha, Abir; Abid, Islem

    2013-03-01

    The best known physiologic function of secreted phospholipase A2 (sPLA2) group IIA (sPLA2-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA2-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye's innate immune defense system. The antibacterial properties of sPLA2 have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA2 on several Gram-positive bacteria. Our results showed that the sPLA2 displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca(2+) is crucial for the activity of dromedary tear sPLA2 and to a less extent Mg(2+) ions. Given the presence of sPLA2 in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.

  3. The inhibition of lipoprotein-associated phospholipase A2 exerts beneficial effects against atherosclerosis in LDLR-deficient mice

    Institute of Scientific and Technical Information of China (English)

    Miao-miao HU; Jie ZHANG; Wen-yi WANG; Wen-yu WU; Yan-ling MA; Wei-hai CHEN; Yi-ping WANG

    2011-01-01

    Aim:To investigate the effects of darapladib,a specific inhibitor of lipoprotein-associated phospholipase A2 (Ip-PLA2),on inflammation and atherosclerotic formation in the low density lipoprotein receptor (LDLR)-deficient mice.Methods:Six-week-old LDLR-deficient mice were fed an atherogenic high-fat diet for 17 weeks and then randomly divided into two groups.One group was administered darapladib (50 mg·kg1·d1;p0) for 6 weeks.The other group was administered saline as a control.Serum lipid levels were measured using the corresponding kits,and three inflammatory markers -- interleukin-6 (IL-6),C reactive protein (hs-CRP),and platelet activating factor (PAF) - were determined using ELISA.Atherosclerotic plaque areas were stained with Sudan IV,and inflammatory gene expression at the lesions was evaluated using quantitative real-time PCR.Results:The body weight and serum lipid level between the two groups were similar at the end of the dietary period.The serum Ip-PLA2 activity,hs-CRP and IL-6 levels,however,were significantly reduced in the darpladib group.The inhibition of Ip-PLA2 did not alter the serum PAF level.Furthermore,the plaque area,from the aortic arch to the abdominal aorta,was significantly reduced in the darpladib group.Additionally,the expression of inflammatory genes monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) was significantly reduced at the lesions in the darapladib group.Conclusion:Inhibition of Ip-PLA2 by darapladib decreases the inflammatory burden and atherosclerotic plaque formation in LDLR-deficient mice,which may be a new strategy for the treatment of atherosclerosis.

  4. Secretory phospholipase A2-mediated progression of hepatotoxicity initiated by acetaminophen is exacerbated in the absence of hepatic COX-2

    International Nuclear Information System (INIS)

    We have previously reported that among the other death proteins, hepatic secretory phospholipase A2 (sPLA2) is a leading mediator of progression of liver injury initiated by CCl4 in rats. The aim of our present study was to test the hypothesis that increased hepatic sPLA2 released after acetaminophen (APAP) challenge mediates progression of liver injury in wild type (WT) and COX-2 knockout (KO) mice. COX-2 WT and KO mice were administered a normally non lethal dose (400 mg/kg) of acetaminophen. The COX-2 KO mice suffered 60% mortality compared to 100% survival of the WT mice, suggesting higher susceptibility of COX-2 KO mice to sPLA2-mediated progression of acetaminophen hepatotoxicity. Liver injury was significantly higher at later time points in the KO mice compared to the WT mice indicating that the abatement of progression of injury requires the presence of COX-2. This difference in hepatotoxicity was not due to increased bioactivation of acetaminophen as indicated by unchanged cyp2E1 protein and covalently bound 14C-APAP in the livers of KO mice. Hepatic sPLA2 activity and plasma TNF-α were significantly higher after APAP administration in the KO mice. This was accompanied by a corresponding fall in hepatic PGE2 and lower compensatory liver regeneration and repair (3H-thymidine incorporation) in the KO mice. These results suggest that hindered compensatory tissue repair and poor resolution of inflammation for want of beneficial prostaglandins render the liver very vulnerable to sPLA2-mediated progression of liver injury. These findings are consistent with the destructive role of sPLA2 in the progression and expansion of tissue injury as a result of continued hydrolytic breakdown of plasma membrane phospholipids of perinecrotic hepatocytes unless mitigated by sufficient co-induction of COX-2.

  5. Group IVA phospholipase A2-associated production of MMP-9 in macrophages and formation of atherosclerotic lesions.

    Science.gov (United States)

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

    Science.gov (United States)

    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.

  7. Study on the correlation between the concentration of plasma lipoprotein-associated phospholipase A2 and coronary heart disease

    Institute of Scientific and Technical Information of China (English)

    Jin-Ming Cen; Jie Cheng; Qing-Yuan Xiong; Bai-Qiang Mei; Wei-Biao Cai; Xi-Li Yang

    2015-01-01

    Objective: This study explores the correlation between plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) and coronary heart disease (CHD) by comparing the level of plasma Lp-PLA2 in the plasma of patients with different types of CHD. Methods: Blood samples were collected from 56 patients diagnosed with CHD by the Department of Cardiology of the First People's Hospital of Foshan and 34 healthy subjects from February 2013 to January 2014. We measured the concentration of plasma Lp-PLA2 and determined the levels of total cholesterol (Tch), triglyceride (TG), apolipoprotein A1 (Apo-A1), apolipoprotein B (Apo-B), high density lipoprotein-cholesterol (HDL-c), low density lipoprotein-cholesterol (LDL-c), lipoprotein a (Lp(a)), glucose (Glu), and high-sensitivity C-reactive protein (hs-CRP). The concentration of plasma Lp-PLA2 in the healthy control group and each subgroup of CHD patients were compared and analyzed for correlations of plasma Lp-PLA2 between the patients in different CHD subgroups and several laboratory indicators. Results: The concentration of plasma Lp-PLA2 in each subgroup of CHD was significantly higher than in the control group (P Conclusions: The concentration of plasma Lp-PLA2 in patients with CHD was higher than that in the control group. The concentration of plasma Lp-PLA2 in the subgroups of CHD patients varied greatly from each other. The inflammatory response of atherosclerosis might be resulted from the synergy of plasma Lp-PLA2 and hs-CRP. Copyright © 2015, Chinese Medical Association Production. Production and hosting by Elsevier B.V. on behalf of KeAi Com-munications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

  8. Cytosolic phospholipase A2 activation correlates with HER2 overexpression and mediates estrogen-dependent breast cancer cell growth.

    LENUS (Irish Health Repository)

    Caiazza, Francesco

    2010-05-01

    Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) catalyzes the hydrolysis of membrane glycerol-phospholipids to release arachidonic acid as the first step of the eicosanoid signaling pathway. This pathway contributes to proliferation in breast cancer, and numerous studies have demonstrated a crucial role of cyclooxygenase 2 and prostaglandin E(2) release in breast cancer progression. The role of cPLA(2)alpha activation is less clear, and we recently showed that 17beta-estradiol (E2) can rapidly activate cPLA(2)alpha in MCF-7 breast cancer cells. Overexpression or gene amplification of HER2 is found in approximately 30% of breast cancer patients and correlates with a poor clinical outcome and resistance to endocrine therapy. This study reports the first evidence for a correlation between cPLA(2)alpha enzymatic activity and overexpression of the HER2 receptor. The activation of cPLA(2)alpha in response to E2 treatment was biphasic with the first phase dependent on trans-activation through the matrix metalloproteinase-dependent release of heparin-bound epidermal growth factor. EGFR\\/HER2 heterodimerization resulted in downstream signaling through the ERK1\\/2 cascade to promote cPLA(2)alpha phosphorylation at Ser505. There was a correlation between HER2 and cPLA(2)alpha expression in six breast cancer cell lines examined, and inhibition of HER2 activation or expression in the SKBR3 cell line using herceptin or HER2-specific small interfering RNA, respectively, resulted in decreased activation and expression of cPLA(2)alpha. Pharmacological blockade of cPLA(2)alpha using a specific antagonist suppressed the growth of both MCF-7 and SKBR3 cells by reducing E2-induced proliferation and by stimulating cellular apoptosis and necrosis. This study highlights cPLAalpha(2) as a potential target for therapeutic intervention in endocrine-dependent and endocrine-independent breast cancer.

  9. Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

    Science.gov (United States)

    Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

    2015-10-01

    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.

  10. Secretory Phospholipases A2 in Durum Wheat (Triticum durum Desf.: Gene Expression, Enzymatic Activity, and Relation to Drought Stress Adaptation

    Directory of Open Access Journals (Sweden)

    Daniela Trono

    2013-03-01

    Full Text Available Phospholipases A2 (PLA2s are known to mediate signaling cascades during plant growth and development, as well as biotic and abiotic stress responses. In this context, the present study provides extensive characterization of specific PLA2s in durum wheat, and assesses their involvement in durum wheat response to drought stress. In durum wheat leaves, four full-length expressed sequences encoding putative PLA2s were isolated and characterized as belonging to the class of secretory PLA2s (sPLA2s: TdsPLA2I, TdsPLA2II, TdsPLA2III and TdsPLA2IV. PLA2 activity was also detected, the characteristics of which resemble those of previously characterized plant sPLA2s: strong preference for phospholipids; requirement for millimolar Ca2+ concentrations; optimal activity at basic pH; heat stability; and inhibition by the reducing agent dithiothreitol. With drought stress imposed at both the vegetative and reproductive stages, accumulation of TdsPLA2I and TdsPLA2III transcripts, and to a lesser extent of TdsPLA2IV transcript, paralleled increased PLA2 activity; both transcript levels and enzymatic activity decreased as a consequence of stress recovery. Consistently, free fatty acid analysis of drought-stressed leaves revealed increased linoleate, linolenate and palmitate contents, which were reversed by plant re-watering. Overall, these findings strongly suggest that there are inducible sPLA2 isoforms in durum wheat that have roles in orchestrating the plant response to drought stress.

  11. Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers

    NARCIS (Netherlands)

    Demel, R.A.; Geurts van Kessel, W.S.M.; Zwaal, R.F.A.; Roelofsen, B.; Deenen, L.L.M. van

    1975-01-01

    The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes. The phospholipases which cannot hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A2 from

  12. On the role of protein disulfide isomerase in the retrograde cell transport of secreted phospholipases A2.

    Directory of Open Access Journals (Sweden)

    Jernej Oberčkal

    Full Text Available Following the finding that ammodytoxin (Atx, a neurotoxic secreted phospholipase A2 (sPLA2 in snake venom, binds specifically to protein disulfide isomerase (PDI in vitro we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA sPLA2. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI, a three-dimensional model of the complex between Atx and human PDI (hPDI was constructed. The Atx binding site on hPDI is situated between domains b and b'. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA2s have the same fold as Atx. The first three sPLA2s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA2 in the Atx-hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA2s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA2 and hPDI is however different from that of the other sPLA2s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA2s confirmed the model-based expectation that GV sPLA2 was a more effective inhibitor than GX sPLA2, thus validating our model. The results suggest a role of hPDI in the (pathophysiology of some snake venom and mammalian sPLA2s by assisting the retrograde transport of these molecules from the cell surface. The sPLA2-hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (pathophysiology of sPLA2s in relation to their action intracellularly.

  13. Functional analysis of two PLA2G2A variants associated with secretory phospholipase A2-IIA levels.

    Directory of Open Access Journals (Sweden)

    Holly J Exeter

    Full Text Available BACKGROUND: Secretory phospholipase A2 group IIA (sPLA2-IIA has been identified as a biomarker of atherosclerosis in observational and animal studies. The protein is encoded by the PLA2G2A gene and the aim of this study was to test the functionality of two PLA2G2A non-coding SNPs, rs11573156 C>G and rs3767221 T>G where the rare alleles have been previously associated with higher and lower sPLA2-IIA levels respectively. METHODOLOGY/PRINCIPAL FINDINGS: Luciferase assays, electrophoretic mobility shift assays (EMSA, and RNA expression by RT-PCR were used to examine allelic differences. For rs3767221 the G allele showed ∼55% lower luciferase activity compared to the T allele (T = 62.1 (95% CI 59.1 to 65.1 G = 27.8 (95% CI 25.0 to 30.6, p = 1.22×10⁻³⁵, and stronger EMSA binding of a nuclear protein compared to the T-allele. For rs11573156 C >G there were no luciferase or EMSA allelic differences seen. In lymphocyte cell RNA, from individuals of known rs11573156 genotype, there was no allelic RNA expression difference for exons 5 and 6, but G allele carriers (n = 7 showed a trend to lower exon 1-2 expression compared to CC individuals. To take this further, in the ASAP study (n = 223, an rs11573156 proxy (r² = 0.91 showed ∼25% higher liver expression of PLA2G2A (1.67×10⁻¹⁷ associated with the G allele. However, considering exon specific expression, the association was greatly reduced for exon 2 (4.5×10⁻⁵ compared to exons 3-6 (10⁻¹⁰ to 10⁻²⁰, suggesting rs11573156 G allele-specific exon 2 skipping. CONCLUSION: Both SNPs are functional and provide useful tools for Mendelian Randomisation to determine whether the relationship between sPLA2-IIA and coronary heart disease is causal.

  14. Modulation of the Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus cascavella Induced by Naringin

    OpenAIRE

    Toyama, Marcos H; Ricardo Aparicio; Soares, Veronica C G; Fagundes, Fábio H. R.; Diz-Filho, Eduardo B. S.; Simone C. B. Oliveira; Marcelo L. Santos; Camila A. Cotrim; Toyama, Daniela O

    2011-01-01

    In this work we have characterized the action of the naringin, a flavonoid found in grapefruit and known for its various pharmacological effects, which include antioxidant blood lipid lowering and anticancer activity, on the structure and biochemical activities of a secretory phospholipase A (sPLA2) from Crotalus durissus cascavella, an important protein involved in the releasinge of arachidonic acid in phospholipid membranes. sPLA2 was incubated with naringin (mol:mol) at 37 °C and a discret...

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

    OpenAIRE

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

    2016-01-01

    Background Alzheimer’s disease (AD) is a severe neuroinflammatory disease. CD4+Foxp3+ regulatory T cells (Tregs) modulate various inflammatory diseases via suppressing Th cell activation. There are increasing evidences that Tregs have beneficial roles in neurodegenerative diseases. Previously, we found the population of Treg cells was significantly increased by bee venom phospholipase A2 (bvPLA2) treatment in vivo and in vitro. Methods To examine the effects of bvPLA2 on AD, bvPLA2 was admini...

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

    OpenAIRE

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

    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 focall...

  17. Reduction of cytosolic phospholipase A2α upregulation delays the onset of symptoms in SOD1G93A mouse model of amyotrophic lateral sclerosis

    OpenAIRE

    Solomonov, Yulia; Hadad, Nurit; Levy, Rachel

    2016-01-01

    Background Amyotrophic lateral sclerosis (ALS) is a fatal multifactorial neurodegenerative disease characterized by selective death of motor neurons in the cortex, brainstem, and spinal cord. Cytosolic phospholipase A2 alpha (cPLA2α) upregulation and activation in the spinal cord of patients with sporadic ALS and in the spinal cord of human mutant SOD1G93A (hmSOD1) transgenic mice were recently reported. Methods cPLA2α upregulation in the brainstem and spinal cord was reduced by brain infusio...

  18. Male Mice That Do Not Express Group VIA Phospholipase A2 Produce Spermatozoa with Impaired Motility and Have Greatly Reduced Fertility*

    OpenAIRE

    Bao, Shunzhong; Miller, David J.; Ma, Zhongmin; Wohltmann, Mary; Eng, Grace; Ramanadham, Sasanka; Moley, Kelle; Turk, John

    2004-01-01

    The Group VIA Phospholipase A2 (iPLA2β) is the first recognized cytosolic Ca2+-independent PLA2 and has been proposed to participate in arachidonic acid (20:4) incorporation into glycerophosphocholine lipids, cell proliferation, exocytosis, apoptosis, and other processes. To study iPLA2β functions, we disrupted its gene by homologous recombination to generate mice that do not express iPLA2β. Heterozygous iPLA2β+/− breeding pairs yield a Mendelian 1:2:1 ratio of iPLA2β+/+, iPLA2β+/−, and iPLA2...

  19. Distinct expression pattern of the full set of secreted phospholipases A2 in human colorectal adenocarcinomas: sPLA2-III as a biomarker candidate

    OpenAIRE

    Mounier, C M; Wendum, D; Greenspan, E; Fléjou, J-F; Rosenberg, D W; Lambeau, G.

    2008-01-01

    Recent studies suggest that secreted phospholipases A2 (sPLA2s) represent attractive potential tumour biomarkers and therapeutic targets for various cancers. As a first step to address this issue in human colorectal cancer, we examined the expression of the full set of sPLA2s in sporadic adenocarcinomas and normal matched mucosa from 21 patients by quantitative PCR and immunohistochemistry. In normal colon, PLA2G2A and PLA2G12A were expressed at high levels, PLA2G2D, PLA2G5, PLA2G10 and PLA2G...

  20. Correlation of the inhibitory activity of phospholipase A2 snake venom and the antioxidant activity of Colombian plant extracts

    Directory of Open Access Journals (Sweden)

    Jaime A. Pereañez

    2010-12-01

    Full Text Available Snakebite continues to be a significant health problem in many countries of Latin America. Even though, there has been an improvement in the antivenom therapy, the local effects caused by myotoxic phospholipases A2 (PLA2 present in the venoms, still persist. In search for alternatives to antagonize the PLA2 activity of Bothrops asper's venom, 36 extracts belonging to seventeen families of vascular plants and bryophytes were screened. A significant inhibition of the enzymatic activity of PLA2 present in B. asper's whole venom was seen in eleven of these extracts. In addition, the antioxidant activity of all the extracts was evaluated. The results evidenced a significant statistical correlation between extracts with an inhibitory effect against PLA2 and those with an antioxidant activity. Moreover, the amount of phenols was quantified finding a relationship between the bioactivity and the presence of these compounds. Nine extracts were screened against a fraction of the venom rich in basic PLA2 (Fx-V B. asper, exhibiting an inhibitory effect on PLA2 activity of this fraction in a range from 30-80%. This activity was supported by the inhibition that these extracts presented on the cytotoxicity caused by Fx-V B. asper on murine skeletal muscle C2C12 myoblasts. The results obtained, could point to minimize efforts in the search of PLA2 inhibitors by focusing in samples with known antioxidant properties.Veneno de cobra continua a ser um problema importante de saúde em muitos países da América Latina. Apesar dos avanços na terapia antiveneno, os efeitos locais causados por fosfolipases A2 miotóxica (PLA2 presentes no veneno, ainda persistem. Em busca de alternativas para antagonizar a atividade da PLA2 do veneno de Bothrops asper, foram selecionados 36 extratos pertencentes a dezessete famílias de plantas vasculares e briófitas. Uma inibição significativa da atividade enzimática de PLA2 presente no veneno de B. asper foi observada em onze

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

    Directory of Open Access Journals (Sweden)

    C. L. S. Guimarães

    2014-01-01

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

  2. "Self" and "non-self" in the control of phytoalexin biosynthesis: plant phospholipases A2 with alkaloid-specific molecular fingerprints.

    Science.gov (United States)

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

    2015-02-01

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

  3. Structural and functional characterization of myotoxin I, a Lys49 phospholipase A2 homologue from the venom of the snake Bothrops atrox.

    Science.gov (United States)

    Núñez, Vitelbina; Arce, Viviana; Gutiérrez, José María; Lomonte, Bruno

    2004-07-01

    A new myotoxin was isolated from the venom of Bothrops atrox from Colombia. B. atrox myotoxin I is a homodimer, with a subunit molecular mass of 13,826, and a pI of 8.9. Its complete nucleotide sequence was obtained by cDNA cloning, indicating a mature product of 122 residues that belongs to the family of Lys49 phospholipase A(2) (PLA(2)) homologues, a subgroup of catalytically inactive proteins within the group IIA. Accordingly, the toxin was devoid of phospholipase and anticoagulant activities, in vitro. In mice, it induced conspicuous local myonecrosis, edema, and a systemic interleukin-6 response. In vitro, it was cytolytic upon myoblasts, and weakly bactericidal. The toxin showed highest homology with other Lys49 PLA(2)s, both in its primary and three-dimensional modeled structure, although with an evident difference in the C-terminal region. Unlike Lys49 proteins of American crotalids having 121 residues, this toxin presents an insertion (Asn) between positions 118 and 119. Despite several substitutions within the C-terminal region 115-129 between B. atrox myotoxin I and B. asper myotoxin II, antibodies against synthetic peptide 115-129 of the latter were strongly cross-reactive to the former, indicating the antigenic conservation of this site, known to be critical for the membrane-damaging activities of Lys49 myotoxins. PMID:15225567

  4. Blockade of Human Group X Secreted Phospholipase A2 (GX-sPLA2)-induced Airway Inflammation and Hyperresponsiveness in a Mouse Asthma Model by a Selective GX-sPLA2 Inhibitor*

    OpenAIRE

    Henderson, William R.; Oslund, Rob C.; BOLLINGER, JAMES G.; Ye, Xin; Tien, Ying-tzang; Xue, Jun; Gelb, Michael H

    2011-01-01

    Group X (GX) phospholipase A2, a member of a large group of secreted phospholipases A2 (sPLA2s), has recently been demonstrated to play an important in vivo role in the release of arachidonic acid and subsequent formation of eicosanoids. In a Th2 cytokine-driven mouse asthma model, deficiency of mouse GX (mGX)-sPLA2 significantly impairs development of the asthma phenotype. In this study, we generated mGX-sPLA2−/− mice with knock-in of human GX (hGX)-sPLA2 (i.e. hGX-sPLA2+/+ knock-in mice) to...

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

    Science.gov (United States)

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

    2007-05-22

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

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

    Science.gov (United States)

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

    2015-10-01

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

  7. [Anti-phospholipase A2 receptor (anti-PLA2R) antibodies and idiopathic membranous nephropathy: which role in diagnosis and prognosis of this disease?].

    Science.gov (United States)

    Netti, Giuseppe Stefano; Ranieri, Elena

    2014-01-01

    The discovery of the M-type phospholipase A2 receptor (PLA2R) as a major antigen in idiopathic membranous nephropathy (iMN) was a breakthrough in understanding the pathogenesis of this disease, establishing iMN as an autoimmune disease. Subsequent studies confirmed that detection of circulating antibodies against PLA2R was positive in approximately 70% of incident iMN patients. We discuss several studies that have suggested the potential role of measuring PLA2R antibodies for clinical practice. Recently, it has been shown that the presence of PLA2R antibodies supported a diagnosis of iMN, changes in antibody levels were related to clinical disease activity, disappearance of antibodies preceded and predicted subsequent decrease of proteinuria and high titers of antibodies were associated with a low likelihood spontaneous remission.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Aristides Quintero Rueda

    2013-01-01

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

  10. Purification and characterization of a platelet aggregation inhibitor acidic phospholipase A2 from Indian saw-scaled viper (Echis carinatus) venom.

    Science.gov (United States)

    Kemparaju, K; Krishnakanth, T P; Veerabasappa Gowda, T

    1999-12-01

    An acidic phospholipase A2 (EC-I-PLA2) has been purified from the Indian saw-scaled viper (Echis carinatus) venom through a combination of column chromatography and electrophoresis. EC-I-PLA2 has a molecular weight of 16000 by SDS-PAGE. It was focussed between pH 4.2 and 4.8 by isoelectro focussing. EC-I-PLA2 was non-lethal to mice and devoid of neurotoxicity, myotoxicity, anticoagulant activity and cytotoxicity. It induced mild oedema in the foot pads of mice. The purified PLA2 inhibited ADP, collagen and epinephrine induced human platelet aggregation and the inhibition was both dose and time dependent. PMID:10519645

  11. Modulation of the Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus cascavella Induced by Naringin

    Directory of Open Access Journals (Sweden)

    Marcos H. Toyama

    2011-01-01

    Full Text Available In this work we have characterized the action of the naringin, a flavonoid found in grapefruit and known for its various pharmacological effects, which include antioxidant blood lipid lowering and anticancer activity, on the structure and biochemical activities of a secretory phospholipase A (sPLA2 from Crotalus durissus cascavella, an important protein involved in the releasinge of arachidonic acid in phospholipid membranes. sPLA2 was incubated with naringin (mol:mol at 37 °C and a discrete reduction in the UV scanning signal and a modification of the circular dichroism spectra were observed after treatment with naringin, suggesting modifications of the secondary structure of the protein. This flavonoid was able to decrease enzymatic activity and some pharmacological effects, such as myonecrosis, platelet aggregation, and neurotoxic activity caused by sPLA2, however, the inflammatory effect was not affected by naringin. In addition, small angle X-ray scattering (SAXS data were collected for sPLA2 and naringin-treated sPLA2 to evaluate possible modifications of the protein structure. These structural investigations have shown that sPLA2 is an elongated dimer in solution and after treatment with naringin a conformational change in the dimeric configuration was observed. Our results suggest that structural modification may be correlated with the loss of enzymatic activity and alterations in pharmacological properties.

  12. Changes in wetting properties of silica surface treated with DPPC in the presence of phospholipase A2 enzyme

    International Nuclear Information System (INIS)

    Wetting properties of silica plates contacted with dipalmitoylphosphatidylcholine (DPPC) or DPPC/enzyme (phospholipase PLA2) in NaCl solution were determined by thin layer wicking and with a help of Washburn equation. The wicking experiments were performed both for bare plates and the silica plates precontacted overnight with the probe liquid saturated vapors the silica plates, as well as untreated and DPPC (or DPPC/enzyme) treated. Adsorption of DPPC on original silica plates increases a bit hydrophobic character of silica surface in such a way that hydrocarbon chains are directed outwards and the polar part towards the silica surface. However, after the enzyme action the products of DPPC hydrolysis by PLA2 (palmitic acid and lysophosphatidylcholine) increase again hydrophilic character of silica surface (an increase in acid-base interactions, γsAB). The changes of silica surface wettability are evidently dependent on the time of enzyme contacting with DPPC in NaCl solution. Although, the changes of total surface free energy of silica after treatment with DPPC/enzyme solution are minor about 2-6 mJ/m2, the changes of the electron-donor (γs-) and Lifshitz-van der Waals (γsLW) component of the surface free energy are noticeable. Despite, these results are somehow preliminary, it seems that thin layer wicking method is an interesting tool for investigation of the effect of adsorbed DPPC on hydrophobicity/hydrophilicity of silica surface and influence of enzyme PLA2 action.

  13. CoaTx-II, a new dimeric Lys49 phospholipase A2 from Crotalus oreganus abyssus snake venom with bactericidal potential: Insights into its structure and biological roles.

    Science.gov (United States)

    Almeida, J R; Lancellotti, M; Soares, A M; Calderon, L A; Ramírez, D; González, W; Marangoni, S; Da Silva, S L

    2016-09-15

    Snake venoms are rich and intriguing sources of biologically-active molecules that act on target cells, modulating a diversity of physiological functions and presenting promising pharmacological applications. Lys49 phospholipase A2 is one of the multifunctional proteins present in these complex secretions and, although catalytically inactive, has a variety of biological activities, including cytotoxic, antibacterial, inflammatory, antifungal activities. Herein, a Lys49 phospholipase A2, denominated CoaTx-II from Crotalus oreganus abyssus, was purified and structurally and pharmacologically characterized. CoaTx-II was isolated with a high degree of purity by a combination of two chromatographic steps; molecular exclusion and reversed-phase high performance liquid chromatography. This toxin is dimeric with a mass of 13868.2 Da (monomeric form), as determined by mass spectrometry. CoaTx-II is rich in Arg and Lys residues and displays high identity with other Lys49 PLA2 homologues, which have high isoelectric points. The structural model of dimeric CoaTx-II shows that the toxin is non-covalently stabilized. Despite its enzymatic inactivity, in vivo CoaTx-II caused local muscular damage, characterized by increased plasma creatine kinase and confirmed by histological alterations, in addition to an inflammatory activity, as demonstrated by mice paw edema induction and pro-inflammatory cytokine IL-6 elevation. CoaTx-II also presents antibacterial activity against gram negative (Pseudomonas aeruginosa 31NM, Escherichia coli ATCC 25922) and positive (Staphyloccocus aureus BEC9393 and Rib1) bacteria. Therefore, data show that this newly purified toxin plays a central role in mediating the degenerative events associated with envenomation, in addition to demonstrating antibacterial properties, with potential for use in the development of strategies for antivenom therapy and combating antibiotic-resistant bacteria. PMID:27530662

  14. Simvastatin but not bezafibrate decreases plasma lipoprotein-associated phospholipase A(2) mass in type 2 diabetes mellitus : Relevance of high sensitive C-reactive protein, lipoprotein profile and low-density lipoprotein (LDL) electronegativity

    NARCIS (Netherlands)

    Constantinides, Alexander; de Vries, Rindert; van Leeuwen, Jeroen J. J.; Gautier, Thomas; van Pelt, L. Joost; Tselepis, Alexandros D.; Lagrost, Laurent; Dullaart, Robin P. F.

    2012-01-01

    Objective: Plasma lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) levels predict incident cardiovascular disease, impacting Lp-PLA(2) as an emerging therapeutic target. We determined Lp-PLA(2) responses to statin and fibrate administration in type 2 diabetes mellitus, and assessed relationship

  15. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    Directory of Open Access Journals (Sweden)

    Nidiane D R Prado

    Full Text Available Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II, two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs and immunoglobulin frameworks (FRs of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718 were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607 neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.

  16. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments

    Science.gov (United States)

    Prado, Nidiane D. R.; Pereira, Soraya S.; da Silva, Michele P.; Morais, Michelle S. S.; Kayano, Anderson M.; Moreira-Dill, Leandro S.; Luiz, Marcos B.; Zanchi, Fernando B.; Fuly, André L.; E. F. Huacca, Maribel; Fernandes, Cleberson F.; Calderon, Leonardo A.; Zuliani, Juliana P.; Soares, Andreimar M.; Stabeli, Rodrigo G.; F. C. Fernandes, Carla

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  17. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    Science.gov (United States)

    Prado, Nidiane D R; Pereira, Soraya S; da Silva, Michele P; Morais, Michelle S S; Kayano, Anderson M; Moreira-Dill, Leandro S; Luiz, Marcos B; Zanchi, Fernando B; Fuly, André L; Huacca, Maribel E F; Fernandes, Cleberson F; Calderon, Leonardo A; Zuliani, Juliana P; Pereira da Silva, Luiz H; Soares, Andreimar M; Stabeli, Rodrigo G; Fernandes, Carla F C

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  18. LPS—induced activation of phospholipase A2 phospholipase C and protein kinase C of murine macrophage—like cell lines (J774 and P388D1)

    Institute of Scientific and Technical Information of China (English)

    CHANGZHONGLIANG; MICHAELNOVOTNEY; 等

    1992-01-01

    A murine macrophage-like cell line J774,acquired,in response to LPS,an ability to kill tumor necrosis factor(TNF)-insensitive target P815 mastocytoma cells whereas another cell line,P388D1 did not ,LPS triggered signaling mechanisms between the two cell lines were compared with an aim to inquire about the possible nature of the above-mentioned difference,The results whowed that two cell lines respond to LPS-treatment by parallel activation of both phospholipases C and A2 (PLC and PLA2) to approximately the same extent.The maximum response of toth enzymes of J774 cells was noted within 10 min the treatment whereas that of P388D1 cells required more than 20 min,The other properties of LPS-responsive enzymes studied were similar between two cell lines,including Activation of PLC and PLA2 and PKC in macrophages by LPS. Ca2+ augmentation of enzyme activation,participation of guanine nucleotide binding(G) proteins in the initial activation preocesses,and inhibition of enzyme activation by the prior treatment of cells with choleraor pertussis toxinsetc.Moreover,LPS-triggered activation of PLC and PLA2 was found to be followed by the increase of PKC activities in both cell lines.Inspite of these similarities.J774 cells possessed both basic and acidicforms of PKC activities,while P 388 D1 cells owned only PKC of basic form,Nevertheless,the question why J774 cells but not P388D1 cells,can acquire the tumoricidal activity,aganist P815,cells following LPStreatment rematins to be answered.

  19. Changes in wetting properties of alumina surface treated with DPPC in the presence of phospholipase A2 enzyme.

    Science.gov (United States)

    Wiącek, Agnieszka Ewa

    2011-10-01

    Wetting properties of commercial Al(2)O(3) plates contacted with dipalmitoylphosphatidylcholine (DPPC) or DPPC+enzyme (phospholipase PLA(2)) in NaCl solution were determined by thin layer wicking and with the help of Washburn equation. Van Oss et al.'s approach to interfacial free energy interactions was applied to determining the solid surface free energy components. Wicking experiments were performed both for bare and alumina plates precontacted overnight with the probe liquid saturated vapours, as well as the untreated and DPPC (or DPPC+PLA(2)) treated alumina plates. For this purpose the penetration rates of n-octane, water and formamide were measured. From these experiments it resulted that original alumina surface is strongly polar with electron-donor interactions originating from the surface hydroxyl groups. Adsorption of DPPC on Al(2)O(3) plates slightly increased the hydrophobic character of the alumina surface (considerable decrease of the electron-donor, γ(s)(-) parameter and γ(s)(AB) component was visible) in such a way that the hydrocarbon chains were directed outwards and the polar part towards the alumina surface. However, after the enzyme action the products of DPPC hydrolysis by PLA(2) (palmitic acid and lysophosphatidylcholine) increased again the hydrophilic character of Al(2)O(3) surface (a minor increase in γ(s)(AB) component and drastic increase of the electron-donor γ(s)(-) parameter was noticeable). After treatment with DPPC or DPPC+enzyme PLA(2) solution the changes of the total surface free energy of alumina and its Lifshits-van der Waals (γ(s)(LW)) component were in the range 7-10 mJ/m(2), but the most considerable and delivering more interesting information were the changes of the electron-donor (γ(s)(-)) parameter ranging from 27 to 35 mJ/m(2). Moreover, the changes of the alumina surface wettability were dependent on the time of the enzyme contacting with DPPC in NaCl solution. On the basis of the obtained results it seems that

  20. Diverse regulation of retinal pigment epithelium phagocytosis of photoreceptor outer segments by calcium-independent phospholipase A₂, group VIA and secretory phospholipase A₂, group IB

    DEFF Research Database (Denmark)

    Zhan, Chen; Wang, Jinmei; Kolko, Miriam

    2012-01-01

    the role of iPLA(2)-VIA in RPE phagocytosis of POS, experiments with iPLA(2)-VIA vector transfection, iPLA(2)-VIA(-/-) knockout (KO) mice, and iPLA(2)-VIA inhibition by bromoenol lactone (BEL) were done. Exogenous addition of sPLA(2)-IB was used to investigate the role of sPLA(2)-IB in RPE phagocytosis...... and in RPE from KO mice. Exogenous addition of enzymatically active and inactive sPLA(2)-IB reduced phagocytosis in ARPE-19 and primary mouse RPE cells. Finally, sPLA(2)-IB did not seem to affect the iPLA(2)-VIA promoter. CONCLUSION: The present study confirms the involvement of iPLA(2)-VIA in efficient RPE...

  1. Heterologous expression of Lactobacillus casei phospholipase A2 in Escherichia coli%Lactobacillus casei磷脂酶A2基因在大肠杆菌中的重组表达

    Institute of Scientific and Technical Information of China (English)

    刘丹丹; 张梁; 顾正华; 丁重阳; 石贵阳

    2014-01-01

    The fragment of PLA2 gene was amplified from Lactobacillus casei by PCR. Using pET-28a(+) as vector, the recombinant plasmid pET-pla2 was constructed. Phospholipase A2 was successfully expressed in E.coli DE3. After optimazation, the maximum activity of PLA2 reached 2�8 U/mL. Then, the protein was purified by Ni-chelating column, SDS-PAGE analysis showed that the molecular weight of the recombinant phospholipase A2 was 1�7×104 . Finally, the optimal activity determined at 37℃ and pH 8 was 110 U/mg.%以Lactobacillus casei染色体基因组为模板,PCR扩增获得磷脂酶A2基因pla2,以pET 28a(+)为载体构建重组表达质粒pET 28a(+) pla2。通过IPTG诱导实现磷脂酶A2在E.coli DE3中的重组表达。对诱导条件初步优化后,重组菌酶活最大可达2�8 U/mL。通过Ni 螯合柱对目的蛋白进行纯化,SDS PAGE分析重组磷脂酶A2相对分子质量为1�7×104。通过酶学性质分析,最适温度为37℃,最适pH 8,比酶活为110 U/mg。

  2. EXPRESSION OF A BEE-VENOM PHOSPHOLIPASE A2 FROM APIS CERANA CERANA IN ESCHERICHIA COLI%中华蜜蜂蜂毒磷脂酶A2基因在大肠杆菌中的表达

    Institute of Scientific and Technical Information of China (English)

    沈立荣; 程家安; 张传溪

    2004-01-01

    将中华蜜蜂蜂毒磷脂酶A2(AcPLA2)蛋白成熟肽编码区基因(495 bp)克隆至表达载体-pETBlue-1,在大肠杆菌Tuner(DE3)plac I中诱导表达,经SDS--PAGE电泳检测,表达产物分子量为15kD,约占细菌总蛋白的百分之四点六;用意大利蜜蜂蜂毒磷脂酶A2(AmPLA2)纯品制备的兔源多克隆抗体为一抗作Westerm blot,表达产物显示类似于天然纯AmPLA2的特异性印迹,证实AcPLA2基因已在大肠杆菌中得到表达.%The venomous phospholipase A2 (AcPLA2) coding reading region of the Chinese honeybee (Apis cerana cerana),which is composed of 405 bp encoding a mature glycosylated peptide with 134 amino residues , was transformed into the expression vector pETblue-1. Then the recombinant vector was introduced into Escherichia coli Tuner (DE3) placⅠfor expression. Analysis result of SDS-PAGE showed that the expression products had a protein band of about 15 kD. Detection of western blot using ant-European honeybee (Apis mellifera)phospholipase A2 (AmPLA2) polyclonal serum as the first antibody showed that the expression products appeared a special blot same as the native AmPLA2. The result demonstrated that the AcPLA2 peptide had been expressed in E. coli. and the AcPLA2 has the similar antigenicity as the AmPLA2.

