Sample records for phospholipase a2 group

  1. Biallelic Mutations in PLA2G5, Encoding Group V Phospholipase A2, Cause Benign Fleck Retina

    PubMed Central

    Sergouniotis, Panagiotis I.; Davidson, Alice E.; Mackay, Donna S.; Lenassi, Eva; Li, Zheng; Robson, Anthony G.; Yang, Xu; Kam, Jaimie Hoh; Isaacs, Timothy W.; Holder, Graham E.; Jeffery, Glen; Beck, Jonathan A.; Moore, Anthony T.; Plagnol, Vincent; Webster, Andrew R.

    2011-01-01

    Flecked-retina syndromes, including fundus flavimaculatus, fundus albipunctatus, and benign fleck retina, comprise a group of disorders with widespread or limited distribution of yellow-white retinal lesions of various sizes and configurations. Three siblings who have benign fleck retina and were born to consanguineous parents are the basis of this report. A combination of homozygosity mapping and exome sequencing helped to identify a homozygous missense mutation, c.133G>T (p.Gly45Cys), in PLA2G5, a gene encoding a secreted phospholipase (group V phospholipase A2). A screen of a further four unrelated individuals with benign fleck retina detected biallelic variants in the same gene in three patients. In contrast, no loss of function or common (minor-allele frequency>0.05%) nonsynonymous PLA2G5 variants have been previously reported (EVS, dbSNP, 1000 Genomes Project) or were detected in an internal database of 224 exomes (from subjects with adult onset neurodegenerative disease and without a diagnosis of ophthalmic disease). All seven affected individuals had fundoscopic features compatible with those previously described in benign fleck retina and no visual or electrophysiological deficits. No medical history of major illness was reported. Levels of low-density lipoprotein were mildly elevated in two patients. Optical coherence tomography and fundus autofluorescence findings suggest that group V phospholipase A2 plays a role in the phagocytosis of photoreceptor outer-segment discs by the retinal pigment epithelium. Surprisingly, immunohistochemical staining of human retinal tissue revealed localization of the protein predominantly in the inner and outer plexiform layers. PMID:22137173

  2. Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture.

    PubMed

    Levin, W; Daniel, R F; Stoner, C R; Stoller, T J; Wardwell-Swanson, J A; Angelillo, Y M; Familletti, P C; Crowl, R M

    1992-02-01

    Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2). The purified recombinant protein has an apparent molecular weight of 16 kDa, identical to that of purified human placental or synovial fluid phospholipase A2, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Application of the purified protein onto several different gel filtration columns resulted in elution of the protein at molecular weights corresponding to 3.1-4.7 kDa, suggesting an interaction of the protein with the column resins. However, analytical ultracentrifugation experiments revealed that the protein behaves as a monomer (13.8-14.2 kDa) over a protein concentration range of approximately 10 micrograms/ml to 5 mg/ml. With autoclaved Escherichia coli membranes as substrate, the recombinant protein has catalytic properties (pH optimum, effects of bovine serum albumin, sodium chloride concentration, and requirement for calcium) similar to those of the protein purified from human placenta.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Site-specific epsilon-NH2 monoacylation of pancreatic phospholipase A2. 2. Transformation of soluble phospholipase A2 into a highly penetrating "membrane-bound" form.

    PubMed

    Van der Wiele, F C; Atsma, W; Roelofsen, B; van Linde, M; Van Binsbergen, J; Radvanyi, F; Raykova, D; Slotboom, A J; De Haas, G H

    1988-03-08

    Long-chain lecithins present in bilayer structures like vesicles or membranes are only very poor substrates for pancreatic phospholipases A2. This is probably due to the fact that pancreatic phospholipases A2 cannot penetrate into the densely packed bilayer structures. To improve the weak penetrating properties of pancreatic phospholipases A2, we prepared and characterized a number of pancreatic phospholipase A2 mutants that have various long acyl chains linked covalently to Lys116 in porcine and to Lys10 in bovine phospholipase A2 [Van der Wiele, F.C., Atsma, W., Dijkman, R., Schreurs, A.M.M., Slotboom, A.J., & De Haas, G.H. (1988) Biochemistry (preceding paper in this issue)]. When monomolecular surface layers of L- and D-didecanoyllecithin were used, it was found that the introduction of caprinic, lauric, palmitic, and oleic acid at Lys116 in the porcine enzyme increases its penetrating power from 13 to about 17, 20, 32, and 22 dyn/cm, respectively, before long lag periods were obtained. Incorporation of a palmitoyl moiety at Lys10 in the bovine enzyme shifted the penetrating power from 11 to about 25 dyn/cm. Only the best penetrating mutant, viz., porcine phospholipase A2 having a palmitoyl moiety at Lys116, was able to cause complete leakage of 6-carboxyfluorescein entrapped in small unilamellar vesicles of egg lecithin under nonhydrolytic conditions. Similarly, only this latter palmitoylphospholipase A2 completely hydrolyzed all lecithin in the outer monolayer of the human erythrocyte at a rate much faster than Naja naja phospholipase A2, the most powerful penetrating snake venom enzyme presently known.

  6. Group IIA secretory phospholipase A2 (GIIA) mediates apoptotic death during NMDA receptor activation in rat primary cortical neurons.

    PubMed

    Chiricozzi, Elena; Fernandez-Fernandez, Seila; Nardicchi, Vincenza; Almeida, Angeles; Bolaños, Juan Pedro; Goracci, Gianfrancesco

    2010-03-01

    Phospholipases A(2) (PLA(2)) participate in neuronal death signalling pathways because of their ability to release lipid mediators, although the contribution of each isoform and mechanism of neurotoxicity are still elusive. Using a novel fluorogenic method to assess changes in a PLA(2) activity by flow cytometry, here we show that the group IIA secretory phospholipase A(2) isoform (GIIA) was specifically activated in cortical neurons following stimulation of N-methyl-d-aspartate glutamate receptor subtype (NMDAR). For activation, GIIA required Ca(2+) and reactive oxygen/nitrogen species, and inhibition of its activity fully prevented NMDAR-mediated neuronal apoptotic death. Superoxide, nitric oxide or peroxynitrite donors stimulated GIIA activity, which mediated neuronal death. Intriguingly, we also found that GIIA activity induced mitochondrial superoxide production after NMDAR stimulation. These results reveal a novel role for GIIA in excitotoxicity both as target and producer of superoxide in a positive-loop of activation that may contribute to the propagation of neurodegeneration.

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  8. 2-Oxoamide inhibitors of cytosolic group IVA phospholipase A2 with reduced lipophilicity.

    PubMed

    Antonopoulou, Georgia; Magrioti, Victoria; Kokotou, Maroula G; Nikolaou, Aikaterini; Barbayianni, Efrosini; Mouchlis, Varnavas D; Dennis, Edward A; Kokotos, George

    2016-10-01

    Cytosolic GIVA phospholipase A2 (GIVA cPLA2) initiates the eicosanoid pathway of inflammation and thus inhibitors of this enzyme constitute novel potential agents for the treatment of inflammatory diseases. Traditionally, GIVA cPLA2 inhibitors have suffered systemically from high lipophilicity. We have developed a variety of long chain 2-oxoamides as inhibitors of GIVA PLA2. Among them, AX048 was found to produce a potent analgesic effect. We have now reduced the lipophilicity of AX048 by replacing the long aliphatic chain with a chain containing an ether linked aromatic ring with in vitro inhibitory activities similar to AX048. Copyright © 2016 Elsevier Ltd. All rights reserved.

  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

    PubMed Central

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

    2014-01-01

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

  10. 40 CFR 721.4585 - Lecithins, phospholipase A2-hydrolyzed.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Lecithins, phospholipase A2-hydrolyzed... Substances § 721.4585 Lecithins, phospholipase A2-hydrolyzed. (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substances identified generically as lecithins...

  11. Origin and evolution of group XI secretory phospholipase A2 from flax (Linum usitatissimum) based on phylogenetic analysis of conserved domains.

    PubMed

    Gupta, Payal; Saini, Raman; Dash, Prasanta K

    2017-07-01

    Phospholipase A 2 (PLA 2 ) belongs to class of lipolytic enzymes (EC 3.1.1.4). Lysophosphatidic acid (LPA) and free fatty acids (FFAs) are the products of PLA 2 catalyzed hydrolysis of phosphoglycerides at sn-2 position. LPA and FFA that act as second mediators involved in the development and maturation of plants and animals. Mining of flax genome identified two phospholipase A 2 encoding genes, viz., LusPLA 2 I and LusPLA 2 II (Linum usitatissimum secretory phospholipase A 2 ). Molecular simulation of LusPLA 2 s with already characterized plant sPLA 2 s revealed the presence of conserved motifs and signature domains necessary to classify them as secretory phospholipase A 2 . Phylogenetic analysis of flax sPLA 2 with representative sPLA 2 s from other organisms revealed that they evolved rapidly via gene duplication/deletion events and shares a common ancestor. Our study is the first report of detailed phylogenetic analysis for secretory phospholipase A 2 in flax. Comparative genomic analysis of two LusPLA 2 s with earlier reported plant sPLA 2 s, based on their gene architectures, sequence similarities, and domain structures are presented elucidating the uniqueness of flax sPLA 2 .

  12. The molecular biology of the group VIA Ca2+-independent phospholipase A2.

    PubMed

    Ma, Z; Turk, J

    2001-01-01

    The group VIA PLA2 is a member of the PLA2 superfamily. This enzyme, which is cytosolic and Ca2+-independent, has been designated iPLA2beta to distinguish it from another recently cloned Ca2+-independent PLA2. Features of iPLA2beta molecular structure offer some insight into possible cellular functions of the enzyme. At least two catalytically active iPLA2beta isoforms and additionalsplicing variants are derived from a single gene that consists of at least 17 exons located on human chromosome 22q13.1. Potential tumor suppressor genes also reside at or near this locus. Structural analyses reveal that iPLA2beta contains unique structural features that include a serine lipase consensus motif (GXSXG), a putative ATP-binding domain, an ankyrin-repeat domain, a caspase-3 cleavage motif DVTD138Y/N, a bipartite nuclear localization signal sequence, and a proline-rich region in the human long isoform. iPLA2beta is widely expressed among mammalian tissues, with highest expression in testis and brain. iPLA2beta prefers to hydrolyze fatty acid at the sn-2 fatty acid substituent but also exhibits phospholipase A1, lysophospholipase, PAF acetylhydrolase, and transacylase activities. iPLA2beta may participate in signaling, apoptosis, membrane phospholipid remodeling, membrane homeostasis, arachidonate release, and exocytotic membrane fusion. Structural features and the existence of multiple splicing variants of iPLA2beta suggest that iPLA2beta may be subject to complex regulatory mechanisms that differ among cell types. Further study of its regulation and interaction with other proteins may yield insight into how its structural features are related to its function.

  13. Inhibition of (/sup 3/H)nitrendipine binding by phospholipase A/sub 2/

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Goldman, M.E.; Pisano, J.J.

    Phospholipase A/sub 2/ from several sources inhibited (/sup 3/H)nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC/sub 50/ values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A/sub 2/ was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A/sub 2/ enzymatic activity, shifted the bee venom phospholipase A/sub 2/ dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A/sub 2/ (10 ng/ml) for 15 min caused a 2-foldmore » increase in the K/sub d/ without changing the B/sub max/ compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the K/sub d/ but significantly decreased the B/sub max/ to 71% the value for untreated membranes. (/sup 3/H)Nitrendipine, preincubated with bee venom phospholipase A/sub 2/, was recovered and found to be fully active, indicating that the phospholipase A/sub 2/ did not modify the ligand. It is concluded that phospholipase A/sub 2/ acts on the membrane at or near the (/sup 3/H)nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site. 33 references, 3 figures, 1 table.« less

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

    PubMed Central

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

    2017-01-01

    Abstract Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein’s ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients’ fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2. The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance. PMID:28007986

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

    PubMed Central

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

    2012-01-01

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

  16. The role of negatively charged lipids in lysosomal phospholipase A2 function

    PubMed Central

    Abe, Akira; Shayman, James A.

    2009-01-01

    Lysosomal phospholipase A2 (LPLA2) is characterized by increased activity toward zwitterionic phospholipid liposomes containing negatively charged lipids under acidic conditions. The effect of anionic lipids on LPLA2 activity was investigated. Mouse LPLA2 activity was assayed as C2-ceramide transacylation. Sulfatide incorporated into liposomes enhanced LPLA2 activity under acidic conditions and was weakened by NaCl or increased pH. Amiodarone, a cationic amphiphilic drug, reduced LPLA2 activity. LPLA2 exhibited esterase activity when p-nitro-phenylbutyrate (pNPB) was used as a substrate. Unlike the phospholipase A2 activity, the esterase activity was detected over wide pH range and not inhibited by NaCl or amiodarone. Presteady-state kinetics using pNPB were consistent with the formation of an acyl-enzyme intermediate. C2-ceramide was an acceptor for the acyl group of the acyl-enzyme but was not available as the acyl group acceptor when dispersed in liposomes containing amiodarone. Cosedimentation of LPLA2 with liposomes was enhanced in the presence of sulfatide and was reduced by raising NaCl, amiodarone, or pH in the reaction mixture. LPLA2 adsorption to negatively charged lipid membrane surfaces through an electrostatic attraction, therefore, enhances LPLA2 enzyme activity toward insoluble substrates. Thus, anionic lipids present within lipid membranes enhance the rate of phospholipid hydrolysis by LPLA2 at lipid-water interfaces.—Abe, A., and J. A. Shayman. The role of negatively charged lipids in lysosomal phospholipase A2 function. PMID:19321879

  17. Signal-activated phospholipase regulation of leukocyte chemotaxis.

    PubMed

    Cathcart, Martha K

    2009-04-01

    Signal-activated phospholipases are a recent focus of the rapidly growing field of lipid signaling. The extent of their impact on the pathways regulating diverse cell functions is beginning to be appreciated. A critical step in inflammation is the attraction of leukocytes to injured or diseased tissue. Chemotaxis of leukocytes, a requisite process for monocyte and neutrophil extravasation from the blood into tissues, is a critical step for initiating and maintaining inflammation in both acute and chronic settings. Recent studies have identified new important and required roles for two signal-activated phospholipases A2 (PLA2) in regulating chemotaxis. The two intracellular phospholipases, cPLA2alpha (Group IVA) and iPLA2beta (Group VIA), act in parallel to provide distinct lipid mediators at different intracellular sites that are both required for leukocytes to migrate toward the chemokine monocyte chemoattractant protein-1. This review will summarize the separate roles of these phospholipases as well as what is currently known about the influence of two other classes of intracellular signal-activated phospholipases, phospholipase C and phospholipase D, in regulating chemotaxis in eukaryotic cells, but particularly in human monocytes. The contributions of these phospholipases to chemotaxis both in vitro and in vivo will be highlighted.

  18. Cyclopentanoid analogs of phosphatidylcholine: susceptibility to phospholipase A2.

    PubMed

    Lister, M D; Hancock, A J

    1988-10-01

    Six isomers of dipalmitoylcyclopentanetriol phosphocholine (cyclopentano-lecithin) were tested as potential substrates for phospholipase A2. Since each of these analogs possesses a configuration that mimics a narrow range of conformations of a glycerophospholipid molecule, the analogs were used to assess the enzyme's conformational requirements. Studies showed that all of the analogs containing the phosphocholine at the C-1 (or C-3) position could be hydrolyzed, while only one of the three analogs that contains the polar head group at the C-2 position was susceptible. Kinetic studies, however, revealed that only the all-trans-(1,3/2-1P)-cyclopentano-lecithin gave initial rates of hydrolysis that were measurable by pH-stat. Acyl group specificity of the enzyme towards the all-trans isomer was determined with an analog was acyl groups were distinguishable. The synthesis of this mixed-acid-cyclopentano-PC is described herein. When this analog was enzymatically assayed, results unequivocally showed the enzyme to be specific for C-2 acyl hydrolysis. This specificity, and data showing that the all-trans analog is stereospecifically hydrolyzed, indicate that it is acted on in an analogous manner to dipalmitoylphosphatidylcholine. These studies indicate that although the configuration of the analog is not necessarily a prerequisite for hydrolysis, there does appear to be an optimal spatial orientation for enzymatic activity. The analogy between the susceptibilities of all-trans-(1,3/2-1P)-cyclopentano-lecithin and glycero-lecithin suggests that the conformation of the glycero-lecithin during phospholipase A2-mediated hydrolysis may be best simulated by the all-trans orientation of C-O bonds in the artificial substrate.

  19. Biochemical characterization of a phospholipase A2 from Photobacterium damselae subsp. piscicida.

    PubMed

    Hsu, Po-Yuan; Lee, Kuo-Kau; Lee, Pei-Shan; Hu, Chih-Chuang; Lin, Cheng-Hui; Liu, Ping-Chung

    2013-01-01

    Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of fish photobacteriosis (pasteurellosis) in cultured cobia (Rachycentron canadum) in Taiwan. A component was purified from the extracellular products (ECP) of the bacterium strain 9205 by fast protein liquid chromatography (FPLC) and identified as a phospholipase. An N-terminal sequence of 10 amino acid residues, QDQPNLDPGK, was determined by mass spectroscopy (MS) and found to be identical with that of another Phdp phospholipase (GenBank accession no. BAB85814) at positions 21 to 30. The corresponding gene sequence of the phospholipase (GenBank accession no. AB071137) was employed to design primers for amplification of the sequence by the polymerase chain reaction (PCR). The PCR products were transformed into Escherichia coli, and a recombinant protein product was obtained which was purified as a His-tag fusion protein by Ni-metal affinity chromatography. A single 43-kDa band was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Phosphatidylcholine was degraded by this protein to lysophosphatidylcholine and a fatty acid. These products were characterized by thin-layer (TLC) and gas chromatography (GC), respectively, allowing the identification of the protein as a phospholipase A2. The recombinant protein had maximum enzymatic activity between pH 4 and 7, and at 40 degrees C. The activity was inhibited by Zn(2+) and Cu(2+), activated by Ca(2+) and Mg(2+), and completely inactivated by dexamethasone and p-bromophenacyl bromide. A rabbit antiserum against the recombinant protein neutralized the phospholipase A2 activity in the ECP of Phdp strain 9205 and the recombinant protein itself. The recombinant protein was toxic to cobia of about 5 g weight with an LD50 value between 2 and 4 microg protein/g fish. The results revealed phospholipase A2 as a fish toxin in the ECP of Phdp strain 9205.

  20. Functional interaction between Cerebratulus lacteus cytolysin A-III and phospholipase A/sub 2/

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, J.; Blumenthal, K.M.

    A study on the interaction between bee venom phospholipase A/sub 2/ and Cerebratulus lacteus cytolysin A-III, a major hemolysin secreted by this organism has been carried out. The hemolytic activity of A-III in phosphate-buffered saline is increased 5-fold in the presence of phospholipase A/sub 2/ from bee venom. Dansylphosphatidylethanolamine (DPE) labeled, phosphatidylcholine-containing liposomes and human erythrocyte membranes were employed to study the interaction between these two proteins. In DPE-liposomes, A-III alone had no effect on DPE fluorescence nor did it enhance either the phospholipase A/sub 2/-dependent fluorescence increase or blue shift in emission maximum, indicating that the cytolysis is notmore » a major phospholipase A/sub 2/-activator. However, when DPE was incorporated into erythrocyte membranes, A-III alone induced a 40% fluorescence increase and a 5 nm blue shift, implying a transient activation of an endogenous phospholipase A/sub 2/. Further studies using synthetic lysophosphatidylcholine and free fatty acids demonstrated that the hemolytic activity of A-III is potentiated by free fatty acids, a product of phospholipid degradation catalyzed by phospholipase A/sub 2/. Subsequent analysis of this phenomenon by gel filtration chromatography, analytical ultracentrifugation, chemical cross-linking, and measurement of (/sup 14/C)oleic acid binding by the cytolysin demonstrated that binding of oleic acid to A-III causes aggregation of the toxin molecules to a tetrameric form which has a higher ..cap alpha..-helix content and a greater activity than the monomer.« less

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

    PubMed Central

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

    1980-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1989-02-01

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

  4. Phospholipase activities associated with the tonoplast from Acer pseudoplatanus cells: identification of a phospholipase A1 activity.

    PubMed

    Tavernier, E; Pugin, A

    1995-02-15

    The study of phospholipase activities associated with the tonoplast of Acer pseudoplatanus was performed in vitro with sn-2-[14C]acylphosphatidylcholine (PC) as a substrate. The hydrolysis of radiolabelled PC into [14C]phosphatidic acid and [14C]lyso-PC demonstrated the presence of phospholipase D and A1 activities, respectively, associated with the tonoplast of Acer pseudoplatanus. The vacuolar sap did not show any significant phospholipase activity. In a second step, the properties of the phospholipase A1 activity was studied using tonoplast endogenous PC labelled in vivo with [14C]choline as a substrate. The phospholipase A1 showed an optimal activity at pH about 6-6.5, did not necessarily require divalent cations, but was stimulated by Mg2+ and particularly by Ca2+. This work presents the first evidence for the presence of phospholipases A1 in plant cells.

  5. Glutaraldehyde pretreatment blocks phospholipase A2 modulation of adrenergic receptors.

    PubMed

    Cohen, R M; McLellan, C; Dauphin, M; Hirata, F

    1985-01-07

    Treatment of rat cerebral cortical membranes with phospholipase A2 affects, in a parallel fashion, beta-, alpha 1- and alpha 2-adrenergic receptor binding, but not the affinity of these receptors for their respective ligands. Pretreatment of membranes with 0.1 percent glutaraldehyde blocks the effects of phospholipase A2 on adrenergic receptor binding. The results support the hypothesis that desensitization or "masking" of adrenergic receptors may involve changes in membrane lipid composition. Furthermore, glutaraldehyde may prove a useful tool in the investigation of the dynamic roles of lipids in receptor function and more specifically, their regulation and coupling to physiological events.

  6. Postprandial lysophospholipid suppresses hepatic fatty acid oxidation: the molecular link between group 1B phospholipase A2 and diet-induced obesity

    PubMed Central

    Labonté, Eric D.; Pfluger, Paul T.; Cash, James G.; Kuhel, David G.; Roja, Juan C.; Magness, Daniel P.; Jandacek, Ronald J.; Tschöp, Matthias H.; Hui, David Y.