  3. The mediation of the central histaminergic system in the pressor effect of intracerebroventricularly injected melittin, a phospholipase A2 activator, in normotensive rats.

    Science.gov (United States)

    Altinbas, Burcin; Topuz, Bora B; Yilmaz, Mustafa S; Aydin, Cenk; Savci, Vahide; Jochem, Jerzy; Aydin, Sami; Yalcin, Murat

    2012-01-01

    Melittin is a polypeptide component of bee venom that leads to an increase in arachidonic acid release and subsequently in prostaglandin synthesis by activating phospholipase A(2). Recently we demonstrated that centrally or peripherally administrated melittin caused pressor effect and central thromboxane A(2) (TXA(2)) and cholinergic system mediated these effects of melittin. Also centrally injected histamine leads to pressor and bradycardic response by activating central histamine receptors in normotensive rats and central cholinergic system involved the effects of histamine. The present study demonstrates an involvement of the central histaminergic system in melittin-induced cardiovascular effect in normotensive rats. Experiments were carried out in male Sprague Dawley rats. Intracerebroventricularly (i.c.v.) injected melittin (0.5, 1 and 2 nmol) caused dose- and time-dependent increases in mean arterial pressure (MAP) and decrease in heart rate (HR) as we reported previously. Moreover, H(2) receptor antagonist ranitidine (50 nmol; i.c.v.) almost completely and H(3)/H(4) receptor antagonist thioperamide (50 nmol; i.c.v.) partly blocked melittin-evoked cardiovascular effects, whereas H(1) receptor blocker chlorpheniramine (50 nmol; i.c.v.) had no effect. Also centrally injected melittin was accompanied by 28% increase in extracellular histamine concentration in the posterior hypothalamus, as shown in microdialysis studies. In conclusion, results show that centrally administered melittin causes pressor and bradycardic response in conscious rats. Moreover, according to our findings, there is an involvement of the central histaminergic system in melittin-induced cardiovascular effects.

  4. Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

    Science.gov (United States)

    Iyer, Janaki Krishnamurthy; Shih, Norrapat; Majumder, Munmi; Mattaparthi, Venkata Satish Kumar; Mukhopadhyay, Rupak; Doley, Robin

    2016-01-01

    In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity. PMID:27089306

  5. Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity.

    Directory of Open Access Journals (Sweden)

    Maitreyee Sharma

    Full Text Available In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0 and neutral pH (pH 7.0 and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48 was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity.

  6. Correlation of Secreted Phospholipase A2 - Ⅰ B, Anti - phospholipase A2 Receptor Antibody with Idiopathic Membranous Nephropathy among Adult Patients%成人特发性膜性肾病与分泌型磷脂酶A2-ⅠB及抗磷脂酶A2受体抗体的相关性研究

    Institute of Scientific and Technical Information of China (English)

    杨雪芬; 潘阳彬; 丁国华; 万建新; 马屹茕; 杨红霞

    2015-01-01

    目的:探讨成人特发性膜性肾病( IMN)与分泌型磷脂酶A2-ⅠB( sPLA2-ⅠB)、抗磷脂酶A2受体抗体(Anti-PLA2R AB)的相关性。方法选取2013年7—12月武汉大学人民医院、福建医科大学附属第一医院经肾活检证实的成人IMN、继发性膜性肾病( SMN)患者共30例,其中IMN 20例、SMN 10例,另选取同时期体检健康的志愿者5例为对照组。根据24 h尿蛋白总量分为低蛋白尿组7例、中蛋白尿组11例、高蛋白尿组17例。酶联免疫吸附试验( ELISA)法检测血清sPLA2-ⅠB水平;免疫组化法检测肾脏sPLA2-ⅠB表达;间接免疫荧光法检测血清Anti-PLA2R AB表达。结果 IMN组和SMN组血清sPLA2-ⅠB水平高于对照组( P<0.05);IMN组与SMN组血清sPLA2-ⅠB水平比较,差异无统计学意义( P>0.05)。IMN和SMN患者血清sPLA2-ⅠB水平与年龄、24 h尿蛋白总量呈正相关(r值分别为0.475和0.581,P值分别为0.008和0.002),与血清总蛋白、清蛋白呈负相关(r值分别为-0.487和-0.473,P 值分别为0.009和0.011);多元线性回归分析结果显示,年龄、24 h 尿蛋白总量与血清sPLA2-ⅠB水平呈独立正相关(P<0.05)。高蛋白尿组血清sPLA2-ⅠB水平、sPLA2-ⅠB阳性区域面积占肾小球区域面积比例高于低蛋白尿组和中蛋白尿组,中蛋白尿组血清sPLA2-ⅠB水平、sPLA2-ⅠB阳性区域面积占肾小球区域面积比例高于低蛋白尿组(P<0.05)。IMN组Anti-PLA2R AB表达阳性率60%(12/20)高于SMN组的20%(2/10)(P=0.039)。结论年龄、24 h尿蛋白总量与血清sPLA2-ⅠB水平呈独立正相关,血清sPLA2-ⅠB水平可能与IMN病情有一定相关性;IMN患者血清Anti-PLA2R AB阳性率高于SMN患者,提示Anti-PLA2R AB可能是IMN的特异性抗体,且有助于IMN与SMN的鉴别诊断。%Objective To investigate the correlation of secreted phospholipase A2 -ⅠB( sPLA2-ⅠB)and anti-phospholipase

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

    Directory of Open Access Journals (Sweden)

    Martín Rubén

    2012-07-01

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

  8. cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

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    F.H.R. Fagundes

    2010-03-01

    Full Text Available To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR, we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49, from the venom of Crotalus durissus collilineatus (Cdc PLA2. The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC. The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.

  9. Enhanced expression of the M-type phospholipase A2 receptor in glomeruli correlates with serum receptor antibodies in primary membranous nephropathy.

    Science.gov (United States)

    Hoxha, Elion; Kneißler, Ursula; Stege, Gesa; Zahner, Gunther; Thiele, Ina; Panzer, Ulf; Harendza, Sigrid; Helmchen, Udo M; Stahl, Rolf A K

    2012-10-01

    The M-type phospholipase A2 receptor (PLA2R) is the major target antigen in idiopathic membranous nephropathy with detectable autoantibodies in the serum of up to 70% of patients. In retrospective studies, the PLA2R-autoantibody titer in the serum was sometimes negative indicating their measurement alone may be inconclusive. In order to better differentiate between primary and secondary membranous nephropathy, we conducted a prospective study that included 88 patients with a histologic diagnosis of membranous nephropathy. Immunohistochemical analysis for PLA2R was faintly positive in kidneys from normal individuals and patients with various other glomerular injuries. In 61 of the 88 patients, PLA2R expression was strongly positive in glomeruli, and in 60 of these patients PLA2R autoantibodies were also detected in the serum. The 27 patients negative for serum PLA2R autoantibodies were faintly positive for PLA2R staining in glomeruli and in 15 of these patients a secondary cause was found. The remaining 12 patients have a yet undetected secondary cause of membranous nephropathy or have different glomerular antigens other than PLA2R. Thus, increased staining for PLA2R in glomeruli of renal biopsies tightly correlates with the presence of PLA2R autoantibodies in the serum and this may help discriminate between primary and secondary membranous nephropathy.

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

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    Matthew Lewin

    2016-08-01

    Full Text Available 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.

  11. Purification and partial characterization of phospholipases A2 from Bothrops asper (barba amarilla snake venom from Chiriguaná (Cesar, Colombia

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    J. Ramírez-Avila

    2004-01-01

    Full Text Available Components with phospholipase A2 activity were isolated by gel filtration and cationic exchange chromatography from the venom of Bothrops asper snakes from Chiriguaná, Colombia (9°22´N; 73°37´W. Five fractions were obtained by the gel filtration, and PLA2 activity was found in fraction 3 (F3. In the cationic exchange chromatography, F3 showed eight components with PLA2 activity. Six of these components appeared as one band in polyacrylamide gel electrophoresis (SDS-PAGE. Fractions II and VII exhibited an optimal activity at pH 9 and 52ºC. The optimum calcium concentration for fraction II was 48 mM and for fraction VII, 384 mM. Both fractions showed thermal stability. Fraction II was stable at pH values between 2.5 and 9, and fraction VII, between 2.5 and 8. The Michaelis Menten constant (K M was 3.5x10-3 M for fraction II and 1.6x10-3 M for fraction VII. The molecular weight was 16,000 Dalton for fraction II and 17,000 Dalton for fraction VII. Both isoenzymes did not show any toxic activity (DL50 at 5.3 and 4 µg/g. The two fractions showed different kinetic constant (K M, calcium requirement, and substrate specificity for haemolytic activity.

  12. Three metabolites from an entomopathogenic bacterium, Xenorhabdus nematophila, inhibit larval development of Spodoptera exigua (Lepidoptera: Noctuidae) by inhibiting a digestive enzyme, phospholipase A2

    Institute of Scientific and Technical Information of China (English)

    Jaehyun Kim; Yonggyun Kim

    2011-01-01

    An entomopathogenic bacterium, Xenorhabdus nematophila, has been known to induce significant immunosuppression of target insects by inhibiting immune-associated phospholipase A2 (PLA2), which subsequently shuts down biosynthesis of eicosanoids that are critical in immune mediation in insects. Some metabolites originated from the bacterial culture broth have been identified and include benzylideneacetone, proline-tyrosine and acetylated phenylalanine-glycine-valine, which are known to inhibit enzyme activity of PLA2 extracted from hemocyte and fat body. This study tested their effects on digestive PLA2 of the beet armyworm, Spodoptera exigua. Young larvae fed different concentrations of the three metabolites resulted in significant adverse effects on larval development even at doses below 100 μg/mL. In particular, they induced significant reduction in digestive efficiency of ingested food. All three metabolites significantly inhibited catalytic activity of digestive PLA2 extracted from midgut lumen of the fifth instar larvae at a low micromolar range. These results suggest that the inhibitory activities of the three bacterial metabolites on digestive PLA2 of S. exigua midgut may explain some of their oral toxic effects.

  13. Polydatin up-regulates clara cell secretory protein to suppress phospholipase A2 of lung induced by LPS in vivo and in vitro

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    Jie Chen

    2011-07-01

    Full Text Available Abstract Background Lung injury induced by lipopolysaccharide (LPS remains one of the leading causes of morbidity and mortality in children. The damage to membrane phospholipids leads to the collapse of the bronchial alveolar epithelial barrier during acute lung injury (ALI/acute respiratory distress syndrome (ARDS. Phospholipase A2 (PLA2, a key enzyme in the hydrolysis of membrane phospholipids, plays an important traumatic role in pulmonary inflammation, and Clara cell secretory protein (CCSP is an endogenous inhibitor of PLA2. Our previous study showed that polydatin (PD, a monocrystalline extracted from a traditional Chinese medicinal herb (Polygonum cuspidatum Sieb, et Zucc, reduced PLA2 activity and sPLA2-IIA mRNA expression and mitigated LPS-induced lung injury. However, the potential mechanism for these effects has not been well defined. We have continued to investigate the effect of PD on LPS-induced expression of CCSP mRNA and protein in vivo and in vitro. Results Our results suggested that the CCSP mRNA level was consistent with its protein expression. CCSP expression was decreased in lung after LPS challenge. In contrast, PD markedly increased CCSP expression in a concentration-dependent manner. In particular, CCSP expression in PD-pretreated rat lung was higher than in rats receiving only PD treatment. Conclusion These results indicated that up-regulation of CCSP expression causing inhibition of PLA2 activation may be one of the crucial protective mechanisms of PD in LPS-induced lung injury.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  16. Epithelium specific ETS transcription factor, ESE-3, of Protobothrops flavoviridis snake venom gland transactivates the promoters of venom phospholipase A2 isozyme genes.

    Science.gov (United States)

    Nakamura, Hitomi; Murakami, Tatsuo; Hattori, Shosaku; Sakaki, Yoshiyuki; Ohkuri, Takatoshi; Chijiwa, Takahito; Ohno, Motonori; Oda-Ueda, Naoko

    2014-12-15

    Protobothrops flavoviridis (habu) (Crotalinae, Viperidae) is a Japanese venomous snake, and its venom contains the enzymes with a variety of physiological activities. The phospholipases A2 (PLA2s) are the major components and exert various toxic effects. They are expressed abundantly in the venom gland. It is thought that the venom gland-specific transcription factors play a key role for activation of PLA2 genes specifically expressed in the venom gland. Thus, the full-length cDNA library for P. flavoviridis venom gland after milking of the venom was made to explore the transcription factors therein. As a result, three cDNAs encoding epithelium-specific ETS transcription factors (ESE)-1, -2, and -3 were obtained. Among them, ESE-3 was specifically expressed in the venom gland and activated the proximal promoters of venom PLA2 genes, which are possibly regarded as the representatives of the venom gland-specific protein genes in P. flavoviridis. Interestingly, the binding specificity of ESE-3 to the ETS binding motif located near TATA box is well correlated with transcriptional activities for the venom PLA2 genes. This is the first report that venom gland-specific transcription factor could actually activate the promoters of the venom protein genes.

  17. Purification, crystallization and preliminary crystallographic analysis of a natural complex of phospholipase A2 from Echis carinatus (saw-scaled viper).

    Science.gov (United States)

    Nagpal, A; Chandra, V; Kaur, P; Singh, T P

    1999-06-01

    A novel complex of phospholipase A2 complexed with another venom protein has been isolated and purified from saw-scaled viper (Echis carinatus) venom. The molecular weights of the two components are 16 and 14 kDa, respectively. The complex was purified using an Affigel blue column and an anion-exchange (DEAE Sephacel) column. Long diamond-shaped crystals were obtained by hanging-drop vapour diffusion. The protein complex was dissolved at a concentration of 10 mg ml-1 in 20 mM sodium cacodylate, 1 mM CaCl2 and 2% dioxane at pH 6.0. The reservoir contained the same buffer with 7%(w/v) PEG 4000. Crystals appeared within 2-3 weeks. Native data to 2.9 A resolution have been obtained at 291 K. The crystals belong to the monoclinic space group P21 with unit-cell parameters a = 74.47, b = 47.87, c = 106.39 A, beta = 104.5 degrees and contain two molecules per asymmetric unit. Structure determination by molecular replacement is in progress. PMID:10329797

  18. Structure of an acidic phospholipase A2 from Indian saw-scaled viper (Echis carinatus) at 2.6 A resolution reveals a novel intermolecular interaction.

    Science.gov (United States)

    Jasti, Jayasankar; Paramasivam, M; Srinivasan, A; Singh, T P

    2004-01-01

    The crystal structure of an acidic phospholipase A(2) from the venom of Echis carinatus (saw-scaled viper; scPLA(2)) has been determined at 2.6 A resolution and refined to a crystallographic R factor of 0.192. Although the overall structure of scPLA(2) is essentially similar to those of other group II acidic PLA(2)s from different species, it shows unique features in several parts. Particularly noteworthy is the C-terminal part, which folds differently to those of other group II PLA(2)s. This part is considered to be responsible for inhibition of the platelet-aggregation activity. The calcium-binding loop is tightly organized with sevenfold coordination. Another striking feature of scPLA(2) is the involvement of Asn79 O(delta1) of a symmetry-related molecule in a coordination linkage with Ca(2+) of the calcium-binding loop. This is the first observation of an internal metal ion participating in an intermolecular interaction. The beta-wing of a molecule is deeply inserted into the hydrophobic channel of another molecule and forms several intermolecular interactions. This results in the formation of an infinite chain of molecules. These chains are stacked in an antiparallel arrangement in the crystals. PMID:14684894

  19. Immunocytochemical localization of secretory phospholipase A(2)-like protein in the pituitary gland and surrounding tissue of the bullfrog, Rana catesbeiana.

    Science.gov (United States)

    Yaoi, Y; Kikuyama, S; Hayashi, H; Hanaoka, Y; Sakai, M; Tanaka, S

    2001-05-01

    Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A(2) (PLA(2)) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA(2)-like protein were observed predominately in these secretory granules. These findings support the view that this PLA(2)-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin. (J Histochem Cytochem 49:631-637, 2001) PMID:11304801

  20. Cytosolic phospholipase A2-alpha expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours.

    LENUS (Irish Health Repository)

    Caiazza, F

    2012-02-01

    BACKGROUND: The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA(2)alpha expression correlated with EGFR\\/HER2 over-expression in a small number of breast cancer cell lines. METHODS: The importance of differential cPLA(2)alpha activity in clinical breast cancer was established by relating the expression of cPLA(2)alpha in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters. RESULTS: High cPLA(2)alpha mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA(2)alpha expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA(2)alpha expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up. CONCLUSION: This study shows a role of cPLA(2)alpha in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker.

  1. Flavocoxid Inhibits Phospholipase A2, Peroxidase Moieties of the Cyclooxygenases (COX, and 5-Lipoxygenase, Modifies COX-2 Gene Expression, and Acts as an Antioxidant

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    Bruce P. Burnett

    2011-01-01

    Full Text Available The multiple mechanisms of action for flavocoxid relating to arachidonic acid (AA formation and metabolism were studied in vitro. Flavocoxid titrated into rat peritoneal macrophage cultures inhibited cellular phospholipase A2 (PLA2 (IC50 = 60 μg/mL. In in vitro enzyme assays, flavocoxid showed little anti-cyclooxygenase (CO activity on COX-1/-2 enzymes, but inhibited the COX-1 (IC50 = 12.3 and COX-2 (IC50 = 11.3 μg/mL peroxidase (PO moieties as well as 5-lipoxygenase (5-LOX (IC50 = 110 μg/mL. No detectable 5-LOX inhibition was found for multiple traditional and COX-2 selective NSAIDs. Flavocoxid also exhibited strong and varied antioxidant capacities in vitro and decreased nitrite levels (IC50 = 38 μg/mL in rat peritoneal macrophages. Finally, in contrast to celecoxib and ibuprofen, which upregulated the cox-2 gene, flavocoxid strongly decreased expression. This work suggests that clinically favourable effects of flavocoxid for management of osteoarthritis (OA are achieved by simultaneous modification of multiple molecular pathways relating to AA metabolism, oxidative induction of inflammation, and neutralization of reactive oxygen species (ROS.

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

    Science.gov (United States)

    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.

  3. Anti-parasitic effect on Toxoplasma gondii induced by BnSP-7, a Lys49-phospholipase A2 homologue from Bothrops pauloensis venom.

    Science.gov (United States)

    Borges, Isabela Pacheco; Castanheira, Letícia Eulalio; Barbosa, Bellisa Freitas; de Souza, Dayane Lorena Naves; da Silva, Rafaela José; Mineo, José Roberto; Tudini, Kelly Aparecida Yoneyama; Rodrigues, Renata Santos; Ferro, Eloísa Amália Vieira; de Melo Rodrigues, Veridiana

    2016-09-01

    Toxoplasmosis affects a third of the global population and presents high incidence in tropical areas. Its great relevance in public health has led to a search for new therapeutic approaches. Herein, we report the antiparasitic effects of BnSP-7 toxin, a Lys49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, on Toxoplasma gondii. In an MTT assay, BnSP-7 presented significant cytotoxicity against host HeLa cells at higher doses (200 μg/mL to 50 μg/mL), whereas lower doses (25 μg/mL to 1.56 μg/mL) produced low cytotoxicity. Furthermore, the toxin showed no effect on T. gondii tachyzoite viability when evaluated by trypan blue exclusion, but decreased both adhesion and parasite proliferation when tachyzoites were treated before infection. We also measured cytokines in supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BnSP-7, which revealed enhancement of only MIF and IL-6 cytokines levels in supernatants of HeLa cells after BnSP-7 treatment. Our results showed that the BnSP-7 PLA2 exerts an anti-Toxoplasma effect at a lower dose than that required to induce cytotoxicity in HeLa cells, and also modulates the immune response of host cells. In this sense, the anti-parasitic effect of BnSP-7 PLA2 demonstrated in the present study opens perspectives for use of this toxin as a tool for future studies on toxoplasmosis. PMID:27212627

  4. Humanized-single domain antibodies (VH/VHH) that bound specifically to Naja kaouthia phospholipase A2 and neutralized the enzymatic activity.

    Science.gov (United States)

    Chavanayarn, Charnwit; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Bangphoomi, Kunan; Sookrung, Nitat; Chaicumpa, Wanpen

    2012-07-01

    Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

  5. An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor.

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    Behnert, Astrid; Fritzler, Marvin J; Teng, Beina; Zhang, Meifeng; Bollig, Frank; Haller, Hermann; Skoberne, Andrej; Mahler, Michael; Schiffer, Mario

    2013-01-01

    The phospholipase A2 receptor (PLA2R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA2R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

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

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    Kei Hihara

    Full Text Available 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.

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

    Science.gov (United States)

    Hihara, Kei; Iyoda, Masayuki; 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.

  8. An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor.

    Directory of Open Access Journals (Sweden)

    Astrid Behnert

    Full Text Available The phospholipase A2 receptor (PLA2R was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN. Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA utilizing indirect immunofluorescence (IIF on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA platform. Since reactive domains of PLA2R (i.e. epitopes could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

  9. Clinical usefulness of autoantibodies to M-type phospholipase A2 receptor (PLA2R) for monitoring disease activity in idiopathic membranous nephropathy (IMN).

    Science.gov (United States)

    Radice, Antonella; Trezzi, Barbara; Maggiore, Umberto; Pregnolato, Francesca; Stellato, Tiziana; Napodano, Pietro; Rolla, Davide; Pesce, Gianpaola; D'Amico, Marco; Santoro, Domenico; Londrino, Francesco; Ravera, Federica; Ortisi, Giuseppe; Sinico, Renato Alberto

    2016-02-01

    Autoantibodies to M-type phospholipase A2 receptor (PLA2R) are specific markers of idiopathic membranous nephropathy (IMN). They can differentiate IMN from other glomerular diseases and primary from secondary forms of MN. Preliminary data suggest that anti-PLA2R antibody titer correlates with disease activity but more solid evidence is needed. To evaluate the performance of anti-PLA2R antibody for monitoring nephropathy activity, 149 anti-PLA2R antibody measurements were performed during the follow-up of 42 biopsy proven IMN consecutive patients. Patients were enrolled either at time of diagnosis (33 cases, inception cohort) or after diagnosis (9 patients, non-inception cohort). Anti-PLA2R detection was performed using the highly sensitive transfected cell-based indirect immunofluorescence (IIFT). Over the follow-up there was a linear time-trend of decreasing proteinuria (PPLA2R antibody levels (P=0.002). There was a statistically significant association between changes in PLA2R antibody levels and the clinical course of PLA2R-positive IMN. The positive PLA2R serum antibody status was linearly associated with increasing proteinuria and decreasing serum albumin over time, compared with negative antibody status. Moreover, the strong correlation between the clinical conditions and PLA2R antibody levels allowed the prediction of prevalence distribution of patients with active disease, partial and complete remission. Over the course of the follow-up, the probability of halving proteinuria increased 6.5 times after disappearance of PLA2R antibodies. Our data suggest that the serial evaluation of anti-PLA2R antibodies could help in optimal timing and duration of the immunosuppressive therapy, reducing over(under)-treatment and associated side-effects.

  10. Distinct expression pattern of the full set of secreted phospholipases A2 in human colorectal adenocarcinomas: sPLA2-III as a biomarker candidate

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    Mounier, C M; Wendum, D; Greenspan, E; Fléjou, J-F; Rosenberg, D W; Lambeau, G

    2008-01-01

    Recent studies suggest that secreted phospholipases A2 (sPLA2s) represent attractive potential tumour biomarkers and therapeutic targets for various cancers. As a first step to address this issue in human colorectal cancer, we examined the expression of the full set of sPLA2s in sporadic adenocarcinomas and normal matched mucosa from 21 patients by quantitative PCR and immunohistochemistry. In normal colon, PLA2G2A and PLA2G12A were expressed at high levels, PLA2G2D, PLA2G5, PLA2G10 and PLA2G12B at moderate levels, and PLA2G1B, PLA2G2F and PLA2G3 at low levels. In adenocarcinomas from left and right colon, the expression of PLA2G3 was increased by up to 40-fold, while that of PLA2G2D and PLA2G5 was decreased by up to 23- and 14-fold. The variations of expression for sPLA2-IID, sPLA2-III and sPLA2-V were confirmed at the protein level. The expression pattern of these sPLA2s appeared to be linked respectively to the overexpression of interleukin-8, defensin α6, survivin and matrilysin, and downregulation of SFRP-1 and RLPA-1, all these genes being associated to colon cancer. This original sPLA2 profile observed in adenocarcinomas highlights the potential role of certain sPLA2s in colon cancer and suggests that sPLA2-III might be a good candidate as a novel biomarker for both left and right colon cancers. PMID:18212756

  11. Plasma Lipoprotein-Associated Phospholipase A2 Levels Correlated with the Cardio-Ankle Vascular Index in Long-Term Type 2 Diabetes Mellitus Patients

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    Kazuhiko Kotani

    2016-04-01

    Full Text Available The circulating levels of lipoprotein-associated phospholipase A2 (Lp-PLA2 can be a simple, but practical and useful marker of cardiovascular disease (CVD. As limited studies are available in patients with diabetes mellitus (DM, further studies are needed to establish the clinical application of Lp-PLA2 in DM practice. The present study investigated the correlation between Lp-PLA2 and the cardio-ankle vascular index (CAVI, a recent marker of arterial stiffness, in DM patients according to their diabetes duration. Clinical data, including the plasma Lp-PLA2 mass and CAVI values, were collected from CVD-free type 2 DM female patients (n = 65, mean age 62 years, mean hemoglobin A1c 7.0%. The Lp-PLA2 level of patients with a diabetes duration of <10 years (n = 40:20.2 IU/mL was not significantly different from that of patients with a diabetes duration of ≥10 years (n = 25:20.5 IU/mL, while the CAVI level was significantly higher in patients with ≥10 years (9.0 than in those with <10 years (8.1; p < 0.05. A stepwise multiple regression analysis found a positive correlation between the Lp-PLA2 and CAVI levels (β = 0.43, p < 0.01 in patients with a diabetes duration of ≥10 years. This correlation between Lp-PLA2 and CVAI suggests the possible use of Lp-PLA2 in DM patients with long-term disease. Further studies on Lp-PLA2 are warranted in DM practice in relation to the disease duration.

  12. Synergism between basic Asp49 and Lys49 phospholipase A2 myotoxins of viperid snake venom in vitro and in vivo.

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    Diana Mora-Obando

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

  13. Role of epidermal growth factor receptor transactivation in the activation of cytosolic phospholipase A(2) in leptin protection of salivary gland acinar cells against ethanol cytotoxicity.

    Science.gov (United States)

    Slomiany, B L; Slomiany, A

    2009-06-01

    A pleiotropic hormone, leptin, secreted into saliva by the acinar cells of salivary glands is an important mediator of the processes of oral mucosal defense. Here, we report on the role of epidermal growth factor receptor (EGFR) transactivation in the signaling events that mediate leptin protection of sublingual salivary gland acinar cells against ethanol cytotoxicity. We show that the protective effect of leptin against ethanol cytotoxicity was associated with the increased EGFR protein tyrosine kinase and cytosolic phospholipase A(2) (cPLA(2)) activity, and characterized by a marked increase in matrix metalloproteinase MMP-9 and arachidonic acid (AA) release, and PGE(2) generation. The loss in countering capacity of leptin against ethanol cytotoxicity was attained with JAK inhibitor AG490, Src inhibitor PP2, and EGFR inhibitor AG1478, as well as ERK inhibitor PD98059. Moreover, the agents evoked also the inhibition in leptin-induced up-regulation in cPLA(2) activity, AA release, and PGE(2) generation. The changes caused by leptin in EGFR phosphorylation, MMP-9, and cPLA(2) activation were susceptible to suppression by metalloprotease inhibitor GM6001, but the production of MMP-9 was not affected by EGFR inhibitor AG1478 or PKC inhibitor Ro318220. These findings point to the involvement of MMP-9 in the event of leptin-induced EGFR transactivation that results in the signaling cascade leading to cPLA(2) activation and up-regulation in PGE(2) generation, thus providing new insights into the mechanism of oral mucosal protection against ethanol toxicity.

  14. Transcriptional Regulation of the Group IIA Secretory Phospholipase A2 Gene by C/EBPδ in Rat liver and its Relationship to Hepatic Gluconeogenesis during Sepsis

    Science.gov (United States)

    Yang, Rei-Cheng; Hsu, Chin; Lee, Tzu-Ying; Kuo, Kung-Kai; Wu, Shou-Mei; Chen, Yen-Hsu; Ho, Mei-Ling; Yao, Xing-Hai; Liu, Chia-Hsiung; Liu, Maw-Shung

    2014-01-01

    Background The present study was undertaken to test hypothesis that altered transcription of secretory Phospholipase A2 (sPLA2) gene in rat liver is regulated by CCAAT/enhancer binding protein δ (C/EBPδ), and to assess its relationship to hepatic gluconeogenesis during the progression of sepsis. Methods Sepsis was induced by Cecal Ligation and Puncture (CLP). Experiments were divided into three groups, control, early sepsis (9 h after CLP), and late sepsis (18 h after CLP). Results DNA mobility and super shift assays reveal that C/EBP complexes in the liver consisted of at least three isoforms: C/EBPα, C/EBPβ, and C/EBPδ; and various C/EBP isoforms were capable of interacting with each other. Hepatocyte transfection experiments demonstrate that under normal conditions, binding of C/EBPδ to sPLA2 gene enhanced sPLA2 promoter activity and the binding resulted in an increase in hepatic gluconeogenesis. Under pathological conditions such as sepsis, binding of C/EBPδ to sPLA2 promoter increased during early and late phases of sepsis, and the increases in C/EBPδ binding correlated with increases in sPLA2 mRNA abundance and sPLA2 protein levels. Under otherwise the identical experimental conditions, hepatic gluconeogenesis was reduced during early and late phases of sepsis and the sepsis-induced reductions in liver gluconeogenesis were aggravated by binding of C/EBPδ to sPLA2 gene. Conclusions These results link C/EBPδ binding to altered sPLA2 promoter, and to hepatic gluconeogenesis under normal and pathological conditions. It is suggested that C/EBPδ-sPLA2- hepatic gluconeogenesis may function as a signalling axis affecting glucose homeostasis during the progression of sepsis. PMID:25035816

  15. Anti-parasitic effect on Toxoplasma gondii induced by BnSP-7, a Lys49-phospholipase A2 homologue from Bothrops pauloensis venom.

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    Borges, Isabela Pacheco; Castanheira, Letícia Eulalio; Barbosa, Bellisa Freitas; de Souza, Dayane Lorena Naves; da Silva, Rafaela José; Mineo, José Roberto; Tudini, Kelly Aparecida Yoneyama; Rodrigues, Renata Santos; Ferro, Eloísa Amália Vieira; de Melo Rodrigues, Veridiana

    2016-09-01

    Toxoplasmosis affects a third of the global population and presents high incidence in tropical areas. Its great relevance in public health has led to a search for new therapeutic approaches. Herein, we report the antiparasitic effects of BnSP-7 toxin, a Lys49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, on Toxoplasma gondii. In an MTT assay, BnSP-7 presented significant cytotoxicity against host HeLa cells at higher doses (200 μg/mL to 50 μg/mL), whereas lower doses (25 μg/mL to 1.56 μg/mL) produced low cytotoxicity. Furthermore, the toxin showed no effect on T. gondii tachyzoite viability when evaluated by trypan blue exclusion, but decreased both adhesion and parasite proliferation when tachyzoites were treated before infection. We also measured cytokines in supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BnSP-7, which revealed enhancement of only MIF and IL-6 cytokines levels in supernatants of HeLa cells after BnSP-7 treatment. Our results showed that the BnSP-7 PLA2 exerts an anti-Toxoplasma effect at a lower dose than that required to induce cytotoxicity in HeLa cells, and also modulates the immune response of host cells. In this sense, the anti-parasitic effect of BnSP-7 PLA2 demonstrated in the present study opens perspectives for use of this toxin as a tool for future studies on toxoplasmosis.

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

    Science.gov (United States)

    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.

  17. Bee venom phospholipase A2 suppresses allergic airway inflammation in an ovalbumin-induced asthma model through the induction of regulatory T cells.

    Science.gov (United States)

    Park, Soojin; Baek, Hyunjung; Jung, Kyung-Hwa; Lee, Gihyun; Lee, Hyeonhoon; Kang, Geun-Hyung; Lee, Gyeseok; Bae, Hyunsu

    2015-12-01

    Bee venom (BV) is one of the alternative medicines that have been widely used in the treatment of chronic inflammatory diseases. We previously demonstrated that BV induces immune tolerance by increasing the population of regulatory T cells (Tregs) in immune disorders. However, the major component and how it regulates the immune response have not been elucidated. We investigated whether bee venom phospholipase A2 (bvPLA2) exerts protective effects that are mediated via Tregs in OVA-induced asthma model. bvPLA2 was administered by intraperitoneal injection into control and OVA-challenged mice. The Treg population, total and differential bronchoalveolar lavage fluid (BALF) cell count, Th2 cytokines, and lung histological features were assessed. Treg depletion was used to determine the involvement of Treg migration and the reduction of asthmatic symptoms. The CD206-dependence of bvPLA2-treated suppression of airway inflammation was evaluated in OVA-challenged CD206(-/-) mice. The bvPLA2 treatment induced the Tregs and reduced the infiltration of inflammatory cells into the lung in the OVA-challenged mice. Th2 cytokines in the bronchoalveolar lavage fluid (BALF) were reduced in bvPLA2-treated mice. Although bvPLA2 suppressed the number of inflammatory cells after OVA challenge, these effects were not observed in Treg-depleted mice. In addition, we investigated the involvement of CD206 in bvPLA2-mediated immune tolerance in OVA-induced asthma model. We observed a significant reduction in the levels of Th2 cytokines and inflammatory cells in the BALF of bvPLA2-treated OVA-induced mice but not in bvPLA2-treated OVA-induced CD206(-/-) mice. These results demonstrated that bvPLA2 can mitigate airway inflammation by the induction of Tregs in an OVA-induced asthma model. PMID:26734460

  18. Critical role of TLR2 and MyD88 for functional response of macrophages to a group IIA-secreted phospholipase A2 from snake venom.

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    Elbio Leiguez

    Full Text Available The snake venom MT-III is a group IIA secreted phospholipase A2 (sPLA2 enzyme with functional and structural similarities with mammalian pro-inflammatory sPLA2s of the same group. Previously, we demonstrated that MT-III directly activates the innate inflammatory response of macrophages, including release of inflammatory mediators and formation of lipid droplets (LDs. However, the mechanisms coordinating these processes remain unclear. In the present study, by using TLR2-/- or MyD88-/- or C57BL/6 (WT male mice, we report that TLR2 and MyD88 signaling have a critical role in MT-III-induced inflammatory response in macrophages. MT-III caused a marked release of PGE2, PGD2, PGJ2, IL-1β and IL-10 and increased the number of LDs in WT macrophages. In MT-III-stimulated TLR2-/- macrophages, formation of LDs and release of eicosanoids and cytokines were abrogated. In MyD88-/- macrophages, MT-III-induced release of PGE2, IL-1β and IL-10 was abrogated, but release of PGD2 and PGJ2 was maintained. In addition, COX-2 protein expression seen in MT-III-stimulated WT macrophages was abolished in both TLR2-/- and MyD88-/- cells, while perilipin 2 expression was abolished only in MyD88-/- cells. We further demonstrated a reduction of saturated, monounsaturated and polyunsaturated fatty acids and a release of the TLR2 agonists palmitic and oleic acid from MT-III-stimulated WT macrophages compared with WT control cells, thus suggesting these fatty acids as major messengers for MT-III-induced engagement of TLR2/MyD88 signaling. Collectively, our findings identify for the first time a TLR2 and MyD88-dependent mechanism that underlies group IIA sPLA2-induced inflammatory response in macrophages.

  19. Critical role of TLR2 and MyD88 for functional response of macrophages to a group IIA-secreted phospholipase A2 from snake venom.

    Science.gov (United States)

    Leiguez, Elbio; Giannotti, Karina Cristina; Moreira, Vanessa; Matsubara, Márcio Hideki; Gutiérrez, José María; Lomonte, Bruno; Rodríguez, Juan Pablo; Balsinde, Jesús; Teixeira, Catarina

    2014-01-01

    The snake venom MT-III is a group IIA secreted phospholipase A2 (sPLA2) enzyme with functional and structural similarities with mammalian pro-inflammatory sPLA2s of the same group. Previously, we demonstrated that MT-III directly activates the innate inflammatory response of macrophages, including release of inflammatory mediators and formation of lipid droplets (LDs). However, the mechanisms coordinating these processes remain unclear. In the present study, by using TLR2-/- or MyD88-/- or C57BL/6 (WT) male mice, we report that TLR2 and MyD88 signaling have a critical role in MT-III-induced inflammatory response in macrophages. MT-III caused a marked release of PGE2, PGD2, PGJ2, IL-1β and IL-10 and increased the number of LDs in WT macrophages. In MT-III-stimulated TLR2-/- macrophages, formation of LDs and release of eicosanoids and cytokines were abrogated. In MyD88-/- macrophages, MT-III-induced release of PGE2, IL-1β and IL-10 was abrogated, but release of PGD2 and PGJ2 was maintained. In addition, COX-2 protein expression seen in MT-III-stimulated WT macrophages was abolished in both TLR2-/- and MyD88-/- cells, while perilipin 2 expression was abolished only in MyD88-/- cells. We further demonstrated a reduction of saturated, monounsaturated and polyunsaturated fatty acids and a release of the TLR2 agonists palmitic and oleic acid from MT-III-stimulated WT macrophages compared with WT control cells, thus suggesting these fatty acids as major messengers for MT-III-induced engagement of TLR2/MyD88 signaling. Collectively, our findings identify for the first time a TLR2 and MyD88-dependent mechanism that underlies group IIA sPLA2-induced inflammatory response in macrophages. PMID:24718259

  20. Bee venom phospholipase A2 protects against acetaminophen-induced acute liver injury by modulating regulatory T cells and IL-10 in mice.

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    Hyunseong Kim

    Full Text Available 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.

  1. High density lipoprotein suppresses lipoprotein associated phospholipase A2 in human monocytes-derived macrophages through peroxisome proliferator-activated receptor-γ pathway

    Institute of Scientific and Technical Information of China (English)

    HAN Guan-ping; REN Jing-yi; QIN Li; SONG Jun-xian; WANG Lan; CHEN Hong

    2012-01-01

    Background Lipoprotein-associated phospholipase A2 (Lp-PLA2) is mainly secreted by macrophages,serving as a specific marker of atherosclerotic plaque and exerting pro-atherogenic effects.It is known that high-density lipoprotein (HDL) plays an important role against atherosclerosis by inhibiting pro-inflammatory factors,however,the relationship between HDL and Lp-PLA2 remains elusive.Methods In this study,reverse transcription-polymerase chain reaction (RT-PCR),Western blotting,and a platelet-activating factor (PAF) acetylhydrolase assay were performed to determine the Lp-PLA2 mRNA level,protein expression and activity in human monocyte-derived macrophages upon HDL treatment of different concentrations and durations.To investigate the underlying mechanism of HDL-induced Lp-PLA2 action,pioglitazone,a peroxisome proliferator-activated receptor-y (PPARy) ligand,was introduced to human monocyte-derived macrophages and mRNA and protein levels of Lp-PLA2,as well as its activity,were determined.Results Lp-PLA2 mRNA levels,protein expression and activity were significantly inhibited in response to HDL treatment in a dose and time dependent manner in human monocyte-derived macrophages.Pioglitazone treatment (1-10 ng/ml) upregulated the Lp-PLA2 mRNA level,protein expression and activity in human monocyte-derived macrophages,while the effects were markedly reversed by HDL.In addition,pioglitazone resulted in a significant increase in PPARY phosphorylation in human monocyte-derived macrophages,which could be inhibited by HDL.Conclusion These findings indicate that HDL suppresses the expression and activity of Lp-PLA2 in human monocyte-derived macrophages,and the underlying mechanisms may be mediated through the PPARY pathway.

  2. Endothelial PKCα-MAPK/ERK-phospholipase A2 pathway activation as a response of glioma in a triple culture model. A new role for pericytes?

    Science.gov (United States)

    Anfuso, Carmelina Daniela; Motta, Carla; Giurdanella, Giovanni; Arena, Valeria; Alberghina, Mario; Lupo, Gabriella

    2014-04-01

    In view of understanding the molecular mechanisms through which angiogenic switch on happens in the early phases of reciprocal interaction between tumor and cells constituting microvessel, a triple culture model in which endothelial cells (EC), pericytes (PC) and glioma C6 cells were cultured together. In the present work, we observed that C6 enhanced EC proliferation. This effect was reduced by cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2), cyclooxygenase-2 (COX-2), PI3-K, MEK-1, and ERK1/2 inhibitors and by siRNAs against both PLA2s. In EC, C6 induced an increase in iPLA2, cPLA2 and COX-2 total protein expression. Moreover, the increase in endothelial cPLA2 phosphorylation was attenuated by kinase inhibitors. Both EC proliferation and signal protein phosphorylation were attenuated when PC were in triple culture. In EC/C6 supernatants, and, in a lesser extent, in EC/PC co-cultures, an enhancement in prostaglandins E2 (PGE2) was found. The presence of PC in triple-cultures caused a decrease in production of PGE2 respect to EC/C6 double-cultures. In all systems, AACOCF3 and BEL significantly reduced PGE2 secretion. In Matrigel-based assays, emerging branch points from EC cell bodies and tubule-like structures were observed. C6 conditioned EC/PC co-cultures in constituting poorly organized tubules. Transfection of EC with c- and iPLA2 siRNA strongly reduced in vitro tubulogenesis. Data here reported indicate that PKCα, ERK kinase phosphorylation, PLA2s and COX-2 activation, and PGE2 production in EC stimulated by tumor cells are coincident phenomena and could represent therapeutic targets in chemoprevention of glioma. Moreover, PC exhibited an important "modulating" role in the initial stages of angiogenesis driven by a brain tumor.

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

    Science.gov (United States)

    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 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. PMID:25913570

  4. Changes of gastric and intestinal blood flow, serum phospholipase A2 and interleukin-1β in rats with acute necrotizing pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Jian-Xin Zhang; Sheng-Chun Dang; Jian-Guo Qu; Xue-Qing Wang; Guo-Zuo Chen

    2005-01-01

    AIM: To explore the relationship between gastric and intestinal microcirculatory impairment and inflammatory mediators released in rats with acute necrotizing pancreatitis (ANP).METHODS: A total of 64 rats were randomized into control group and ANP group. ANP model was induced by injection of 5% sodium taurocholate under the pancreatic membrane.Radioactive biomicrosphere technique was used to measure the gastric and intestinal tissue blood flow at 2 and 12 h after the induction of ANP, meanwhile serum phospholipase A2 (PLA2) activities and interleukin-1β levels were determined. Pathologic changes in pancreas, gastric and intestinal mucosae were studied. RESULTS: The gastric blood flow in ANP group (0.62±0.06 (P<0.01) at 2 and 12 h after induction of ANP. The intestinal blood flow in ANP group (0.80±0.07 and (P<0.01). Serum PLA2 activities (94.29±9.96 and 103.71± 14.40) U/L and IL-1β levels (0.78±0.13 and 0.83±0.20) μg/L in ANP group were higher than those in control group (65.27±10.52 and 66.63±9.81) U/L, (0.32±0.06 and 0.33±0.07) μg/L (P<0.01). At 2 and 12 h after introduction of the model, typical pathologic changes were found in ANP. Compared with control group, the gastric and intestinal mucosal pathologic changes were aggravated significantly (P<0.01) at 12 h after induction of ANP. Gastric and intestinal mucosal necrosis, multiple ulcer and hemorrhage occurred.CONCLUSION: Decrease of gastric and intestinal blood flow and increase of inflammatory mediators occur simultaneously early in ANP, both of them are important pathogenic factors for gastric and intestinal mucosal injury in ANP.

  5. Prostaglandins from Cytosolic Phospholipase A2α/Cyclooxygenase-1 Pathway and Mitogen-activated Protein Kinases Regulate Gene Expression in Candida albicans-infected Macrophages.

    Science.gov (United States)

    Yun, Bogeon; Lee, HeeJung; Jayaraja, Sabarirajan; Suram, Saritha; Murphy, Robert C; Leslie, Christina C

    2016-03-25

    In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2(cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. InC. albicans-infected cPLA2α(-/-)or COX-1(-/-)macrophages, expression ofI l10,Nr4a2, and Ptgs2 was lower, and expression ofTnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α(-/-)or COX-1(-/-)macrophages. InC. albicans-infected cPLA2α(+/+)macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2 Activation of ERKs and p38, but not JNKs, by C. albicansacted synergistically with prostaglandins to induce expression of Il10,Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved inIl10,Nr4a2,Ptgs2, andTnfα expression, was not mediated by cAMP/PKA because it was similar inC. albicans-infected wild type and cPLA2α(-/-)or COX-1(-/-)macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation inC. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.

  6. Variations of protein profiles and calcium and phospholipase A2 concentrations in thawed bovine semen and their relation to acrosome reaction

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    V. Alonso Marques

    2000-12-01

    Full Text Available Just as calcium plays an integral role in acrosome capacitation and reaction, several spermatozoon proteins have been reported as binding to the ovum at fertilization. We examined the relationship between thawed bovine semen protein profiles, seminal plasma calcium ion concentration, spermatozoon phospholipase A2 (PLA2 activity and acrosome reaction. Electrophoretic profile analysis of spermatozoa and bovine seminal plasma proteins (total and membrane revealed qualitative and quantitative differences among bulls. Variations in PLA2 and seminal plasma calcium concentration indicated genetic diversity among individuals. A 15.7-kDa membrane protein was significantly correlated (r = 0.71 with acrosome reaction, which in turn has been associated with in vivo fertility.Várias proteínas que constituem o espermatozóide têm sido relatadas como sendo proteínas que se ligam ao óvulo no momento da fertilização, bem como íons cálcio têm um papel importante na capacitação e reação acrossômica. Baseado nisto, este estudo teve como objetivo analisar e correlacionar proteínas do sêmen congelado bovino de diferentes raças, concentração de íons cálcio no plasma seminal e atividade da fosfolipase A2 do espermatozóide com a reação acrossômica, visando encontrar fatores que influenciem no processo de fertilização bovina. Análises do perfil eletroforético das proteínas (totais e de membrana do espermatozóide e do plasma seminal bovino revelaram variabilidade protéica entre indivíduos na qual diferenças qualitativas e quantitativas foram identificadas. A quantificação da fosfolipase A2, bem como da concentração de cálcio no plasma seminal revelaram diversidade genética entre touros. Uma proteína de 15,7 kDa apresentou correlação significativa (0.71 com a reação acrossômica, que pode estar diretamente relacionada com a fertilização in vivo e deste modo outros experimentos podem ser realizados a fim de investigar a

  7. Phospholipase A2 receptor in idiopathic membranous nephropathy%磷脂酶A2受体与特发性膜性肾病

    Institute of Scientific and Technical Information of China (English)

    秦华章

    2015-01-01

    特发性膜性肾病(IMN)是一种以肾小球毛细血管袢上皮侧免疫复合物沉积为特征的肾小球疾病,其致病抗体抗M型磷脂酶A2受体1(PLA2R1)抗体的发现使得IMN的诊断、预后判断和病情监测等方面取得突破性的进展.全基因组关联研究发现HLA-DQA1和PLA2R1是IMN的易感基因,进一步明确IMN的遗传学背景.最近研究发现PLA2R上引发免疫反应的主要抗原表位.本文就PLA2R与IMN的研究进展作一综述.

  8. Molecular characterization of a novel phospholipase A_2 from Clonorchis sinensis%华支睾吸虫磷脂酶A_2基因生物学特性的初步研究

    Institute of Scientific and Technical Information of China (English)

    马长玲; 胡旭初; 胡凤玉; 李艳文; 赵俊红; 郑小凌; 余新炳

    2009-01-01

    Objective To clone and express the novel gene phospholipase Az of Clonorchis sinensis (CsPLA_2) for molecular characterization. Methods Using the full length cDNA plasmid clone (No. Cs005f08) as a template, the coding region of phospholipase A_2 was amplified by PCR, cloned into the prokaryotic expression vector pET28a ( + ), and then expressed in Escherichia coli BL21 by IPTG induction. The recombinant protein was detected by SDS-PAGE. The excretory-secretory properties of this protein were analyzed by Western blotting. Immunohistochemical localization of CsPLA_2 in the living adults of C. sinensis was done using fluorescence microscopy. Results A cDNA clone encoding a novel phospholipase A_2(307 amino acids) was isolated from a C. sinensis adult cDNA library. Analysis using bioinformatics software showed that the possible sequence shared conserved features of phospholipase A_2 and its active site. In a search of GenBank, the signal sequence cleavage site of CsPLA_2 was after 36v.i. The predicted CsPLA_2 amino acid sequence had 45% identity and 52% similarity to Triboliumcastaneum. The prokaryotic expressing vector (pET28a-CsLysoPLA) constructed was successfully indentified by PCR, restriction enzyme digestion, and sequencing. The coding sequence of the gene was highly expressed in E. coli. Western blot analysis indicated that it belonged to excretory-secretory proteins of the adults. Immunohistochemistry suggested that the CsPLAz was markedly localized in the ventral sucker, intestine, and testis of the adults. Conclusion The novel CsPLA_2, a component of excretory-secretory proteins, was localized in the ventral sucker, intestine, and testis of adults. The coding region of CsPLA_2 was successfully expressed in E. coli.%目的 对新发现的华支睾吸虫磷脂酶A2(CsPLA_2)基因进行生克隆、原核表达,确定该蛋白是否为成虫分泌/排泄蛋白(excretory-secretory protein,ESP). 方法 利用多种生物信息学分析软件,从华支睾吸虫全

  9. Retrospective Study of Phospholipase A2 Receptor and IgG Subclasses in Glomerular Deposits in Chinese Patients with Membranous Nephropathy.

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    Hong-Rui Dong

    Full Text Available The research work in the past years showed that detection of phospholipase A2 receptor (PLA2R antigen and its dominant IgG4 autoantibody in glomerular deposits of patients with membranous nephropathy (MN was useful for the differentiation between primary MN (PMN and secondary MN (SMN, but so far such research data from large Chinese patient series is little. Here, we are going to report a research work in a Chinese cohort.This study enrolled 179 patients with PMN, 40 patients with membranous lupus nephritis (LN-MN, 26 patients with hepatitis B virus-associated MN (HBV-MN, 2 patients with malignancy-associated MN (M-MN and one patient with IgG4-related MN (IgG4-MN. PLA2R and IgG subclasses in glomerular deposits of these patients were examined by immunofluorescence and/or immunohistochemical staining, and the potential value of the above examinations for differential diagnosis of PMN and SMN was evaluated.Glomerular PLA2R deposition was present in 92.2% patients with PMN and 7.7% patients with HBV-MN, but none of the patients with LN-MN. Predominant/codominant IgG4 deposition was found in 93.3% patients with PMN and 11.5% patients with HBV-MN, but none of the patients with LN-MN. The two M-MN patients both had glomerular PLA2R and predominant/codominant IgG4 deposition. The one IgG4-MN patient had deeply staining IgG4 but no PLA2R in glomeruli.The glomerular PLA2R and predominant/codominant IgG4 deposition is frequently observed in Chinese patients with PMN. Immunofluorescence and immunohistochemical staining of renal biopsy tissue for detection of glomerular PLA2R and IgG subclasses deposition can help to distinguish PMN from LN-MN and most of HBV-MN.

  10. Upregulation of group IB secreted phospholipase A(2) and its M-type receptor in rat ANTI-THY-1 glomerulonephritis.

    Science.gov (United States)

    Beck, S; Beck, G; Ostendorf, T; Floege, J; Lambeau, G; Nevalainen, T; Radeke, H H; Gurrieri, S; Haas, U; Thorwart, B; Pfeilschifter, J; Kaszkin, M

    2006-10-01

    Treatment of rat glomerular mesangial cell (GMC) cultures with pancreatic secreted phospholipase A(2) (sPLA(2)-IB) results in an enhanced expression of sPLA(2)-IIA and COX-2, possibly via binding to its specific M-type sPLA(2) receptor. In the current study, we have investigated the expression and regulation of sPLA(2)-IB and its receptor during glomerulonephritis (GN). In vivo we used the well-established rat model of anti-Thy 1.1 GN (anti-Thy 1.1-GN) to study the expression of sPLA(2)-IB and the M-type sPLA(2) receptor by immunohistochemistry. In addition, in vitro we determined the interkeukin (IL)-1beta-regulated mRNA and protein expression in primary rat glomerular mesangial and endothelial cells as well as in rat peripheral blood leukocytes (PBLs). Shortly after induction of anti-Thy 1.1-GN, sPLA(2)-IB expression was markedly upregulated in the kidney at 6-24 h. Within glomeruli, the strongest sPLA(2)-IB protein expression was detected on infiltrated granulocytes and monocytes. However, at the same time, the M-type receptor was also markedly upregulated on resident glomerular cells. In vitro, the most prominent cytokine-stimulated secretion of sPLA(2)-IB was observed in monocytes isolated from rat PBLs. Treating glomerular endothelial cells (GECs) with cytokines elicited only weak sPLA(2)-IB expression, but treatment of these cells with exogenous sPLA(2)-IB resulted in a marked expression of the endogenous sPLA(2)-IB. Mesangial cells did not express sPLA(2)-IB at all. The M-type sPLA(2) receptor protein was markedly upregulated on cytokine-stimulated mesangial and endothelial cells as well as on lymphocytes and granulocytes. During anti-Thy 1.1 rat GN, sPLA(2)-IB and the M-type sPLA(2) receptor are induced as primary downstream genes stimulated by inflammatory cytokines. Subsequently, both sPLA(2)-IB and the M-type sPLA(2) receptor are involved in the autocrine and paracrine amplification of the inflammatory process in different resident and infiltrating

  11. Admission lipoprotein-associated phospholipase A2 activity is not associated with long-term clinical outcomes after ST-segment elevation myocardial infarction.

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    Pier Woudstra

    Full Text Available BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2 activity is a biomarker predicting cardiovascular diseases in a real-world. However, the prognostic value in patients undergoing primary percutaneous coronary intervention (pPCI for ST-segment elevation myocardial infarction (STEMI on long-term clinical outcomes is unknown. METHODS: Lp-PLA2 activity was measured in samples obtained prior to pPCI from consecutive STEMI patients in a high-volume intervention center from 2005 until 2007. Five years all-cause mortality was estimated with the Kaplan-Meier method and compared among tertiles of Lp-PLA2 activity during complete follow-up and with a landmark at 30 days. In a subpopulation clinical endpoints were assessed at three years. The prognostic value of Lp-PLA2, in addition to the Thrombolysis In Myocardial Infarction or multimarker risk score, was assessed in multivariable Cox regression. RESULTS: The cohort (n = 987 was divided into tertiles (low 179 nmol/min/mL. Among the tertiles differences in baseline characteristics associated with long-term mortality were observed. However, no significant differences in five years mortality in association with Lp-PLA2 activity levels were found; intermediate versus low Lp-PLA2 (HR 0.97; CI 95% 0.68-1.40; p = 0.88 or high versus low Lp-PLA2 (HR 0.75; CI 95% 0.51-1.11; p = 0.15. Both in a landmark analysis and after adjustments for the established risk scores and selection of cases with biomarkers obtained, non-significant differences among the tertiles were observed. In the subpopulation no significant differences in clinical endpoints were observed among the tertiles. CONCLUSION: Lp-PLA2 activity levels at admission prior to pPCI in STEMI patients are not associated with the incidence of short and/or long-term clinical endpoints. Lp-PLA2 as an independent and clinically useful biomarker in the risk stratification of STEMI patients still remains to be proven.

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

    Science.gov (United States)

    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. PMID:27319010

  13. Inhibition of cytosolic Phospholipase A2 prevents prion peptide-induced neuronal damage and co-localisation with Beta III Tubulin

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    Last Victoria

    2012-08-01

    Full Text Available Abstract Background Activation of phospholipase A2 (PLA2 and the subsequent metabolism of arachidonic acid (AA to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease. Here we report the effects of the prion peptide fragment HuPrP106-126 on the PLA2 cascade in primary cortical neurons and translocation of cPLA2 to neurites. Results Exposure of primary cortical neurons to HuPrP106-126 increased the levels of phosphorylated cPLA2 and caused phosphorylated cPLA2 to relocate from the cell body to the cellular neurite in a PrP-dependent manner, a previously unreported observation. HuPrP106-126 also induced significant AA release, an indicator of cPLA2 activation; this preceded synapse damage and subsequent cellular death. The novel translocation of p-cPLA2 postulated the potential for exposure to HuPrP106-126 to result in a re-arrangement of the cellular cytoskeleton. However p-cPLA2 did not colocalise significantly with F-actin, intermediate filaments, or microtubule-associated proteins. Conversely, p-cPLA2 did significantly colocalise with the cytoskeletal protein beta III tubulin. Pre-treatment with the PLA2 inhibitor, palmitoyl trifluoromethyl ketone (PACOCF3 reduced cPLA2 activation, AA release and damage to the neuronal synapse. Furthermore, PACOCF3 reduced expression of p-cPLA2 in neurites and inhibited colocalisation with beta III tubulin, resulting in protection against PrP-induced cell death. Conclusions Collectively, these findings suggest that cPLA2 plays a vital role in the action of HuPrP106-126 and that the colocalisation of p-cPLA2 with beta III tubulin could be central to the progress of neurodegeneration caused by prion peptides. Further work is needed to define exactly how PLA2 inhibitors protect neurons from peptide-induced toxicity and how this relates to intracellular structural changes occurring in neurodegeneration.

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

    Science.gov (United States)

    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.

  15. Identification and characterization of a phospholipase A2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities.

    Science.gov (United States)

    Perumal Samy, Ramar; Gopalakrishnakone, P; Bow, Ho; Puspharaj, Peter N; Chow, Vincent T K

    2010-12-01

    Phospholipase A(2) (PLA(2)), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA(2), named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA(2), EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA(2)s from other snake venoms. The purified PLA(2) EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA(2) (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region

  16. Time-dependent changes of glial fibriliary acidic protein and cytosolic phospholipase A2 in hippocampal area of focal cerebral ischemia/reperfusion in rats

    Institute of Scientific and Technical Information of China (English)

    Qingzhou Cheng; Xingui Ming

    2006-01-01

    BACKGROUND: Interaction between astrocyte and neuron may two-dimensionally influence on ischemic injury; however, glial fibriliary acidic protein (GFAP) and cytosolic phospholipase A2 (cPLA2) are both important markers to reflect changes of astrocyte and neuron after cerebral ischemia, respectively.OBJECTIVE: To observe the changes of GFAP and positive cPLA2 cells in hippocampal area of model rats with focal cerebral ischemia in various phases of cerebral ischemia/reperfusion.DESIGN: Randomized contrast observation.SETTTNG: Department of Basic Medical Science, Medical College of Wuhan Polytechnic University; Faculty of Human Anatomy and Histology & Embryology, Medical College of Wuhan University.MATERIALS: The experiment was carried out in the Department of Basic Medical Science, Medical College of Wuhan Industry College from May to June 2004. A total of 28 healthy SD rats of either gender and weighing 200-250 g were provided by Animal Department of Medical College of Jianghan University.METHODS: All 28 rats were randomly divided into 7 groups, including sham operation group, 2-, 6-, 12-,24- and 48-reperfusion groups, and triphenyltetrazolium chloride (TTC) group, with 4 in each group. Two hours after ischemia, ischemia/reperfusion models were established in left middle cerebral artery (MCA);common carotid artery was ligated and line cork was inserted into it with the depth of (1.8±0.5) cm. Rats in sham operation group were inserted with the depth of 1.0 cm, and other operations were as the same as those in 2-hour ischemia/reperfusion groups. Models in TTC group were established as the same as those in 2-hour ischemia/24-hour reperfusion group, and they were used to evaluate the therapeutic effect.Changes of GFAP and cPLA2 in hippocampal area in various phases were detected with immunohistochemical method.MATN OUTCOME MEASURES: Changes of GFAP and positive cPLA2 cells in hippocampal area of rats with focai cerebral ischemia in various phases of ischemia

  17. Cytosolic phospholipase A2 alpha amplifies early cyclooxygenase-2 expression, oxidative stress and MAP kinase phosphorylation after cerebral ischemia in mice

    Directory of Open Access Journals (Sweden)

    Koehler Raymond C

    2010-07-01

    Full Text Available Abstract Background The enzyme cytosolic phospholipase A2 alpha (cPLA2α has been implicated in the progression of cerebral injury following ischemia and reperfusion. Previous studies in rodents suggest that cPLA2α enhances delayed injury extension and disruption of the blood brain barrier many hours after reperfusion. In this study we investigated the role of cPLA2α in early ischemic cerebral injury. Methods Middle cerebral artery occlusion (MCAO was performed on cPLA2α+/+ and cPLA2α-/- mice for 2 hours followed by 0, 2, or 6 hours of reperfusion. The levels of cPLA2α, cyclooxygenase-2, neuronal morphology and reactive oxygen species in the ischemic and contralateral hemispheres were evaluated by light and fluorescent microscopy. PGE2 content was compared between genotypes and hemispheres after MCAO and MCAO and 6 hours reperfusion. Regional cerebral blood flow was measured during MCAO and phosphorylation of relevant MAPKs in brain protein homogenates was measured by Western analysis after 6 hours of reperfusion. Results Neuronal cPLA2α protein increased by 2-fold immediately after MCAO and returned to pre-MCAO levels after 2 hours reperfusion. Neuronal cyclooxygenase-2 induction and PGE2 concentration were greater in cPLA2α+/+ compared to cPLA2α-/- ischemic cortex. Neuronal swelling in ischemic regions was significantly greater in the cPLA2α+/+ than in cPLA2α-/- brains (+/+: 2.2 ± 0.3 fold vs. -/-: 1.7 ± 0.4 fold increase; P 2α+/+ ischemic core than in cPLA2α-/- (+/+: 7.12 ± 1.2 fold vs. -/-: 3.1 ± 1.4 fold; P 2α+/+, but not cPLA2α-/-, had disruption of neuron morphology and decreased PGE2 content. Phosphorylation of the MAPKs-p38, ERK 1/2, and MEK 1/2-was significantly greater in cPLA2a+/+ than in cPLA2α-/- ischemic cortex 6 hours after reperfusion. Conclusions These results indicate that cPLA2α modulates the earliest molecular and injury responses after cerebral ischemia and have implications for the potential clinical

  18. Asp-49 is not an absolute prerequisite for the enzymic activity of low-M(r) phospholipases A2: purification, characterization and computer modelling of an enzymically active Ser-49 phospholipase A2, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper).

    Science.gov (United States)

    Polgár, J; Magnenat, E M; Peitsch, M C; Wells, T N; Clemetson, K J

    1996-11-01

    Several studies have shown that Asp-49 is the residue that controls calcium binding in, and so plays a critical role in the calcium-mediated activation of, low-M(r) group I-III phospholipases A2 (PLA2s). The present paper provides experimental evidence that Asp-49 is not an absolute prerequisite for the enzymic activity of PLA2s, and that proteins with amino acid(s) other than Asp at position 49 can exhibit significant phospholipase activity. The purification, complete amino acid sequence and characterization of ecarpholin S, a PLA2 from Echis carinatus sochureki (saw-scaled viper) venom, is described. This single-chain, 122-amino-acid, basic (pI 7.9) protein is a group II PLA2. Although Asp-49 is replaced by Ser and Tyr-28 by Phe (both of these positions being involved in the Ca(2+)-binding site of PLA2s), the lipolysis of soybean phosphatidylcholine and egg yolk in the presence of 10 mM CaCl2 was 1.5 times and 2.9 times greater respectively with ecarpholin S than with recombinant human group II PLA2. The Ca(2+)-dependencies of the enzymic activities of ecarpholin S and rPLA2 were found to be similar. Ecarpholin S added to washed platelets induced aggregation; the presence of Ca2+ was a prerequisite for this platelet-aggregating effect. Computer modelling of the Ca(2+)-binding site of Ser-49 PLA2 compared with the Asp-49 and Lys-49 forms, for which crystallographic data exist, shows that the Ca(2+)-binding site is sterically blocked by Lys-49 but not by Ser-49; in the latter, the Ser hydroxy group may replace the Asp carboxylate in stabilization of Ca2+ binding. Sequence comparisons of ecarpholin S and other low-M(r) PLA2s predicts the presence of a Ser-49 group in the protein family of low-M(r) PLA2s that is distinct from the Asp-49 and Lys-49 groups. PMID:8921006

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

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    Dong Ju Son

    2014-08-01

    Full Text Available PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum and long pepper (Piper longum, was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2, COX-1, COX-2, and thromboxane A2 (TXA2 synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PGE2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms.

  20. Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases

    NARCIS (Netherlands)

    Zwaal, R.F.A.; Roelofsen, B.; Comfurius, P.; Deenen, L.L.M. van

    1975-01-01

    1. 1. The action of eight purified phospholipases on intact human erythrocytes has been investigated. Four enzymes, e.g. phospholipases A2 from pancreas and Crotalus adamanteus, phospholipase C from Bacillus cereus, and phospholipase D from cabbage produce neither haemolysis nor hydrolysis of phosph

  1. Colourimetric Determination of Phospholipase Activities in Balamuthia mandrillaris

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    Syed Razi Haider

    2007-01-01

    Full Text Available Balamuthia mandrillaris is a recently identified protozoan pathogen that can cause fatal granulomatous encephalitis however the pathogenesis and pathophysiology associated with Balamuthia encephalitis remain unclear. We have recently isolated B. mandrillaris from a 33-years old male who died of encephalitis. Using this isolate, we demonstrated for the first time that B. mandrillaris exhibited phospholipase activities. More specifically, B. mandrillaris exhibited phospholipase A2 and phospholipase D activities. For the first time we used colourimetric technique based on spectrophotometer and designed phospholipases assays to determine these phospholipase activities. The functional role of phospholipases was determined in in vitro assays using human brain microvascular endothelial cells (HBMEC. We observed that PLA2-specific inhibitor i.e., cytidine 5'-diphosphocholine significantly inhibited B. mandrillaris binding to HBMEC. Similarly PLD inhibitor i.e., compound 48/80 inhibited B. mandrillaris binding to HBMEC. Moreover, both inhibitors inhibited B. mandrillaris-mediated HBMEC cytotoxicity. Overall these results clearly demonstrate that phospholipases are important virulence determinants in B. mandrillaris. Further studies will identify the precise role of phospholipases in the pathogenesis of B. mandrillaris, which may help develop therapeutic interventions. Using a novel spectrophotometric-based assay we demonstrated for the first time that B. mandrillaris exhibit phospholipase activities.

  2. Calcium-independent NO-synthase activity and nitrites/nitrates production in transient focal cerebral ischaemia in mice

    OpenAIRE

    Grandati, M; Verrecchia, C; Revaud, M L; Allix, M.; Boulu, R. G.; Plotkine, M.

    1997-01-01

    The temporal changes in constitutive NO-synthase (cNOS) and in calcium-independent NO-synthase activities were studied in mice subjected to 2 h of transient focal cerebral ischaemia. The changes in brain nitrites/nitrates (NOx) content were also studied.NOS activities were measured by the conversion of L-[14C]-arginine to L-[14C]-citrulline. Brain NOx contents were investigated by the Griess colourimetric method.cNOS activity in the infarcted cortical area was significantly reduced after 6 h ...

  3. Secreted phospholipase A2 and glutathione peroxidase activities in chicken PSE (pale, soft, exudative meatAtividades de fosfolipase A2 secretada e glutationa peroxidase em filés PSE (pale, soft, exudative de frango

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    Adriana Lourenço Soares

    Full Text Available The excessive release of calcium from the sarcoplasmic reticulum during the installation of PSE (Pale, Soft, Exudative meat, leads to increase of phospholipase A2 (PLA2 activity and consequently causes a higher lipid oxidation. In contrast, glutathione peroxidase (GSH-Px is a selenium-dependent antioxidant enzyme that prevents the oxidative damage. The aim of this work was to investigate the secreted PLA2 (sPLA2 and GSH-PX activities in PSE poultry meat. Breast meat samples (Pectoralis major m. (n=24 were obtained from commercial slaughterhouse. Samples were classified as PSE and Control Meat based on pH and L* values, fillets with pH1h30? 6.0 and L24h* ?53.0 as PSE and fillets with pH1h30> 6.0 and L24h*0,05. The GSH-Px activity was approximately 24,4% higher (p?0,05 for Control meat when compared to PSE meat. The sPLA2 activity of PSE fillets did not changed, however PSE fillets present the enzymatic system of antioxidant defense compromised with lower GSH-PX activity.A liberação excessiva de cálcio do retículo sarcoplasmático durante a instalação de carnes PSE (Pale, Soft, Exudative leva a um aumento da atividade da fosfolipase A2 (PLA2, promovendo uma maior oxidação lipídica. Por outro lado, a glutationa peroxidase (GSH-Px é uma enzima antioxidante selênio dependente, prevenindo danos oxidativos. O objetivo deste trabalho foi investigar as atividades da PLA2 secretada (sPLA2 e da GSH-PX em filés PSE de frango. Filés de frango (Pectoralis major m. (n=24 foram obtidos de um frigorífico comercial. As amostras foram classificadas em PSE e Controle, com base nos valores de pH e L*, filés com pH1h30 ? 6.0 e L24h* ? 53,0 como PSE e filés com pH1h30> 6,0 e L24h* 0,05. A atividade da GSH-Px foi aproximadamente 24,4% maior (p?0,05 para os filés Controle quando comparado com filés PSE. A atividade da sPLA2 em filés PSE não foi alterada, entretanto filés PSE apresentam o sistema enzimático de defesa antioxidante comprometido com

  4. Secreted phospholipase A2 and glutathione peroxidase activities in chicken PSE (pale, soft, exudative meatAtividades de fosfolipase A2 secretada e glutationa peroxidase em filés PSE (pale, soft, exudative de frango

    Directory of Open Access Journals (Sweden)

    Adriana Lourenço Soares

    2012-02-01

    Full Text Available The excessive release of calcium from the sarcoplasmic reticulum during the installation of PSE (Pale, Soft, Exudative meat, leads to increase of phospholipase A2 (PLA2 activity and consequently causes a higher lipid oxidation. In contrast, glutathione peroxidase (GSH-Px is a selenium-dependent antioxidant enzyme that prevents the oxidative damage. The aim of this work was to investigate the secreted PLA2 (sPLA2 and GSH-PX activities in PSE poultry meat. Breast meat samples (Pectoralis major m. (n=24 were obtained from commercial slaughterhouse. Samples were classified as PSE and Control Meat based on pH and L* values, fillets with pH1h30? 6.0 and L24h* ?53.0 as PSE and fillets with pH1h30> 6.0 and L24h*0,05. The GSH-Px activity was approximately 24,4% higher (p?0,05 for Control meat when compared to PSE meat. The sPLA2 activity of PSE fillets did not changed, however PSE fillets present the enzymatic system of antioxidant defense compromised with lower GSH-PX activity.A liberação excessiva de cálcio do retículo sarcoplasmático durante a instalação de carnes PSE (Pale, Soft, Exudative leva a um aumento da atividade da fosfolipase A2 (PLA2, promovendo uma maior oxidação lipídica. Por outro lado, a glutationa peroxidase (GSH-Px é uma enzima antioxidante selênio dependente, prevenindo danos oxidativos. O objetivo deste trabalho foi investigar as atividades da PLA2 secretada (sPLA2 e da GSH-PX em filés PSE de frango. Filés de frango (Pectoralis major m. (n=24 foram obtidos de um frigorífico comercial. As amostras foram classificadas em PSE e Controle, com base nos valores de pH e L*, filés com pH1h30 ? 6.0 e L24h* ? 53,0 como PSE e filés com pH1h30> 6,0 e L24h* 0,05. A atividade da GSH-Px foi aproximadamente 24,4% maior (p?0,05 para os filés Controle quando comparado com filés PSE. A atividade da sPLA2 em filés PSE não foi alterada, entretanto filés PSE apresentam o sistema enzimático de defesa antioxidante comprometido com

  5. PhTX-II a Basic Myotoxic Phospholipase A2 from Porthidium hyoprora Snake Venom, Pharmacological Characterization and Amino Acid Sequence by Mass Spectrometry

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    Salomón Huancahuire-Vega

    2014-10-01

    Full Text Available A monomeric basic PLA2 (PhTX-II of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom.

  6. Reminiscence of phospholipase B in Penicillium notatum

    OpenAIRE

    SAITO, Kunihiko

    2014-01-01

    Since the phospholipase B (PLB) was reported as a deacylase of both lecithin and lysolecithin yielding fatty acids and glycerophosphocholine (GPC), there was a question as to whether it is a single enzyme or a mixture of a phospholipase A2 (PLA2) and a lysophospholipase (LPL). We purified the PLB in Penicillium notatum and showed that it catalyzed deacylation of sn-1 and sn-2 fatty acids of 1,2-diacylphospholipids and also sn-1 or sn-2 fatty acids of 1- or 2-monoacylphospholipids (lysophospho...