    2010-01-01

    Decrease in fat catabolic rate on consuming a high-fat diet contributes to diet-induced obesity. This study used group 1B phospholipase A2 (Pla2g1b)-deficient mice, which are resistant to hyperglycemia, to test the hypothesis that Pla2g1b and its lipolytic product lysophospholipid suppress hepatic fat utilization and energy metabolism in promoting diet-induced obesity. The metabolic consequences of hypercaloric diet, including body weight gain, energy expenditure, and fatty acid oxidation, were compared between Pla2g1b+/+ and Pla2g1b−/− mice. The Pla2g1b−/− mice displayed normal energy balance when fed chow, but were resistant to obesity when challenged with a hypercaloric diet. Obesity resistance in Pla2g1b−/− mice is due to their ability to maintain elevated energy expenditure and core body temperature when subjected to hypercaloric diet, which was not observed in Pla2g1b+/+ mice. The Pla2g1b−/− mice also displayed increased postprandial hepatic fat utilization due to increased expression of peroxisome proliferator-activated receptor (PPAR)-α, PPAR-δ, PPAR-γ, cd36/Fat, and Ucp2, which coincided with reduced postprandial plasma lysophospholipid levels. Lysophospholipids produced by Pla2g1b hydrolysis suppress hepatic fat utilization and down-regulate energy expenditure, thereby preventing metabolically beneficial adaptation to a high-fat diet exposure in promoting diet-induced obesity and type 2 diabetes.—Labonté, E. D., Pfluger, P. T., Cash, J. G., Kuhel, D. G., Rojas, J. C., Magness, D. P., Jandacek, R. J., Tschöp, M. H., Hui, D. Y. Postprandial lysophospholipid suppresses hepatic fatty acid oxidation: the molecular link between group 1B phospholipase A2 and diet-induced obesity. PMID:20215528

  7. Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A{sub 2}

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Santos, K. F.; Murakami, M. T.; Cintra, A. C. O.

    2007-04-01

    Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A{sub 2} and a catalytically inactive acidic phospholipase A{sub 2} analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A{sub 2} and a catalytically inactive acidic phospholipase A{sub 2} analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained.more » The crotoxin complex crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 Å, and diffracted to 1.75 Å resolution. The crystal of the phospholipase A{sub 2} domain belongs to the hexagonal space group P6{sub 1}22 (or its enantiomorph P6{sub 5}22), with unit-cell parameters a = b = 38.7, c = 286.7 Å, and diffracted to 2.6 Å resolution. The crotapotin crystal diffracted to 2.3 Å resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.« less

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

    PubMed

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

    2002-10-01

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

  9. Probing phospholipase a(2) with fluorescent phospholipid substrates.

    PubMed

    Wichmann, Oliver; Gelb, Michael H; Schultz, Carsten

    2007-09-03

    The Foerster resonance energy transfer-based sensor, PENN, measures intracellular phospholipase A(2) (PLA(2)) activity in living cells and small organisms. In an attempt to modify the probe for the detection of particular isoforms, we altered the sn-2 fatty acid in such a way that either one or three of the Z double bonds in arachidonic acid were present in the sensor molecule. Arachidonic-acid-mimicking fatty acids were prepared by copper-mediated coupling reactions. Probes with a single double bond in the 5-position exhibited favorable substrate properties for secretory PLA(2)s. In vitro experiments with the novel unsaturated doubly labeled phosphatidylethanolamine derivatives showed preferred cleavage of the sensor PENN2 (one double bond) by the physiologically important group V sPLA(2), while the O-methyl-derivative PMNN2 was accepted best by the isoform from hog pancreas. For experiments in living cells, we demonstrated that bioactivation via S-acetylthioethyl (SATE) groups is essential for probe performance. Surprisingly, membrane-permeant versions of the new sensors that contained double bonds, PENN2 and PENN3, were only cleaved to a minor extent in HeLa cells while the saturated form, PENN, was well accepted.

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

    PubMed Central

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

    2011-01-01

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

  11. Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms.

    PubMed

    Angulo, Yamileth; Lomonte, Bruno

    2005-01-01

    Group II phospholipase A(2) (PLA(2)) myotoxins isolated from Viperidae/Crotalidae snake venoms induce a rapid cytolytic effect upon diverse cell types in vitro. Previous studies suggested that this effect could be more pronounced on skeletal muscle myotubes than on other cell types, including undifferentiated myoblasts. This study utilized the murine skeletal muscle C2C12 cell line to investigate whether differentiated myotubes are more susceptible than myoblasts, and if this characteristic is specific for the group II myotoxic PLA(2)s. The release of lactic dehydrogenase was quantified as a measure of cytolysis, 3 h after cell exposure to different group II PLA(2)s purified from Bothrops asper, Atropoides nummifer, Cerrophidion godmani, and Bothriechis schlegelii venoms. In addition, susceptibility to lysis induced by synthetic melittin and group III PLA(2) from bee (Apis mellifera) venom, as well as by anionic, cationic, and neutral detergents, was comparatively evaluated on the two cultures. Myotubes were significantly more susceptible to group II PLA(2) myotoxins, but not to the other agents tested, under the same conditions. Moreover, the increased susceptibility of myotubes over myoblasts was also demonstrated with two cytolytic synthetic peptides, derived from the C-terminal region of Lys49 PLA(2) myotoxins, that reproduce the action of their parent proteins. These results indicate that fusion and differentiation of myoblasts into myotubes induce changes that render these cells more susceptible to the toxic mechanism of group II PLA(2) myotoxins, but not to general perturbations of membrane homeostasis. Such changes are likely to involve myotoxin acceptor site(s), which remain(s) to be identified.

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

    PubMed

    Lee, Gihyun; Bae, Hyunsu

    2016-02-22

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

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

    PubMed Central

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

    2013-01-01

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

  14. Phospholipase A2 superfamily members play divergent roles after spinal cord injury

    PubMed Central

    López-Vales, Rubèn; Ghasemlou, Nader; Redensek, Adriana; Kerr, Bradley J.; Barbayianni, Efrosini; Antonopoulou, Georgia; Baskakis, Constantinos; Rathore, Khizr I.; Constantinou-Kokotou, Violetta; Stephens, Daren; Shimizu, Takao; Dennis, Edward A.; Kokotos, George; David, Samuel

    2011-01-01

    Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be preventable as it occurs, secondary to the trauma. We show that the phospholipase A2 (PLA2) superfamily plays important roles in SCI. PLA2 enzymes hydrolyze membrane glycerophospholipids to yield a free fatty acid and lysophospholipid. Some free fatty acids (arachidonic acid) give rise to eicosanoids that promote inflammation, while some lysophospholipids (lysophosphatidylcholine) cause demyelination. We show in a mouse model of SCI that two cytosolic forms [calcium-dependent PLA2 group IVA (cPLA2 GIVA) and calcium-independent PLA2 group VIA (iPLA2 GVIA)], and a secreted form [secreted PLA2 group IIA (sPLA2 GIIA)] are up-regulated. Using selective inhibitors and null mice, we show that these PLA2s play differing roles. cPLA2 GIVA mediates protection, whereas sPLA2 GIIA and, to a lesser extent, iPLA2 GVIA are detrimental. Furthermore, completely blocking all three PLA2s worsens outcome, while the most beneficial effects are seen by partial inhibition of all three. The partial inhibitor enhances expression of cPLA2 and mediates its beneficial effects via the prostaglandin EP1 receptor. These findings indicate that drugs that inhibit detrimental forms of PLA2 (sPLA2 and iPLA2) and up-regulate the protective form (cPLA2) may be useful for the treatment of SCI.—López-Vales, R., Ghasemlou, N., Redensek, A., Kerr, B. J., Barbayianni, E., Antonopoulou, G., Baskakis, C., Rathore, K. I., Constantinou-Kokotou, V., Stephens, D., Shimizu, T., Dennis, E. A., Kokotos, G., David, S. Phospholipase A2 superfamily members play divergent roles after spinal cord injury. PMID:21868473

  15. M-type phospholipase A2 receptor autoantibodies and renal function in patients with primary membranous nephropathy.

    PubMed

    Hoxha, Elion; Harendza, Sigrid; Pinnschmidt, Hans; Panzer, Ulf; Stahl, Rolf A K

    2014-11-07

    Loss of renal function in patients with primary membranous nephropathy cannot be reliably predicted by laboratory or clinical markers at the time of diagnosis. M-type phospholipase A2 receptor autoantibodies have been shown to be associated with changes in proteinuria. Their eventual effect on renal function, however, is unclear. In this prospective, open, multicenter study, the potential role of M-type phospholipase A2 receptor autoantibodies levels on the increase of serum creatinine in 118 consecutive patients with membranous nephropathy and positivity for serum M-type phospholipase A2 receptor autoantibodies was analyzed. Patients were included in the study between April of 2010 and December of 2012 and observed until December of 2013. The clinical end point was defined as an increase of serum creatinine by ≥ 25% and serum creatinine reaching ≥ 1.3 mg/dl. Patients were divided into tertiles according to their M-type phospholipase A2 receptor autoantibody levels at the time of inclusion in the study: tertile 1 levels=20-86 units/ml (low), tertile 2 levels=87-201 units/ml (medium), and tertile 3 levels ≥ 202 units/ml (high). The median follow-up time of all patients in the study was 27 months (interquartile range=18-33 months). The clinical end point was reached in 69% of patients with high M-type phospholipase A2 receptor autoantibodies levels (tertile 3) but only 25% of patients with low M-type phospholipase A2 receptor autoantibodies levels. The average time to reach the study end point was 17.7 months in patients with high M-type phospholipase A2 receptor autoantibodies levels and 30.9 months in patients with low M-type phospholipase A2 receptor autoantibodies levels. A multivariate Cox regression analysis showed that high M-type phospholipase A2 receptor autoantibodies levels-in addition to men and older age-are an independent predictor for progressive loss of renal function. High M-type phospholipase A2 receptor autoantibodies levels were associated

  16. Group X Phospholipase A2 Stimulates the Proliferation of Colon Cancer Cells by Producing Various Lipid Mediators

    PubMed Central

    Surrel, Fanny; Jemel, Ikram; Boilard, Eric; Bollinger, James G.; Payré, Christine; Mounier, Carine M.; Talvinen, Kati A.; Laine, Veli J. O.; Nevalainen, Timo J.; Gelb, Michael H.

    2009-01-01

    Among mammalian secreted phospholipases A2 (sPLA2s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA2 [mouse (m)GX] is one of the most highly expressed PLA2 in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA2s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA2 inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA2α and M-type sPLA2 receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA2 mitogenic effects. Together, our results indicate that group X sPLA2 may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression. PMID:19602573

  17. Group X phospholipase A2 stimulates the proliferation of colon cancer cells by producing various lipid mediators.

    PubMed

    Surrel, Fanny; Jemel, Ikram; Boilard, Eric; Bollinger, James G; Payré, Christine; Mounier, Carine M; Talvinen, Kati A; Laine, Veli J O; Nevalainen, Timo J; Gelb, Michael H; Lambeau, Gérard

    2009-10-01

    Among mammalian secreted phospholipases A2 (sPLA(2)s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA(2) [mouse (m)GX] is one of the most highly expressed PLA(2) in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA(2)s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA(2) inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA(2)alpha and M-type sPLA(2) receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA(2) mitogenic effects. Together, our results indicate that group X sPLA(2) may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression.

  18. The secretory phospholipase A2 group IIA: a missing link between inflammation, activated renin-angiotensin system, and atherogenesis?