  7. The relationship between anti-phospholipase A2 receptor antibody and idiopathic membranous nephropathy%抗磷脂酶A2受体抗体与特发性膜性肾病的关系

    Institute of Scientific and Technical Information of China (English)

    林伟锋; 李航; 李雪梅; 秦岩; 苏颖; 于阳; 管音; 段琳; 李艳

    2015-01-01

    目的 探讨抗磷脂酶A2受体(PLA2R)抗体对特发性膜性肾病(IMN)的诊断和病情活动监测价值.方法 选择2012年1月至2014年3月北京协和医院行肾活检并确诊的IMN 233例,以同期46例非IMN肾脏疾病为对照,采用定量ELISA检测肾活检时血清抗PLA2R抗体滴度.绘制抗PLA2R抗体诊断IMN的ROC曲线.结果 抗PLA2R抗体在IMN中总敏感性为60.0%,特异性为100.0%,检测前未行免疫抑制治疗者的阳性率为71.3%,其中25例狼疮性肾炎患者抗体均阴性.肾病范畴蛋白尿者抗体阳性率为68.3%,非肾病范畴蛋白尿者抗体阳性率为41.7%(P<0.05);血白蛋白低于30 g/L者抗体阳性率为67.3%,高于血白蛋白>30 g/L者(44.6%;P<0.05).抗体滴度水平越高,则低白蛋白血症越严重(P<0.05),肾病范畴蛋白尿的比例越高(P<0.05).抗PLA2R抗体诊断IMN的AUCRoc为0.800.结论 抗PLA2R抗体对IMN诊断具有较高的敏感性和特异性,诊断准确性良好.抗体阳性率受免疫抑制治疗、病情活动等因素影响.抗PLA2R抗体可反映IMN的活动性.%Objective To explore the value of anti-phospholipase A2 receptor (PLA2R) antibody in the diagnosis and disease activity monitoring of idiopathic membranous nephropathy (IMN).Methods A total of 233 patients with IMN proven by kidney biopsy at Peking Union Medical College Hospital from January 2012 to March 2014 were enrolled in this study.Another 46 patients with non-IMN kidney diseases at the same period were selected as control group.Serum titer of anti-PLA2R antibody was measured by quantitative enzyme-linked immuno sorbent assay (ELISA) at the time of renal biopsy.Clinical data were reviewed and retrospectively analyzed.The diagnostic accuracy of anti-PLA2R antibody in IMN was estimated by ROC curve.Results The total sensitivity of anti-PLA2R antibody was 60.0% in IMN.However,the sensitivity increased to 71.3% in patients who did not receive immuno-suppression therapies

  8. Expression and Renaturation of Streptomyces violaceoruber 2917 Phospholipase A2 Protein in E.coil Rosetta(DE3)%紫红链霉菌2917磷脂酶A2在大肠杆菌Rosetta(DE3)中表达与复性

    Institute of Scientific and Technical Information of China (English)

    杨波; 邓贝; 刘志刚; 陈威; 李维琳; 胡征

    2013-01-01

    The gene encoding the secreted phospholipase A2 of Streptomyces violaceoruber 2917 was chemical syn-thesized without changing the amino acid sequence and cloned into pGEX-6P-1 to construct the expression vector pGEX-6P-1-PLA2. Then, this recombinant plasmid was introduced into E. coli Rosetta (DE3) to obtain engineering bacterium. The fusion recombinant protein was expressed as inclusion body after induction of isopropy-β-D-thio-galactosic (IPTG). The inclusion bodies were then harvested and refolded by means of pulsed-dilution method. Sub-sequently, the enzymatic activity of the refolded phospholipase A2 was determined. SDS-PAGE analysis showed that the phospholipase A2 was highly expressed in the E. coli cells, accounting for around 30% of total cell proteins. Enzymatic activity assay showed that this refolded protein displayed a activity of 0.15U/mL of enzyme. This study laid a foundation for the further studies of renaturation of the enzyme, and also its industrial application.%  通过化学合成紫红链霉菌2917分泌型磷脂酶A2全基因序列后插入融合表达载体pGEX-6P-1中,经酶切和测序鉴定无误后转化E.coli Rosetta(DE3)菌株,用异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,获得目的蛋白后对包涵体形式的重组蛋白进行稀释复性,同时对复性后的蛋白进行酶学研究.结果表明磷脂酶A2基因在大肠杆菌中获得了高效表达(占总细胞蛋白30%),复性后酶液酶活性为0.15U/mL。这将为包涵体的复性研究以及磷脂酶A2结构和功能的研究打下良好的基础。

  9. Effect of improving glycemic control in patients with type 2 diabetes mellitus on low-density lipoprotein size, electronegative low-density lipoprotein and lipoprotein-associated phospholipase A2 distribution.

    Science.gov (United States)

    Sánchez-Quesada, José L; Vinagre, Irene; de Juan-Franco, Elena; Sánchez-Hernández, Juan; Blanco-Vaca, Francisco; Ordóñez-Llanos, Jordi; Pérez, Antonio

    2012-07-01

    The aim of this study was to determine the effect of intensified hypoglycemic therapy in patients with type 2 diabetes mellitus on the distribution of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity between high-density lipoprotein and low-density lipoprotein (LDL) and its relation with the lipid profile and other qualitative properties of LDL. Forty-two patients with type 2 diabetes on the basis of poor glycemic control and normal or near normal LDL cholesterol were recruited. Lifestyle counseling and pharmacologic hypoglycemic therapy were intensified to improve glycemic control, but lipid-lowering therapy was unchanged. At 4 ± 2 months, glycosylated hemoglobin had decreased by a mean of 2.1%, but the only effect on the lipid profile were statistically significant decreases in nonesterified fatty acids and apolipoprotein B concentration. LDL size increased and the proportion of electronegative LDL decreased significantly. In parallel, total Lp-PLA2 activity decreased significantly, promoting a redistribution of Lp-PLA2 activity toward a higher proportion in high-density lipoprotein. Improvements in glycemic control led to more marked changes in Lp-PLA2 activity and distribution in patients with diabetes who had not received previous lipid-lowering therapy. In conclusion, optimizing glycemic control in patients with type 2 diabetes promotes atheroprotective changes, including larger LDL size, decreased electronegative LDL, and a higher proportion of Lp-PLA2 activity in high-density lipoprotein. PMID:22481012

  10. An Asp49 Phospholipase A2 from Snake Venom Induces Cyclooxygenase-2 Expression and Prostaglandin E2 Production via Activation of NF-κB, p38MAPK, and PKC in Macrophages

    Directory of Open Access Journals (Sweden)

    Vanessa Moreira

    2014-01-01

    Full Text Available Phospholipases A2 (PLA2 are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PGE2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2. Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.

  11. Phospholipase A2 - nexus of aging, oxidative stress, neuronal excitability and functional decline of the aging nervous system? Insights from a snail model system of neuronal aging and age-associated memory impairment.

    Directory of Open Access Journals (Sweden)

    Petra Maria Hermann

    2014-12-01

    Full Text Available TThe aging brain can undergo a range of changes varying from subtle structural and physiological changes causing only minor functional decline under healthy normal aging conditions, to severe cognitive or neurological impairment associated with extensive loss of neurons and circuits due to age-associated neurodegenerative disease conditions. Understanding how biological aging processes affect the brain and how they contribute to the onset and progress of age-associated neurodegenerative diseases is a core research goal in contemporary neuroscience. This review focuses on the idea that changes in intrinsic neuronal electrical excitability associated with (peroxidation of membrane lipids and activation of phospholipase A2 (PLA2 enzymes are an important mechanism of learning and memory failure under normal aging conditions. Specifically, in the context of this special issue on the Biology of cognitive aging we (1 portray the opportunities offered by the identifiable neurons and behaviorally characterized neural circuits of the freshwater snail Lymnaea stagnalis in neuronal aging research and (2 recapitulate recent insights indicating a key role of lipid peroxidation-induced PLA2 as instruments of aging, oxidative stress and inflammation in age-associated neuronal and memory impairment in this model system. The findings are discussed in view of accumulating evidence suggesting involvement of analogous mechanisms in the etiology of age-associated dysfunction and disease of the human and mammalian brain.

  12. 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

    Directory of Open Access Journals (Sweden)

    Mónica Acevedo

    2015-01-01

    Full Text Available 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.

  13. Pre-transplant phospholipase A2 receptor autoantibody concentration is associated with clinically significant recurrence of membranous nephropathy post-kidney transplantation.

    Science.gov (United States)

    Gupta, Gaurav; Fattah, Hasan; Ayalon, Rivka; Kidd, Jason; Gehr, Todd; Quintana, Luis F; Kimball, Pamela; Sadruddin, Salima; Massey, H Davis; Kumar, Dhiren; King, Anne L; Beck, Laurence H

    2016-04-01

    Previous studies that have assessed the association of pre-transplant antiphospholipase A2 receptor autoantibody (PLA2R-Ab) concentration with a recurrence of membranous nephropathy (rMN) post-kidney transplant have yielded variable results. We tested 16 consecutive transplant patients with a history of iMN for pre-transplant PLA2R-Ab. Enzyme-linked immunosorbent assay titers (Euroimmun, NJ, USA) >14 RU/mL were considered positive. A receiver operating characteristic (ROC) analysis was performed after combining data from Quintana et al. (n = 21; Transplantation February 2015) to determine a PLA2R-Ab concentration which could predict rMN. Six of 16 (37%) patients had biopsy-proven rMN at a median of 3.2 yr post-transplant. Of these, five of six (83%) had a positive PLA2R-Ab pre-transplant with a median of 82 RU/mL (range = 31-1500). The only patient who had rMN with negative PLA2R-Ab was later diagnosed with B-cell lymphoma. One hundred percent (n = 10) of patients with no evidence of rMN (median follow-up = five yr) had negative pre-transplant PLA2R-Ab. In a combined ROC analysis (n = 37), a pre-transplant PLA2R-Ab > 29 RU/mL predicted rMN with a sensitivity of 85% and a specificity of 92%. Pre-transplant PLA2R-Ab could be a useful tool for the prediction of rMN. Patients with rMN in the absence of PLA2R-Ab should be screened for occult malignancy and/or alternate antigens.

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

    Science.gov (United States)

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

    2016-08-01

    The deposition of IgG4 of antibodies against phospholipase A2 receptor (anti-PLA2R) is predominating in the kidneys of patients with idiopathic membranous nephropathy, while its predictive value has not been determined. It was a retrospective study, and 438 patients were included. Serum samples of two time points [before intervention (baseline) and after 1.5-year treatment (endpoint)] were detected for total and IgG4 anti-PLA2R. IgG4 PLA2R was a useful predictor for achieving CR, but there was a high heterogeneity; (2) there was significant correlation between the baseline and decrease in IgG4 subclass and the achievement of CR; (3) bi-negativity of IgG4 has a high accuracy of predicting CR compared with total antibodies; (4) in patients of bi-positivity, those achieving CR showed lower MASP-1/2, MBL, C3a, C5a, FB, Ba and Bb than patients failing to achieve CR; (5) the titers of endpoint and decrease in Ba and Bb were associated with improvement of 24 h-UP in those of bi-positivity; and (6) the decrease in Ba was a significant factor for achieving CR in those of bi-positivity. Continuous IgG4 negativity was a useful tool to predict the achievement of CR; however, in patients of continuous IgG4 positivity, those with lower activation of lectin and alternative pathways would still more probably achieve CR.

  15. Expression pattern of three-finger toxin and phospholipase A2 genes in the venom glands of two sea snakes, Lapemis curtus and Acalyptophis peronii: comparison of evolution of these toxins in land snakes, sea kraits and sea snakes

    Directory of Open Access Journals (Sweden)

    Fry Bryan G

    2007-09-01

    Full Text Available Abstract Background Snake venom composition varies widely both among closely related species and within the same species, based on ecological variables. In terrestrial snakes, such variation has been proposed to be due to snakes' diet. Land snakes target various prey species including insects (arthropods, lizards (reptiles, frogs and toads (amphibians, birds (aves, and rodents (mammals, whereas sea snakes target a single vertebrate class (fishes and often specialize on specific types of fish. It is therefore interesting to examine the evolution of toxins in sea snake venoms compared to that of land snakes. Results Here we describe the expression of toxin genes in the venom glands of two sea snakes, Lapemis curtus (Spine-bellied Sea Snake and Acalyptophis peronii (Horned Sea Snake, two members of a large adaptive radiation which occupy very different ecological niches. We constructed cDNA libraries from their venom glands and sequenced 214 and 192 clones, respectively. Our data show that despite their explosive evolutionary radiation, there is very little variability in the three-finger toxin (3FTx as well as the phospholipase A2 (PLA2 enzymes, the two main constituents of Lapemis curtus and Acalyptophis peronii venom. To understand the evolutionary trends among land snakes, sea snakes and sea kraits, pairwise genetic distances (intraspecific and interspecific of 3FTx and PLA2 sequences were calculated. Results show that these proteins appear to be highly conserved in sea snakes in contrast to land snakes or sea kraits, despite their extremely divergent and adaptive ecological radiation. Conclusion Based on these results, we suggest that streamlining in habitat and diet in sea snakes has possibly kept their toxin genes conserved, suggesting the idea that prey composition and diet breadth may contribute to the diversity and evolution of venom components.

  16. Humanized-Single Domain Antibodies (VH/VHH that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Wanpen Chaicumpa

    2012-07-01

    Full Text Available Naja kaouthia (monocled cobra venom contains many isoforms of secreted phospholipase A2 (sPLA2. The PLA2 exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/VHH that bound specifically to the P3 and P5 were selected from a humanized-camel VH/VHH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3 produced humanized-VHH, while another clone (P3-7 produced humanized-VH. At the optimal venom:antibody ratio, the VH/VHH purified from the E. coli homogenates neutralized PLA2 enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/VHH covered the areas around the PLA2 catalytic groove and inserted their Complementarity Determining Regions (CDRs into the enzymatic cleft. It is envisaged that the VH/VHH would ameliorate/abrogate the principal toxicity of the venom PLA2 (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis, if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

  17. Molecular modeling of Gly80 and Ser80 variants of human group IID phospholipase A2 and their receptor complexes: potential basis for weight loss in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Khan, Mohd Imran; Gupta, Ashish Kumar; Kumar, Domada Ratna; Kumar, Manoj; Ethayathulla, Abdul Samarth; Hariprasad, Gururao

    2016-09-01

    Weight loss is a well known systemic manifestation of chronic obstructive pulmonary disease (COPD). A Gly80Ser mutation on human group IID secretory phospholipase A2 (sPLA2) enhances expression of the cytokines that are responsible for weight loss. In this study, we seek to establish a structural correlation of wild type sPLA2 and the Gly80Ser mutation with function. sPLA2 with glycine and serine at the 80th positions and the M-type receptor were modelled. The enzymes were docked to the receptor and molecular dynamics was carried out to 70 ns. Structural analysis revealed the enzymes to comprise three helices (H1-H3), two short helices (SH1 and SH2), and five loops including a calcium binding loop (L1-L5), and to be stabilized by seven disulfide bonds. The overall backbone folds of the two models are very similar, with main chain RMSD of less than 1 Å. The active site within the substrate binding channel shows a catalytic triad of water-His67-Asp112, showing a hydrogen bonded network. Major structural differences between wild type and mutant enzymes were observed locally at the site of the mutation and in their global conformations. These differences include: (1) loop-L3 between H2 and H3, which bears residue Gly80 in the wild type, is in a closed conformation with respect to the channel opening, while in the mutant enzyme it adopts a relatively open conformation; (2) the mutant enzyme is less compact and has higher solvent accessible surface area; and (3) interfacial binding contact surface area is greater, and the quality of interactions with the receptor is better in the mutant enzyme as compared to the wild type. Therefore, the structural differences delineated in this study are potential biophysical factors that could determine the increased potency of the mutant enzyme with macrophage receptor for cytokine secreting function, resulting in exacerbation of cachexia in COPD. PMID:27585677

  18. Single and Multiple Dose Pharmacokinetics, Pharmacodynamics and Safety of the Novel Lipoprotein-Associated Phospholipase A2 Enzyme Inhibitor Darapladib in Healthy Chinese Subjects: An Open Label Phase-1 Clinical Trial.

    Directory of Open Access Journals (Sweden)

    Chaoying Hu

    Full Text Available Darapladib is a lipoprotein-associated phospholipase A2 (Lp-PLA2 inhibitor. This study evaluated the pharmacokinetics, pharmacodynamics and safety of darapladib in healthy Chinese subjects.Twenty-four subjects received darapladib 160 mg orally, approximately 1 hour after a standard breakfast, as a single dose and once daily for 28 days. Non-compartmental methods were used to determine the single and multiple dose pharmacokinetics of darapladib and its metabolite SB-553253. Repeat dose Lp-PLA2 activity and safety were evaluated.Systemic exposure (AUC(0-T, Cmax geometric mean (CVb% of darapladib was higher after multiple-dosing (519 ng.h/mL (33.3%, 34.4 ng/mL (49.9% compared to single-dose administration (153 ng.h/mL (69.0%, 17.9 ng/mL (55.2%. The steady-state accumulation ratio was less than unity (Rs = 0.80, indicating time-dependent pharmacokinetics of darapladib. Darapladib steady-state was reached by Day 14 of once daily dosing. Systemic exposure to SB-553253 was lower than darapladib with median (SB-553253: darapladib ratios for AUC(0-τ of 0.0786 for single dose and 0.0532 for multiple dose administration. On Day 28, pre-dose and maximum inhibition of Lp-PLA2 activity was approximately 70% and 75% relative to the baseline value, respectively and was dependent of darapladib concentration. The most common adverse events (≥ 21% subjects were abnormal faeces, abnormal urine odour, diarrhoea and nasopharyngitis.Darapladib 160 mg single and repeat doses were profiled in healthy Chinese subjects. Single dose systemic exposure to darapladib in healthy Chinese subjects was consistent with that observed previously in Western subjects whereas steady-state systemic exposure was approximately 65% higher in Chinese than Western subjects. The Lp-PLA2 activity and adverse event profile were similar in healthy Chinese and previous reports in Western subjects. Ethnic-specific dose adjustment of darapladib is not considered necessary for the Chinese

  19. Molecular modeling of Gly80 and Ser80 variants of human group IID phospholipase A2 and their receptor complexes: potential basis for weight loss in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Khan, Mohd Imran; Gupta, Ashish Kumar; Kumar, Domada Ratna; Kumar, Manoj; Ethayathulla, Abdul Samarth; Hariprasad, Gururao

    2016-09-01

    Weight loss is a well known systemic manifestation of chronic obstructive pulmonary disease (COPD). A Gly80Ser mutation on human group IID secretory phospholipase A2 (sPLA2) enhances expression of the cytokines that are responsible for weight loss. In this study, we seek to establish a structural correlation of wild type sPLA2 and the Gly80Ser mutation with function. sPLA2 with glycine and serine at the 80th positions and the M-type receptor were modelled. The enzymes were docked to the receptor and molecular dynamics was carried out to 70 ns. Structural analysis revealed the enzymes to comprise three helices (H1-H3), two short helices (SH1 and SH2), and five loops including a calcium binding loop (L1-L5), and to be stabilized by seven disulfide bonds. The overall backbone folds of the two models are very similar, with main chain RMSD of less than 1 Å. The active site within the substrate binding channel shows a catalytic triad of water-His67-Asp112, showing a hydrogen bonded network. Major structural differences between wild type and mutant enzymes were observed locally at the site of the mutation and in their global conformations. These differences include: (1) loop-L3 between H2 and H3, which bears residue Gly80 in the wild type, is in a closed conformation with respect to the channel opening, while in the mutant enzyme it adopts a relatively open conformation; (2) the mutant enzyme is less compact and has higher solvent accessible surface area; and (3) interfacial binding contact surface area is greater, and the quality of interactions with the receptor is better in the mutant enzyme as compared to the wild type. Therefore, the structural differences delineated in this study are potential biophysical factors that could determine the increased potency of the mutant enzyme with macrophage receptor for cytokine secreting function, resulting in exacerbation of cachexia in COPD.

  20. A Novel Genetic Approach to Investigate the Role of Plasma Secretory Phospholipase A2 (sPLA2)-V Isoenzyme in Coronary Heart Disease: A Modified Mendelian Randomization Analysis Using PLA2G5 Expression Levels

    Science.gov (United States)

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

    2014-01-01

    Background Secretory phospholipase A2 (sPLA2) enzymes are considered to play a role in atherosclerosis. sPLA2 activity encompasses several sPLA2 isoenzymes, including sPLA2-V. While observational studies show strong association between elevated sPLA2 activity and CHD, no assay to measure sPLA2-V levels exists and the only evidence linking the sPLA2-V isoform to atherosclerosis progression comes from animal studies. In the absence of an assay that directly quantifies sPLA2-V levels, we used PLA2G5 mRNA levels in a novel, modified Mendelian randomization approach to investigate the hypothesized causal role of sPLA2-V in coronary heart disease (CHD) pathogenesis. Methods and Results Using data from the Advanced Study of Aortic Pathology, we identified the single nucleotide polymorphism (SNP) in PLA2G5 showing strongest association with PLA2G5 mRNA expression levels, as a proxy for sPLA2-V levels. We tested the association of this SNP with sPLA2 activity and CHD events in four prospective and 14 case-control studies with 27,230 events and 70,500 controls. rs525380C>A showed the strongest association with PLA2G5 mRNA expression (P=5.1×10−6). There was no association of rs525380C>A with plasma sPLA2 activity (difference in geometric mean of sPLA2 activity per rs525380 A-allele 0.4% (95%CI: −0.9%, 1.6%), P=0.56). In meta-analyses, the odds ratio for CHD per A allele was 1.02 (95% CI: 0.99, 1.04; P=0.20). Conclusions This novel approach for SNP selection for this modified Mendelian randomization analysis showed no association between rs525380 (the lead SNP for PLA2G5 expression, a surrogate for sPLA2-V levels) and CHD events. The evidence does not support a causal role for sPLA2-V in CHD. PMID:24563418

  1. Mammalian phospholipase C.

    Science.gov (United States)

    Kadamur, Ganesh; Ross, Elliott M

    2013-01-01

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

  2. Spreading depression induces expression of calcium-independent protein kinase C subspecies in ischaemia-sensitive cortical layers: regulation by N-methyl-D-aspartate receptors and glucocorticoids.

    Science.gov (United States)

    Koponen, S; Keinänen, R; Roivainen, R; Hirvonen, T; Närhi, M; Chan, P H; Koistinaho, J

    1999-01-01

    Spreading depression is a wave of sustained depolarization challenging the energy metabolism of the cells without causing irreversible damage. In the ischaemic brain, sreading depression-like depolarization contributes to the evolution of ischaemia to infarction. The depolarization is propagated by activation of N-methyl-D-aspartate receptors, but changes in signal transduction downstream of the receptors are not known. Because protein phosphorylation is a general mechanism whereby most cellular processes are regulated, and inhibition of N-methyl-D-aspartate receptors or protein kinase C is neuroprotective, the expression of protein kinase C subspecies in spreading depression was examined. Cortical treatment with KCl induced an upregulation of protein kinase Cdelta and zeta messenger RNA at 4 and 8 h, whereas protein kinase Calpha, beta, gamma and epsilon did not show significant changes. The gene induction was the strongest in layers 2 and 3, and was followed by an increased number of protein kinase Cdelta-immunoreactive neurons. Protein kinase Cdelta and zeta inductions were inhibited by pretreatment with an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate, which also blocked spreading depression propagation, and with dexamethasone, which acted without blocking the propagation. Quinacrine, a phospholipase A2 inhibitor, reduced only protein kinase C5 induction. In addition, N(G)(-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, did not influence protein kinase Cdelta or zeta induction, whereas 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist, and the cyclo-oxygenase inhibitors indomethacin and diclophenac tended to increase gene expression. The data show that cortical spreading depression induces Ca2(+)-independent protein kinase C subspecies delta and zeta, but not Ca(2+)-dependent subspecies, through activation of N-methyl-D-aspartate receptors and

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

    Science.gov (United States)

    Eggers, Arnold E

    2012-12-01

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

  4. 分泌型磷脂酶A2与冠心病的关系%Correlation of circulating levels of phospholipase A2 with coronary artery syndrome

    Institute of Scientific and Technical Information of China (English)

    傅慎文; 于路; 傅国胜

    2010-01-01

    目的 探讨分泌型磷脂酶A2(sPLA2)与冠心病的关系.方法 选取心内科住院患者262例,正常者89例(对照组),稳定型心绞痛患者63例(SAP组),急性冠脉综合征患者110例(ACS组).经桡动脉途径行冠状动脉造影计算各组研究对象的冠状动脉病变积分(CAS),并采用ELISA法检测其血清sPLA2,比较分析组间差异以及sPLA2与CAS、冠心病相关因素等的关系.结果 ACS组sPLA2均显著高于SAP组及对照组(均P0.05). Cox回归分析结果示,sPLA2是冠心病的独立危险因素(P0.05).结论 sPLA2可以作为冠状动脉粥样斑块不稳定的一个预测指标.其水平升高提示病情较重、预后不良.

  5. Recurrent membranous nephropathy with phospholipase A2 receptor antibody%肾移植术后抗磷脂酶A2受体抗体阳性的复发性膜性肾病

    Institute of Scientific and Technical Information of China (English)

    程东瑞; 倪雪峰; 陈惠萍

    2015-01-01

    40岁男性患者,既往临床表现肾病综合征,自体肾活检诊断膜性肾病(MN),抗磷脂酶A2受体(PLA2R)抗体滴度阳性,起病后7年进入慢性肾衰竭行血液透析(HD)治疗,HD治疗5个月后接受亲属活体肾移植,术前抗PLA2R抗体阳性.移植术后6个月尿蛋白阳性,抗PLA2R抗体滴度升高,移植肾活检提示MN复发.以抗CD20单克隆抗体(美罗华)375 mg/m2治疗后,患者蛋白尿转阴,抗PLA2R抗体滴度下降.

  6. Correlation between indirect immunofluorescence and enzyme linked immunosorbent assay in detecting anti-phospholipase A2 receptor antibody%IIF与ELISA在抗磷脂酶A2受体抗体检测中相关性

    Institute of Scientific and Technical Information of China (English)

    符克英; 蔡俊宏; 符生苗; 韩叶光; 王茹; 张培

    2015-01-01

    目的 分析间接免疫荧光法(IIF)与酶联免疫吸附法(ELISA)对抗磷脂酶A2受体抗体(anti-PLA2R)检测的相关性.方法 应用IIF与ELISA检测特发性膜性肾病患者血液中的anti-PLA2R,并分析两种检测方法的相关性和临床应用的可行性.结果 IIF法检测血液中的anti-PLA2R敏感性为76.7%,特异性为98.3%;ELISA法检测血液中的an-ti-PLA2R,敏感性为73.3%,特异性为95.0%,两种方法敏感性和特异性比较差异无统计学意义(D0.05);两种方法相关性分析结果发现,其阳性符合率为93.6%,阴性符合率为95.9%,总符合率为95.0%,Kappa一致性检验的Kappa为0.895.结论 IIF法与ELISA法检测血液中anti-PLA2R的结果相关性高,均可成为体外诊断特发性膜性肾病的特异性指标,两者有互补性,能为不同条件的实验室提供选择.

  7. Messenger molecules of the phospholipase signaling system have dual effects on vascular smooth muscle contraction.

    Science.gov (United States)

    Vidulescu, Cristina; Mironneau, J.; Mironneau, Chantal; Popescu, L. M.

    2000-01-01

    Background and methods. In order to investigate the role of phospholipases and their immediately derived messengers in agonist-induced contraction of portal vein smooth muscle, we used the addition in the organ bath of exogenous molecules such as: phospholipases C, A(2), and D, diacylglycerol, arachidonic acid, phosphatidic acid, choline. We also used substances modulating activity of downstream molecules like protein kinase C, phosphatidic acid phosphohydrolase, or cyclooxygenase. Results. a) Exogenous phospholipases C or A(2), respectively, induced small agonist-like contractions, while exogenous phospholipase D did not. Moreover, phospholipase D inhibited spontaneous contractions. However, when added during noradrenaline-induced plateau, phospholipase D shortly potentiated it. b) The protein kinase C activator, phorbol dibutyrate potentiated both the exogenous phospholipase C-induced contraction and the noradrenaline-induced plateau, while the protein kinase C inhibitor 1-(-5-isoquinolinesulfonyl)-2-methyl-piperazine relaxed the plateau. c) When added before noradrenaline, indomethacin inhibited both phasic and tonic contractions, but when added during the tonic contraction shortly potentiated it. Arachidonic acid strongly potentiated both spontaneous and noradrenaline-induced contractions, irrespective of the moment of its addition. d) In contrast, phosphatidic acid inhibited spontaneous contractile activity, nevertheless it was occasionally capable of inducing small contractions, and when repetitively added during the agonist-induced tonic contraction, produced short potentiations of the plateau. Pretreatment with propranolol inhibited noradrenaline-induced contractions and further addition of phosphatidic acid augmented this inhibition. Choline augmented the duration and amplitude of noradrenaline-induced tonic contraction and final contractile oscillations. Conclusions. These data suggest that messengers produced by phospholipase C and phospholipase A(2

  8. M 型磷脂酶 A2受体抗体及免疫球蛋白 IgG 亚型在乙型肝炎病毒相关膜性肾病中的检测∗%The clinical value of plasma M type phospholipase A2 receptor antibody and IgG subtypes deposition in diagnosis of hepatitis B virus-associated membranous nephropathy

    Institute of Scientific and Technical Information of China (English)

    李隽; 卓莉; 高红梅; 芦建华; 邹古明; 李文歌

    2015-01-01

    Objective To investigate the different expressions of plasma M type phospholipase A2 receptor antibody and IgG subtypes deposition of kidney tissues in idiopathic membranous nephropathy and hepatitis B virus-associated membranous nephropa-thy,and to evaluate the significance of plasma M type phospholipase A2 receptor antibody and IgG subtypes in diagnosis of hepatitis B virus-associated membranous nephropathy.Methods Plasma samples were obtained from patients with idiopathic membranous nephropathy,hepatitis B virus-associated membranous nephropathy and minimal change disease,respectively,before immunosup-pressive therapy.Concentration of plasma M type phospholipase A2 receptor antibody was detected by sandwich ELISA and concen-tration of IgG subtypes were measured by immunofluorescence.Results Concentration of plasma M type phospholipase A2 receptor antibody was (15.4±7.2)μg/mL in idiopathic membranous nephropathy group,higher than that in the hepatitis B virus-associated membranous nephropathy group (10.3±5.7)μg/mL (P <0.01),between idiopathic membranous nephropathy group and hepatitis B virus-associated membranous nephropathy group.There was no distinct difference of IgG subtypes deposition in glomerlar capil-lary wall.Conclusion There is obvious clinical significance of concentration of plasma M type phospholipase A2 receptor antibody in differential diagnosis of idiopathic membranous nephropathy and hepatitis B virus-associated membranous nephropathy,while no distinct significance of IgG subtypes deposition.%目的:通过比较原发性膜性肾病(IMN)和乙型肝炎病毒相关膜性肾病(HBV-MN)患者血清中 M 型磷脂酶 A2受体抗体(PLA2R)水平及肾活检组织中 IgG 亚型沉积情况,探讨 PLA2R 抗体和 IgG 亚型对 HBV-MN 诊断的意义。方法应用ELISA 法检测了10例 IMN 患者和25例 HBV-MN 患者血清中浓度,以同期7例微小病变肾病(MCD)患者血清检测作为阴性对照。通过免疫荧

  9. Reminiscence of phospholipase B in Penicillium notatum.

    Science.gov (United States)

    Saito, Kunihiko

    2014-01-01

    Since the phospholipase B (PLB) was reported as a deacylase of both lecithin and lysolecithin yielding fatty acids and glycerophosphocholine (GPC), there was a question as to whether it is a single enzyme or a mixture of a phospholipase A2 (PLA2) and a lysophospholipase (LPL). We purified the PLB in Penicillium notatum and showed that it catalyzed deacylation of sn-1 and sn-2 fatty acids of 1,2-diacylphospholipids and also sn-1 or sn-2 fatty acids of 1- or 2-monoacylphospholipids (lysophospholipids). Further, it also has a monoacyllipase activity. The purified PLB is a glycoprotein with m.w. of 91,300. The sugar moiety is M9 only and the protein moiety consists of 603 amino acids. PLB, different from PLA2, shows other enzymatic activities, such as transacylase, lipase and acylesterase. PLB activity is influenced by various substances, e.g. detergents, deoxycholate, diethylether, Fe(3+), and endogenous protease. Therefore, PLB might have broader roles than PLA2 in vivo. The database shows an extensive sequence similarity between P. notatum PLB and fungal PLB, cPLA2 and patatin, suggesting a homologous relationship. The catalytic triad of cPLA2, Ser, Asp and Arg, is also present in P. notatum PLB. Other related PLBs, PLB/Lipases are discussed. PMID:25391318

  10. Serum anti-phospholipase A2 receptor antibody detection in the diagnosis of idiopathic membranous nephropathy%血清抗磷脂酶A2受体抗体在特发性膜性肾病诊断中意义

    Institute of Scientific and Technical Information of China (English)

    刘莎莎; 张丽; 李素华; 米娜瓦尔·玉努斯; 刘健; 桑晓红

    2016-01-01

    目的 探讨血清抗磷脂酶A2受体(anti-phospholipase A2 receptor,PLA2R)抗体诊断特发性膜性肾病(idiopathic membranous nephropathy,IMN)的价值.方法 76例肾脏病患者,肾穿刺活检组织病理证实IMN患者31例,非IMN患者45例.采用间接免疫荧光法检测其抗PLA2R抗体水平;以肾穿刺活检组织病理检查结果为金标准,计算抗PLA2R抗体诊断IMN灵敏度、特异度、阳性预测值、阴性预测值、阳性似然比、阴性似然比、Kappa系数.结果 31例IMN患者中,抗PLA2R抗体阳性28例、阴性3例,45例非IMN患者抗PLA2R抗体均为阴性;与肾穿刺活检组织病理进行对照,抗PLA2R抗体诊断IMN的灵敏度为90.32%,特异度为100.00%,假阴性率为9.68%,假阳性率为0,阳性预测值为100.00%,阴性预测值为93.75%,阳性似然比为0,阴性似然比为9.68%,Kappa系数为0.917.结论 抗PLA2R抗体作为IMN的特异性标志物,可用于IMN的诊断和鉴别诊断.

  11. M型磷脂酶A2受体基因单核苷酸多态性与膜性肾病的相关性%Association of single nucleotide polymorphism in M-type phospholipase A2 receptor gene with membranous nephropathy

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    周广宇; 刘锋; 张文龙

    2013-01-01

    目的 探讨M型磷脂酶A2受体(M-type phospholipase A2 receptor,PLA2R)基因单核苷酸多态性与膜性肾病(membranous nephropathy,MN)的相关性.方法 选取145例特发性膜性肾病(idiopathic membranous nephropathy,IMN)患者、53例继发性MN患者和232名正常对照.用聚合酶链反应-限制性片段长度多态性分析PLA2R基因rs35771982位点的单核苷酸多态;用Western印迹法检测IMN患者血清抗PLA2R抗体.结果 三组人群PLA2R基因rs35771982位点的基因型和等位基因频率差异均有统计学意义(P=0.004; P<0.001);IMN组CC基因型和C等位基因的频率显著高于正常对照组(P=0.002; P<0.001)和继发性MN组(P=0.011; P=0.001).CC基因型是IMN的风险因素(OR=8.927,95%CI:2.107-37.821,P=0.003).CC基因型患者血清Alb水平明显低于CG和GG基因型患者(P<0.001),24h尿蛋白量和抗PLA2R抗体阳性率明显高于其它基因型患者(P<0.001,P=0.010).结论 中国汉族人群PLA2R基因rs35771982位点CC基因型和C等位基因是IMN的易感因素,CC基因型与病情和血清抗PLA2R抗体相关,表明IMN患者抗PLA2R自身抗体的产生可能与rs35771982位点的基因突变相关.%Objective To assess the association between single nucleotide polymorphism in M-type phospholipase A2 receptor (PLA2R)gene and membranous nephropathy (MN) in a Chinese Han population.Methods A total of 430 non-related Chinese Hans were enrolled,which included 145 patients with idiopathic membranous nephropathy (IMN),53 patients with secondary MN and 232 normal controls (NC).The polymorphism of rs35771982 in PLA2R gene was determined with polymerase chain reaction-restriction fragment length polymorphism assay.Serum anti-PLA2R antibodies were detected by Western blotting.Results The genotypic and allelic frequencies for rs35771982 was significantly different among the three groups (P=0.004; P<0.001).CC genotype and C allele were significantly more common in IMN group compared with NC group (P=0

  12. Effect of different cyclic stretching strengths on expressions of phospholipase A2 and cyclooxygenase in human tenocytes in vitro%不同牵伸强度对体外人肌腱细胞磷脂酶A2/环氧合酶表达的影响

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    谢美明; 唐康来; 邓银栓; 李辉; 周建波; 谭晓康; 陈磊; 许建中

    2011-01-01

    Objective To investigate the effect of different cyclic strengths on expressions of phospholipase A2 (PLA2) and cyclooxygenase (COX) in human tenocytes. Methods Human tenocytes were uniaxially stretched with different stretching intensity (4%, 8% and 12%) under 0.5 Hz for four hours.Non-stretched tenocytes were applied to the control group.The expressions of cytosolic PLA2(cPLA2), COX1 and COX2 were measured by Western blot and RT-PCR.The secretion of secretory PLA2 (sPLA2) was measured by ELISA. Results The mRNA expressions of cPLA2, COX1 and COX2 in control group, 4%, 8% and 12% stretch groups showed an increase trend.But protein expressions of cPLA2 and COX1 in 4% stretch group were increased insignificantly compared with the control group (P > 0.05).Protein expressions of cPLA2 and COX1 in 8% and 12% stretch groups were increased more significantly compared with the control group (P < 0.01).The COX2 expression in 4%,8% and 12% stretch groups showed statistical difference compared with that in the control group (P <0.01) and the difference increased with stretch intensity.There was no different expression of sPLA2 between 4% stretch group and control group (P = 0.260).However, expression of sPLA2 was increased markedly in 8% and 12% stretch groups (P < 0.01). ConclusionsThe expressions of human tendnocytes PLA2, COX1 and COX2 in vitro are positively correlated with stretch intensity.PLA2/COX system may be a new molecule target in clinical treatment of tendinopathy.%目的 探讨不同循环牵伸强度对体外人肌腱细胞磷脂酶A2(phospholipase A2,PLA2)和环氧合酶(cyclooxygenase,COX)表达的影响。 方法 取4~6代人肌腱细胞在0.5 Hz、4h条件下施加不同牵伸强度(4%、8%、12%),未牵伸组设为对照组。静置4h后,用RT-PCR、Western blot检测胞浆性PLA2(cytosolic PLA.2,cPLA2)、COX1、COX2的表达,ELISA测定分泌性PLA2 (secretory PLA2,sPLA2)的表达。 结果

  13. Activation of phospholipase A2, changes of free ca2+ concertration and protection of nimodipine in rats with acute cerebral ischemia injury%急性缺血性脑损伤后脑组织磷脂酶A2活性和脑细胞游离钙离子浓度变化及尼莫地平的保护作用

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    王兴勇; 李晓文; 卢仲毅; 匡凤梧; 许峰

    2005-01-01

    2 mRNA表达水平很低.缺血后120 min后脑组织中出现Ⅱ类A型分泌型磷脂酶A2 mRNA表达,胞浆型磷脂酶A2mRNA表达水平明显增强.尼莫地平干预组与缺血组比较,Ⅱ类A型分泌型磷脂酶A2 mRNA表达水平无明显降低,胞浆型磷脂酶A2mRNA表达水平有所降低. 结论:尼莫地平可降低缺血后脑细胞游离Ca2+浓度,脑组织中磷脂酶A2的活性和脑含水量,但不能明显抑制脑缺血后Ⅱ类A型分泌型磷脂酶A2mRNA及胞浆型磷脂酶A2mRNA的表达.%BACKGROUND: Activated by Ca2+, phospholipase A2 will aggravate the influx of Ca2+ or the release of intracellular Ca2+, and then forms a vicious circle, which results in a continuous increase in free calcium level and leads to server injury in neural cells.OBJECTIVE: To discuss the protective effects of nimodipine on acute ischemic brain injury caused by activation of phospholipase A2.DESIGN: A completely randomized controlled trial.SETTING: Intensive Care Unit (ICU) of Children's Hospital, Chongqing Medical University.MATERIALS: From January 2001 to October 2003, it was completed at the ICU of Children' s Hospital, Chongqing Medical University. Thirty male rats were selected and divided into sham operation group, ischemia group and nimodipine treated group randomly, with 10 rats in each group.METHODS: In sham operation group, the right common carotid artery was identified by blunt dissection without ligation under anesthesia in rats. In ischemia group, at 30 minutes before cerebral ischemia, 2 mL saline was injected intraperitoneally. In nimodipine treated group, at 30 minutes before cerebral ischemia, 0.2 g/L nimodipine (2 mg/kg) was injected intraperitoneally. In all the three groups, the duration between ischemia and decollation was 120 minutes. Rats were decollated under anesthesia and their brains were taken out to assess the activity of phospholipase A2, the free calcium level in brain cells, the brain water content and the changes in mRNA levels

  14. Effect of Probucol Combined with Atorvastatin on Plasma Level of Lipoprotein-Associated phospholipase A2 in Patients with Acute Coronary Syndrome%普罗布考联合阿托伐他汀对急性冠脉综合征血脂蛋白相关脂酶A2的影响

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    杨丽; 刘寅; 刘婷; 陈倩

    2012-01-01

    Objective: To evaluate the effect of probucol and atorvastatin combined medication on blood levels of liquids and Lipoprotein-associated phospholipase A2 (Lp-PLA2) in patients with acute coronary syndrome(ACS). Methods: A total of 94 patients with ACS diagnosed by coronary angiography (CAG) were randomly divided into two groups: single medication group (re=48) and combined medication group (n=46). Patients were given atorvastatin 20 mg/d or atorvastatin 20 mg/d with probucol 500 mg/d in two groups of patients. The plasma levels of Lp-PLA2, total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C) were detected and compared before and 6-8 weeks after the medication two groups of patients. Results: There were no significant differences in levels of TC,TG,LDL-C,HDL-C and Lp_PLA2 before treatment between two groups of patients^ > 0.05). The values of TC, TG, LDL-C and Lp-PLA2 were significantly decreased after the treatment in patients of both two groups (P 0.05). Conclusion: The values of TC, LDL-C and Lp-PLA2 can be decreased by single atorvastatin medication or probucol and atorvastatin combined medication, but there was more significant effect by combined medication in ACS patients. The combined treatment plays an important role in stabilizing the plaques and anti-atherosclerosis in ACS patients.%目的:评价普罗布考和阿托伐他汀联合应用对急性冠脉综合征(ACS)患者血脂及脂蛋白相关磷脂酶A2(Lp-PLA2)的影响.方法:将94例经冠脉造影证实的ACS患者随机分为2组:单药组48例,予以阿托伐他汀(20 mg/d)治疗;联合组46例,予以阿托伐他汀(20 mg/d)和普罗布考(500 mg/d)联合治疗.分别于治疗前和治疗后6~8周检测血Lp-PLA2和血总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平并进行比较分析.结果:2组治疗前TC、TG、LDL-C、HDL-C、Lp-PLA2水平

  15. 特发性膜性肾病肾组织磷酯酶A2受体的检测及其临床意义%Detection and the clinical significance of phospholipase A2 receptor in idiopathic membranous nephropathy tissues

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    刘红; 骆伟丽; 龚劭敏; 丁小强

    2015-01-01

    目的 探讨特发性膜性肾病(idiopathic membranous nephropathy,IMN)患者肾组织磷酯酶A2受体(phospholipaseA2 receptor,PLA2 R)的检测情况并分析其临床意义.方法 回顾性分析101例经临床和肾脏病理确诊为IMN并随访1年以上的患者(在本单位肾活检前均未接受激素或免疫抑制剂治疗);13例临床和病理确诊为系统性红斑狼疮Ⅴ型、乙型肝炎、干燥综合征相关等继发性膜性肾病及20例非膜性肾病患者作为对照组.检测各组患者肾组织PLA2R及IgG分型的结果,并分析PLA2R阳性及阴性IMN患者治疗前后尿蛋白、肾功能的变化.结果 86.14% IMN患者肾组织PLA2R阳性,而继发性膜性肾病患者肾组织PLA2R阳性率仅为23.08%(vs.IMN组,P<0.001).83.75% IMN患者肾组织PLA2R阳性伴IgG4中度以上阳性;继发性膜性肾病患者中,无一例肾组织PLA2R阳性同时伴IgG4中度以上阳性(vs.IMN组,P<0.01).47例肾组织PLA2R阳性的IMN患者予激素或激素联合免疫抑制剂治疗,随访6个月以上,85.11%患者尿蛋白缓解,而9例肾组织PLA2R阴性的IMN患者尿蛋白缓解率只有30.0%(vs.IMN PLA2R阳性组,P<0.01).结论 IMN患者肾组织PLA2R阳性率高.肾组织PLA2R阳性伴IgG4中度以上阳性是鉴别IMN和继发性膜性肾病的重要指标.激素或/和免疫抑制剂可以更有效地降低肾组织PLA2R阳性IMN患者的蛋白尿.肾组织PLA2R阳性可能可以作为预测激素或免疫抑制剂治疗有效与否的重要指标.

  16. Significance of anti-phospholipase A2 receptor antibody in the diagnosis of idiopathic membranous nephropathy%抗磷脂酶A2受体抗体检测在特发性膜性肾病诊断中的意义

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    符克英; 蔡俊宏; 符生苗; 韩叶光; 王茹; 张培

    2015-01-01

    Objective To discuss the significance of anti-phospholipase A2 receptor antibody (anti-PLA2R) in diagnosing idiopathic membranous nephropathy. Methods Anti-PLA2R in the blood was detected by indirect im-munofluorescence in the patients with idiopathic membranous nephropathy (IMN group), and then compared with those in the patients with secondary membranous nephropathy (secondary membranous nephropathy group) and healthy blood donors (healthy control group). The relationship between anti-PLA2R and pathological diagnosis of re-nal tissue was analyzed. Results For detection of anti-PLA2R in blood by indirect immunofluorescence, the sensibility was 76.7%and the specificity was 96.7%. Only one case was positive in the secondary membranous nephropathy group, and none was positive in the healthy control group. Correlation analysis between anti-PLA2R and pathological diagno-sis showed a positive coincidence rate of 97.9%, negative coincidence rate of 80.8%, total coincidence rate of 87.5%, and the Kappa of consistency test of 0.752, P<0.01. Conclusion Anti-PLA2R in blood detected by indirect immunoflu-orescence could be used as the specific indicator for diagnosing idiopathic membranous nephropathy in vitro.%目的:探讨血液中抗磷脂酶A2受体抗体(anti-PLA2R)在特发性膜性肾病诊断中的意义。方法应用间接免疫荧光方法检测特发性膜性肾病患者血液中的anti-PLA2R,以继发性膜性肾病患者及健康献血员作为对照,分析其与肾活检组织中病理诊断的相关性。结果间接免疫荧光方法检测血液中的anti-PLA2R敏感性为76.7%、特异性为96.7%。继发性膜性肾病组仅检出1例阳性,正常组未检出阳性。血液anti-PLA2R与病理诊断的相关性分析发现其阳性符合率为97.9%,阴性符合率为80.8%,总符合率为87.5%,Kappa一致性检验,Kappa=0.752,P<0.01。结论间接免疫荧光方法检测血液中的anti-PLA2R可成为体外诊断特发性膜性肾病的特异性指标。

  17. 血浆脂蛋白磷脂酶A2水平对冠心病合并2型糖尿病患者的影响%The influence of plasma lipoprotein-associated phospholipase A2 in patients with both coronary heart disease and type 2 diabetes

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    欧阳新根

    2014-01-01

    Objective To investigate the influence of plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) in patients with both coronary heart disease (CHD) and type 2 diabetes (T2D). Methods Seventy cases of patients with CHD and T2D diagnosed and treated in Dongguan Shijie Hospital from January 2012 to January 2014 were used as the experimental group,whereas another 70 cases of T2D patients without CHD that were treated in the same hospital in the same period were used as control group.The patients in the two groups had similar gender ratio,age,and pathologic phases without any significantly difference(P > 0.05).The plasma Lp-PLA2 activity of all these patients was detected using the sandwich ELISA method. Results The average Lp-PLA2 activity of patients in the experimental group was significantly higher than that of patients in the control group,and it could be affected by factors,such as obesity,gender, metabolic disorders of high density lipoprotein (HDLP) and low-density lipoprotein (LDLP),as well as smoking. Conclusion Lp-PLA2 is involved in various pathophysiological processes and its activity could be affected by several factors,which indicate that it could be a reasonable strategy to control the progress of CHD and T2D by reasonably controlling these risk factors.%目的:探讨血浆脂蛋白磷脂酶A2水平对冠心病合并2型糖尿病患者的影响。方法选取2012年1月~2014年1月间在我院接受诊断和治疗的70例冠心病合并2型糖尿病患者作为实验组,同时选取同期入院的70例2型糖尿病患者作为对照组,对照组患者无冠心病。两组患者在性别、年龄、病程等方面差异不明显(P>0.05),具有可比性。用ELISA双抗体夹心法检测患者血浆Lp-PLA2活性。结果实验组的Lp-PLA2平均活性明显高于对照组;肥胖因素、性别因素、高密度脂蛋白代谢紊乱、低密度脂蛋白代谢紊乱、吸烟因素都可以明显影响Lp-PLA2平均活性。结论 LP-PLA2

  18. Rabbit IgG antibodies against Phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom Los anticuerpos IgG de conejos anti-fosfolipasa A2 de Crotalus durissus terrificus neutralizan la actividad letal del veneno

    Directory of Open Access Journals (Sweden)

    Juan P. Rodríguez

    2006-12-01

    Full Text Available Crotalus durissus terrificus (C.d.t. (South American rattlesnake venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2 and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75. Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg with Freund adjuvant. Groups of six mice (20 + 2 g were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms.El veneno de Crotalus durissus terrificus (C.d.t. (Cascabel de Sud América posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2 y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75. Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del

  19. Expression and function of secretory type Ⅱ phospholipase A2 in lung of severe acute pancreatitis of rats%重症急性胰腺炎肺组织Ⅱ型分泌型磷脂酶A2的表达及功能改变

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    张雪梅; 陈海龙; 王朝晖

    2012-01-01

    [目的]探讨重症急性胰腺炎(SAP)时肺组织Ⅱ型分泌型磷脂酶A2(sPLA2 -Ⅱ)的表达及功能改变.[方法]将SD大鼠随机分为假手术组(SO组,n=10)、模型组(SAP组,n=10).SO组仅行剖腹术,翻动胰腺;SAP组用去氧胆酸钠胰管逆行注射建立SAP合并肺损伤模型.2组动物在术后24 h测pH、PaQ、PaCO2、血淀粉酶、sPLA2 -Ⅱ,肺湿/干比值.应用RT-PCR、western-blot观察肺组织sPLA2-Ⅱ表达,并观察胰、肺组织病理变化.[结果]SAP组血淀粉酶、sPLA2、肺湿/干比值显著高于SO组(P<0.05).SAP组PaO2、pH显著低于SO组(P<0.05),PaCO2、sPLA -Ⅱ显著高于SO组(P<0.05).[结论]AAP时肺组织sPLA2 -Ⅱ表达增高,可能是急性肺损伤的发病机制之一.%[Objective] To investigate the expression of secretory type Ⅱ phospholipase A2 (sPLA2-Ⅱ )in lung in rats with severe acute pancreatitis. [Methods]SD rats were randomly divided into 2 groups of sham operation group(SO,n=10)and SAP model group(SAP,n=10). Severe acute pancreatitis was induced in SAP. Sham operation was only made in SO group. Serum amylase( AMY) levels, Ph, PaO2, PaCO2, sPLA2 and lung wet/dry ratio( W/D) were determined. sPLA2-Ⅱ mRNA expression in lung was detected by RT-PCR. The sPLA2- Ⅱ protein expression in lung was detected by western-blot. The pathologic changes of pancreas and lung were observed 24 hours after establishment of the model. [Results]Serum levels of AMY([7144.19 ±727. 91]U/ L),sPLA2([45.13±6.05]nmol·min-1·ml-1),W/D(8. 57±2.45)and PaCO2([47. 57±2. 55] mmHg)in SAP were remarkably higher than those in SO([1193. 41±192. 54]U/L, [29. 94± 6. 39]nmol·min-1·ml-1 ,[3. 70±0. 90],[27.69±1.02]mmHg,P<0.05). The levels of PaO2 ([79. 24± 5. 84] mmHg)and pH(7. 269±0. 054)in SAP were lower than that in SO([96. 78± 3. 81] mmHg,7. 391±0. 054,P<0. 05). The expression of sPLA2-Ⅱ in lung in SAP was significantly increased than in SO( P<3. 05). The pathologic changes of pancreas and lung in SO

  20. Periodontal therapy improved lipoprotein-associated phospholipase A2 and lipid levels in serum of chronic periodontitis subjects with hyperlipidemia%牙周基础治疗降低高血脂伴牙周炎患者血清脂蛋白相关磷脂酶A2及血脂水平

    Institute of Scientific and Technical Information of China (English)

    周爽英; 段晋瑜; 欧阳翔英

    2012-01-01

    目的 观察牙周基础治疗对牙周炎伴高血脂患者血清脂蛋白相关磷脂酶A2(lipoprotein-associated phospholipase A2,LP-PLA2)和血脂水平的影响,探讨通过牙周基础治疗降低高血脂患者心血管疾病风险的可能性.方法 选择40例慢性牙周炎伴高血脂患者[男性20例,女性20例,平均年龄(56.1±10.3)岁],对所有患者进行牙周基础治疗前和治疗后3个月的牙周临床指标、血清LP-PLA2水平、血脂水平及白细胞计数测定,治疗前后数据比较采用Wilcoxon符号秩检验或配对t检验,样本率的比较采用x2检验.结果 牙周基础治疗3个月后,牙周探诊深度[M(Q25,Q75)]从3.93(3.63,4.48) mm下降为2.47 (2.06,2.92) mm,临床附着丧失[M(Q25,Q75)]从5.14(4.51,5.46) mm下降为4.03(3.38,4.33) mm,菌斑指数[M(Q25,Q75)]从1.28(0.94,1.61)下降为0.77(0.65,1,13),探诊出血阳性位点百分比[M(Q25,Q75)]从99.82%(86.25%,100.00%)下降为33.49%(29.45%,43.30%),与治疗前相比差异均有统计学意义(P <0.001);血清LP-PLA2水平[(30.06 ±9.96) mg/L]、血脂总胆固醇[M(Q25,Q75)][5.57(5.26,6.25) mmol/L]、三酰甘油[M(Q25,Q75)][1.79(1.55,2.01) mmol/L]水平及白细胞计数[M(Q25,Q75)][5.90(5.40,6.55)×103个/L]均较治疗前[分别为(23.80±14.76) mg/L、5.36(5.16,5.86) mmol/L、1.57 (1.34,1.79) mmol/L及5.45(5.13,5.80)×103个/L]显著降低(P<0.01);高密度脂蛋白水平[M(Q25,Q75)][1.23(1.13,1.53) mmol/L]较治疗前[1.12(0.97,1.49) mmol/L]显著升高(P=0.001).血清LP-PLA2水平的减低值(△LP-PLA2)与探诊深度的降低值(△PD)呈显著正相关(r=0.367,P=0.020).结论 牙周基础治疗对牙周炎伴高血脂患者血清LP-PLA2水平和白细胞计数有显著降低作用,同时还可改善高血脂人群的血脂水平;提示牙周基础治疗可能是降低高血脂伴牙周炎人群心血管风险的途径之一.%Objective To observe the effect of non-surgical periodontal therapy on levels of lipoprotein

  1. Diagnostic Value of Serum M-type Phospholipase A2 Receptor Antibody in Patients with Idiopathic Membranous Nephropathy%血清抗M型磷脂酶A2受体抗体对特发性膜性肾病的诊断价值

    Institute of Scientific and Technical Information of China (English)

    邱杰山; 胡良峰; 张丽红; 李青华; 沈水娟

    2016-01-01

    Objective ToinvestigatethediagnosticvalueofserumM-typephospholipaseA2receptorantibody (anti-PLA2 R)in patients with idiopathic membranous nephropathy(IMN)and the correlation between anti-PLA2Randurineprotein.Methods Atotalof76patientswithrenalbiopsy-provedglomerulardiseasewere enrolled in this study,including 20 cases with IMN,20 cases with lupus nephritis (LN),16 cases with hepatitis B virus associated nephropathy (HBV-GN),and 20 cases with IgA nephropathy (IgAN).Eight healthy controls were also enrolled. The parameters of urinary albumin/creatinine ratio (UAlb/Cr),24 hour urinary protein secretion level (24hPro),serum creatinine (Scr),blood urea nitrogen (BUN)and serum albumin (Alb)were measured. The serum anti-PLA2 R levels were detected by a ELISA kit. The levels of serum anti-PLA2R were compared among the five groups,and the correlation between serum anti-PLA2R with UAlb/Cr and 24hPro were analyzed. ROC curve was plotted to determine the diagnostic sector of serum anti-PLA2RinpredictingIMN.Results Thelevelsofserumanti-PLA2RinpatientswithIMN[(23.66± 4. 06)μg/L] were higher than the LN group [(17. 92 ±3. 13 )μg/L],HBV-GN group [(14. 33 ± 4. 43)μg/L],IgAN group [(13. 68 ±1. 81)μg/L],and the control group [(11. 65 ±3. 08)μg/L]. Correlation analysis found that the serum anti-PLA2 R levels are positively correlated with UAlb/Cr (r =0. 795,P<0. 001)and 24hPro (r=0. 531,P=0. 016)in patients with IMN. ROC curve showed that the area under the curve was 0. 938 (95% CI:0. 881 -0. 996).The clinical diagnostic sector of anti-PLA2R indiagnosingIMNwas19.07μg/l(sensitivity95.0%,specificity79.1%).Conclusions Serumanti-PLA2 R levels are positively correlated with urine protein in patients with IMN,and it may be a novel serum biomarker for predicting IMN with high sensitivity and specificity.%目的:探讨血清抗M型磷脂酶A2受体抗体(M-type phospholipase A2 receptor antibody,anti-PLA2 R)对特发性膜性肾病(idiopathic membranous nephropathy,IMN

  2. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells

    DEFF Research Database (Denmark)

    Nyegaard, Steffen; Novakovic, Valerie A.; Rasmussen, Jan Trige;

    2013-01-01

    Secretory phospholipase A2 (sPLA2) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2’s do not bind to plasma membranes of quies......% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2...

  3. The risk factors and the correlation of lipoprotein associated phospholipase A2 ( Lp-PLA2 ) in patients of cerebral infarction with leukoaraiosis (LA)%脑梗死患者伴脑白质疏松症的相关危险因素及与脂蛋白相关磷脂酶A2相关性的研究

    Institute of Scientific and Technical Information of China (English)

    贵永堃; 吕晓红

    2011-01-01

    目的 探讨脑梗死患者伴脑白质疏松症(LA)的相关危险因素及与脂蛋白相关磷脂酶A2( LP - PLA2)的相关性.方法 选取178例脑梗死患者,依其影像学表现将其分为脑梗死合并LA组(83例)和脑梗死不合并LA组(95例),对比分析两组的Lp-PLA2含量以及临床、辅助检查指标.结果 (1)脑梗死患者共178例,LA的发病率46.63%.(2)单因素分析结果显示:在LA例组和无LA组年龄、Lp-PLA2、高血压病史及腔隙性脑梗死之间的差异具有统计学意义;多因素Logistic回归分析结果筛选出3个具有统计学意义的指标:Lp-PLA2、年龄和腔隙性脑梗死.(3)性别、糖尿病史、高脂血症、吸烟史、饮酒史、冠心病史及脑血管狭窄发生率在LA组和无LA组的差异无统计学意义.(4)两组高脂血症患者的LDL、甘油三脂、胆固醇水平及两组糖尿病患者的空腹血糖值差异无统计学意义,脑血管狭窄分为单纯前循环病变、单纯后循环病变及前后循环均有病变后,两组间构成比差异无统计学意义.结论 年龄、Lp-PLA2、腔隙性脑梗死是LA独立的危险因素.颅内外大血管狭窄与LA无确切的相关性.高血压是LA重要的危险因素.糖尿病、高血脂、吸烟史、饮酒史、冠心病史是LA和缺血性脑卒中的共同危险因素.%Objective To study the related risk factors and the correlation of lipoprotein associated phospholipase A2 (Lp-PLA2)in patients of cerebral infarction with Jeukoaraiosis( LA). Methods 178 cerebral infarction patients were divided into a cerebral infarction with leukoaraiosis group( LA group)and a cerebral infarction without leukoaraiosis group(no LA group). Their Lp-PLA2 levels and clinical,laboratory examinations were compared. Results The incidence of LA was 46.63% in 178 patients with cerebral infarction. Univariate analysis showed there were significant differences between LA group and no LA group in age,Lp-PLA2 .history of hypertension and

  4. Clonaje y caracterización molecular in silico de un transcrito de fosfolipasa A2 aislado del veneno de la serpiente peruana Lachesis muta Molecular cloning and characterization in silico of phospholipase A2 transcripto isolated from Lachesis muta peruvian snake venom

    Directory of Open Access Journals (Sweden)

    Karim L. Jimenez

    2010-12-01

    Full Text Available Objetivo. Aislar y caracterizar in silico un transcrito del gen de fosfolipasa A2 (PLA2 aislado del veneno de Lachesis muta de la Amazonía peruana. Materiales y métodos. Se amplificó el transcrito del gen sPLA2 mediante la técnica de RT-PCR a partir de RNA total utilizando cebadores específicos, el producto de DNA amplificado se insertó en el vector pGEM para su posterior secuenciación. Mediante análisis bioinformático de la secuencia nucleotídica se determinó un marco de lectura abierta de 414 nucleótidos que codifica 138 aminoácidos, incluyendo16 aminoácidos del péptido señal, el peso molecular y el pI fueron de 13 976 kDa y 5,66 respectivamente. Resultados. La secuencia aminoacídica denominada Lm-PLA2- Perú, contiene Asp49, así como Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 importantes para la actividad enzimática. La comparación de Lm-PLA2-Perú con las secuencias aminoacídicas de los bancos de datos mostró 93% de similitud con las sPLA2 de Lachesis stenophrys y más del 80% con otras sPLA2 de venenos de la familia Viperidae. El análisis filogenético de la secuencia nucleotídica del transcrito del gen sPLA2 indica que Lm-PLA2-Perú se agrupa con otras sPLA2 [Asp49] ácidas previamente aisladas del veneno de Bothriechis schlegelii con un 89% de identidad. El modelaje tridimensional de Lm-PLA2-Perú, presenta una estructura característica de sPLA2 del Grupo II formada por tres hélices-α, una lámina-β, una hélice corta y un lazo de unión con calcio. Conclusión. La secuencia nucleotídica corresponde al primer transcripto del gen de PLA2 clonado a partir del veneno de la serpiente Lachesis muta, que habita en la selva del Perú.Objective. Isolate and characterize in silico gene phospholipase A2 (PLA2 isolated from Lachesis muta venom of the Peruvian Amazon. Material and methods. Technique RT-PCR from total RNA was using specific primers, the amplified DNA product was inserted into the pGEM vector for

  5. 血清抗M型磷脂酶A2受体抗体对成年人特发性膜性肾病诊治的指导作用%Role of M _ type Phospholipase A2 Receptor Antibody in the Diagnosis and Treatment of Idiopathic Membranous Nephropathy in Adults

    Institute of Scientific and Technical Information of China (English)

    鲁欢; 汤水福; 陈刚毅; 苏保林; 吴兴波; 王超; 涂海涛; 罗月中

    2016-01-01

    Objective To evaluate the role of M _ type phospholipase A2 receptor antibody( anti-PLA2R) in the diagnosis and treatment of idiopathic membranous nephropathy(IMN)in adults. Methods From January 2014 to May 2015, we enrolled 58 patients who were definitely diagnosed with glomerular disease by renal biopsy in the First Affiliated Hospital of Guangzhou University of Chinese Medicine. Among them,there were 31 IMN patients(IMN group)and 27 non _ IMN patients (non _ IMN group). ELISA method was used to detect serum anti-PLA2R,in order to determine anti-PLA2R was positive or negative. Comparison was made between IMN group and non _ IMN group in the general data,laboratory examination indexes〔24 h UTP,Alb,Scr,urine RBC( HPF),UA,PT,INR,APTT and FIB〕,pathological indexes( mesangial proliferation, crescent formation,glomerulosclerosis,tubular atrophy/ fibrosis,renal interstitial inflammatory cell infiltration,renal interstitial vascular disease and immunofluorescence deposition) and clinical outcomes and the same comparison was made between anti-PLA2R positive and anti-PLA2R negative. Results In IMN group,there were 21 patients with anti-PLA2R positive with a positive rate of 67. 7%. In non _ IMN group,there was one patient with anti-PLA2R positive with a positive rate of 3. 7% . IMN group was higher in age,the optical density of serum anti-PLA2R and lower in urine RBC(HPF),PT,INR and APTT than non _ IMN group(P 0. 05). Patients with serum anti-PLA2R positive were higher than patients with anti-PLA2R negative in the optical density of serum anti-PLA2R(P 0. 05). Spearman correlation analysis showed that the optical density of serum anti-PLA2R had no correlation with MAP,UTP,Alb and Scr(rs = 0. 097, _ 0. 208,0. 266, _ 0. 358;P > 0. 05). Patients with anti-PLA2R positive and patients with anti-PLA2R negative were not significantly different in mesangial proliferation,crescent formation, glomerulosclerosis,tubular atrophy / fibrosis,renal interstitial inflammatory cell

  6. 老年膜性肾病患者抗M型磷脂酶A2受体抗体检测的临床意义%Clinical significance of detection for anti-M-type phospholipase A2 receptor antibody in elderly patients with membranous nephropathy

    Institute of Scientific and Technical Information of China (English)

    周广宇; 张文龙; 张博; 庄振起

    2015-01-01

    Objective To detect serum level of anti-M-type phospholipase A2 receptor (PLA2R) antibody in elderly patients with idiopathic membranous nephropathy (IMN) and to explore its clinical significance in IMN disease.Methods A total of 134 patients with renal biopsy-proved glomerular diseases were enrolled in this study,including 42 elderly cases with IMN,45 non-elderly cases with IMN,19 elderly cases with minimal change nephropathy (MCN),12 elderly cases with IgA nephropathy (IgAN),8 elderly cases with hepatitis B-associated membranous nephropathy (HBV-MN) and 8 elderly cases with focal segmental glomerulosclerosis (FSGS).Western blotting was used to detect serum anti-PLA2R antibody and the correlation of anti-PLA2R antibody with laboratory parameters in elderly IMN patients was analyzed.Results The elderly and non-elderly patients with IMN showed that the positive rate of anti-PLA2R antibody was 71.4% and 73.3%,respectively (P> 0.05).The positive rate of anti-PLA2R antibody in elderly IgA nephropathy,HBV-MN and MCN patients was 16.7 %,12.5% and 5.3% respectively.Anti-PLA2R antibody was not detected in serum from elderly FSGS patients.The positive rates of serum anti-PLA2R antibody were significantly higher in both elderly and non-elderly IMN patients than in elderly patients with secondary MN and other types of glomerlonephritis (P < 0.01).Furthermore,anti-PLA2R autoantibody level was positively correlated with 24-hour urine protein level and negatively correlated with the concentration of serum albumin in elderly patients with IMN (P<0.01).Conclusions Anti-PLA2R antibody is a sensitive serological marker of IMN.Detection for anti-PLA2R antibody might be helpful for the diagnosis of both elderly and non-elderly IMN and for the monitor of IMN disease severity.%目的 检测老年膜性肾病患者血清抗M型磷脂酶A2受体(PLA2R)抗体,并探讨其临床意义. 方法 应用蛋白免疫印迹法(Western blot)检测42例老

  7. Correlation of M-type phospholipase A2 receptor genetic polymorphism with idiopathic membranous nephropathy%特发性膜性肾病与M型磷脂酶A2受体基因多态性的相关性

    Institute of Scientific and Technical Information of China (English)

    周广宇; 孙延霞; 周立祥; 于晶; 郭莹; 尹敏

    2013-01-01

    目的 研究M型磷脂酶A2受体(PLA2R)的基因多态性与中国东北汉族人群特发性膜性肾病(IMN)的相关性.方法 应用聚合酶链反应-限制性片段长度多态性(PCRRFLP)技术检测95例IMN患者(IMN组)及232例健康体检者(HC组)PLA2R基因rs35771982和rs3828323两个位点的基因型和等位基因频率.结果 IMN组和HC组间性别、体质量指数(BMI)相匹配.IMN组的平均年龄、Scr、总胆固醇(TC)和24 h尿蛋白量均显著高于HC组(均P< 0.01);血清Alb和eGFR显著低于HC组(P<0.01).IMN组rs35771982位点CC基因型和C等位基因的频率均显著高于HC组(x2=13.658,P=0.001;x2=15.315,P=9.10×10-5).而两组间rs3828323位点基因型和等位基因频率的差异无统计学意义.rs35771982位点CC基因型与年龄、性别、BMI、血压、血清Alb、TC、Scr、eGFR及24 h尿蛋白量等指标无相关性.rs35771982位点基因型、年龄、TC、Scr及eGFR与IMN的发病相关,rs35771982位点的CC基因型是IMN的危险因素(OR=4.408,95% CI 1.488~ 13.058).结论 中国汉族人群PLA2R rs35771982位点基因多态性可能与IMN易感性相关,而rs3828323位点基因多态性与IMN无相关.rs35771982位点CC基因型是IMN的危险因素.%Objective To investigate the correlation of M-type phospholipase A2 receptor (PLA2R) genetic polymorphism in two single nucleotide polymorphisms (SNPs) with idiopathic membranous nephropathy (IMN) of Chinese Han population in Northeast China.Methods A total of 327 individuals were enrolled in the study including 95 adult patients with biopsy-proved IMN (IMN group) followed up for (25.4±11.6) months and 232 healthy people identified by healthy examination in China-Japan Union Hospital of Jilin University (HC group).Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect the genotype and allele frequency of rs35771982 and rs3828323 site in PLA2R gene.The x2 test was performed to compare the distribution

  8. Study on phospholipase A2 activation and antagonizing effect of fructose-1,6-diphosphate in endotoxin-induced acute lung injury in rabbits%内毒素致兔急性肺损伤时磷脂酶A2激活及1,6-二磷酸果糖拮抗作用的研究

    Institute of Scientific and Technical Information of China (English)

    汪建新; 薛庆亮; 江宏

    2006-01-01

    Objective To study the changes in activity of phospholipase A2 (PLA2) in the course of endotoxin (ET) induced acute lung injury (ALI) inrabbits and the antagonizing effect of fructose-1, 6-diphosphate (FDP), in order to evaluate the therapeutic effect of FDP on ET-induced ALI. Methods Flapeared white rabbits were randomly assigned to three groups: control group (group A), ET challenge group (group B) and treatment group (ET challenged followedby FDP, group C). Group A animals were injected with saline (2ml/kg) as control. Group B animals were injected with ET (500μg/kg) solution followed by saline. Total amount of liquid was 2ml/kg. Group C animals were given the same amount of ET solution followed by injection of FDP (300mg/kg) solution. Total amount of liquid was also 2 ml/kg. During the experiment, respiratory rate, heart rate, blood pressure, arterial blood gases and the plasma PLA2 activity were determined at 0h, 0. 5h, 2h, 4h and 6h respectively. The rabbits were sacrificed at 6h, pulmonary PLA2 activity was assessed, and the pathologic changes in pulmonary tissues were examined with light microscope and transmission electron microscope. Results Compared with group A,rabbits of group B manifested the typical characters of ALI after ET injection, and the PLA2 activity in both serum and pulmonary tissue was much higher than those of group A (P<0. 01).Values of the PLA2 activity in group C were between those of the two former groups. At the rame time, obvious pathological changes indicating lung injury were observed in group B and only mild pathological changes could be discerned in group C. Conclusion Activation of PLA2 activity is an important factor in pathogenesis of ET-induced ALI. FDP can antagonize the PLA2 activity and protect rabbits from early ET induced ALI to a certain extent.%目的研究内毒素(ET)致大耳白兔急性肺损伤(ALI)过程中磷脂酶A2的变化和1,6-二磷酸果糖(FDP)的拮抗作用,探讨FDP对ET所致ALI的治疗作用.