    PubMed

    Divchev, Dimitar; Schieffer, Bernhard

    2008-01-01

    Inflammation, lipid peroxidation and chronic activation of the renin-angiotensin system (RAS) are hallmarks of the development of atherosclerosis. Recent studies have suggested the involvement of the pro-inflammatory secretory phospholipase A(2) (sPLA(2))-IIA in atherogenesis. This enzyme is produced by different cell types through stimulation by pro-inflammatory cytokines. It is detectable in the intima and in media smooth muscle cells, not only in atherosclerotic lesions but also in the very early stages of atherogenesis. sPLA(2)-IIA can hydrolyse the phospholipid monolayers of low density lipoproteins (LDL). Such modified LDL show increased affinity to proteoglycans. The modified particles have a greater tendency to aggregate and an enhanced ability to insert cholesterol into cells. This modification may promote macrophage LDL uptake leading to the formation of foam cells. Furthermore, sPLA(2)-IIA is not only a mediator for localized inflammation but may be also used as an independent predictor of adverse outcomes in patients with stable coronary artery disease or acute coronary syndromes. An interaction between activated RAS and phospholipases has been indicated by observations showing that inhibitors of sPLA(2) decrease angiotensin (Ang) II-induced macrophage lipid peroxidation. Meanwhile, various interactions between Ang II and oxLDL have been demonstrated suggesting a central role of sPLA(2)-IIA in these processes and offering a possible target for treatment. The role of sPLA(2)-IIA in the perpetuation of atherosclerosis appears to be the missing link between inflammation, activated RAS and lipid peroxidation.

  19. Phospholipase B activity of a purified phospholipase A from Vipera palestinae venom.

    PubMed

    Shiloah, J; Klibansky, C; de Vries, A; Berger, A

    1973-05-01

    Phospholipase was isolated (in two fractions) from Vipera palestinae venom and it was shown to possess phospholipase A activity (hydrolyzing diacyl-sn-glycerophosphorylcholines, e.g., lecithin, in the 2-position) as well as lysophospholipase (phospholipase B) activity (hydrolyzing 1-monoacyl-sn-glycerophosphorylcholines, e.g., lysolecithin, yielding free fatty acid and glycerophosphorylcholine). Each of the two purified enzyme fractions was homogeneous as judged by electrophoresis on acrylamide gel and by immunodiffusion and immunoelectrophoresis, and both had essentially equal activities. The ratio of the specific activity, at various purification stages, to the specific activity of the whole venom was the same for A activity (substrate lecithin) as for B activity (substrate lysolecithin). The enzyme has a molecular weight of 16,000, six S-S bridges, and no free thiol groups. At pH 7, dimerization was observed in the ultracentrifuge. A dissociation constant of about 10(-5) m was estimated. The amino acid composition for both fractions (140 amino acid residues) was found to be essentially the same. The A activity had a pH optimum at 9; B activity was low at this pH but increased steadily beyond pH 10.5. For the hydrolysis of lysolecithin the Lineweaver-Burk plot was found to be linear, giving K(m) = 1.1 mm and k(cat) = 0.55 sec(-1) at 37 degrees C and pH 10. 2-Deoxylysolecithin was also hydrolyzed by the enzyme at pH 10, with k(cat) = 0.01 sec(-1) (zero-order kinetics in the range 0.5-2.5 mm). For lecithin these constants could not be determined, but at 0.25 mm substrate the hydrolysis rate (at pH 9) of lecithin was about 1000 times the hydrolysis rate of lysolecithin (at pH 10).

  20. Structural characterization of alpha-terminal group of natural rubber. 2. Decomposition of branch-points by phospholipase and chemical treatments.

    PubMed

    Tarachiwin, Lucksanaporn; Sakdapipanich, Jitladda; Ute, Koichi; Kitayama, Tatsuki; Tanaka, Yasuyuki

    2005-01-01

    The treatment of deproteinized natural rubber (DPNR) latex with phospholipases A(2), B, C, and D decreased significantly the long-chain fatty acid ester contents in DPNR and also the molecular weight and Higgins' k' constant, except for phospholipase D treatment. This indicates the presence of phospholipid molecules in NR, which combine rubber molecules together. Transesterification of DPNR resulted in the decomposition of the functional group at the terminal chain-end (alpha-terminal), including phospholipids and formed linear rubber molecules. The addition of small amounts of ethanol into the DPNR solution reduced the molecular weight and shifted the molecular weight distribution (MWD) comparable to that of transesterified DPNR (TE-DPNR). The addition of diammonium hydrogen phosphate into DPNR-latex in order to remove Mg2+ ions yielded a slight decrease in molecular weight and a slight shift in MWD. The phospholipids are expected to link with mono- and diphosphate groups at the alpha-terminal by hydrogen bonding and/or ionic linkages. The decrease in the molecular weight and Huggins' k' constant of DPNR demonstrates the formation of linear molecules after decomposition of branch-points by this treatment, showing that phospholipids participate in the branching formation of NR. The branch-points formed at the alpha-terminus are postulated to originate predominantly by the association of phospholipids via micelle formation of long-chain fatty acid esters and hydrogen bonding between polar headgroups of phospholipids.

  1. Phospholipase and Aspartyl Proteinase Activities of Candida Species Causing Vulvovaginal Candidiasis in Patients with Type 2 Diabetes Mellitus.

    PubMed

    Bassyouni, Rasha H; Wegdan, Ahmed Ashraf; Abdelmoneim, Abdelsamie; Said, Wessam; AboElnaga, Fatma

    2015-10-01

    Few research had investigated the secretion of phospholipase and aspartyl proteinase from Candida spp. causing infection in females with type 2 diabetes mellitus. This research aimed to investigate the prevalence of vulvovaginal candidiasis (VVC) in diabetic versus non-diabetic women and compare the ability of identified Candida isolates to secrete phospholipases and aspartyl proteinases with characterization of their genetic profile. The study included 80 females with type 2 diabetes mellitus and 100 non-diabetic females within the child-bearing period. Candida strains were isolated and identified by conventional microbiological methods and by API Candida. The isolates were screened for their extracellular phospholipase and proteinase activities by culturing them on egg yolk and bovine serum albumin media, respectively. Detection of aspartyl proteinase genes (SAP1 to SAP8) and phospholipase genes (PLB1, PLB2) were performed by multiplex polymerase chain reaction. Our results indicated that vaginal candidiasis was significantly higher among the diabetic group versus nondiabetic group (50% versus 20%, respectively) (p = 0.004). C. albicans was the most prevalent species followed by C. glabrata in both groups. No significant association between diabetes mellitus and phospholipase activities was detected (p = 0.262), whereas high significant proteinase activities exhibited by Candida isolated from diabetic females were found (82.5%) (p = 0.000). Non-significant associations between any of the tested proteinase or phospholipase genes and diabetes mellitus were detected (p > 0.05). In conclusion, it is noticed that the incidence of C. glabrata causing VVC is increased. The higher prevalence of vaginal candidiasis among diabetics could be related to the increased aspartyl proteinase production in this group of patients.

  2. Age-Related Changes in Bone Morphology Are Accelerated in Group VIA Phospholipase A2 (iPLA2β)-Null Mice

    PubMed Central

    Ramanadham, Sasanka; Yarasheski, Kevin E.; Silva, Matthew J.; Wohltmann, Mary; Novack, Deborah Veis; Christiansen, Blaine; Tu, Xiaolin; Zhang, Sheng; Lei, Xiaoyong; Turk, John

    2008-01-01

    Phospholipases A2 (PLA2) hydrolyze the sn−2 fatty acid substituent, such as arachidonic acid, from phospholipids, and arachidonate metabolites are recognized mediators of bone modeling. We have previously generated knockout (KO) mice lacking the group VIA PLA2 (iPLA2β), which participates in a variety of signaling events; iPLA2β mRNA is expressed in bones of wild-type (WT) but not KO mice. Cortical bone size, trabecular bone volume, bone mineralizing surfaces, and bone strength are similar in WT and KO mice at 3 months and decline with age in both groups, but the decreases are more pronounced in KO mice. The lower bone mass phenotype observed in KO mice is not associated with an increase in osteoclast abundance/activity or a decrease in osteoblast density, but is accompanied by an increase in bone marrow fat. Relative to WT mice, undifferentiated bone marrow stromal cells (BMSCs) from KO mice express higher levels of PPAR-γ and lower levels of Runx2 mRNA, and this correlates with increased adipogenesis and decreased osteogenesis in BMSCs from these mice. In summary, our studies indicate that age-related losses in bone mass and strength are accelerated in iPLA2β-null mice. Because adipocytes and osteoblasts share a common mesenchymal stem cell origin, our findings suggest that absence of iPLA2β causes abnormalities in osteoblast function and BMSC differentiation and identify a previously unrecognized role of iPLA2β in bone formation. PMID:18349124

  3. Secreted and intracellular phospholipases A2 inhibition by 1-decyl 2-octyl-glycerophosphocholine in rat peritoneal macrophages.

    PubMed

    Boucrot, P; Bobin-Dubigeon, C; Elkihel, L; Letourneux, Y; Jugé, M; Gandemer, G; Petit, J Y

    1998-01-01

    Compounds able to inhibit phospholipases A2 can be considered as potential anti-inflammatory drugs. In this respect, the inhibitory effect of the phospholipid analogue 1-decyl 2-octyl-rac-glycero-3-phosphocholine (decyloctyl-GPC) added to the culture medium of rat peritoneal macrophages stimulated with ionophore A23187 was determined. (a) The substrate of phospholipase A2 1-octadecanoyl 2-[14C]eicosatetraenoyl-sn-glycero-3-phosphocholine ([14C]20:4-GPC) was added to the culture medium. In macrophages + extracellular fluids, its hydrolysis at the 2-position, produced [14C]non-phosphorous lipids which reached 12% of the dose at 0.14 microM, 73% at 0.9 and > 90% at 1.6 microM; in experiments where macrophages and extracellular fluids were analyzed separately, decyloctyl-GPC initially added at 4 microM, significantly inhibited the release of [14C]fatty acids and the eicosanoid synthesis, demonstrating its ability to inhibit secreted and/or intracellular phospholipases A2. (b) Extracellular fluids were separated from the macrophages and incubated with [14C]20:4-GPC: 48% of the dose was hydrolyzed by extracellular fluid-associated secreted phospholipase A2 and decyloctyl-GPC at 3 microM, reduced this hydrolysis by 50%. (c) [3H]arachidonic acid ([3H]20:4) was added to the culture medium and was esterified in the macrophage membrane phospholipids. Activation of intracellular phospholipase A2 induced the release of [3H] fatty acids and eicosanoid synthesis. These releases were inhibited by 50% with decyloctyl-GPC added at 4 microM. (d) [3H]20:4 and [14C]20:4-GPC were added to the culture medium of the macrophages. [3H] and [14C] fatty acids and eicosanoids were released in macrophages or extracellular fluids. They were significantly reduced by the phospholipid analogue added at 4 microM. It is concluded that secreted and intracellular phospholipases A2 were both inhibited by decyloctyl-GPC which extensively reduced the 20:4 release from exogenous and membrane phospholipids

  4. Hydrolysis of short-chain phosphatidylcholines by bee venom phospholipase A2.

    PubMed

    Raykova, D; Blagoev, B

    1986-01-01

    In order to find out the aggregation state of the substrate, preferred by bee venom phospholipase A2 (EC 3.1.1.4), its action on short-chain phosphatidylcholines with two identical (C6-C10) fatty acids has been tested. The rate of hydrolysis as a function of acyl chain length showed a maximum at dioctanoylphosphatidylcholine. The effects of alcohols, NaCl and Triton X-100, which affect the aggregation state of phospholipids in water, were also studied. The addition of n-alcohol led to a significant inhibition of the hydrolysis of the substrates present in micellar form and activated the hydrolysis of substrates which form liposomes. The inhibitory effect increased with increasing length of the aliphatic carbon chain of the alcohol. Triton X-100 at low Triton/phospholipid molar ratios enhanced enzyme activity. These results do not agree with the accepted idea that bee venom phospholipase A2 hydrolyzes short-chain lecithins in their molecularly dispersed form and that micelles cannot act as substrates. The data indicate that short-chain lecithins in the aggregated state are hydrolyzed and that the requirements of bee venom phospholipase A2 for the aggregation state of the substrate are not strict.