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

    Science.gov (United States)

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

    1991-01-01

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

  10. Expression of secretory type Ⅱ phospholipase A2 in acute lung injury following acute pancreatitis and interventional effect of Qingyi decoction(清胰汤) on it%急性胰腺炎肺损伤时肺组织Ⅱ型分泌型磷脂酶A2表达及清胰汤的干预作用

    Institute of Scientific and Technical Information of China (English)

    张雪梅; 陈海龙; 王朝晖

    2010-01-01

    Objective To investigate the expression of secretory type Ⅱ phospholipase A2 (sPLA2-Ⅱ) in lung of rats with acute lung injury (ALI) complicating severe acute pancreatitis (SAP), and the effect of Qingyi decoction (QYT, 清胰汤) on ALI. Methods Thirty Sprague Dawley (SD) rats were randomly divided into three groups: sham operation (SO) group, model group and QYT group, with 10 rats in each group. SAP model was reproduced by reverse injection of sodium deoxycholate into the common bile-pancreatic duct of rats. The pancreas of rats was just exposed in SO group. QYT (10 ml/kg) was gavaged 30 minutes and 12 hours after SAP was induced in QYT group. The blood gas analysis was performed 24 hours after operation. Serum amylase (AMY) levels, sPLA2 and lung wet/dry ratio (W/D) were determined. The sPLA2-Ⅱ mRNA and sPLA2-Ⅱ protein expression in lung were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The pathological changes in lung and pancreas were observed. Results Compared with SO group, the levels of arterial partial pressure of oxygen (PaO2) and pH value in model group were significantly decreased [PaO2 (mm Hg, 1 mm Hg=0.133 kPa): 79.24±5.84 vs. 96.78±3.81, pH value: 7.269±0.054 vs. 7.391±0.054], arterial partial pressure of carbon dioxide (PaCO2), the serum levels of AMY, W/D ratio and the serum levels of sPLA2 were significantly increased [PaCO2 (mm Hg): 47.57±2.55 vs. 27.69±1.02, AMY (U/L): 7 144.19±727.91 vs. 1 193.41±192.54, W/D ratio: 8.57±2.45 vs. 3.70±0.90, sPLA2 (nmol·min-1·ml-1): 45.13±6.05 vs. 29.94±6.39], the expression of sPLA2-Ⅱ mRNA (1.28±0.21 vs. 0.80±0.08) and protein were significantly increased (all P<0.05). Compared with model group, blood PaO2 and pH value were significantly increased [PaO2: (88.16±5.07) mm Hg, pH value: 7.322±0.039], the PaCO2, the serum levels of AMY, W/D ratio and the serum levels of sPLA2 in QYT group were significantly decreased [PaCO2: (33.13±2.14) mm

  11. Expressions and significance of IgG subtypes and M-type phospholipase A2 receptor (PLA2R) in membra-nous nephropathy%IgG亚型及M型磷脂酶A2受体在膜性肾病中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    韩伟霞; 高丽芳; 王莹; 张晓琴; 魏荣; 王晨

    2016-01-01

    Objective To investigate the expressions of IgG subtypes and M-type phospholipase A2 receptor (PLA2R) in membranous nephropathy (MN), and to retrospectively analyze its significance and value of differential di-agnosis in idiopathic membranous nephropathy (IMN) and undetermined atypical membranous nephropathy (UAMN). Methods A total of 120 cases of membranous nephropathy diagnosed by renal biopsy pathology (immunofluores-cence, light microscopy, and electron microscopy) in our hospital between February and September, 2015 were includ-ed in the study. The expressions of IgG subtypes and PLA2R were determined by immunohistochemistry, and retro-spectively analyzed, combined with the clinical data and pathological features. Their value in the differential diagnosis of IMN and UAMN was evaluated. Results ①A total of 120 cases were included, that IMN accounted for 69.2%and UAMN accounted for 30.8%. The average age of onset in the UAMN group was lower than that in the IMN group, and the difference was statistical significance, whereas no statistical difference was found in the level of urinary protein be-tween the two groups. ②The positive rates of IgA, IgM, C3, FRA, and C1q in the UAMN group were higher than those in the IMN group, and there were statistical differences in the expressions of IgA and complement C1q between the two groups. ③Positive IgG4 was mainly expressed in IMN group that the positive rate of IgG4 was significantly higher than the other IgG subtypes, and there was statistical difference between the IMN group and the UAMN group. In the UAMN group, positive IgG1 and IgG2 were highly expressed. The positive rate of IgG4 in the UAMN group was significantly lower than that in the IMN group. ④The positive rate of PLA2R was significantly higher than all IgG subtypes in the IMN group, and there was statistical difference between the IMN group and the UAMN group. ⑤In the evaluation of the value of IMN and UAMN by the differential diagnosis of IgG4 and

  12. 二甲双胍对大鼠非酒精性脂肪肝病的干预作用及对肝细胞脂性凋亡磷脂酶A2-溶血磷脂酰胆碱通路的影响%Metformin prevents non-alcoholic fatty liver disease in rats: role of phospholipase A2/lysophosphatidylcholine lipoapoptosis pathway in hepatocytes

    Institute of Scientific and Technical Information of China (English)

    黄邀; 傅君芬; 施红波; 刘利蕊

    2011-01-01

    血磷脂酰胆碱水平(μmol/L)(549.92±90.78 vs.707.26±92.48),肝脏甘油三酯(mg/g)(11.23±1.70 vs.13.57 ±0.65)、胆固醇(mg/g)(1.97±0.20 vs.2.19±0.15)较高脂饮食组低(P均<0.05),脂肪变性程度显著减轻,sPLA2 mRNA表达显著降低.(3)二甲双胍干预组大鼠肝指数(0.034±0.004 vs.0.026±0.002)、低密度脂蛋白胆固醇(mmol/L)(0.96±0.34vs.0.65±0.22)较普通饮食组高(P<0.05),脂肪变性程度、sPLA2 mRNA表达等差异无统计学意义(P>0.05).结论 二甲双胍对高脂饮食诱导大鼠非酒精性脂肪肝病有干预作用,其作用可能与改善胰岛素抵抗,降低分泌型磷脂酶A2基因表达,降低血清分泌型磷脂酶A2、溶血磷脂酰胆碱水平,减轻炎症反应和保护线粒体功能有关.%Objective To investigate the potential preventive effects of metformin on non-alcoholic fatty liver disease (NAFLD) and roles of phospholipase A2/lysophosphatidylcholine pathway in hepatocyte lipoapoptosis in a rat NAFLD model induced by high-fat diet. Method Male SD rats ( n = 36 ) were randomly divided into three groups with 12 rats in each and treated with different diet and drugs: group Ⅰ:ordinary diet, group Ⅱ: high-fat diet, group Ⅲ: high-fat diet and metformin. Ten weeks later, the rats were sacrificed and their body weight and liver weight were obtained, serum lipid metabolic indexes, insulin resistance indexes and secretory phospholipase A2( sPLA2 ), lysophosphatidylcholine(LPC) levels and other parameters were measured. Phospholipase A2 mRNA expression levels were measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). In addition, the histological changes of liver tissue were analyzed. Result Compared to ordinary diet group,the rat's liver weight (g) ( 16.92 ±2.49 vs. 12.16 ±0.82) ,hepatic exponent ( 0.034 ± 0.004 vs. 0.026 ± 0.002 ), serum alanine aminotransferase (U/L)(45.43 ±9.73 vs. 29.42 ± 6.73), triglyceride(mmol/L) ( 1.22 ±0.24 vs. 0.85 ±0

  13. Bacterial Sphingomyelinases and Phospholipases as Virulence Factors.

    Science.gov (United States)

    Flores-Díaz, Marietta; Monturiol-Gross, Laura; Naylor, Claire; Alape-Girón, Alberto; Flieger, Antje

    2016-09-01

    Bacterial sphingomyelinases and phospholipases are a heterogeneous group of esterases which are usually surface associated or secreted by a wide variety of Gram-positive and Gram-negative bacteria. These enzymes hydrolyze sphingomyelin and glycerophospholipids, respectively, generating products identical to the ones produced by eukaryotic enzymes which play crucial roles in distinct physiological processes, including membrane dynamics, cellular signaling, migration, growth, and death. Several bacterial sphingomyelinases and phospholipases are essential for virulence of extracellular, facultative, or obligate intracellular pathogens, as these enzymes contribute to phagosomal escape or phagosomal maturation avoidance, favoring tissue colonization, infection establishment and progression, or immune response evasion. This work presents a classification proposal for bacterial sphingomyelinases and phospholipases that considers not only their enzymatic activities but also their structural aspects. An overview of the main physiopathological activities is provided for each enzyme type, as are examples in which inactivation of a sphingomyelinase- or a phospholipase-encoding gene impairs the virulence of a pathogen. The identification of sphingomyelinases and phospholipases important for bacterial pathogenesis and the development of inhibitors for these enzymes could generate candidate vaccines and therapeutic agents, which will diminish the impacts of the associated human and animal diseases. PMID:27307578

  14. Ectopic Expression of Arabidopsis Phospholipase A Genes Elucidates Role of Phospholipase Bs in S. cerevisiae Cells

    OpenAIRE

    Zhang, Meng; Zhang, Yan; Giblin, E Michael; Taylor, David C.

    2009-01-01

    In S. cerevisiae neither disruption of the phospholipase B triple knockout mutant (plb1plb2plb3; plb123) nor over-expression of phospholipase Bs (PLBs) result in a phenotype different from wild type. In performing experiments to characterize candidate plant phospholipase (PLA) genes, we found, surprisingly, that ectopic expression of either of two different A. thaliana PLA2 or PLA1 genes in the yeast plb123 mutant completely inhibited cell growth. We proposed that while PLBs might not be esse...

  15. Diagnostic value of serum anti-phospholipase A2 receptor antibodies (PLA2R) in idiopathic membranous nephropathy%磷脂酶 A2受体抗体在特发性膜性肾病中的诊断价值

    Institute of Scientific and Technical Information of China (English)

    牛广华; 高玉洁; 王柏山; 吴丽娜; 郭宏洋; 崔百慧; 张程; 郭鹤

    2015-01-01

    UA分别为262±49、342±92,t=2.409,P=0.026;CYSC分别为0.78±0.21、0.92±0.24, t=1.674,P=0.00。对照组与IgA肾病亚组UREA分别为4.12±1.25、6.69±2.87,t=4.756,P=0.00;UA分别为262±49、361±52, t=4.598,P=0.00;CYSC分别为0.78±0.21、1.30±0.36, t=4.752,P=0.00。对照组与肿瘤相关肾病亚组UREA分别为4.12±1.25、5.02±1.70,t=3.626,P=0.002;UA分别为262±49、289±92, t=0.05,P=0.01;CYSC分别为0.78±0.21、0.98±0.20, t=1.1,P=0.01)。 IMN组与NIMN组中的4个亚组肾功能相关指标比较差异无统计学意义(P>0.05)。(3) IMN组的PLA2R抗体荧光强度与24 h PRO呈正相关(r=0.877, P=0.00),与URIC呈正相关(r=0.766,P=0.00)。(4)IMN组血清中抗PLA2R抗体水平与病理Ⅰ期、Ⅱ期、Ⅲ期分期无相关性( r值分别为0.087、0.194、0.182;P值分别为0.598、0.399、0.667),但抗体阳性率(Ⅰ期37.50%、Ⅱ期84.85%、Ⅲ期85.71%)可随着病情严重程度有递增趋势。结论PLA2R抗体可能是新的IMN的实验室诊断指标。(中华检验医学杂志,2015,38:595-599)%Objective To investigate the diagnostic value and monitoring application of anti-phospholipase A2 receptor antibodies ( PLA2R ) in patients with idiopathic membranous nephropathy ( IMN) .Methods A retrospective study was used .48 patients were diagnosed as idiopathic membranous nephropathy by puncturing kidney from January of 2013 to June of 2014 in the Hospital of Liaoning Traditional Chinese Medical University and Shengjing Hospital of China Medical University were included. 43 patients were diagnosed as non-idiopathic membranous nephropathy ( NIMN ).35 healthy volunteers were selected as control groups.Serum PLA2R were detected by indirect immunofluorescence.The intensity of antibodies fluorescence and the

  16. Research progress in phospholipase%磷脂酶研究进展

    Institute of Scientific and Technical Information of China (English)

    梁丽; 常明; 刘睿杰; 刘元法; 王兴国; 金青哲

    2013-01-01

    The phospholipases.including phospholipase A1,A2,B,C and D.that are a complex and crucially important group of enzymes that exit in organism and can specifically hydrolyze the intramolecular ester bond of p-hosphoglycerides releasing different products. Thus,phospholipases are widely used in phosphoglycerides transformation. The extraction of phospholipases from animal organs by the traditional means cannot meet the current industry requirements,however,the study of microbial source phospholipases and the rapid development of gene engineering not only broaden the sources of them but also improve their properties effectively. The paper reviewed the progress in the screening of the high yield strain and phospholipases molecular modification and transformation.%磷脂酶是在生物体内存在的可以水解甘油磷脂的一类酶,其中主要包括磷脂酶A1、AC、B、C和D,它们特异地作用于磷脂分子内部的各个酯键,形成不同的产物,被广泛用于甘油磷脂的改造.依靠传统手段从动物脏器中提取的磷脂酶已经不能满足目前生产的需求,而微生物来源磷脂酶的筛选及其基因工程发展迅速,显著拓宽了磷脂酶的来源,同时又有效地改善了磷脂酶的性质本文综述了近年磷脂酶高产微生物菌株的选育、磷脂酶的分子改造以及修饰方面取得的进展.

  17. Melittin stimulates fatty acid release through non-phospholipase-mediated mechanisms and interacts with the dopamine transporter and other membrane spanning proteins

    OpenAIRE

    Keith, Dove J; Eshleman, Amy J; Janowsky, Aaron

    2010-01-01

    Phospholipase A2 releases the fatty acid arachidonic acid from membrane phospholipids. We used the purported phospholipase A2 stimulator, melittin, to examine the effects of endogenous arachidonic acid signaling on dopamine transporter function and trafficking. In HEK-293 cells stably transfected with the dopamine transporter, melittin reduced uptake of [3H]dopamine. Additionally, measurements of fatty acid content demonstrated a melittin-induced release of membrane-incorporated arachidonic a...

  18. Phospholipase A2 receptor and sarcoidosis-associated membranous nephropathy.

    Science.gov (United States)

    Stehlé, Thomas; Audard, Vincent; Ronco, Pierre; Debiec, Hanna

    2015-06-01

    Of the glomerulonephritis associated with sarcoidosis, membranous nephropathy (MN) is the most prevalent. Coincidence or a causal relationship between these two diseases is unclear. Here, we present for the first time a high prevalence of PLA2R-related MN among patients with MN associated with active sarcoidosis. Our results suggest some causal link between sarcoidosis and PLA2R-related MN. Detection of anti-PLA2R antibodies in serum or PLA2R antigen in biopsy should not be taken as evidence against a secondary cause, particularly sarcoidosis. This important observation can affect treatment decision in these patients.

  19. Cytosolic phospholipase A2 modulates TLR2 signaling in synoviocytes.

    Directory of Open Access Journals (Sweden)

    Randi M Sommerfelt

    Full Text Available Rheumatoid arthritis (RA is an autoimmune disease characterized by chronic synovitis leading to destruction of cartilage and bone. PLA2 enzymes are key players in inflammation regulating the release of unsaturated fatty acids such as arachidonic acid (AA, a precursor of pro-inflammatory eicosanoids. Several lines of evidence point to toll-like receptors (TLRs as drivers of synovitis and joint destruction in RA. However, few studies have addressed the implication of PLA2 activity downstream TLR activation in the synovium. Here, we aimed to characterize PLA2 enzyme involvement in TLR2-induced signaling in synovial fibroblast-like cells. TLRs1-7 and a range of sPLA2, iPLA2 and cPLA2 enzymes were found to be transcriptionally expressed in cultured synoviocytes. Activation of TLR2/1 and TLR2/6 led to phosphorylation of cPLA2α at Ser505, and induced AA release and PGE2 production; effects that were attenuated by cPLA2α inhibitors. In contrast, sPLA2 inhibitors did not affect AA or PGE2 release. cPLA2α inhibitors furthermore attenuated TLR-induced expression of IL-6, IL-8 and COX2. COX1/2 inhibitors attenuated TLR2/6-induced IL-6 transcription and protein production comparable to cPLA2α inhibition. Moreover, exogenously PGE2 added alone induced IL-6 production and completely rescued IL-6 transcription when added simultaneously with FSL-1 in the presence of a cPLA2α inhibitor. Our results demonstrate for the first time that cPLA2α is involved in TLR2/1- and TLR2/6-induced AA release, PGE2 production and pro-inflammatory cytokine expression in synoviocytes, possibly through COX/PGE2-dependent pathways. These findings expand our understanding of cPLA2α as a modulator of inflammatory molecular mechanisms in chronic diseases such as RA.

  20. Phospholipase Cδ regulates germination of Dictyostelium spores

    NARCIS (Netherlands)

    Dijken, Peter van; Haastert, Peter J.M. van

    2001-01-01

    Background: Many eukaryotes, including plants and fungi make spores that resist severe environmental stress. The micro-organism Dictyostelium contains a single phospholipase C gene (PLC); deletion of the gene has no effect on growth, cell movement and differentiation. In this report we show that PLC

  1. GsAPK, an ABA-activated and calcium-independent SnRK2-type kinase from G. soja, mediates the regulation of plant tolerance to salinity and ABA stress.

    Directory of Open Access Journals (Sweden)

    Liang Yang

    Full Text Available Plant Snf1 (sucrose non-fermenting-1 related protein kinase (SnRK, a subfamily of serine/threonine kinases, has been implicated as a crucial upstream regulator of ABA and osmotic signaling as in many other signaling cascades. In this paper, we have isolated a novel plant specific ABA activated calcium independent protein kinase (GsAPK from a highly salt tolerant plant, Glycine soja (50109, which is a member of the SnRK2 family. Subcellular localization studies using GFP fusion protein indicated that GsAPK is localized in the plasma membrane. We found that autophosphorylation and Myelin Basis Protein phosphorylation activity of GsAPK is only activated by ABA and the kinase activity also was observed when calcium was replaced by EGTA, suggesting its independence of calcium in enzyme activity. We also found that cold, salinity, drought, and ABA stress alter GsAPK gene transcripts and heterogonous overexpression of GsAPK in Arabidopsis alters plant tolerance to high salinity and ABA stress. In summary, we demonstrated that GsAPK is a Glycine soja ABA activated calcium independent SnRK-type kinase presumably involved in ABA mediated stress signal transduction.

  2. GsAPK, an ABA-activated and calcium-independent SnRK2-type kinase from G. soja, mediates the regulation of plant tolerance to salinity and ABA stress.

    Science.gov (United States)

    Yang, Liang; Ji, Wei; Gao, Peng; Li, Yong; Cai, Hua; Bai, Xi; Chen, Qin; Zhu, Yanming

    2012-01-01

    Plant Snf1 (sucrose non-fermenting-1) related protein kinase (SnRK), a subfamily of serine/threonine kinases, has been implicated as a crucial upstream regulator of ABA and osmotic signaling as in many other signaling cascades. In this paper, we have isolated a novel plant specific ABA activated calcium independent protein kinase (GsAPK) from a highly salt tolerant plant, Glycine soja (50109), which is a member of the SnRK2 family. Subcellular localization studies using GFP fusion protein indicated that GsAPK is localized in the plasma membrane. We found that autophosphorylation and Myelin Basis Protein phosphorylation activity of GsAPK is only activated by ABA and the kinase activity also was observed when calcium was replaced by EGTA, suggesting its independence of calcium in enzyme activity. We also found that cold, salinity, drought, and ABA stress alter GsAPK gene transcripts and heterogonous overexpression of GsAPK in Arabidopsis alters plant tolerance to high salinity and ABA stress. In summary, we demonstrated that GsAPK is a Glycine soja ABA activated calcium independent SnRK-type kinase presumably involved in ABA mediated stress signal transduction.

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

    OpenAIRE

    Gargouri Youssef; Ben Ali Yassine; Frikha Fakher; Karray Aida; Bezzine Sofiane

    2011-01-01

    Abstract Background Secretory phospholipase A2 group IIA (IIA PLA2) is a protein shown to be highly expressed in the intestine of mammals. However, no study was reported in birds. Results Chicken intestinal group IIA phospholipase A2 (ChPLA2-IIA) was obtained after an acidic treatment (pH.3.0), precipitation by ammonium sulphate, followed by sequential column chromatographies on Sephadex G-50 and mono-S ion exchanger. The enzyme was found to be a monomeric protein with a molecular mass of aro...

  4. The influence of chronic Helicobacter pylori infection in serum lipoprotein associated phospholipase A2 level and stability of atherosclerotic plaques in patients with carotid atherosclerosis%幽门螺杆菌感染对颈动脉粥样硬化患者血清脂蛋白相关磷脂酶A2水平及颈动脉斑块稳定性的影响

    Institute of Scientific and Technical Information of China (English)

    雷鸣; 夏丹妮; 汤兰桂; 周权; 向攀; 张洪鑫

    2015-01-01

    Objective To explore the influence of Helicobacter pylori (Hp) infection in serum lipoprotein associated phospholipase A2 (Lp-PLA2), carotid intima-media thickness and stability of atherosclerotic plaques in atherosclerosis patients.Methods A total of 393 cases of patients with carotid artery arteriosclerosis confirmed by carotid color uhrasonography, who are informed consent, was selected as objects.The14C urea breath test was used to determine the infection situation of selected objects of helicobacter pylori.Meanwhile, enzyme-linked immunosorbent assay (ELISA) was used to determine the level of serum lipoprotein associated phospholipase A2 (Lp-PLA2).Results Serum Lp-PLA2 levels and carotid intimamedia thickness (IMT) of patients with carotid artery atherosclerosis in Hp infection group were higher than that of Hp non-infection group, and with the degree of Hp infection aggravating in the patients of carotid artery atherosclerosis, their serum Lp-PLA2 levels and carotid IMT were also increased accordingly.F test showed that the differences of serum Lp-PLA2 levels and carotid IMT in different degree of carotid artery atherosclerosis group were statistically significant (P <0.01).The incidence of unstable plaque of Hp infection group was obviously higher than that of the Hp non-infection group in the carotid atherosclerosis with plaques with statistical significance (chi square value =4.744, P =0.029).Multivariate linear regression analysis showed that the possibility of complication of unstable plaques in Hp infection group of carotid artery atherosclerosis was 1.82 times than that of non-infection group.With serum Lp-PLA2 every increasing 1 μg/L, the possibility of instability plaque increased by 2%.Conclusions Hp infection may promote the occurrence and development of carotid artery atherosclerosis by increasing serum level of Lp-PLA2 and changing the stability of atherosclerotic plaques.%目的 探讨幽门螺杆菌(Hp)感染对颈动脉粥样硬化患者

  5. Rice phospholipase A superfamily: organization, phylogenetic and expression analysis during abiotic stresses and development.

    Directory of Open Access Journals (Sweden)

    Amarjeet Singh

    Full Text Available BACKGROUND: Phospholipase A (PLA is an important group of enzymes responsible for phospholipid hydrolysis in lipid signaling. PLAs have been implicated in abiotic stress signaling and developmental events in various plants species. Genome-wide analysis of PLA superfamily has been carried out in dicot plant Arabidopsis. A comprehensive genome-wide analysis of PLAs has not been presented yet in crop plant rice. METHODOLOGY/PRINCIPAL FINDINGS: A comprehensive bioinformatics analysis identified a total of 31 PLA encoding genes in the rice genome, which are divided into three classes; phospholipase A(1 (PLA(1, patatin like phospholipases (pPLA and low molecular weight secretory phospholipase A(2 (sPLA(2 based on their sequences and phylogeny. A subset of 10 rice PLAs exhibited chromosomal duplication, emphasizing the role of duplication in the expansion of this gene family in rice. Microarray expression profiling revealed a number of PLA members expressing differentially and significantly under abiotic stresses and reproductive development. Comparative expression analysis with Arabidopsis PLAs revealed a high degree of functional conservation between the orthologs in two plant species, which also indicated the vital role of PLAs in stress signaling and plant development across different plant species. Moreover, sub-cellular localization of a few candidates suggests their differential localization and functional role in the lipid signaling. CONCLUSION/SIGNIFICANCE: The comprehensive analysis and expression profiling would provide a critical platform for the functional characterization of the candidate PLA genes in crop plants.

  6. Effects of dexamethasone on palate mesenchymal cell phospholipase activity

    International Nuclear Information System (INIS)

    Corticosteroids will induce cleft palate in mice. One suggested mechanism for this effect is through inhibition of phospholipase activity. This hypothesis was tested by measuring the effects of dexamethasone, a synthetic corticosteroid, on phospholipase activity in cultures of palate mesenchymal cells. Palate mesenchymal cells were prelabeled with [3H]arachidonic acid. The cells were subsequently treated with various concentrations of dexamethasone. Concurrently, cultures of M-MSV-transformed 3T3 cells were prepared identically. After treatment, phospholipase activity was stimulated by the addition of serum or epidermal growth factor (EGF), and radioactivity released into the medium was taken as a measure of phospholipase activity. Dexamethasone (1 X 10(-5) or 1 X 10(-4) M) could inhibit serum-stimulated phospholipase activity in transformed 3T3 cells after 1 to 24 hr of treatment. However, no inhibition of activity was measured in palate mesenchymal cells following this period of treatment. Not until 120 hr of treatment with dexamethasone (1 X 10(-4) M) was any significant inhibition of serum-stimulated phospholipase activity observed in palate mesenchymal cells. When EGF was used to stimulate phospholipase activity, dexamethasone (1 X 10(-5) M) caused an increase in phospholipase activity in palate mesenchymal cells. These observations suggested that phospholipase in transformed 3T3 cells was sensitive to inhibition by dexamethasone. However, palate mesenchymal cell phospholipase is only minimally sensitive to dexamethasone, and in certain instances can be enhanced. These results cannot support the hypothesis that corticosteroids mediate their teratogenic effect via inhibition of phospholipase activity

  7. Synthesis of tocopheryl succinate phospholipid conjugates and monitoring of phospholipase A2 activity

    DEFF Research Database (Denmark)

    Pedersen, Palle Jacob; Viart, Helene Marie-France; Melander, Fredrik;

    2012-01-01

    Tocopheryl succinates (TOSs) are, in contrast to tocopherols, highly cytotoxic against many cancer cells. In this study the enzyme activity of secretory phospholipase A2 towards various succinate-phospholipid conjugates has been investigated. The synthesis of six novel phospholipids is described,...

  8. Preservation of bilayer structure in human erythrocytes and erythrocyte ghosts after phospholipase treatment : A 31P-NMR study

    NARCIS (Netherlands)

    Meer, G. van; Kruijff, B. de; Kamp, J.A.F. op den; Deenen, L.L.M. van

    1980-01-01

    1. 1. Fresh human erythrocytes were treated with lytic and non-lytic combinations of phospholipases A2, C and sphingomyelinase. The 31P-NMR spectra of ghosts derived from such erythrocytes show that, in all cases, the residual phospholipids and lysophospholipids remain organized in a bilayer configu

  9. Static magnetic field changes the activity of venom phospholipase of Vipera Lebetina snakes

    International Nuclear Information System (INIS)

    The effect of the static magnetic field (SMF) on the phospholipid activity of the class-A snake venom is studied. The Vipera Lebetina snake venom was subjected during 10 days to 30 minute impact of the CMF daily. It is established that increase in the phospholipase A1 and A2 approximately by 21 and 32 % correspondingly and in the phosphodiesterase C - by 33 % was observed. The decrease in the total protein level of the snake venom by 31.6 ± 2.2 % was noted thereby. It may be assumed that the described phospholipase and phosphoesterase changes may lead to essential shifts in the total metabolic activity of cells and organism as a whole. The activity index of these ferments may serve as an indicator of changes in the environmental magnetic field

  10. 意大利蜜蜂蜂毒磷脂酶A2基因在大肠杆菌中的表达%Expression of a bee-venom phospholipase A2 gene from Apis mellifera in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    沈立荣; 程家安; 张传溪; 吴建祥

    2004-01-01

    将意大利蜜蜂蜂毒磷脂酶A2(AmPLA2)成熟肽编码区基因(405 bp) 插入表达载体pETBlue-1,在大肠杆菌Tuner(DE3)placⅠ中诱导表达,经SDS-PAGE电泳检测,表达产物分子量为15 kD,约占细菌总蛋白的4.3%;用兔抗AmPLA2多克隆血清为第一抗体作Western blot检测,表达产物显示与天然AmPLA2同样的特异性印迹,证实AmPLA2 基因已在大肠杆菌中得到表达.

  11. 风湿骨痛药酒药槌外治法对兔退变腰椎间盘内磷脂酶A2活性的影响%The effect of external treatment of medicated mallet of Fengshi Gutong Herb Liquor on phospholipase A2 activity in the degenerative intervertebral disc in rabbit model

    Institute of Scientific and Technical Information of China (English)

    肖强兵; 白书臣; 何承建; 曾俊华; 李浩; 陈大伟

    2011-01-01

    To study the effect of external treatment of medicated mallet of Fengshi Gutong Herb Liquor on phcspholipase A2 activity in the degenerative intervertebral disc in rabbit model and exploring its mechanism of treatment and prevention on discogenic low back pain. Methods 21 rabbits, after modeled by injuring the intervertebral disc percutaneously under X - ray, were randomly divided into 3 groups: mallet of Fengshi Gutong Herb Liquor group, medicine treatment group, model control group. Treatment was given after 8 weeks after modeling, L4 -5 intervertebral disc was sampled for PLA2 activity detection. Results Compared with model control group, PLA2 activity of both treatment groups were significantly lower (P <0.01). No significant difference was observed in comparison between the external treatment group and medicine treatment group (P > 0.05). Conclusion Mallet of Gutong Herb Liquor mallet of Gutong Herb Liquor can lower the PLA2 activity in intervetebral disc, which may be a mechanism of treating dicogenic back pain.%目的 观察风湿骨痛药酒药槌对兔退变椎间盘内磷脂酶A2(PLA2)活性的影响,探讨其防治椎间盘源性下腰痛的机理.方法 采用在X光机透视定位下经皮穿刺破坏椎间盘的造模方法,将21只实验免随机分为3组:风湿骨痛药酒药槌外治法治疗组、药物治疗组和模型对照组.造模8周后开始治疗,3周后手术取L4-5椎间盘进行PLA2活性检测.结果 与模型组比较,两治疗组PLA2活性明显降低(P<0.01).外治法组与药物治疗组比较,PLA2活性无明显差异(P>0.05).结论 风湿骨痛药酒药槌外治法可降低椎间盘内PLA2活性,这可能是其防治椎间盘源性下腰痛的机制之一.

  12. The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase-D toxin.

    Science.gov (United States)

    Chaves-Moreira, Daniele; Souza, Fernanda N; Fogaça, Rosalvo T H; Mangili, Oldemir C; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Chaim, Olga M; Veiga, Silvio S

    2011-09-01

    Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying recombinant phospholipase-D, a potent hemolytic and phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase-D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium-sensitive fluorescent probe (Fluo-4). This calcium influx was shown to be channel

  13. 成人膜性肾病患者血清抗PLA2R抗体与病情的相关性%Correlation of anti-M-type phospholipase A2 receptor antibody with disease severity in adult patients with idiopathic membranous nephropathy

    Institute of Scientific and Technical Information of China (English)

    周广宇; 金玲; 于晶; 张芝平

    2012-01-01

    目的 分析成人膜性肾病患者血清抗M型磷脂酶A2受体(PLA2R)抗体与特发性膜性肾病( IMN)实验室指标的相关性,探讨抗PLA2R抗体在IMN发病中的作用.方法 选取经肾活检证实的46例肾小球疾病患者,包括20例IMN、7例IgA肾病、6例乙型肝炎病毒相关性膜性肾病( HBV-MN)、6例微小病变性肾病、4例局灶性节段性肾小球硬化、3例Ⅴ型狼疮肾炎.应用Western印迹法检测血清抗PLA2R抗体,并对其与IMN患者血清白蛋白、24 h尿蛋白量、血清总胆固醇和Scr作相关性分析.结果 20例IMN患者中15例血清抗PLA2R抗体阳性,阳性比例为75%;7例IgA肾病患者中1例抗PLA2R抗体阳性,阳性比例为14.29%;6例HBV-MN患者中1例阳性,阳性比例为16.67%;其余患者均为阴性.IMN患者血清抗PLA2R抗体阳性比例显著高于继发性膜性肾病和其他肾小球肾炎(均P<0.01),且抗PLA2R抗体水平与IMN患者尿蛋白量呈正相关(r=0.803,P<0.01);与血清白蛋白呈负相关(r=-0.816,P<0.01).结论 IMN患者血清抗PLA2R抗体阳性比例高,提示抗PLA2R抗体可能是IMN的特异性抗体.该抗体与尿蛋白量呈正相关,提示抗PLA2R抗体可能是IMN的致病性抗体.%Objective To investigate the correlation of serum anti-M-type phospholipase A2 receptor (PLA2R) antibody with laboratory parameters of idiopathic membranous nephropathy (IMN) in adult patients with membranous nephropathy (MN),and to explore the role of anti-PLA2R antibody in the pathogenesis of IMN. Methods Forty-six adult patients with biopsy-proved glomerular diseases were involved in this study,including 20 cases with IMN,7 cases with IgA nephropathy (IgAN),6 cases with hepatitis B-associated membranous nephropathy (HBV-MN),6 cases with minimal change nephropathy (MCN),4 cases with focal segmental glomerulosclerosis (FSGS) and 3 cases with class Ⅴ lupus nephritis.Total RNA was extracted from human glomeruli and was reversely transcribed to the

  14. 特发性膜性肾病中血清磷脂酶A2受体抗体的临床意义%Detection of serum phospholipase A2 receptor antibodies and its clinical significance in patients with idiopathic membranous nephropathy

    Institute of Scientific and Technical Information of China (English)

    韩丹诺; 谌贻璞; 王艳艳; 董鸿瑞; 芮宏亮; 王国勤; 程虹

    2015-01-01

    目的 观察特发性膜性肾病(IMN)患者治疗前、后血清抗磷脂酶A2受体(PLA2R)抗体变化及与病情的关系.方法 以首都医科大学附属北京安贞医院肾内科2013年1月至2014年3月诊断为IMN的52例患者为研究对象,分别用免疫组化及免疫荧光法检测肾小球中PLA2R表达及IgG亚类沉积,用间接免疫荧光及酶联免疫吸附法检测血清抗PLA2R抗体,并进行相关关系分析.结果 治疗前肾小球PLA2R和血清抗体阳性者占96.2%,肾小球IgG4沉积为主者占100%.治疗后完全缓解者血清抗PLA2R抗体全部转阴;部分缓解者一半以上转阴;未缓解者持续阳性.Spearman秩相关分析显示,治疗前抗体水平与尿蛋白及血清胆固醇呈正相关;治疗后与尿蛋白、血清胆固醇及血清三酰甘油呈正相关,与血清白蛋白呈负相关.多元线性回归分析显示,治疗前抗体水平与尿蛋白呈正相关,治疗后与尿蛋白呈正相关而与血清白蛋白负相关.结论 IMN患者的血清抗PLA2R抗体水平与尿蛋白等检验指标及疾病转归密切相关.

  15. 成人特发性膜性肾病血清抗M型磷脂酶A2受体抗体临床意义研究%Clinical significance of autoantibodies against phospholipase A2 receptor in adult patients with idiopathic membranous nephropathy

    Institute of Scientific and Technical Information of China (English)

    宋东旭; 王武涛; 林鹭; 郁胜强

    2015-01-01

    目的 探讨抗M型磷脂酶A2受体(PLA2R)抗体在诊断特发性膜性肾病(IMN)与判断病情中的作用.方法 以2012年6月至2013年6月第二军医大学长征医院肾内科经肾活检确诊为肾小球疾病的342例患者和30名健康成人为研究对象.肾小球疾病包括IMN 72例,存在继发因素的MN 13例,IgA肾病172例,系膜增生性肾炎47例,微小病变型肾病23例,局灶节段性肾小球硬化15例.收集患者及健康人群的临床资料,应用间接免疫荧光法检测其血清抗M型PLA2R抗体,并对抗体阳性和阴性的两组IMN患者年龄、24 h尿蛋白定量、血清白蛋白、血肌酐、尿素氮、总胆固醇、三酰甘油以及大量蛋白尿、低蛋白血症的发生率进行比较.结果 (1)57例(79.2%) IMN患者血清抗PLA2R抗体阳性;5例(38.5%)存在继发因素的MN患者抗体阳性;其他肾小球疾病和健康人群均阴性.(2) IMN血清抗体阳性和阴性两组患者在大量蛋白尿、低蛋白血症的分布方面差异有统计学意义(P<0.05),且高抗体滴度患者24 h尿蛋白定量更高,低蛋白血症更加严重,差异有统计学意义(P<0.01).结论 血清抗M型PLA2R抗体是反映IMN患者病情严重程度的一项重要指标.

  16. 膜性肾病肾小球IgG亚型沉积及磷脂酶A2受体表达的回顾性分析%IgG subclasses and phospholipase A2 receptor in glomerular deposits of membranous nephropathy: a retrospective analysis

    Institute of Scientific and Technical Information of China (English)

    董鸿瑞; 王艳艳; 王国勤; 孙丽君; 程虹; 谌贻璞

    2014-01-01

    目的 研究特发性膜性肾病(IMN)、狼疮性膜性肾病(LN-MN)及乙型肝炎病毒相关性膜性肾病(HBV-MN)患者肾小球IgG亚型分布及磷脂酶A2受体(PLA2R)表达情况.方法 用免疫荧光和(或)免疫组化染色检查2010年6月至2013年10月北京安贞医院肾内科患者肾小球IgG亚型及PLA2R.结果 (1)77例IMN IgG1、IgG2、IgG3及IgC4的阳性率分别为19.5%、37.7%、46.8%及97.4%,IgG,4阳性率显著高于其他亚型(P<0.01)且免疫荧光强度最强.15例LN-MN IgG1、IgG2、IgG3及IgG4的阳性率分别为73.3%、93.3%、66.7%及6.7%,IgG4阳性率显著低于其他亚型(P<0.01).8例HBV-MN IgG1、IgG2、IgG3及IgG4的阳性率分别为12.5%、62.5%、75.0%及12.5%.74例(96.1%)IMN PLA2R阳性,全部LN-MN及HBV-MN患者PLA2R阴性.72例(93.5%)IMN同时有IgG4沉积及PLA2R高表达.结论 IMN患者肾小球以IgG4沉积为主同时伴PLA2R高表达,而LN-MN及HBV-MN则以非IgG4的IgG亚型沉积为主,无PLA2R高表达.该检查可能有助于IMN与此2种继发性MN鉴别.