  5. Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70 kDa protein.

    PubMed

    Gross, W; Yang, W; Boss, W F

    1992-02-19

    Plasma membranes were isolated from carrot (Daucus carota L.) cells grown in suspension culture and treated with phospholipase A2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinase or phosphatidylinositol monophosphate kinase activity released from the membranes after the phospholipase A2 treatment. Treating the plasma membranes with phospholipase C or D did not release PI kinase activity. The phospholipase A2-released PI kinase was activated over 2-fold by a heat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the Vmax but does not affect the Km of the phospholipase A2-released PI kinase.

  6. New phospholipase A1-producing bacteria from a marine fish.

    PubMed

    Nishihara, Masaaki; Kamata, Masazumi; Koyama, Tomoyuki; Yazawa, Kazunaga

    2008-01-01

    Phospholipase A1 is a hydrolytic enzyme that catalyzes the removal of the acyl group from position 1 of glycerophospholipids to form 2-acyl lysophospholipids. Lysophospholipids are used in foods, cosmetics, and pharmaceuticals as surfactants. Novel forms of phospholipase A1 that function at low temperatures are desirable for use in lipophilic systems in food processing. However, there is currently little variety in the available sources of phospholipase A1. Given this situation, we screened the intestinal contents of marine animals for phospholipase A1-producing bacteria. Colonies that formed a halo on K28CP screening medium and that grew in K28 medium were cultured in liquid K28 medium, and the supernatant was retrieved for analysis. Phosphatidylcholine was added to the culture supernatant, and the product of the reaction was analyzed by using TLC. For culture supernatants that were able to generate lysophosphatidylcholine, synthetic phosphatidylcholines were added, and the site of the reaction was determined by analyzing the fatty acid compositions of the lysophosphatidylcholines generated by GLC. A bacterial isolate from a flatfish, which we named HFKI0020, was found to have phospholipase A1 activity at low temperatures. We determined that the isolate HFKI0020 is closely related to Pseudomonas by using 16S rDNA sequence analysis and by characterizing the isolate with respect to its physiologic and biochemical properties. From the intestinal contents of a marine fish, we successfully isolated a bacterium that secretes phospholipase A1 that is active at low temperatures.

  7. Molecular and functional characterization of polymorphisms in the secreted phospholipase A2 group X gene: relevance to coronary artery disease.

    PubMed

    Gora, Sarah; Perret, Claire; Jemel, Ikram; Nicaud, Viviane; Lambeau, Gérard; Cambien, François; Ninio, Ewa; Blankenberg, Stefan; Tiret, Laurence; Karabina, Sonia-Athina

    2009-07-01

    Among secreted phospholipases A2 (sPLA2s), human group X sPLA2 (hGX sPLA2) is emerging as a novel attractive therapeutic target due to its implication in inflammatory diseases. To elucidate whether hGX sPLA2 plays a causative role in coronary artery disease (CAD), we screened the human PLA2G10 gene to identify polymorphisms and possible associations with CAD end-points in a prospective study, AtheroGene. We identified eight polymorphisms, among which, one non-synonymous polymorphism R38C in the propeptide region of the sPLA2. The T-512C polymorphism located in the 5' untranslated region was associated with a decreased risk of recurrent cardiovascular events during follow-up. The functional analysis of the R38C polymorphism showed that it leads to a profound change in expression and activity of hGX sPLA2, although there was no detectable impact on CAD risk. Due to the potential role of hGX sPLA2 in inflammatory processes, these polymorphisms should be investigated in other inflammatory diseases.

  8. Discovery of Ecopladib, an indole inhibitor of cytosolic phospholipase A2alpha.

    PubMed

    Lee, Katherine L; Foley, Megan A; Chen, Lihren; Behnke, Mark L; Lovering, Frank E; Kirincich, Steven J; Wang, Weiheng; Shim, Jaechul; Tam, Steve; Shen, Marina W H; Khor, Soopeang; Xu, Xin; Goodwin, Debra G; Ramarao, Manjunath K; Nickerson-Nutter, Cheryl; Donahue, Frances; Ku, M Sherry; Clark, James D; McKew, John C

    2007-03-22

    The synthesis and structure-activity relationship of a series of indole inhibitors of cytosolic phospholipase A2alpha (cPLA2alpha, type IVA phospholipase) are described. Inhibitors of cPLA2alpha are predicted to be efficacious in treating asthma as well as the signs and symptoms of osteoarthritis, rheumatoid arthritis, and pain. The introduction of a benzyl sulfonamide substituent at C2 was found to impart improved potency of these inhibitors, and the SAR of these sulfonamide analogues is disclosed. Compound 123 (Ecopladib) is a sub-micromolar inhibitor of cPLA2alpha in the GLU micelle and rat whole blood assays. Compound 123 displayed oral efficacy in the rat carrageenan air pouch and rat carrageenan-induced paw edema models.

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

    PubMed Central

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

    2014-01-01

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

  10. Activities of native and tyrosine-69 mutant phospholipases A2 on phospholipid analogues. A reevaluation of the minimal substrate requirements.

    PubMed

    Kuipers, O P; Dekker, N; Verheij, H M; de Haas, G H

    1990-06-26

    The role of Tyr-69 of porcine pancreatic phospholipase A2 in substrate binding was studied with the help of proteins modified by site-directed mutagenesis and phospholipid analogues with a changed head-group geometry. Two mutants were used containing Phe and Lys, respectively, at position 69. Modifications in the phospholipids included introduction of a sulfur at the phosphorus (thionophospholipids), removal of the negative charge at phosphorus (phosphatidic acid dimethyl ester), and reduction (phosphonolipids) or extension (diacylbutanetriol choline phosphate) of the distance between the phosphorus and the acyl ester bond. Replacement of Tyr-69 by Lys reduces enzymatic activity, but the mutant enzyme retains both the stereospecificity and positional specificity of native phospholipase A2. The Phe-69 mutant not only hydrolyzes the Rp isomer of thionophospholipids more efficiently than the wild-type enzyme, but the Sp thiono isomer is hydrolyzed too, although at a low (approximately 4%) rate. Phosphonolipids are hydrolyzed by native phospholipase A2 about 7 times more slowly than natural phospholipids, with retention of positional specificity and a (partial) loss of stereospecificity. The dimethyl ester of phosphatidic acid is degraded efficiently in a calcium-dependent and positional-specific way by native phospholipase A2 and by the mutants, indicating that a negative charge at phosphorus is not an absolute substrate requirement. The activities on the phosphatidic acid dimethyl ester of native enzyme and the Lys-69 mutant are lower than those on the corresponding lecithin, in contrast to the Phe-69 mutant, which has equal activities on both substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. 40 CFR 721.4585 - Lecithins, phospholipase A2-hydrolyzed.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) Release to water. Requirements as specified in § 721.90 (a)(4), (b)(4), and (c)(4) (where N = 10 ppb). (b..., phospholipase A2-hydrolyzed (PMN P-93-333) is subject to reporting under this section for the significant new... communication program. Requirements as specified in § 721.72 (a), (b), (c), (d), (f), (g)(3)(i), and (g)(3)(ii...

  12. Brown spider phospholipase-D containing a conservative mutation (D233E) in the catalytic site: identification and functional characterization.

    PubMed

    Vuitika, Larissa; Gremski, Luiza Helena; Belisário-Ferrari, Matheus Regis; Chaves-Moreira, Daniele; Ferrer, Valéria Pereira; Senff-Ribeiro, Andrea; Chaim, Olga Meiri; Veiga, Silvio Sanches

    2013-11-01

    phospholipase-D recombinant toxin contains a conservative mutation (D233E) on the catalytic site. 2-LiRecDT7 shares high identity level with isoforms of Loxosceles genus. 3-LiRecDT7 is a recombinant protein immunodetected by specific antibodies to native and recombinant phospholipase-D toxins. 4-LiRecDT7 shows sphingomyelinase-D activity in a concentration-dependent manner, but less intense than other isoforms. 5-LiRecDT7 induces dermonecrosis and inflammatory response in rabbit skin. 6-LiRecDT7 increases vascular permeability in mice. 7-LiRecDT7 triggers direct complement-independent hemolysis in erythrocytes. © 2013 Wiley Periodicals, Inc.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

    Lee, Gihyun; Bae, Hyunsu

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  16. Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils.

    PubMed

    Hallstrand, Teal S; Lai, Ying; Hooper, Kathryn A; Oslund, Rob C; Altemeier, William A; Matute-Bello, Gustavo; Gelb, Michael H

    2016-01-01

    Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1β increased the

  17. Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice.

    PubMed

    Escoffier, Jessica; Jemel, Ikram; Tanemoto, Akemi; Taketomi, Yoshitaka; Payre, Christine; Coatrieux, Christelle; Sato, Hiroyasu; Yamamoto, Kei; Masuda, Seiko; Pernet-Gallay, Karin; Pierre, Virginie; Hara, Shuntaro; Murakami, Makoto; De Waard, Michel; Lambeau, Gérard; Arnoult, Christophe

    2010-05-01

    Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization.

  18. Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice

    PubMed Central

    Escoffier, Jessica; Jemel, Ikram; Tanemoto, Akemi; Taketomi, Yoshitaka; Payre, Christine; Coatrieux, Christelle; Sato, Hiroyasu; Yamamoto, Kei; Masuda, Seiko; Pernet-Gallay, Karin; Pierre, Virginie; Hara, Shuntaro; Murakami, Makoto; De Waard, Michel; Lambeau, Gérard; Arnoult, Christophe

    2010-01-01

    Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization. PMID:20424324

  19. Lack of genetic association between the phospholipase A2 gene and bipolar mood disorder in a European multicentre case-control study.

    PubMed

    Dikeos, Dimitris G; Papadimitriou, George N; Souery, Daniel; Del-Favero, Jurgen; Massat, Isabelle; Blackwood, Douglas; Cichon, Sven; Daskalopoulou, Eugenia; Ivezic, Sladjana; Kaneva, Radka; Karadima, Georgia; Lorenzi, Cristina; Milanova, Vihra; Muir, Walter; Nöthen, Markus; Oruc, Lilijana; Rietschel, Marcella; Serretti, Alessandro; Van Broeckhoven, Christine; Soldatos, Constantin R; Stefanis, Costas N; Mendlewicz, Julien

    2006-08-01

    The possible association between phospholipase A2 gene and bipolar mood disorder was examined in 557 bipolar patients and 725 controls (all personally interviewed), recruited from seven countries (Belgium, Bulgaria, Croatia, Germany, Greece, Italy, and UK). The frequencies of the eight alleles that were identified did not differ between patients and control individuals in the whole population, while the power to detect an association based on our sample was relatively high. Some differences were noted among the various ethnic groups, but no significant trends existed, suggesting that population stratification by country may not be responsible for a type II error. On the basis of these results, mutations of the phospholipase A2 gene, at least in the region close to the polymorphism examined between exons 1 and 2, are not involved in the pathogenesis of bipolar mood disorder.