  17. Clinical significance of anti-M type phospholipase A2 receptor antibody in treatment of idiopathic membranous nephropathy%抗M型磷脂酶A2受体抗体在特发性膜性肾病治疗中的临床意义

    Institute of Scientific and Technical Information of China (English)

    袁莉; 施岚; 徐玉音; 吴亚君; 吴建华; 曹英杰; 黄新忠; 陈晓岚; 施辉

    2015-01-01

    目的 探讨抗M型磷脂酶A2受体(PLA2R)抗体在特发性膜性肾病(IMN)治疗中的临床意义.方法 纳入2013年4月至2014年6月间在南通大学附属医院住院并行肾活检的61例初治肾脏病患者,采用间接免疫荧光法检测血清抗PLA2R抗体的表达,并收集相关临床资料进行分析.结果 61例患者中,19例IMN患者血清抗PLA2R抗体阳性率为89.5%(17/19),42例非IMN患者血清抗PLA2R抗体阳性率为7.14% (3/42).17例抗PLA2R抗体呈阳性的IMN患者的抗体滴度为1 000±680,高于抗PLA2R抗体阳性非IMN患者的抗体滴度(P<0.05);就诊时IMN患者抗PLA2R抗体滴度与病程、尿蛋白量、血清白蛋白、血肌酐或内生肌酐清除率以及胆固醇水平无相关性(P>0.05).共14例IMN患者随访观察6个月,治疗早期抗PLA2R抗体降低幅度较尿蛋白更明显,同一时间点完全缓解者抗PLA2R抗体滴度阴性,治疗无效者抗体滴度最高;与高滴度组相比,低滴度组达到部分缓解的时间短[(1.16±0.41)个月vs(2.85±1.86)个月,P<0.05];相同用药方案治疗1个月后发现抗PLA2R抗体滴度与疗效呈负相关.结论 间接免疫荧光法检测血清抗PLA2R抗体在IMN中具有较高的阳性率.抗PIA2R抗体低滴度患者较早达到缓解,抗体持续高滴度提示可能需要更换治疗方案.抗PLA2R抗体对IMN的临床诊断、鉴别诊断以及判断IMN病情活动、制定治疗方案具有重要的临床意义和指导价值.

  18. Application of Immunohistochemistry and Immunofluorescence Staining in Detection of Phospholipase A2 Receptor on Paraffin Section of Renal Biopsy Tissue%免疫组织化学和免疫荧光染色在肾活检组织石蜡切片磷脂酶A2受体检测中的应用

    Institute of Scientific and Technical Information of China (English)

    董鸿瑞; 王艳艳; 王国勤; 孙丽君; 程虹; 谌贻璞

    2015-01-01

    目的 评估免疫组织化学和免疫荧光染色方法在肾活检组织石蜡切片磷脂酶A2受体(PLA2R)检测中的应用情况,寻找在肾组织中检测PLA2R准确、快捷的实验方法.方法 对193例肾活检组织石蜡切片进行PLA2R免疫组织化学染色,抗原修复方法为高压锅热修复加胰蛋白酶双修复法,其中包括特发性膜性肾病(IMN) 139例、膜型狼疮性肾炎15例、乙型肝炎病毒相关性膜性肾病8例、IgA肾病18例和微小病变13例.将其中22例肾组织PLA2R阳性IMN患者的肾组织石蜡切片分别给予高压锅热修复、高压锅热修复加胰蛋白酶双修复、水浴热修复和水浴热修复加胰蛋白酶双修复等4种抗原修复法,然后行PLA2R免疫组织化学染色,对染色效果进行比较.再将其中15例肾组织PLA2R阳性IMN患者的肾组织石蜡切片分别给予水浴热修复加胰蛋白酶双修复、蛋白酶K修复、胃蛋白酶修复等3种抗原修复法,然后行PLA2R免疫荧光染色,对染色效果进行比较.结果 193例标本中,IMN患者PLA2R免疫组织化学染色结果阳性率为90.6% (126/139);其他54例非IMN患者均为阴性.采用高压锅热修复加胰蛋白酶双修复法和水浴热修复加胰蛋白酶双修复时,22例患者的PLA2R免疫组织化学染色结果均为阳性;而采用高压锅热修复法或水浴热修复时,仅部分患者为阳性.采用水浴热修复加胰蛋白酶双修复、蛋白酶K修复和胃蛋白酶修复时,15例患者的PLA2R免疫荧光染色结果均为阳性,但水浴热修复加胰蛋白酶双修复为弥漫球性着色,荧光强度2+~3+,而蛋白酶K修复和胃蛋白酶修复为局灶节段性着色,荧光强度3+ ~4+.结论 肾组织PLA2R免疫组织化学染色可有效鉴别IMN与继发性MN.行免疫组织化学染色和免疫荧光染色时,首选水浴热修复加胰蛋白酶双修复法.

  19. Primary phospholipase C and brain disorders.

    Science.gov (United States)

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

    2016-05-01

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

  20. Oligomerization Inhibits Legionella pneumophila PlaB Phospholipase A Activity*

    OpenAIRE

    Kuhle, Katja; Krausze, Joern; Curth, Ute; Rössle, Manfred; Heuner, Klaus; Lang, Christina; Flieger, Antje

    2014-01-01

    The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity withi...

  1. Association between phospholipase production by Malassezia pachydermatis and skin lesions.

    Science.gov (United States)

    Cafarchia, C; Otranto, D

    2004-10-01

    An evaluation was made of the phospholipase activities of Malassezia pachydermatis strains isolated from healthy dogs versus those from dogs with dermatitis and otitis. A high percentage of strains of M. pachydermatis obtained from lesion sites (93.9%) produced phospholipase, compared to the strains obtained from healthy skin of the same dog with localized lesions (41.4%) and healthy dogs (10.6%). PMID:15472366

  2. Association between Phospholipase Production by Malassezia pachydermatis and Skin Lesions

    OpenAIRE

    Cafarchia, C.; OTRANTO, D.

    2004-01-01

    An evaluation was made of the phospholipase activities of Malassezia pachydermatis strains isolated from healthy dogs versus those from dogs with dermatitis and otitis. A high percentage of strains of M. pachydermatis obtained from lesion sites (93.9%) produced phospholipase, compared to the strains obtained from healthy skin of the same dog with localized lesions (41.4%) and healthy dogs (10.6%).

  3. Molecular diversity of phospholipase D in angiosperms

    Directory of Open Access Journals (Sweden)

    Cvrčková Fatima

    2002-02-01

    Full Text Available Abstract Background The phospholipase D (PLD family has been identified in plants by recent molecular studies, fostered by the emerging importance of plant PLDs in stress physiology and signal transduction. However, the presence of multiple isoforms limits the power of conventional biochemical and pharmacological approaches, and calls for a wider application of genetic methodology. Results Taking advantage of sequence data available in public databases, we attempted to provide a prerequisite for such an approach. We made a complete inventory of the Arabidopsis thaliana PLD family, which was found to comprise 12 distinct genes. The current nomenclature of Arabidopsis PLDs was refined and expanded to include five newly described genes. To assess the degree of plant PLD diversity beyond Arabidopsis we explored data from rice (including the genome draft by Monsanto as well as cDNA and EST sequences from several other plants. Our analysis revealed two major PLD subfamilies in plants. The first, designated C2-PLD, is characterised by presence of the C2 domain and comprises previously known plant PLDs as well as new isoforms with possibly unusual features-catalytically inactive or independent on Ca2+. The second subfamily (denoted PXPH-PLD is novel in plants but is related to animal and fungal enzymes possessing the PX and PH domains. Conclusions The evolutionary dynamics, and inter-specific diversity, of plant PLDs inferred from our phylogenetic analysis, call for more plant species to be employed in PLD research. This will enable us to obtain generally valid conclusions.

  4. Stalling autophagy: a new function for Listeria phospholipases

    Directory of Open Access Journals (Sweden)

    Ivan Tattoli

    2014-01-01

    Full Text Available Listeria monocytogenes is a Gram-positive bacterial pathogen that induces its own uptake in non-phagocytic cells. Following invasion, Listeria escapes from the entry vacuole through the secretion of a pore-forming toxin, listeriolysin O (LLO that acts to damage and disrupt the vacuole membrane. Listeria then replicates in the cytosol and is able to spread from cell-to-cell using actin-based motility. In addition to LLO, Listeria produces two phospholipase toxins, a phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB and a broad-range phospholipase C (PC-PLC, encoded by plcA, which contribute to bacterial virulence. It has long been recognized that secretion of PI- and PC-PLC enables the disruption of the double membrane vacuole during cell-to-cell spread, and those phospholipases have also been shown to augment LLO-dependent escape from the entry endosome. However, a specific role for Listeria phospholipases during the cytosolic stage of infection has not been previously reported. In a recent study, we demonstrated that Listeria PI-PLC and PC-PLC contribute to the bacterial escape from autophagy through a mechanism that involves direct inhibition of the autophagic flux in the infected cells [Tattoli et al. EMBO J (2013, 32, 3066-3078].

  5. Disruption of the Phospholipase D Gene Attenuates the Virulence of Aspergillus fumigatus

    OpenAIRE

    Li, Xianping; Gao, Meihua; Han, Xuelin; Tao, Sha; Zheng, Dongyu; Cheng, Ying; Yu, Rentao; Han, Gaige; Schmidt, Martina; Han, Li

    2012-01-01

    Aspergillus fumigatus is the most prevalent airborne fungal pathogen that induces serious infections in immunocompromised patients. Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, knowledge of the impact of phospholipases on A. fumigatus virulence is rather limited. In this study, disruption of the pld gene encoding phospholipase D (PLD), an important member of the phospholipase protei...

  6. Overexpression of Patatin-Related Phospholipase AIIIβ Altered the Content and Composition of Sphingolipids in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Maoyin eLi

    2014-10-01

    Full Text Available In plants, fatty acids are primarily synthesized in plastids and then transported to the endoplasmic reticulum for synthesis of most of the complex membrane lipids, including glycerolipids and sphingolipids. The first step of sphingolipid synthesis, which uses a fatty acid and a serine as substrates, is critical for sphingolipid homeostasis; its disruption leads to an altered plant growth. Phospholipase As (PLAs have been implicated in the trafficking of fatty acids from plastids to the endoplasmic reticulum. Previously, we found that overexpression of a patatin-related phospholipase, pPLAIIIβ, resulted in a smaller plant size and altered anisotropic cell expansion. Here, we determined the content and composition of sphingolipids in pPLAIIIβ-knockout and overexpression plants (pPLAIIIβ-KO and -OE. 3-keto-sphinganine, the product of the first step of sphingolipid synthesis, had a 26% decrease in leaves of pPLAIIIβ-KO while a 52% increase in pPLAIIIβ-OE compared to wild type (WT. The levels of free long-chain base species, dihydroxy-C18:0 and trihydroxy-18:0 (d18:0 and t18:0, were 38% and 97% higher, respectively, in pPLAIIIβ-OE than in WT. The level of complex sphingolipids ceramide d18:0-16:0 and t18:1-16:0 had a 2-fold increase in pPLAIIIβ-OE. The level of hydroxyceramide d18:0-h16:0 was 72% higher in pPLAIIIβ-OE compared to WT. The levels of several species of glucosylceramide and glycosylinositolphosphoceramide tended to be higher in pPLAIIIβ-OE than in WT. The total content of the complex sphingolipids showed a slightly higher in pPLAIIIβ-OE than in WT. These results revealed an involvement of phospholipase-mediated lipid homeostasis in plant growth.

  7. Alopecia in a viable phospholipase C delta 1 and phospholipase C delta 3 double mutant.

    Directory of Open Access Journals (Sweden)

    Fabian Runkel

    Full Text Available BACKGROUND: Inositol 1,4,5trisphosphate (IP(3 and diacylglycerol (DAG are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia, whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. METHODOLOGY/PRINCIPAL FINDINGS: We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3(mNab that resulted from the insertion of an intracisternal A particle (IAP into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3(mNab alleles are phenotypically normal. However, the presence of one Plcd3(mNab allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9olt1Pas and the Plcd3(mNab mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. CONCLUSIONS/SIGNIFICANCE: The Plcd3(mNab mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface.

  8. Anti-phospholipase A₂ receptor antibodies in recurrent membranous nephropathy.

    Science.gov (United States)

    Kattah, A; Ayalon, R; Beck, L H; Sethi, S; Sandor, D G; Cosio, F G; Gandhi, M J; Lorenz, E C; Salant, D J; Fervenza, F C

    2015-05-01

    About 70% of patients with primary membranous nephropathy (MN) have circulating anti-phospholipase A2 receptor (PLA2R) antibodies that correlate with disease activity, but their predictive value in post-transplant (Tx) recurrent MN is uncertain. We evaluated 26 patients, 18 with recurrent MN and 8 without recurrence, with serial post-Tx serum samples and renal biopsies to determine if patients with pre-Tx anti-PLA2R are at increased risk of recurrence as compared to seronegative patients and to determine if post-Tx changes in anti-PLA2R correspond to the clinical course. In the recurrent group, 10/17 patients had anti-PLA2R at the time of Tx versus 2/7 patients in the nonrecurrent group. The positive predictive value of pre-Tx anti-PLA2R for recurrence was 83%, while the negative predictive value was 42%. Persistence or reappearance of post-Tx anti-PLA2R was associated with increasing proteinuria and resistant disease in 6/18 cases; little or no proteinuria occurred in cases with pre-Tx anti-PLA2R and biopsy evidence of recurrence in which the antibodies resolved with standard immunosuppression. Some cases with positive pre-Tx anti-PLA2R were seronegative at the time of recurrence. In conclusion, patients with positive pre-Tx anti-PLA2R should be monitored closely for recurrent MN. Persistence or reappearance of antibody post-Tx may indicate a more resistant disease.

  9. Characterization and partial purification of phospholipase D from human placenta

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hansen, Harald S.

    1995-01-01

    We report the existence in the human placenta of a phosphatidylcholine- hydrolyzing phospholipase D (PLD) activity, which has been characterized and partially purified. Triton X-100 effectively solubilized PLD from the particulate fraction of human placenta in a dose-dependent manner. However...

  10. G protein activation stimulates phospholipase D signaling in plants

    NARCIS (Netherlands)

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

    1995-01-01

    We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than w

  11. Phospholipase C delta regulates germination of Dictyostelium spores

    NARCIS (Netherlands)

    Van Dijken, P.; Van Haastert, PJM

    2001-01-01

    Background: Many eukaryotes including plants and fungi make spores that resist severe environmental stress. The micro-organism Dictyostelium contains a single phospholipase C gene (PLC); deletion of the gene has no effect on growth cell movement and differentiation. In this report we show that PLC i

  12. Presenilin dependence of phospholipase C and protein kinase C signaling

    DEFF Research Database (Denmark)

    Dehvari, Nodi; Cedazo-Minguez, Angel; Isacsson, Ola;

    2007-01-01

    -stimulated phospholipase C (PLC) activity which was gamma-secretase dependent. To further evaluate the dependence of PLC on PSs we measured PLC activity and the activation of variant protein kinase C (PKC) isoforms in mouse embryonic fibroblasts (MEFs) lacking either PS1, PS2, or both. PLC activity and PKCalpha...

  13. Reduction of phospholipase D activity during coxsackievirus infection.

    NARCIS (Netherlands)

    Duijsings, D.; Wessels, E.; Emst-de Vries, S.E. van; Melchers, W.J.G.; Willems, P.H.G.M.; Kuppeveld, F.J.M. van

    2007-01-01

    During enterovirus infection, host cell membranes are rigorously rearranged and modified. One ubiquitously expressed lipid-modifying enzyme that might contribute to these alterations is phospholipase D (PLD). Here, we investigated PLD activity in coxsackievirus-infected cells. We show that PLD activ

  14. PI-PLC: Phosphoinositide-Phospholipase C in Plant Signaling

    NARCIS (Netherlands)

    T. Munnik

    2014-01-01

    Historically, phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate the second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG), which release Ca2+ from intracellular stores and activate member

  15. Substrate-enzyme interactions and catalytic mechanism in phospholipase C

    DEFF Research Database (Denmark)

    Byberg, J R; Jørgensen, Flemming Steen; Hansen, S;

    1992-01-01

    Based on the high-resolution X-ray crystallographic structure of phospholipase C from Bacillus cereus, the orientation of the phosphatidylcholine substrate in the active site of the enzyme is proposed. The proposal is based on extensive calculations using the GRID program and molecular mechanics...

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

    Science.gov (United States)

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

    2015-01-01

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

  17. Thyroxine signal transduction in liver cells involves phospholipase C and phospholipase D activation. Genomic independent action of thyroid hormone

    Directory of Open Access Journals (Sweden)

    Krasilnikova Oksana A

    2001-04-01

    Full Text Available Abstract Background Numerous investigations demonstrate a novel role of thyroid hormone as a modulator of signal transduction. Protein kinase C (PKC is critical to the mechanism by which thyroid hormones potentiate both the antiviral and immunomodulatory actions of IFNγ in different cells and regulate the exchange of signalling phospholipids in hepatocytes. Because nothing is known about accumulation of PKC modulator - diacylglycerol in cells treated with T4, we examined the nongenomic effect of thyroid hormones on DAG formation and phospholipase activation in liver cells. Results The results obtained provide the first demonstration of phospholipase C, phospholipase D and protein kinase C nongenomic activation and diacylglycerol (DAG accumulation by L-T4 in liver cells. The experiments were performed in either the [14C]CH3COOH-labeled rat liver slices or isolated hepatocytes pre-labeled by [14C]oleic acid. L-T4 activates the DAG production in a concentration- and time-dependent manner. DAG formation in stimulated cells is biphasic and short-lived event: there is an initial, rapid rise in DAG concentration and then a slower accumulation that can be sustained for a few minutes. The early phase of L-T4 generated DAG only is accompanied by phosphatidylinositol 4,5-bisphosphate level decrease and inositol 1,4,5-trisphosphate formation while the second phase is abolished by PKC inhibitor l,(5-isoquinolinesulphonyl2methylpiperasine dihydrochloride (H7 and propranolol. The second phase of DAG production is accompanied by free choline release, phosphatidylcholine content drop and phosphatidylethanol (Peth formation. Inhibitor of phospholipase-C-dependent phosphoinositide hydrolysis, neomycin sulfate, reduced the Peth as well as the DAG response to L-T4. Conclusions The present data have indicated the DAG signaling in thyroid hormone-stimulated liver cells. L-thyroxine activates a dual phospholipase pathway in a sequential and synchronized manner

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

    Science.gov (United States)

    Rahier, Renaud; Noiriel, Alexandre; Abousalham, Abdelkarim

    2016-01-01

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

  19. The effect of group X secreted phospholipase A2 on fertilization outcome is specific and not mimicked by other secreted phospholipases A2 or progesterone.

    Science.gov (United States)

    Abi Nahed, Roland; Escoffier, Jessica; Revel, Charlaine; Jeammet, Louise; Payré, Christine; Ray, Pierre F; Hennebicq, Sylviane; Lambeau, Gerard; Arnoult, Christophe

    2014-04-01

    Mouse group X sPLA2 (mGX) is an acrosomal protein playing an important role in fertilization and controlling acrosome reaction (AR) occurring during capacitation. We demonstrated previously that sperm from mGX knock-out mice had a severely impaired fertilization potential in vitro. We also showed that treatment of wild-type sperm with recombinant mGX during capacitation improved fertilization outcome. This interesting property suggests that sPLA2s could be used to improve fertilization in assisted reproductive technologies (ART). However the molecular mechanism explaining the mGX-dependent enhancing effect on fertilization outcome remains unclear so far. Interestingly, like progesterone (P4), mGX is a very potent activator of AR and the role of mGX-induced AR in fertilization outcome was not evaluated so far. To assess the role of sPLA2-induced AR in IVF, we first tested the potency of 9 mouse and 2 human sPLA2s and P4 to trigger AR of mouse sperm. We then tested the ability of 6 of these molecules (mouse Group IIA, mouse Group IID, mouse Group X, human Group V, human Group X and P4) to improve the yield of 2-cell embryos obtained by IVF in mouse. We showed that in the mouse neither P4 nor any of the other sPLA2s tested were able to mimic the IVF improvement produced by mGX-treatment. These results demonstrate that sPLA2s are not commutable in the context of mouse sperm fertility, indicating that their utilisation in other species, is subjected to the identification of probably unique species-specific active sPLA2.

  20. Phospholipases and the network of auxin signal transduction with ABP1 and TIR1 as two receptors: a comprehensive and provocative model

    Directory of Open Access Journals (Sweden)

    Günther F. E. Scherer

    2012-04-01

    Full Text Available Phospholipase D (PLD, secreted phospholipase A2 (sPLA2 and patatin-related phospholipase A (pPLA are important elements in auxin signal transduction. PLDζ2 has a function in auxin transport. PLD's potential link to upstream receptors ABP1 or TIR1, and to cytosolic calcium as an activator of PLDζ2, is outlined. A link from PLDζ2 to activation of PINOID, a kinase activating PIN proteins, is suggested. The activation mechanism of sPLA2, also involved in auxin transport-related functions, is unknown. New experiments show that not only ABP1 but also pPLA isoforms are tied to rapid activation of early auxin-induced genes, the functional domain of the other auxin receptor TIR1. Post-translational activation mechanisms for pPLAs are suggested to be tied to ABP1. We propose pPLAs and PLDζ2 are mediators in auxin signaling. The downstream targets of regulation by ABP1 as the receptor we propose to be primarily PIN proteins. This coordinates gene expression regulation by TIR1 in the nucleus. A clear separation of cytosolic mechanisms of auxin signalling is suggested with ABP1 as receptor, and phospholipases A and D and PIN proteins as downstream targets on the on hand, and TIR1 and regulation of early auxin-induced genes on the other. At the same time, this separation is coordinated by auxin transport creating the auxin concentration in the nucleus suitable for gene regulation.

  1. Phospholipase gene expression during Paracoccidioides brasiliensis morphological transition and infection

    Directory of Open Access Journals (Sweden)

    Deyze Alencar Soares

    2013-09-01

    Full Text Available Phospholipase is an important virulence factor for pathogenic fungi. In this study, we demonstrate the following: (i the Paracoccidioides brasiliensis pld gene is preferentially expressed in mycelium cells, (ii the plb1 gene is mostly up-regulated by infection after 6 h of co-infection of MH-S cells or during BALB/c mice lung infection, (iii during lung infection, plb1, plc and pld gene expression are significantly increased 6-48 h post-infection compared to 56 days after infection, strongly suggesting that phospholipases play a role in the early events of infection, but not during the chronic stages of pulmonary infection by P. brasiliensis.

  2. Phospholipase C-ε Regulates Epidermal Morphogenesis in Caenorhabditis elegans

    OpenAIRE

    Vázquez-Manrique, Rafael P.; Nagy, Anikó I.; Legg, James C.; Bales, Olivia A.M.; Ly, Sung; Baylis, Howard A.

    2008-01-01

    Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP3)-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP3 production is stimulated is unknown. IP3 is produced by the action of phospholipase C (PLC). We therefore sur...

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

    OpenAIRE

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

    1995-01-01

    Dopamine and D1 agonists and NE all increase phosphatidyl inositol-specific phospholipase C (PLC) activity, but whereas dopamine produces a natriuresis, NE has an antinatriuretic effect. To determine if catecholamines differentially regulate the expression of PLC isoforms, we infused fenoldopam, a D1 agonist, or pramipexole, a D1/D2 agonist, intravenously or infused fenoldopam or NE into the renal artery of anesthetized rats. After 3-4 h of infusion, when the expected natriuresis (fenoldopam ...

  4. Aberrant accumulation of phospholipase C-delta in Alzheimer brains.

    OpenAIRE

    Shimohama, S.; Homma, Y.; Suenaga, T.; Fujimoto, S; Taniguchi, T; Araki, W.; Yamaoka, Y; Takenawa, T.; Kimura, J

    1991-01-01

    Since phosphoinositide-specific phospholipase C (PLC) is one of the key molecules in signal transduction, the authors assessed its involvement in Alzheimer's disease (AD). Immunostaining of a specific antibody against the PLC isozyme, PLC-delta, demonstrated that this enzyme was abnormally accumulated in neurofibrillary tangles (NFT), the neurites surrounding senile plaque (SP) cores, and neuropil threads in AD brains. Western blot analysis confirmed that PLC-delta was concentrated in the pai...

  5. Enzymkinetik von Phospholipase C und Aggregationsverhalten von Gentransfer-Komplexen

    OpenAIRE

    Galneder, Reinhard Josef

    2007-01-01

    Im Rahmen dieser Arbeit wurde ein Experiment zur Laserfallen-kontrollierten Mikroelektrophorese aufgebaut und getestet, das zeitaufgelöste Messungen des Oberflächenpotentials an einzelnen kolloidalen Teilchen ermöglichte. Mit dieser Methode konnte im folgenden die Enzymkinetik von Phospholipase C mit einer bisher nicht erreichten Zeitauflösung von ca. 1 sec gemessen werden. Dazu wurden Silika-Kugeln mit einem Radius von 500 nm mit einer Lipidmembran aus neutralem Phosphatidylcholin und dem ne...

  6. Phospholipase C in Beijing strains of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    N Faramarzi

    2010-12-01

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

  7. Phospholipase C in Beijing strains of Mycobacterium tuberculosis

    Science.gov (United States)

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

    2010-01-01

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

  8. Melittin stimulates phosphoinositide hydrolysis and placental lactogen release: Arachidonic acid as a link between phospholipase A sub 2 and phospholipase C signal-transduction pathways

    Energy Technology Data Exchange (ETDEWEB)

    Zeitler, P.; Handwerger, S. (Univ. of Cincinnati College of Medicine, OH (USA)); Wu, Y.Q. (Duke Univ. Medical Center, Durham, NC (USA))

    1991-01-01

    Previous investigations from this laboratory have implicated both phospholipase A{sub 2} and phospholipase C in the regulation of human placental lactogen release from human trophoblast. To study further the role of endogenous phospholipase A{sub 2} and the relationship between phospholipase A{sub 2} activation and phosphoinositide metabolism, the authors examined hPL and ({sup 3}H)-inositol release from trophoblast cells in response to agents that stimulate or inhibit the endogenous enzyme. Melittin stimulated rapid, dose-dependent, and reversible increases in the release of hPL, prostaglandin E, and ({sup 3}H)-inositol. Mepacrine inhibited this stimulation. However, mepacrine had no effect on the stimulation of hPL and ({sup 3}H)-inositol release by exogenous arachidonic acid (AA). These results indicate that the stimulation by melittin of phosphoionsitide metabolism and hPL release is mediated by initial activation of phospholipase A{sub 2}. Furthermore, the results support the possibility that AA, released as a consequence of phospholipase A{sub 2} activation, can act as a second messenger linking the two phospholipase pathways.

  9. AGN 190383, a novel phospholipase inhibitor with topical anti-inflammatory activity.

    Science.gov (United States)

    De Vries, G W; Lee, G; Amdahl, L; Wenzel, M; Garst, M; Wheeler, L A

    1991-09-01

    AGN 190383 is a 5-hydroxy-2(5H)-furanone ring analog of the marine natural product manoalide. When applied topically, AGN 190383 inhibits phorbol ester induced mouse ear edema. It is a potent inhibitor of bee venom phospholipase A2 and blocks the release of arachidonic acid from calcium ionophore A23187 stimulated human neutrophils. AGN 190383 also inhibits both hormone-operated and depolarization-dependent calcium mobilization in GH3 cells, as well as fMLP stimulated increases in free cytosolic calcium in human PMNs. Furthermore, it is also able to block the release of the neutral protease elastase from stimulated neutrophils. The effects of AGN 190383 on arachidonic acid metabolism and leukocyte function may account, in part, for its anti-inflammatory activity in vivo.

  10. Novel Metagenome-Derived, Cold-Adapted Alkaline Phospholipase with Superior Lipase Activity as an Intermediate between Phospholipase and Lipase

    OpenAIRE

    Lee, Mi-Hwa; Oh, Ki-Hoon; Kang, Chul-Hyung; Kim, Ji-Hoon; Oh, Tae-Kwang; Ryu, Choong-Min; Yoon, Jung-Hoon

    2012-01-01

    A novel lipolytic enzyme was isolated from a metagenomic library obtained from tidal flat sediments on the Korean west coast. Its putative functional domain, designated MPlaG, showed the highest similarity to phospholipase A from Grimontia hollisae CIP 101886, though it was screened from an emulsified tricaprylin plate. Phylogenetic analysis showed that MPlaG is far from family I.6 lipases, including Staphylococcus hyicus lipase, a unique lipase which can hydrolyze phospholipids, and is more ...

  11. Roles of Cytoplasmic Phospholipase in Expression of the Antimicrobial Activity of Host Macrophages against Mycobacterium tuberculosis Infection

    OpenAIRE

    佐野, 千晶; Yasumoto, Ko; 多田納, 豊; 清水, 利朗; 山部, 清子; 富岡, 治明

    2010-01-01

    We studied roles of phospholipase A2 (PLA2) isozymes, including type IIA secretory PLA2 (sPLA2-IIA), type IV cytosolic, Ca2+-dependent PLA2 (cPLA2) , type V secretory PLA2 (sPLA2-V) , and type VI cytosolic, Ca2+-independent PLA2 (iPLA2) , in macrophage ( Mφ) antimicrobial activity against Mycobacterium tuberculosis (Mtb) H37Ra (avirulent) strain and Mφ mRNA expression of these PLA2 isotypes in response to infection with the microorganisms. First, a cPLA2 inhibitor arachidonyl trifluorometh...

  12. Dynamic surface activity of a fully synthetic phospholipase-resistant lipid/peptide lung surfactant.

    Directory of Open Access Journals (Sweden)

    Frans J Walther

    Full Text Available BACKGROUND: This study examines the surface activity and resistance to phospholipase degradation of a fully-synthetic lung surfactant containing a novel diether phosphonolipid (DEPN-8 plus a 34 amino acid peptide (Mini-B related to native surfactant protein (SP-B. Activity studies used adsorption, pulsating bubble, and captive bubble methods to assess a range of surface behaviors, supplemented by molecular studies using Fourier transform infrared (FTIR spectroscopy, circular dichroism (CD, and plasmon resonance. Calf lung surfactant extract (CLSE was used as a positive control. RESULTS: DEPN-8+1.5% (by wt. Mini-B was fully resistant to degradation by phospholipase A(2 (PLA(2 in vitro, while CLSE was severely degraded by this enzyme. Mini-B interacted with DEPN-8 at the molecular level based on FTIR spectroscopy, and had significant plasmon resonance binding affinity for DEPN-8. DEPN-8+1.5% Mini-B had greatly increased adsorption compared to DEPN-8 alone, but did not fully equal the very high adsorption of CLSE. In pulsating bubble studies at a low phospholipid concentration of 0.5 mg/ml, DEPN-8+1.5% Mini-B and CLSE both reached minimum surface tensions <1 mN/m after 10 min of cycling. DEPN-8 (2.5 mg/ml+1.5% Mini-B and CLSE (2.5 mg/ml also reached minimum surface tensions <1 mN/m at 10 min of pulsation in the presence of serum albumin (3 mg/ml on the pulsating bubble. In captive bubble studies, DEPN-8+1.5% Mini-B and CLSE both generated minimum surface tensions <1 mN/m on 10 successive cycles of compression/expansion at quasi-static and dynamic rates. CONCLUSIONS: These results show that DEPN-8 and 1.5% Mini-B form an interactive binary molecular mixture with very high surface activity and the ability to resist degradation by phospholipases in inflammatory lung injury. These characteristics are promising for the development of related fully-synthetic lipid/peptide exogenous surfactants for treating diseases of surfactant deficiency or dysfunction.

  13. Increased phospholipase a activity and formation of communicative contacts between Acanthamoeba castellanii cells

    NARCIS (Netherlands)

    Hax, W.M.A.; Demel, R.A.; Spies, F.; Vossenberg, J.B.J.; Linnemans, W.A.M.

    1974-01-01

    1. 1. Exogenous 1-palmitoyl-sn-glycero-3-phosphorylcholine becomes incorporated into the membrane of A. castellanii within 2 min of incubation. 2. 2. Homogenates of A. castellanii are shown to contain phospholipase A activity. 3. 3. The phospholipase A activity is dependent on the population density

  14. 实验性急性出血坏死性胰腺炎引发胰性脑组织损害及纳屈酮治疗作用%The releationship between ox ygen free radical and Phospholipase A2 and therapeutic effect of Naltrexone on exp erimental pancreatic cerebral harm induced by AHNP in Rats

    Institute of Scientific and Technical Information of China (English)

    赵海平; 王万祥; 刘淑平; 欧阳晓晖; 杨成旺; 孔广忠; 寿乃延

    2000-01-01

    目的:探讨大鼠急性出血坏死性胰腺炎(AHNP)引发胰性脑组织损害(PCH)机理及评价纳屈酮治疗效果.方法:应用5%牛磺胆酸钠逆行胰胆管注射诱发大鼠AHNP并发脑组织损害模型 .实验分为对照组、PCH组及纳屈酮(NTX)治疗组;分别于6、12、24h检测血浆及脑组织丙二醛(MDA)、磷脂酶A2(PLA2)、超氧化物歧化酶(SOD)、胰腺和脑组织病理学改变.结果:PCH组6、12、24h血浆及脑组织较对照组:MDA、PLA2升高;SOD下降.PCH组光镜下6、12、24h分别出现脑组织水肿、点状出血、脱髓鞘改变.NTX治疗组较PCH组:血浆及脑组织MDA、PLA2下降及SOD升高;而且胰腺及脑组织损害减轻.结论:大鼠AHNP模型可引发PCH;NTX可降低血浆及脑组织氧自由基、PLA2 ,同时增加SOD,进而减轻脑组织损害.

  15. Influence of (phospho)lipases on properties of mica supported phospholipid layers

    International Nuclear Information System (INIS)

    The effect of enzymes: lipase from Candida cylindracea (LCc), phospholipase A2 from hog pancreas (PLA2) and phospholipase C from Bacillus cereus (PLC) to modulate wetting properties of solid supported phospholipid bilayers was studied via advancing and receding contact angle measurements of water, formamide and diiodomethane, and calculation of the surface free energy and its components from van Oss et al. (LWAB) and contact angle hysteresis (CAH) approaches. Simultaneously, topography of the studied layers was determined by Atomic Force Microscopy (AFM). The investigated lipid bilayers were transferred on mica plates from subphase of pure water by means of Langmuir-Blodgett and Langmuir-Schaefer techniques. The investigated phospolipid layers were: saturated DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), unsaturated DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and their mixture DPPC/DOPC. The obtained results revealed that the lipid membrane degradation by the enzymes caused increase in its surface free energy due to the amphiphilic hydrolysis products, which may accumulate in the lipid bilayer. In result activity of the enzymes may increase and then break down the bilayer structure takes place. It is likely that after dissolution of the hydrolysis reaction products in the bulk phase, patches of bare mica surface are accessible, which contribute to the apparent surface free energy changes. Comparison of AFM images and the free energy changes of the layers gives better insight into changes of their properties. The observed gradual increase in the layer surface free energy allows controlling of the hydrolysis process to obtain the surfaces of defined properties.

  16. Vasopressin-stimulated Ca2+ spiking in vascular smooth muscle cells involves phospholipase D.

    Science.gov (United States)

    Li, Y; Shiels, A J; Maszak, G; Byron, K L

    2001-06-01

    Physiological concentrations of [Arg(8)]vasopressin (AVP; 10-500 pM) stimulate oscillations of cytosolic free Ca2+ concentration (Ca2+ spikes) in A7r5 vascular smooth muscle cells. We previously reported that this effect of AVP was blocked by a putative phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (5 microM). In the present study, the products of PLA2, arachidonic acid (AA), and lysophospholipids were found to be ineffective in stimulating Ca2+ spiking, and inhibitors of AA metabolism did not prevent AVP-stimulated Ca2+ spiking. Thin layer chromatography was used to monitor the release of AA and phosphatidic acid (PA), which are the products of PLA2 and phospholipase D (PLD), respectively. AVP (100 pM) stimulated both AA and PA formation, but only PA formation was inhibited by ONO-RS-082 (5 microM). Exogenous PLD (type VII; 2.5 U/ml) stimulated Ca2+ spiking equivalent to the effect of 100 pM AVP. AVP stimulated transphosphatidylation of 1-butanol (a PLD-catalyzed reaction) but not 2-butanol, and 1-butanol (but not 2-butanol) completely prevented AVP-stimulated Ca2+ spiking. Protein kinase C (PKC) inhibition, which completely prevents AVP-stimulated Ca2+ spiking, did not inhibit AVP-stimulated phosphatidylbutanol formation. These results suggest that AVP-stimulated Ca2+ spiking depends on activation of PLD rather than PLA2 and that PKC activation may be downstream of PLD in the signaling cascade.