  20. A MIDGUT DIGESTIVE PHOSPHOLIPASE A2 IN LARVAL MOSQUITOES, AEDES ALBOPICTUS AND CULEX QUINQUEFASCIATUS

    USDA-ARS?s Scientific Manuscript database

    Phospholipase A2 (PLA2) is a secretory digestive enzyme that hydrolyzes ester bond at sn-2 position of dietary phospholipids, creating free fatty acid and lysophopholipid. The free fatty acids (arachidonic acid) are absorbed into midgut cells. Aedes albopictus and Culex quinquefasciatus digestive PL...

  1. Cloning and characterization of a basic phospholipase A2 homologue from Micrurus corallinus (coral snake) venom gland.

    PubMed

    de Oliveira, Ursula Castro; Assui, Alessandra; da Silva, Alvaro Rossan de Brandão Prieto; de Oliveira, Jane Silveira; Ho, Paulo Lee

    2003-09-01

    During the cloning of abundant cDNAs expressed in the Micrurus corallinus coral snake venom gland, several putative toxins, including a phospholipase A2 homologue cDNA (clone V2), were identified. The V2 cDNA clone codes for a potential coral snake toxin with a signal peptide of 27 amino acid residues plus a predicted mature protein with 119 amino acid residues. The deduced protein is highly similar to known phospholipases A2, with seven deduced S-S bridges at the same conserved positions. This protein was expressed in Escherichia coli as a His-tagged protein that allowed the rapid purification of the recombinant protein. This protein was used to generate antibodies, which recognized the recombinant protein in Western blot. This antiserum was used to screen a large number of venoms, showing a ubiquitous distribution of immunorelated proteins in all elapidic venoms but not in the viperidic Bothrops jararaca venom. This is the first description of a complete primary structure of a phospholipase A2 homologue deduced by cDNA cloning from a coral snake.

  2. Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta.

    PubMed

    Ma, Z; Ramanadham, S; Wohltmann, M; Bohrer, A; Hsu, F F; Turk, J

    2001-04-20

    A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.

  3. Crab digestive phospholipase: a new invertebrate member.

    PubMed

    Cherif, Slim; Ben Bacha, Abir; Ben Ali, Yassine; Horchani, Habib; Rekik, Wiem; Gargouri, Youssef

    2010-01-01

    Crab digestive phospholipase (CDPL) was purified from the hepatopancreas of Carcinus mediterraneus crabs. Homogeneous enzyme was obtained after two chromatography steps: anion exchange and size exclusion HPLC column. Homogeneous CDPL has a molecular mass of 14 kDa as determined by SDS/PAGE analysis. Unlike known digestive phospholipases like porcine PLA(2) (PPPL), CDPL displayed its maximal activity at 50 degrees C and not at 37 degrees C. A specific activity of 40 U/mg for the purified CDPL was measured using PC as substrate under optimal conditions (pH 8 and 50 degrees C) in the presence of 8 mM sodium deoxycholate (NaDC) and 10 mM CaCl(2). In contrast to PPPL, purified CDPL was completely inactivated at 60 degrees C. The N-terminal sequence was determined by automatic Edman degradation. No similarity between 12 N-terminal amino acid residues of CDPL was found with those of known digestive phospholipases. CDPL appears to be a new member of invertebrate phospholipases, and it is potentially useful for treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds which can be used in the food industry.

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

    PubMed Central

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

    2016-01-01

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

  5. Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles.

    PubMed

    Horrevoets, A J; Hackeng, T M; Verheij, H M; Dijkman, R; de Haas, G H

    1989-02-07

    The substrate specificity of Escherichia coli outer membrane phospholipase A was analyzed in mixed micelles of lipid with deoxycholate or Triton X-100. Diglycerides, monoglycerides, and Tweens 40 and 85 in Triton X-100 are hydrolyzed at rates comparable to those of phospholipids and lysophospholipids. p-Nitrophenyl esters of fatty acids with different chain lengths and triglycerides are not hydrolyzed. The minimal substrate characteristics consist of a long acyl chain esterified to a more or less hydrophilic headgroup as is the case for the substrate monopalmitoylglycol. Binding occurs via the hydrocarbon chain of the substrate; diacyl compounds are bound three to five times better than monoacyl compounds. When acting on lecithins, phospholipase A1 activity is six times higher than phospholipase A2 activity or 1-acyl lysophospholipase activity. Activity on the 2-acyl lyso compound is about two times less than that on the 1-acyl lysophospholipid. The enzyme therefore has a clear preference for the primary ester bond of phospholipids. In contrast to phospholipase A1 activity, phospholipase A2 activity is stereospecific. Only the L isomer of a lecithin analogue in which the primary acyl chain was replaced by an alkyl ether group is hydrolyzed. The D isomer of this analogue is a competitive inhibitor, bound with the same affinity as the L isomer. On these ether analogues the enzyme shows the same preference for the primary acyl chain as with the natural diester phospholipids. Despite its broad specificity, the enzyme will initially act as a phospholipase A1 in the E. coli envelope where it is embedded in phospholipids.

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

    PubMed

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

    2013-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  8. Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Samanta, Uttamkumar; Kirby, Stephen D.; Srinivasan, Prabhavathi

    The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P{submore » R} and P{sub S} stereoisomers at the P-chiral center. The tabun complex displayed only the P{sub R} stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.« less

  9. Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes.

    PubMed

    Samanta, Uttamkumar; Kirby, Stephen D; Srinivasan, Prabhavathi; Cerasoli, Douglas M; Bahnson, Brian J

    2009-08-15

    The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P(R) and P(S) stereoisomers at the P-chiral center. The tabun complex displayed only the P(R) stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.

  10. Group X secreted phospholipase A2 proenzyme is matured by a furin-like proprotein convertase and releases arachidonic acid inside of human HEK293 cells.

    PubMed

    Jemel, Ikram; Ii, Hiromi; Oslund, Rob C; Payré, Christine; Dabert-Gay, Anne-Sophie; Douguet, Dominique; Chargui, Khaoula; Scarzello, Sabine; Gelb, Michael H; Lambeau, Gérard

    2011-10-21

    Among mammalian secreted phospholipases A(2) (sPLA(2)s), group X sPLA(2) has the most potent hydrolyzing activity toward phosphatidylcholine and is involved in arachidonic acid (AA) release. Group X sPLA(2) is produced as a proenzyme and contains a short propeptide of 11 amino acids ending with a dibasic motif, suggesting cleavage by proprotein convertases. Although the removal of this propeptide is clearly required for enzymatic activity, the cellular location and the protease(s) involved in proenzyme conversion are unknown. Here we have analyzed the maturation of group X sPLA(2) in HEK293 cells, which have been extensively used to analyze sPLA(2)-induced AA release. Using recombinant mouse (PromGX) and human (ProhGX) proenzymes; HEK293 cells transfected with cDNAs coding for full-length ProhGX, PromGX, and propeptide mutants; and various permeable and non-permeable sPLA(2) inhibitors and protease inhibitors, we demonstrate that group X sPLA(2) is mainly converted intracellularly and releases AA before externalization from the cell. Most strikingly, the exogenous proenzyme does not elicit AA release, whereas the transfected proenzyme does elicit AA release in a way insensitive to non-permeable sPLA(2) inhibitors. In transfected cells, a permeable proprotein convertase inhibitor, but not a non-permeable one, prevents group X sPLA(2) maturation and partially blocks AA release. Mutations at the dibasic motif of the propeptide indicate that the last basic residue is required and sufficient for efficient maturation and AA release. All together, these results argue for the intracellular maturation of group X proenzyme in HEK293 cells by a furin-like proprotein convertase, leading to intracellular release of AA during secretion.

  11. Group X Secreted Phospholipase A2 Proenzyme Is Matured by a Furin-like Proprotein Convertase and Releases Arachidonic Acid inside of Human HEK293 Cells*

    PubMed Central

    Jemel, Ikram; Ii, Hiromi; Oslund, Rob C.; Payré, Christine; Dabert-Gay, Anne-Sophie; Douguet, Dominique; Chargui, Khaoula; Scarzello, Sabine; Gelb, Michael H.; Lambeau, Gérard

    2011-01-01

    Among mammalian secreted phospholipases A2 (sPLA2s), group X sPLA2 has the most potent hydrolyzing activity toward phosphatidylcholine and is involved in arachidonic acid (AA) release. Group X sPLA2 is produced as a proenzyme and contains a short propeptide of 11 amino acids ending with a dibasic motif, suggesting cleavage by proprotein convertases. Although the removal of this propeptide is clearly required for enzymatic activity, the cellular location and the protease(s) involved in proenzyme conversion are unknown. Here we have analyzed the maturation of group X sPLA2 in HEK293 cells, which have been extensively used to analyze sPLA2-induced AA release. Using recombinant mouse (PromGX) and human (ProhGX) proenzymes; HEK293 cells transfected with cDNAs coding for full-length ProhGX, PromGX, and propeptide mutants; and various permeable and non-permeable sPLA2 inhibitors and protease inhibitors, we demonstrate that group X sPLA2 is mainly converted intracellularly and releases AA before externalization from the cell. Most strikingly, the exogenous proenzyme does not elicit AA release, whereas the transfected proenzyme does elicit AA release in a way insensitive to non-permeable sPLA2 inhibitors. In transfected cells, a permeable proprotein convertase inhibitor, but not a non-permeable one, prevents group X sPLA2 maturation and partially blocks AA release. Mutations at the dibasic motif of the propeptide indicate that the last basic residue is required and sufficient for efficient maturation and AA release. All together, these results argue for the intracellular maturation of group X proenzyme in HEK293 cells by a furin-like proprotein convertase, leading to intracellular release of AA during secretion. PMID:21878635

  12. [Inhibition of phospholipase A2 of peritoneal macrophages in rats by 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine].

    PubMed

    Boucrot, P; Khettab, E N; Petit, J Y; Welin, L

    1993-01-01

    The 1-O-stearoyl-2-O-[3H] arachidonyl-sn-glycero-3-phosphocholine, introduced in the culture medium, was taken up by the peritoneal macrophages activated by the ionophore A 23187. After intracellular phospholipase A2 activity, the [3H] arachidonic acid was found in cells and in extracellular fluids. It also reached the eicosanoid synthesis. When it was introduced in the culture medium with the tritiated phospholipid, the 1, 2 di-O-hexadecyl-rac-glycero-3-phosphocholine, which has a non hydrolysable alkylated structure in the 2 position of the glycerol, inhibited the intracellular phospholipase A2, then contributed to lower the eicosanoid synthesis.

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

    NASA Astrophysics Data System (ADS)

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

    2007-08-01

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

  14. Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells.

    PubMed

    Abdel-Latif, A A; Ding, K H; Akhtar, R A; Yousufzai, S Y

    1996-09-01

    In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.2 nM) than in activating PLC (EC50 = 1.5 nM) or PLA2 (EC50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA2 was stimulated first (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.

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

    PubMed Central

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

    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/neutralizing potential of ethanolic extract of A. paniculata on the isolated inflammatory PLA2 (VRV-PL-VIIIa) from Daboii rusellii pulchella (belonging to Group IIA inflammatory secretory PLA2 [sPLA2]) and its associated edema-induced activities in Swiss albino mice. A. paniculata extract dose dependently inhibited the Group IIA sPLA2 enzymatic activity with an IC50 value of 10.3 ± 0.5 μg/ml. Further, the extract dose dependently inhibited the edema formation, when co-injected with enzyme indicating that a strong correlation exists between lipolytic and pro-inflammatory activities of the enzyme. In conclusion, results of this study shows that the ethanolic extract of A. paniculata effectively inhibits Group IIA sPLA2 and its associated inflammatory activities, which substantiate its anti-inflammatory properties. The results of the present study warranted further studies to develop bioactive compound (s) in ethanolic extract of A. paniculata as potent therapeutic agent (s) for inflammatory diseases. SUMMARY This study emphasis the anti-inflammatory effect of A. paniculata by inhibiting the inflammatory Group IIA sPLA2 and its associated inflammatory activities such as edema. It was found that there is a strong correlation between lipolytic activity and pro-inflammatory activity inhibition. Therefore, the study suggests that the extract processes potent anti-inflammatory agents, which could be developed as a potential therapeutic agent against inflammatory and related diseases

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

    PubMed Central

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

    2012-01-01

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

  17. Synthetic and natural inhibitors of phospholipases A2: their importance for understanding and treatment of neurological disorders.

    PubMed

    Ong, Wei-Yi; Farooqui, Tahira; Kokotos, George; Farooqui, Akhlaq A

    2015-06-17

    Phospholipases A2 (PLA2) are a diverse group of enzymes that hydrolyze membrane phospholipids into arachidonic acid and lysophospholipids. Arachidonic acid is metabolized to eicosanoids (prostaglandins, leukotrienes, thromboxanes), and lysophospholipids are converted to platelet-activating factors. These lipid mediators play critical roles in the initiation, maintenance, and modulation of neuroinflammation and oxidative stress. Neurological disorders including excitotoxicity; traumatic nerve and brain injury; cerebral ischemia; Alzheimer's disease; Parkinson's disease; multiple sclerosis; experimental allergic encephalitis; pain; depression; bipolar disorder; schizophrenia; and autism are characterized by oxidative stress, inflammatory reactions, alterations in phospholipid metabolism, accumulation of lipid peroxides, and increased activities of brain phospholipase A2 isoforms. Several old and new synthetic inhibitors of PLA2, including fatty acid trifluoromethyl ketones; methyl arachidonyl fluorophosphonate; bromoenol lactone; indole-based inhibitors; pyrrolidine-based inhibitors; amide inhibitors, 2-oxoamides; 1,3-disubstituted propan-2-ones and polyfluoroalkyl ketones as well as phytochemical based PLA2 inhibitors including curcumin, Ginkgo biloba and Centella asiatica extracts have been discovered and used for the treatment of neurological disorders in cell culture and animal model systems. The purpose of this review is to summarize information on selective and potent synthetic inhibitors of PLA2 as well as several PLA2 inhibitors from plants, for treatment of oxidative stress and neuroinflammation associated with the pathogenesis of neurological disorders.

  18. Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

    PubMed Central

    Burch, R M; Connor, J R; Axelrod, J

    1988-01-01

    Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

  19. Legionella phospholipases implicated in virulence.

    PubMed

    Kuhle, Katja; Flieger, Antje

    2013-01-01

    Phospholipases are diverse enzymes produced in eukaryotic hosts and their bacterial pathogens. Several pathogen phospholipases have been identified as major virulence factors acting mainly in two different modes: on the one hand, they have the capability to destroy host membranes and on the other hand they are able to manipulate host signaling pathways. Reaction products of bacterial phospholipases may act as secondary messengers within the host and therefore influence inflammatory cascades and cellular processes, such as proliferation, migration, cytoskeletal changes as well as membrane traffic. The lung pathogen and intracellularly replicating bacterium Legionella pneumophila expresses a variety of phospholipases potentially involved in disease-promoting processes. So far, genes encoding 15 phospholipases A, three phospholipases C, and one phospholipase D have been identified. These cell-associated or secreted phospholipases may contribute to intracellular establishment, to egress of the pathogen from the host cell, and to the observed lung pathology. Due to the importance of phospholipase activities for host cell processes, it is conceivable that the pathogen enzymes may mimic or substitute host cell phospholipases to drive processes for the pathogen's benefit. The following chapter summarizes the current knowledge on the L. pneumophila phospholipases, especially their substrate specificity, localization, mode of secretion, and impact on host cells.

  20. Group X secreted phospholipase A₂ specifically decreases sperm motility in mice.

    PubMed

    Escoffier, Jessica; Pierre, Virginie J; Jemel, Ikram; Munch, Léa; Boudhraa, Zied; Ray, Pierre F; De Waard, Michel; Lambeau, Gérard; Arnoult, Christophe

    2011-10-01

    Different mammalian secreted phospholipases A(2) (sPLA(2) s) are expressed in male reproductive organs and/or in sperm cells but their cellular functions are still not fully characterized. Because several reports indicate a link between cellular lipids and sperm motility, we have investigated the effect of mouse group IIA, IID, IIE, V, and X sPLA(2) s on sperm motility. Among these enzymes, only mouse group X sPLA(2) (mGX sPLA(2) ) acts as a potent inhibitor of sperm motility that decreases track speed (VCL) and lateral displacement of the head (ALH) of both noncapacitated and capacitated sperm. The inhibitory effect of mGX sPLA(2) is dependent on its enzymatic activity because (i) both the proenzyme form of mGX sPLA(2) (pro-mGX) and the H48Q mutant of mGX sPLA(2) have very weak enzymatic activity and are unable to modulate sperm motility and (ii) LY329722, a specific inhibitor of sPLA(2) s, blocks the inhibitory effect of mGX sPLA(2) . Moreover, mGX sPLA(2) exerts a gradual potency on sperm subpopulations with different velocities, an effect which may be linked to the heterogeneity of lipid composition in these sperm subpopulations. Finally, we found that endogenous mGX sPLA(2) released during spontaneous acrosome reaction modulates sperm motility of capacitated sperm. Together, our results suggest a new role of sPLA(2) in sperm physiology where the sPLA2 selects a sperm subpopulation for fertilization based on its effect on sperm motility. Copyright © 2010 Wiley-Liss, Inc.

  1. Development of a potent 2-oxoamide inhibitor of secreted phospholipase A2 guided by molecular docking calculations and molecular dynamics simulations

    PubMed Central

    Vasilakaki, Sofia; Barbayianni, Efrosini; Leonis, Georgios; Papadopoulos, Manthos G.; Mavromoustakos, Thomas; Gelb, Michael H.; Kokotos, George

    2016-01-01

    Inhibition of group IIA secreted phospholipase A2 (GIIA sPLA2) has been an important objective for medicinal chemists. We have previously shown that inhibitors incorporating the 2-oxoamide functionality may inhibit human and mouse GIIA sPLA2s. Herein, the development of new potent inhibitors by molecular docking calculations using the structure of the known inhibitor 7 as scaffold, are described. Synthesis and biological evaluation of the new compounds revealed that the long chain 2-oxoamide based on (S)-valine GK241 led to improved activity (IC50 = 143 nM and 68 nM against human and mouse GIIA sPLA2, respectively). In addition, molecular dynamics simulations were employed to shed light on GK241 potent and selective inhibitory activity. PMID:26970660

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

    PubMed Central

    Sekar, K.; Rajakannan, V.; Gayathri, D.; Velmurugan, D.; Poi, M.-J.; Dauter, M.; Dauter, Z.; Tsai, M.-D.

    2005-01-01

    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% (R free = 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. PMID:16508077

  3. Deposition of lipid, protein, and secretory phospholipase A2 on hydrophilic contact lenses.

    PubMed

    Mochizuki, Hiroshi; Yamada, Masakazu; Hatou, Shin; Kawashima, Motoko; Hata, Seiichiro

    2008-01-01

    Recent studies have shown that low tear phospholipid levels are associated with tear film instability in hydrophilic contact lens wearers. The concentration of secretory phospholipase A2 (sPLA2), the enzyme that hydrolyzes phospholipids, in tears is known to exceed the levels found in serum by four orders of magnitude. This study was performed to determine the levels of sPLA2 from the deposition on two different frequent-replacement contact lens materials. Polymacon and etafilcon A contact lenses worn for 2 weeks by 16 experienced contact lens wearers were used for the analysis. Total lipids were determined by the sulfo-phospho-vanillin reaction. Phospholipids in lipid extracts were estimated by phosphorus determination with ammonium molybdate through enzymatic digestion. Total protein was measured by bicinchoninic acid analysis. Double-antibody sandwich enzyme-linked immunosorbent assay was used to determine sPLA2 concentrations. Total lipid deposition was found to be greater in the polymacon group (66.3+/-16.3 microg/lens) than in the etafilcon A group, although phospholipids were not detected in either group. The etafilcon A group had greater deposition of protein (3.7+/-0.7 mg/lens) than the polymacon group had. The etafilcon A group deposited statistically significantly more group IIa sPLA2 (1.1+/-0.3 microg/lens) than the polymacon group (0.07+/-0.04 microg/lens) did (P<0.001). There was a significant difference in the lipid and protein deposition profiles in the two lenses tested. A significant amount of sPLA2 in the deposition on contact lenses may play a role in tear film instability in hydrophilic contact lens wearers.

  4. Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

    PubMed Central

    Flores-Díaz, Marietta; Monturiol-Gross, Laura; Naylor, Claire

    2016-01-01

    SUMMARY 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

  5. Phospholipase A2 from Bothrops alternatus (víbora de la cruz) venom. Purification and some characteristic properties.

    PubMed

    Nisenbom, H E; Seki, C; Vidal, J C

    1986-01-01

    One single protein species with phospholipase activity has been isolated from Bothrops alternatus venom by a procedure involving gel-filtration on Sephadex G-50 (Step 1), chromatography on SP-Sephadex C-50 (Step 2) and gel-filtration on Sephadex G-75 (Step 3). The purified sample behaved as a homogeneous, monodisperse protein with a molecular weight of 15,000 and isoelectric point of 5.04. The yield in enzyme activity was 48% of the starting material and the apparent purification was 51-fold. When assayed on 1,2-diheptanoyl- or 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine, fatty acids and lysolecithins were the only reaction products, in accordance with the predicted stoichiometry. Studies on positional specificity suggested that the enzyme is a phospholipase A2. The enzyme requires Ca2+ ions for activity and exhibited stereochemical specificity, since the enantiomeric 2, 3-diheptanoyl-sn-glycero-1-phosphorylcholine was not hydrolyzed. Under the experimental conditions employed, reaction products representative of either phospholipase B or C activities could not be detected. After Step 1, the phospholipase activity recovered was higher than the total activity in the crude venom sample, which is explained by the separation of an inhibitor during enzyme purification. The inhibitor was responsible for the initial lag period that characterized the kinetics of the enzyme reaction with crude venom acting on aggregated substrates (lipoprotein, vesicles or micelles), while the rate of hydrolysis of monomeric lecithins was not affected.