  17. Soybean phospholipase D activity determination. A comparison of two methods

    Directory of Open Access Journals (Sweden)

    Ré, E.

    2007-09-01

    Full Text Available Due to a discrepancy between previously published results, two methods to determine the soybean phospholipase D activity were evaluated. One method is based on the extraction of the enzyme from whole soybean flour, quantifying the enzyme activity on the extract. The other method quantifies the enzymatic activity on whole soybean flour without enzyme extraction. In the extraction-based-method, both the extraction time and the number of extractions were optimized. The highest phospholipase D activity values were obtained from the method without enzyme extraction. This method is less complex, requires less running-time and the conditions of the medium in which phospholipase D acts resemble the conditions found in the oil industrySe evaluaron dos métodos para determinar la actividad de la fosfolipasa D en soja debido a que existe discrepancia entre los resultados publicados. Un método se basa en la extracción de la enzima de la harina resultante de la molienda del grano de soja entero, cuantificando la actividad sobre el extracto. En el otro método, la cuantificación se realiza sobre la harina del grano entero molido, sin extraer la enzima. En el método de extracción se optimizaron tanto el tiempo como el número de extracciones. Los mayores valores de actividad de la fosfolipasa D se obtuvieron por el método sin extracción de la enzima. Este método es más simple, exige menos tiempo de ejecución y las condiciones del medio en que actúa la fosfolipasa D se asemejan a las condiciones encontradas en la industria aceitera.

  18. Effect of Atra on the Expression of CyclinD3 and Lipoprotein-associated phospholipase A2 in injury nidus of carotid endothelium in rabbits%ARTA对兔颈动脉内膜损伤病灶CydinD3及LP-PLA2的影响

    Institute of Scientific and Technical Information of China (English)

    胡真真; 董果雄; 张社华; 张滨; 姚如永

    2012-01-01

    目的 观察全反式维甲酸(ARTA)对兔颈动脉内膜损伤与修复病灶内膜增生和细胞周期素CyclinD3及炎性相关因子LP-PLA2表达的影响和机制.方法 将54只健康新西兰大白兔随机分为9组(n=6):正常对照组(A、B、C)、高脂饮食组(A、B、C)、治疗组(A、B、C).普通饮食后正常对照组仅暴露颈动脉;高脂饮食组,暴露颈动脉并损伤内膜;治疗组用ARTA灌胃,其他操作同高脂饮食组.术后1周、2周、4周处死A、B、C组动物,对病变血管行形态学观察和测定,分别用ELISA和免疫组化方法检测颈动脉内膜损伤与修复病灶中LP-PLA2和CyclinD3的表达水平.结果 ①正常对照组LP-PLA2仅有微量表达.CyclinD3胞浆呈弱阳性表达,胞核呈阴性表达.②高脂饮食组在术后7天内膜开始增生,28天后明显增生,出现泡沫细胞、脂质条纹、管腔狭窄.LP-PLA2和CyclinD3表达水平较正常组显著增高.③治疗组LP-PLA2和CyclinD3表达水平明显低于高脂饮食组(P<0.05).内膜的增生和管腔的狭窄显著减轻(P<0.05).结论 ARTA可以通过抑制LP-PLA2和CyclinD3的表达,抑制内膜的炎症反应和血管平滑肌细胞的增生,从而抑制颈动脉内膜损伤病灶的过度增生和管腔狭窄.%Objective To observe the influence of all-trans retinoic acid( ATRA) on the process of neointimal formation and the expression of CyclinD3 and Lipoprotein-associated phos pholipase A2 in injury nidus of carotid endothelium in rabbits. Methods Fifty-six healthy male New-Zealand rabbits were randomly divided in to 9 groups(=6): normal control groups(A, B, C), high-fat groups(A, B, C), treatment groups(A, B, C). There were 6 rabbits in each group. The normal control groups were fed with conventional chow, and underwent surgery without endothelium injury. The high-fat groups were fed with high cholesterol chowand. subjected to the carotid endothelial injury by air desiccation. The treatment groups were administered ATRA

  19. Phospholipase C associated with particulate fractions of bovine brain.

    OpenAIRE

    Lee, K Y; Ryu, S H; Suh, P G; Choi, W C; Rhee, S G

    1987-01-01

    We previously reported that cytosolic fractions of bovine brain contain two immunologically distinct phosphoinositide-specific phospholipases C (PLCs), PLC-I and PLC-II. In this report the subcellular distribution of PLC-I and PLC-II in brain homogenates was measured using RIA. Significant differences were found in the distribution of the two forms of PLC in 100,000 X g supernatants (cytosolic fraction) of brain homogenized in hypotonic buffer and 2 M KCl extracts of washed pellets (particula...

  20. Phospholipase C Is Involved in Kinetochore Function in Saccharomyces cerevisiae

    OpenAIRE

    Lin, Hongyu; Choi, Jae H.; Hasek, Jiri; DeLillo, Nicholas; Lou, Willard; Vancura, Ales

    2000-01-01

    The budding yeast PLC1 gene encodes a homolog of the δ isoform of mammalian phosphoinositide-specific phospholipase C. Here, we present evidence that Plc1p associates with the kinetochore complex CBF3. This association is mediated through interactions with two established kinetochore proteins, Ndc10p and Cep3p. We show by chromatin immunoprecipitation experiments that Plc1p resides at centromeric loci in vivo. Deletion of PLC1, as well as plc1 mutations which abrogate the interaction of Plc1p...

  1. Gestational alterations in phospholipase c coupled muscarinic response

    Energy Technology Data Exchange (ETDEWEB)

    Varol, F.G.; Hadjiconstantinou, M.; Zuspan, F.P.; Neff, N.H. (Ohio State Univ. college of Medicine, Columbus (USA))

    1989-01-01

    In the pregnant rat, carbachol-induced phosphoinositol hydrolysis by myometrium at the placental attachment region progressively decreased toward term, whereas hydrolysis was relatively stable in the myometrium of the non-attachment region. Tritium-quinuclidinyl benzilate binding increased in the myometrium of non-attachment regions as pregnancy progressed. At placental attachment sites binding remained relatively stable until parturition when it increased. Apparently the myometrium associated with the placental attachment site is less sensitive to cholinergic influence during pregnancy compared with the non-attachment site when evaluated by muscarinic activation of phospholipase C or ligand binding.

  2. Phorbol ester and vasopressin activate phospholipase D in Leydig cells

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hansen, Harald S.

    1991-01-01

    In the present study evidence is provided for the existence of phospholipase D (PLD) activity in rat Leydig cells. Leydig cells were cultured and labelled with [H]myristic acid. In the presence of ethanol, phorbol 12-myristate 13-acetate (PMA) stimulated the formation of [H]phosphatidylethanol ([...... support the notion that activation of PLD by PMA is dependent on PKC. Arginine vasopressin (AVP) caused a rapid stimulation of PLD activity in the cells. This activation was inhibited after downregulation of PKC, indicating that the agonist acts by a mechanism similar to that of PMA....

  3. PROFILIN ACTIVATES BACILLUS THURINGIENSIS PHOSPHOINOSITIDE SPECIFIC PHOSPHOLIPASE C

    Directory of Open Access Journals (Sweden)

    Sandeepta Burgula

    2012-08-01

    Full Text Available Many extracellular signaling molecules including hormones, growth factors, neurotransmitters andimmunoglobulins elicit intracellular responses by activating phosphatidylinositol-specific phospholipase C (PI-PLCupon binding to their cell surface receptors. Activated PLC catalyses the hydrolysis of Phosphotidylinositol 4,5-bisphosphate (PIP2 to generate DAG and IP3 , which act as signaling molecules that control various cellular processes.Exploring the mechanism of regulation of PLC activity may lead to understanding various signaling events thatregulate cell growth and differentiation. One of the dramatic effects of profilin is inhibition of PIP2 hydrolysis by PLC-γ in eukaryotic cells. In the present study, the effect of profilin on Phosphotidylinositol specific phospholipase C (PI-PLC purified from Bacillus thuringiensis (Bt was examined. Assay of PI-PLC activity indicated that Bovine profilinactivated the hydrolysis of phosphotidylinositol (PI by BtPI-PLC in a concentration dependent manner under in vitroconditions. A 250 % increase in activity was noted in the presence of profilin but not in presence of phosphoprofilin. Inthe presence of profilin more proteins are observed in the soluble fraction. In conclusion it can be stated that thatprofilin activates bacterial PLC activity towards PI hydrolysis

  4. Role of Phospholipases in Fungal Fitness, Pathogenicity and Drug Development- Lessons from Cryptococcus neoformans

    Directory of Open Access Journals (Sweden)

    Julianne eDjordjevic

    2010-11-01

    Full Text Available Many pathogenic microbes, including many fungi, produce phospholipases which facilitate survival of the pathogen in vivo, invasion and dissemination throughout the host, expression of virulence traits and evasion of host immune defense mechanisms. These phospholipases are either secreted or produced intracellularly and act by physically disrupting host membranes, and/or by affecting fungal cell signaling and production of immunomodulatory effectors. Many of the secreted phospholipases acquire a glycosylphosphatidylinositol (GPI sorting motif to facilitate membrane and/or cell wall association and secretion. This review focuses primarily on the role of two members of the phospholipase enzyme family, phospholipase B (Plb and phosphatidylinositol (PI-specific phospholipase C (PI-PLC/Plc, in fungal pathogenesis and in particular, what has been learnt about their function from studies performed in the model pathogenic yeast, Cryptococcus neoformans. These studies have revealed how Plb has adapted to become an important part of the virulence repertoire of pathogenic fungi and how its secretion is regulated. They have also provided valuable insight into how the intracellular enzyme, Plc1, contributes to fungal fitness and pathogenicity - via a putative role in signal transduction pathways that regulate the production of stress-protecting pigments, polysaccharide capsule, cell wall integrity and adaptation to growth at host temperature. Finally, this review will address the role fungal phospholipases have played in the development of a new class of antifungal drugs, which mimic their phospholipid substrates.

  5. Inhibitors of secreted phospholipase A2 suppress the release of PGE2 in renal mesangial cells.

    Science.gov (United States)

    Vasilakaki, Sofia; Barbayianni, Efrosini; Magrioti, Victoria; Pastukhov, Oleksandr; Constantinou-Kokotou, Violetta; Huwiler, Andrea; Kokotos, George

    2016-07-01

    The upregulation of PGE2 by mesangial cells has been observed under chronic inflammation condition. In the present work, renal mesangial cells were stimulated to trigger a huge increase of PGE2 synthesis and were treated in the absence or presence of known PLA2 inhibitors. A variety of synthetic inhibitors, mainly developed in our labs, which are known to selectively inhibit each of GIVA cPLA2, GVIA iPLA2, and GIIA/GV sPLA2, were used as tools in this study. Synthetic sPLA2 inhibitors, such as GK115 (an amide derivative based on the non-natural amino acid (R)-γ-norleucine) as well as GK126 and GK241 (2-oxoamides based on the natural (S)-α-amino acid leucine and valine, respectively) presented an interesting effect on the suppression of PGE2 formation. PMID:27234891

  6. Liposomes containing alkylated methotrexate analogues for phospholipase A(2) mediated tumor targeted drug delivery

    DEFF Research Database (Denmark)

    Kaasgaard, Thomas; Andresen, Thomas Lars; Jensen, Simon Skøde;

    2009-01-01

    Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO III and HT-29 human colon cancer cells. Both analogues contained a C-16-alkyl chain attached to the gamma-carboxylic acid and one of the analogues had an additional benzyl group attached to the...... tightly anchored to the liposomal carrier. Also, the developed DSC-assay for Studying the anchoring stability of alkylated drugs will be a useful tool in the development of liposomal drug delivery Systems....... of the MTX-liposomes against KATO III and HT-29 cancer cells was found to be independent of sPLA(2) hydrolysis, indicating that the alkylated MTX-analogue was available for cancer cell uptake even in the absence of liposome hydrolysis. Using a DSC based method for assessing the anchoring stability...... of alkylated compounds in liposomes, it was demonstrated that the MTX-analogue partitioned into the water phase and thereby became available for cell uptake. It was concluded that liposomes containing alkylated MTX-analogues show promise as a drug delivery system, although the MTX-analogue needs to be more...

  7. Validation of a phospholipase A2 receptor antibody ELISA in an Australian cohort with membranous glomerulonephritis.

    Science.gov (United States)

    Ong, Lawrence; Silvestrini, Roger; Chapman, Jeremy; Fulcher, David A; Lin, Ming Wei

    2016-04-01

    A commercial PLA2R Ab ELISA was validated by examining its ability to distinguish primary from secondary membranous nephropathy, correlating results with clinical markers of disease activity, and comparing its performance with an indirect immunofluorescence test (IIFT). PLA2R Ab levels were measured in 77 patients with biopsy proven membranous nephropathy, divided into either idiopathic (n = 61) or secondary groups (n = 6). In the idiopathic group, measures of contemporaneous disease activity (proteinuria, serum creatinine) were compared between seropositive and seronegative subjects. ELISA values were then compared with semi-quantitative results from an IIFT using PLA2R transfected HEK293 cells as substrate. The PLA2R Ab ELISA was positive in only 15 of 61 (25%) patients with idiopathic membranous nephropathy (IMN), but there was a significant negative relationship with time since diagnosis. Thus, in a subgroup of patients diagnosed within 6 months of analysis, the sensitivity was 6/15 (55%), rising to 6/8 (75%) in those recently-diagnosed patients who had not been treated. In the entire cohort, there was a significant positive correlation between ELISA values and degree of proteinuria, but our analysis did not control for variation of both variables with time. The PLA2R Ab ELISA also showed very high agreement with IIFT (96%). Therefore, the PLA2R Ab ELISA is a highly specific test for distinguishing primary from secondary membranous nephropathy that is most sensitive in newly diagnosed patients who have not received immunosuppression. Antibody levels correlated with degree of proteinuria, but this relationship was not shown to be independent of time. Both IIFT and ELISA platforms performed comparably.

  8. Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release

    DEFF Research Database (Denmark)

    Arouri, Ahmad; Trojnar, Jakub; Schmidt, Steffen;

    2015-01-01

    models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we...

  9. Secretory phospholipase A2-mediated neuronal cell death involves glutamate ionotropic receptors

    DEFF Research Database (Denmark)

    de Turco, Elena B; Diemer, Nils Henrik; Bazan, Nicolas G;

    2002-01-01

    To define the significance of glutamate ionotropic receptors in sPLA -mediated neuronal cell death we used the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist PNQX. In primary neuronal cell cultures both MK-801 and PNQX inhibited sPLA - and glutamate-induced neuronal death. [ H]A...

  10. Glutamate signalling and secretory phospholipase A2 modulate the release of arachidonic acid from neuronal membranes

    DEFF Research Database (Denmark)

    Rodriguez De Turco, Elena B; Jackson, Fannie R; DeCoster, Mark A;

    2002-01-01

    secretory PLA(2) (sPLA(2)) from bee venom (bv sPLA(2)) and Taipan snake venom (OS2) elicit synergy in inducing neuronal cell death. Low concentrations of sPLA(2) are selective ligands of cell-surface sPLA(2) receptors. We investigated which neuronal arachidonoyl phospholipids are targeted by glutamate...

  11. Renaturation and one step purification of the chicken GIIA secreted phospholipase A2 from inclusion bodies.

    Science.gov (United States)

    Karray, Aida; Amara, Sawsan; Carrière, Frédéric; Gargouri, Youssef; Bezzine, Sofiane

    2014-06-01

    The cDNA coding for a mature protein of 123 amino acids, containing all of the structural features of catalytically active group IIA sPLA2, has been amplified from chicken intestine. The gene has been cloned into the bacterial expression vector pET-21a(+), which allows protein over-expression as inclusion bodies and enables about 3mg/l of pure refolded fully active enzyme to be obtained. Recombinant expression of chicken intestinal sPLA2-IIA (ChPLA2-IIA) in Escherichia coli shows that the enzyme is Ca(2+) dependent, maximally active at pH 8-9, and hydrolyses phosphatidylglycerol versus phosphatidylcholine with a 10-fold preference. Indeed, we report in this work, a comparative kinetic study between the wild type and the recombinant ChPLA2-IIA, on zwitterionic head group phospholipids (DDPC) and negatively charged phospholipids (POPG) using the monomolecular film technique. The ability to express reasonably large amounts of the sPLA2 Group IIA, compared to that obtained with the classical purification will provide a basis for future site directed mutagenesis studies of this important enzyme.

  12. Human secretory phospholipase A(2), group IB in normal eyes and in eye diseases

    DEFF Research Database (Denmark)

    Prause, Jan U; Bazan, Nicolas G; Heegaard, Steffen;

    2007-01-01

    , retinitis pigmentosa and glaucoma were evaluated. RESULTS: Expression of hGIB was found in various cells of the eye. The most abundant expression was found in retinal pigment epithelium (RPE) cells, the inner photoreceptor segments, ganglion cells and the corneal endothelium. We explored diseases involving...

  13. Phospholipase A_2 changes and its significance on brain tissue of rat in severe acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Multiple systemorgan failure is often compli-cated with SAP and PLA2could play an i mportantrole in the study of brain damages.Through thestudy of makingthe rat SAP model inthis study,thesignificance of PLA2on brain damages was surveyedand reported.Materials and methods1 The rat SAP model and its classificationEighty male Sprague-Dawley rats,weight(300±30)g,were randomly divided into4groups:thecontrol group,the sham-operation group,the SAPgroup and the treat ment group of SAP.The ratswere not given food but...

  14. Synthesis of structured phospholipids by immobilized phospholipase A2 catalyzed acidolysis

    DEFF Research Database (Denmark)

    Vikbjerg, Anders Falk; Xu, Xuebing

    2007-01-01

    and maintain catalytic activity; however the final choice of carrier for the continued work was a non-ionic weakly polar macroreticular resin. Response surface methodology was applied to evaluate the influence of substrate ratio, reaction temperature and water addition during acidolysis reaction between...... caprylic acid and soybean phosphatidylcholine (PC). Reaction temperature and water addition had significant effect on acidolysis reaction, however no effect was observed for substrate ratio (mol caprylic acid/mol PC) in range tested. In general an inverse relationship between incorporation of caprylic acid...

  15. Molecular modeling and snake venom phospholipase A2 inhibition by phenolic compounds: Structure-activity relationship.

    Science.gov (United States)

    Alam, Md Iqbal; Alam, Mohammed A; Alam, Ozair; Nargotra, Amit; Taneja, Subhash Chandra; Koul, Surrinder

    2016-05-23

    In our earlier study, we have reported that a phenolic compound 2-hydroxy-4-methoxybenzaldehyde from Janakia arayalpatra root extract was active against Viper and Cobra envenomations. Based on the structure of this natural product, libraries of synthetic structurally variant phenolic compounds were studied through molecular docking on the venom protein. To validate the activity of eight selected compounds, we have tested them in in vivo and in vitro models. The compound 21 (2-hydroxy-3-methoxy benzaldehyde), 22 (2-hydroxy-4-methoxybenzaldehyde) and 35 (2-hydroxy-3-methoxybenzylalcohol) were found to be active against venom-induced pathophysiological changes. The compounds 20, 15 and 35 displayed maximum anti-hemorrhagic, anti-lethal and PLA2 inhibitory activity respectively. In terms of SAR, the presence of a formyl group in conjunction with a phenolic group was seen as a significant contributor towards increasing the antivenom activity. The above observations confirmed the anti-venom activity of the phenolic compounds which needs to be further investigated for the development of new anti-snake venom leads. PMID:26986086

  16. Zona Pellucida Induces Activation of Phospholipase A2 During Acrosomal Exocytosis in Guinea PigSpermatozoa

    Institute of Scientific and Technical Information of China (English)

    袁玉英; 陈文颖; 石其贤; 毛丽珍; 俞淑清; 方昕; E.R.S.Roldan

    2005-01-01

    Objective To investigate whether PLA2 is involved in ZP-stimulated acrosomal exocytosis,if Ca2+ is required for activation of PLA2,and sigal transduction pathways modulating PLA2. Methods Guinea-pig spermatozoa were capacitated and labeled in low Ca2+ medium with [14C]choline chloride or [14C] arachidonic acid,and were then exposed to millimolar Ca2+ and various reagents and stimulated with ZP. Results Precapacitated spermatozoa exposed to millimolar Ca2+ and stimulated with ZP experienced increases in arachidonic acid(AA)and lysophosphatidylcholine (lysoPC) and a parallel decrease in phosphatidylcholine (PC);these chages are indicative of PLA2 activation.Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid,a PLA2 inhibitor, before treatment with ZP.Stimulation with ZP in medium without added Ca2+,or in medium with millimolar Ca2+ and EGTA or La3+ resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin,a G1 protein inhibitor, before stimulation with ZP,blocked the release of AA and lysoPC and acrosomal exocytosis.Exposure of spermatozoa to the DAG kinase inhibitor R59022 before ZP stimulation led to a significant increase in the generation of lysoPC and exocytosis.Conclusion These results indicate very strongly that PLA2 plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA2 activation requires Ca2+ internalization,and that it is regulated by signal transduction pathways involving G proteins and DAG.

  17. Rapamycin-Insensitive Up-Regulation of Adipocyte Phospholipase A2 in Tuberous Sclerosis and Lymphangioleiomyomatosis

    OpenAIRE

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

    2014-01-01

    Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR), and are also seen ...

  18. [Phospholipase, proteinase and hemolytic activities of Candida albicans isolates obtained from clinical specimens].

    Science.gov (United States)

    Yenişehirli, Gülgün; Bulut, Yunus; Tunçoglu, Ebru

    2010-01-01

    This study was aimed to determine the phospholipase, proteinase and hemolytic activities of Candida albicans strains isolated from clinical specimens. A total of 147 C. albicans strains isolated from blood (n = 29), respiratory specimens (n = 44), urine (n = 52), pus (n = 17) and stool (n = 5) were included in the study. Proteinase and phospholipase activities were determined in 81% and 76% of C. albicans isolates, respectively. All C. albicans isolates revealed beta-hemolytic activity on Sabouraud dextrose agar supplemented with 7% fresh sheep blood and 3% glucose. Phospholipase and proteinase positivity were highest among the respiratory isolates. Proteinase activity of respiratory (93%) and blood (83%) isolates were statistically significantly higher than that of urine (77%; p = 0.032), pus (65%; p = 0.007) and stool isolates (60%; p = 0.026). While phospholipase activity showed statistically significant difference between respiratory (84%) and pus (53%) isolates (p = 0.014), no statistically significant difference was determined for blood (79%), urine (75%) and stool (80%) isolates (p > 0.05). Two blood isolates with 4+ proteinase activity and 3 urine isolates with 3+ proteinase activity were phospholipase negative. One urine isolate with 4+ phospholipase activity and 4 with 3+ phospholipase activity were proteinase negative. Phospholipase and proteinase negative 1 isolate from stool and 1 isolate from pus were found to have 4+ hemolytic activity. In conclusion, besides proteinase and phospholipase enzyme activities, hemolytic activity may play an important role for the C.albicans infections. The pathogenetic role of these virulence factors should be evaluated by further clinical studies.

  19. Possible involvement of phospholipase C and protein kinase C in stimulatory actions of L-leucine and its keto acid, alpha-ketoisocaproic acid, on protein synthesis in RLC-16 hepatocytes.

    Science.gov (United States)

    Yagasaki, Kazumi; Morisaki-Tsuji, Naoko; Miura, Atsuhito; Funabiki, Ryuhei

    2002-11-01

    Effects of leucine and related compounds on protein synthesis were studied in RLC-16 hepatocytes. The incorporation of [(3)H] tyrosine into cellular protein was measured as an indexof protein synthesis. In leucine-depleted RLC-16 cells, L-leucineand its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipase A(2) and C canceled stimulatory actions of L-leucine and KIC on protein synthesis, suggesting a possible involvement of either arachidonic acid metabolism by phospholipase A(2), cyclooxygenase or lipoxygenase, or phosphatidylinositol degradation by phospholipase C in the stimulatory actions of L-leucine and KIC.Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of protein kinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in RLC-16 cells via activation of phospholipase C and production of diacylglycerol and inositol triphosphate from phosphatidylinositol, which in turn activate protein kinase C. PMID:19003115

  20. Synergistic activation of vascular TRPC6 channel by receptor and mechanical stimulation via phospholipase C/diacylglycerol and phospholipase A2/¿-hydroxylase/20-HETE pathways

    DEFF Research Database (Denmark)

    Inoue, Ryuji; Jensen, Lars Jørn; Jian, Zhong;

    2009-01-01

    TRPC6 is a non-voltage-gated Ca(2+) entry/depolarization channel associated with vascular tone regulation and remodeling. Expressed TRPC6 channel responds to both neurohormonal and mechanical stimuli, the mechanism for which remains controversial. In this study, we examined the possible interacti......TRPC6 is a non-voltage-gated Ca(2+) entry/depolarization channel associated with vascular tone regulation and remodeling. Expressed TRPC6 channel responds to both neurohormonal and mechanical stimuli, the mechanism for which remains controversial. In this study, we examined the possible...... or Arg8 vasopressin was greatly enhanced by mechanical stimuli via 20-HETE production. Furthermore, myogenic response of pressurized mesenteric artery was significantly enhanced by weak receptor stimulation dependently on 20-HETE production. These results collectively suggest that simultaneous operation...... of receptor and mechanical stimulations may synergistically amplify transmembrane Ca(2+) mobilization through TRPC6 activation, thereby enhancing the vascular tone via phospholipase C/diacylglycerol and phospholipase A(2)/omega-hydroxylase/20-HETE pathways....

  1. Genetic variability and phospholipase production of Malassezia pachydermatis isolated from dogs with diverse grades of skin lesions.

    Science.gov (United States)

    Machado, Mauro L S; Cafarchia, Claudia; Otranto, Domenico; Ferreira, Rafael R; Bianchi, Simone P; Latrofa, Maria S; Parisi, Antonio; Ferreiro, Laerte

    2010-09-01

    Little detailed information is available on the association of Malassezia pachydermatis genotypes and the extent of skin damage that they cause. In the present study, isolates of M. pachydermatis, recovered from the skin of healthy dogs and dogs with dermatitis in Brazil, were characterized on the basis of partial sequencing of the large subunit (LSU), first internal transcribed spacer (ITS-1) and chitin synthase 2 gene (chs-2). The determination of phospholipase production was also included in the investigations. The severity of lesions and hyperpigmentation of dogs with skin disease were evaluated. For each locus, two main sequence types were designated as genotypes A and C. Two other minor sequence types (A2(I)-C2(I)) were also recorded and defined for the ITS-1. Genotype A isolates were the most prevalent, being recovered from healthy and diseased animals. No significant difference was detected among genotypes or ITS-1 sequence types and grades of skin damage or hyperpigmentation in the dogs with skin lesions. The number of M. pachydermatis isolates that produced phospholipase was statistically higher for diseased dogs than for strains found in healthy animals. The present study reveals that multiple genetic variants of M. pachydermatis occur in dogs and that the distribution patterns of particular genotypes on the skin of dogs in Brazil might be related to environmental and ecological factors which maintain distinctive genotype assemblages in specific geographical areas. PMID:20105099

  2. A role for phospholipase D3 in myotube formation.

    Directory of Open Access Journals (Sweden)

    Mary Osisami

    Full Text Available Phospholipase D3 (PLD3 is a non-classical, poorly characterized member of the PLD superfamily of signaling enzymes. PLD3 is a type II glycoprotein associated with the endoplasmic reticulum, is expressed in a wide range of tissues and cells, and undergoes dramatic upregulation in neurons and muscle cells during differentiation. Using an in vitro skeletal muscle differentiation system, we define the ER-tethering mechanism and report that increased PLD3 expression enhances myotube formation, whereas a putatively dominant-negative PLD3 mutant isoform reduces myotube formation. ER stress, which also enhances myotube formation, is shown here to increase PLD3 expression levels. PLD3 protein was observed to localize to a restricted set of subcellular membrane sites in myotubes that may derive from or constitute a subdomain of the endoplasmic reticulum. These findings suggest that PLD3 plays a role in myogenesis during myotube formation, potentially in the events surrounding ER reorganization.

  3. Disruption of the Phospholipase D Gene Attenuates the Virulence of Aspergillus fumigatus

    NARCIS (Netherlands)

    Li, Xianping; Gao, Meihua; Han, Xuelin; Tao, Sha; Zheng, Dongyu; Cheng, Ying; Yu, Rentao; Han, Gaige; Schmidt, Martina; Han, Li

    2012-01-01

    Aspergillus fumigatus is the most prevalent airborne fungal pathogen that induces serious infections in immunocompromised patients. Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, knowledge o

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

    Science.gov (United States)

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

    2013-06-01

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

  5. Identifying New Drug Targets for Potent Phospholipase D Inhibitors: Combining Sequence Alignment, Molecular Docking, and Enzyme Activity/Binding Assays.

    Science.gov (United States)

    Djakpa, Helene; Kulkarni, Aditya; Barrows-Murphy, Scheneque; Miller, Greg; Zhou, Weihong; Cho, Hyejin; Török, Béla; Stieglitz, Kimberly

    2016-05-01

    Phospholipase D enzymes cleave phospholipid substrates generating choline and phosphatidic acid. Phospholipase D from Streptomyces chromofuscus is a non-HKD (histidine, lysine, and aspartic acid) phospholipase D as the enzyme is more similar to members of the diverse family of metallo-phosphodiesterase/phosphatase enzymes than phospholipase D enzymes with active site HKD repeats. A highly efficient library of phospholipase D inhibitors based on 1,3-disubstituted-4-amino-pyrazolopyrimidine core structure was utilized to evaluate the inhibition of purified S. chromofuscus phospholipase D. The molecules exhibited inhibition of phospholipase D activity (IC50 ) in the nanomolar range with monomeric substrate diC4 PC and micromolar range with phospholipid micelles and vesicles. Binding studies with vesicle substrate and phospholipase D strongly indicate that these inhibitors directly block enzyme vesicle binding. Following these compelling results as a starting point, sequence searches and alignments with S. chromofuscus phospholipase D have identified potential new drug targets. Using AutoDock, inhibitors were docked into the enzymes selected from sequence searches and alignments (when 3D co-ordinates were available) and results analyzed to develop next-generation inhibitors for new targets. In vitro enzyme activity assays with several human phosphatases demonstrated that the predictive protocol was accurate. The strategy of combining sequence comparison, docking, and high-throughput screening assays has helped to identify new drug targets and provided some insight into how to make potential inhibitors more specific to desired targets. PMID:26691755

  6. Immunohistochemical localization of hepatopancreatic phospholipase in gastropods mollusc, Littorina littorea and Buccinum undatum digestive cells

    Directory of Open Access Journals (Sweden)

    Zarai Zied

    2011-11-01

    Full Text Available Abstract Background Among the digestive enzymes, phospholipase A2 (PLA2 hydrolyzes the essential dietary phospholipids in marine fish and shellfish. However, we know little about the organs that produce PLA2, and the ontogeny of the PLA2-cells. Accordingly, accurate localization of PLA2 in marine snails might afford a better understanding permitting the control of the quality and composition of diets and the mode of digestion of lipid food. Results We have previously producted an antiserum reacting specifically with mSDPLA2. It labeled zymogen granules of the hepatopancreatic acinar cells and the secretory materials of certain epithelial cells in the depths of epithelial crypts in the hepatopancreas of snail. To confirm this localization a laser capture microdissection was performed targeting stained cells of hepatopancreas tissue sections. A Western blot analysis revealed a strong signal at the expected size (30 kDa, probably corresponding to the PLA2. Conclusions The present results support the presence of two hepatopancreatic intracellular and extracellular PLA2 in the prosobranchs gastropods molluscs, Littorina littorea and Buccinum undatum and bring insights on their localizations.

  7. Mulig terapeutisk intervention med haemmere og fremmere af fosfolipase A 2 -subtyper

    DEFF Research Database (Denmark)

    Zhan, Chen; Andreasen, Jens Rovelt; Prause, Jan Ulrik;

    2007-01-01

    Phospholipase A2 (PLA2) is a group of enzymes discovered more than a century ago in insect and snake poison. Not more than 20 years ago they were identified in vertebrates, and during the last five years, research has accelerated in relation to isolating and cloning several members of this super-...

  8. Calmodulin immunolocalization to cortical microtubules is calcium independent

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, D.D.; Cyr, R.J.

    1992-01-01

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 [mu]M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  9. Calmodulin immunolocalization to cortical microtubules is calcium independent

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, D.D.; Cyr, R.J.

    1992-12-31

    Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 {mu}M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 {mu}M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

  10. Lack of group X secreted phospholipase A2 increases survival following pandemic H1N1 influenza infection

    OpenAIRE

    Alyson A Kelvin; Degousee, Norbert; Banner, David; Stefanski, Eva; Leόn, Albert J.; Angoulvant, Denis; Stéphane G Paquette; Stephen S H Huang; Danesh, Ali; Robbins, Clinton S; Noyan, Hossein; Husain, Mansoor; Lambeau, Gérard; Gelb, Michael; Kelvin, David J.

    2014-01-01

    The role of Group X secreted phospholipase A2 (GX-sPLA2) during influenza infection has not been previously investigated. We examined the role of GX-sPLA2 during H1N1 pandemic influenza infection in a GX-sPLA2 gene targeted mouse (GX−/−) model and found that survival after infection was significantly greater in GX−/− mice than in GX+/+ mice. Downstream products of GX-sPLA2 activity, PGD2, PGE2, LTB4, cysteinyl leukotrienes and Lipoxin A4 were significantly lower in GX−/− mice BAL fluid. Lung ...

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

    Science.gov (United States)

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

    1995-01-01

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

  12. The galactolipase activity of Fusarium solani (phospho)lipase.

    Science.gov (United States)

    Jallouli, Raida; Othman, Houcemeddine; Amara, Sawsan; Parsiegla, Goetz; Carriere, Frédéric; Srairi-Abid, Najet; Gargouri, Youssef; Bezzine, Sofiane

    2015-03-01

    The purified (phospho)lipase of Fusarium solani (FSL), was known to be active on both triglycerides and phospholipids. This study aimed at assessing the potential of this enzyme in hydrolyzing galactolipids. FSL was found to hydrolyze at high rates of synthetic medium chains monogalactosyldiacylglycerol (4658±146U/mg on DiC8-MGDG) and digalactosyldiacylglycerol (3785±83U/mg on DiC8-DGDG) and natural long chain monogalactosyldiacylglycerol extracted from leek leaves (991±85U/mg). It is the microbial enzyme with the highest activity on galactolipids identified so far with a level of activity comparable to that of pancreatic lipase-related protein 2. FSL maximum activity on galactolipids was measured at pH8. The analysis of the hydrolysis product of natural MGDG from leek showed that FSL hydrolyzes preferentially the ester bond at the sn-1 position of galactolipids. To investigate the structure-activity relationships of FSL, a 3D model of this enzyme was built. In silico docking of medium chains MGDG and DGDG and phospholipid in the active site of FSL reveals structural solutions which are in concordance with in vitro tests.

  13. Dual Functions of Phospholipase in Plant Response to Drought

    Institute of Scientific and Technical Information of China (English)

    Yueyun Hong; Suqin Zheng; Xuemin Wang

    2008-01-01

    Phospholipase Dα1 (PLDα1) has been shown to mediate the abscisic acid regulation of stomatal movements.Arabidopsis plants deficient in PLDα1 increased,whereas PLDoLl-overexpressing tobacco decreased,transpirational water loss.In the early stage of drought,the decrease in water loss was associated with a rapid stomatal closure caused by a high level of PLD in PLDα1-overexpressing plants.However,in the late stage of drought,the overexpressing plants displayed more susceptibility to drought than control plants.PLDα1 activity in the overexpressing plants was much higher than that of control plants in which drought also induced an increase in PLDα1 activity.The high level of PLDα1 activity was correlated to membrane degradation in late stages of drought,as demonstrated by ionic leakage and lipid peroxidation.These findings indicate that a high level of PLDα1 expression has different effects on plant response to water deficits.It promotes stomatal closure at earlier stages,but disrupts membranes in prolonged drought stress.These findings are discussed in relation to the understanding of PLD functions and potential applications.

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

    Science.gov (United States)

    Rebecchi, M J; Pentyala, S N

    2000-10-01

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

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

    Science.gov (United States)

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

    2015-12-01

    Phospholipase C (PLC) plays important roles in regulating various biological processes in eukaryotes. Currently, little is known about the function of PLC in filamentous fungi, especially the plant pathogenic fungi. Fusarium graminearum is the causal agent of Fusarium head blight in many cereal crops. BLAST search revealed that Fusarium genome contains six FgPLC genes. Using quantitative RT-PCR, different FgPLC gene expressions in mycelia were analyzed. To investigate the role of FgPLC in F. graminearum biology, a pharmacological study using a known inhibitor of PLC (U7312