  6. A RAPID AND SIMPLE PHOSPHOLIPASE A ASSAY,

    DTIC Science & Technology

    A simple and rapid method for the assay of phospholipase A was developed. As a substrate fresh egg yolk is used which is hydrolyzed by snake venom...phospholipase A at a 10-20 x faster rate than pure lecithin . The released fatty acids, after extraction with appropriate solvents are titrated

  7. Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.

    PubMed Central

    Balsinde, J; Bianco, I D; Ackermann, E J; Conde-Frieboes, K; Dennis, E A

    1995-01-01

    Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca(2+)-independent process and that it is blocked up to 60-70% by a bromoenollactone (BEL) that is a selective inhibitor of a newly discovered Ca(2+)-independent phospholipase A2 (PLA2) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca(2+)-independent PLA2 activity in these cells. This inhibition is specific for the Ca(2+)-independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca(2+)-independent PLA2--namely a bromomethyl ketone and methyl-BEL--do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca(2+)-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction. PMID:7667324

  8. Purification and characterization of a phospholipase by Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum.

    PubMed

    Hsu, Po-Yuan; Lee, Kuo-Kau; Hu, Chih-Chuang; Liu, Ping-Chung

    2014-09-01

    Toxicity of the extracellular products (ECPs) and the lethal attributes of phospholipase secreted by pathogenic Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum was studied. An extracellular lethal toxin in the ECPs was partially purified by using Fast Protein Liquid Chromatography system. A protein band (27 kDa) exhibited phospholipase activity on Native-PAGE (by 0.3% egg yolk agar-overlay), was excised and eluted. The pI value of the purified phospholipase was determined as 3.65 and was determined as a phospholipase C by using the Amplex™ Red phosphatidylcholine -Specific phospholipase C Assay kit. The phospholipase showed maximum activity at temperature around 4-40 °C and maximal activity at pH between 8 and 9. The enzyme was inhibited by ethylenediamine-tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS); but was activated by Ca(2+) and Mg(2+) and inactivated by Zn(2+) and Cu(2+) . Both the ECPs and phospholipase were hemolytic against erythrocytes of cobia and lethal to the fish with LD50 values of 3.25 and 0.91 µg protein g(-1) fish, respectively. In toxicity neutralization test, the rabbit antisera against the phospholipase could neutralize the toxicity of ECPs, indicating that the phospholipase is a major extracellular toxin produced by the bacterium. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP).

    PubMed

    Amara, Sawsan; Delorme, Vincent; Record, Michel; Carrière, Frédéric

    2012-11-01

    Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the presence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351Da, suggesting a covalent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water, HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides>PC>MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Silibinin down-regulates expression of secreted phospholipase A2 enzymes in cancer cells.

    PubMed

    Hagelgans, Albert; Nacke, Brit; Zamaraeva, Maria; Siegert, Gabriele; Menschikowski, Mario

    2014-04-01

    Silibinin, a naturally-occurring flavonoid produced by milk thistle, possesses antioxidant, anti-inflammatory and cancer-preventive activities. In the current study, we examined the effects of silibinin on the expression of secreted phospholipase A2 (sPLA2) enzymes, especially those of group IIA (hGIIA), which play a crucial role in inflammation and carcinogenesis. The effects of silibinin on sPLA2 expressions in human HepG2 hepatoma and PC-3 prostate cancer cells were analyzed using quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay technique. Silibinin inhibited the expression of hGIIA in unstimulated and cytokine-primed HepG2 and PC-3 cells. The mRNA levels of sPLA2 of groups IB, III and V were also significantly decreased by silibinin. Analyses of transcription factor activation suggest that nuclear factor-κB, but not specificity protein 1 (SP1) is implicated in the silibinin-mediated down-regulation of hGIIA. Silibinin exhibits inhibitory effects on basal and cytokine-induced expression of sPLA2s in cancer cells and therefore, may have the potential to protect against up-regulation of hGIIA and other sPLA2 isoforms during inflammation and cancer.

  11. Crystal structure of a complex formed between a snake venom phospholipase A(2) and a potent peptide inhibitor Phe-Leu-Ser-Tyr-Lys at 1.8 A resolution.

    PubMed

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

    2002-10-25

    Phospholipase A(2) is an important enzyme involved in the production of prostaglandins and their related compounds causing inflammatory disorders. Among the several peptides tested, the peptide Phe-Leu-Ser-Tyr-Lys (FLSYK) showed the highest inhibition. The dissociation constant (K(d)) for this peptide was calculated to be 3.57 +/- 0.05 x 10(-9) m. In order to further improve the degree of inhibition of phospholipase A(2), a complex between Russells viper snake venom phospholipase A(2) and a peptide inhibitor FLSYK was crystallized, and its structure was determined by crystallographic methods and refined to an R-factor of 0.205 at 1.8 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) (molecules A and B) and a peptide molecule specifically bound to molecule A only. The two molecules formed an asymmetric dimer. The dimerization caused a modification in the binding site of molecule A. The overall conformations of molecules A and B were found to be generally similar except three regions i.e. the Trp-31-containing loop (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Out of the above three, the most striking difference pertains to the conformation of Trp-31 in the two molecules. The orientation of Trp-31 in molecule A was suitable for the binding of FLSYK, while it disallowed the binding of peptide to molecule B. The structure of the complex clearly shows that the peptide is so placed in the binding site of molecule A that the side chain of its lysine residue interacted extensively with the enzyme and formed several hydrogen bonds in addition to a strong electrostatic interaction with critical Asp-49. The C-terminal carboxylic group of the peptide interacted with the catalytic residue His-48.

  12. Novel Metagenome-Derived, Cold-Adapted Alkaline Phospholipase with Superior Lipase Activity as an Intermediate between Phospholipase and Lipase

    PubMed Central

    Lee, Mi-Hwa; Oh, Ki-Hoon; Kang, Chul-Hyung; Kim, Ji-Hoon; Oh, Tae-Kwang; Ryu, Choong-Min

    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 evolutionarily related to the bacterial phospholipase A1 family. The specific activities of MPlaG against olive oil and phosphatidylcholine were determined to be 2,957 ± 144 and 1,735 ± 147 U mg−1, respectively, which means that MPlaG is a lipid-preferred phospholipase. Among different synthetic esters, triglycerides, and phosphatidylcholine, purified MPlaG exhibited the highest activity toward p-nitrophenyl palmitate (C16), tributyrin (C4), and 1,2-dihexanoyl-phosphatidylcholine (C8). Finally, MPlaG was identified as a phospholipase A1 with lipase activity by cleavage of the sn-1 position of OPPC, interfacial activity, and triolein hydrolysis. These findings suggest that MPlaG is the first experimentally characterized phospholipase A1 with lipase activity obtained from a metagenomic library. Our study provides an opportunity to improve our insight into the evolution of lipases and phospholipases. PMID:22544255

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  14. Plasma glycosylphosphatidylinositol-specific phospholipase D predicts the change in insulin sensitivity in response to a low fat but not a low carbohydrate diet in obese women

    PubMed Central

    Gray, Dona L.; O’Brien, Kevin D.; D’Alessio, David A.; Brehm, Bonnie J.; Deeg, Mark A.

    2013-01-01

    Context Although circulating glycosylphosphatidylinositol-specific phospholipase D, a minor high density lipoprotein-associated protein, is elevated in patients with insulin resistance or high triglycerides, no information is available on the effect of weight loss or changes in insulin sensitivity on circulating glycosylphosphatidylinositol-specific phospholipase D levels. Objective Determine the effect of weight loss and changes in insulin sensitivity on plasma glycosylphosphatidylinositol-specific phospholipase D levels. Participants Forty two non-diabetic obese women. Intervention Three month dietary intervention randomizing patients to a low fat or a low carbohydrate diet. Main outcome measures Plasma glycosylphosphatidylinositol-specific phospholipase D levels and insulin sensitivity as estimated by the homeostasis model assessment. Results The very low carbohydrate diet group lost more weight after 3 months (−7.6 ± 3.2 vs. −4.2 ± 3.5 kg, P < 0.01) although the decrease in insulin resistance was similar between groups. Weight loss with either diet did not alter plasma glycosylphosphatidylinositol-specific phospholipase D levels. However, baseline glycosylphosphatidylinositol-specific phospholipase D levels correlated with the change in insulin sensitivity in response to the low fat diet while baseline insulin sensitivity correlated the change in insulin sensitivity in response to the low carbohydrate diet. Conclusions Plasma GPI-PLD may serve as a clinical tool to determine the effect of a low fat diet on insulin sensitivity. PMID:18328347

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

    PubMed Central

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

    2013-01-01

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

  16. Rice Phospholipase A Superfamily: Organization, Phylogenetic and Expression Analysis during Abiotic Stresses and Development

    PubMed Central

    Singh, Amarjeet; Baranwal, Vinay; Shankar, Alka; Kanwar, Poonam; Ranjan, Rajeev; Yadav, Sandeep; Pandey, Amita; Kapoor, Sanjay; Pandey, Girdhar K.

    2012-01-01

    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 A1 (PLA1), patatin like phospholipases (pPLA) and low molecular weight secretory phospholipase A2 (sPLA2) 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. PMID:22363522

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

    PubMed

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

    2017-03-22

    Novel phospholipase (PLA 2 ) genes from the Sparidae family were cloned. The sequenced PLA 2 revealed an identity with pancreatic PLA 2 group IB. To better understand the structure/function relationships of these enzymes and their evolution, the Diplodus annularis PLA 2 (DaPLA 2 ) was overexpressed in E. coli. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPLA 2 was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 3D structure of DaPLA 2 was constructed to gain insights into the functional properties of sparidae PLA 2 . Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA 2 . Using the monolayer technique, the purified DaPLA 2 was found to be active on various phospholipids ranging from 10 to 20 mN·m -1 , which explained the absence of the hemolytic activity for DaPLA 2 .

  18. Molecular details of secretory phospholipase A2 from flax (Linum usitatissimum L.) provide insight into its structure and function.

    PubMed

    Gupta, Payal; Dash, Prasanta K

    2017-09-11

    Secretory phospholipase A 2 (sPLA 2 ) are low molecular weight proteins (12-18 kDa) involved in a suite of plant cellular processes imparting growth and development. With myriad roles in physiological and biochemical processes in plants, detailed analysis of sPLA 2 in flax/linseed is meagre. The present work, first in flax, embodies cloning, expression, purification and molecular characterisation of two distinct sPLA 2 s (I and II) from flax. PLA 2 activity of the cloned sPLA 2 s were biochemically assayed authenticating them as bona fide phospholipase A 2 . Physiochemical properties of both the sPLA 2 s revealed they are thermostable proteins requiring di-valent cations for optimum activity.While, structural analysis of both the proteins revealed deviations in the amino acid sequence at C- & N-terminal regions; hydropathic study revealed LusPLA 2 I as a hydrophobic protein and LusPLA 2 II as a hydrophilic protein. Structural analysis of flax sPLA 2 s revealed that secondary structure of both the proteins are dominated by α-helix followed by random coils. Modular superimposition of LusPLA 2 isoforms with rice sPLA 2 confirmed monomeric structural preservation among plant phospholipase A 2 and provided insight into structure of folded flax sPLA 2 s.

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

    PubMed Central

    Liao, Hui-Ling; Burns, Jacqueline K.

    2010-01-01

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

  20. Lipoprotein-associated phospholipase A(2) and atherosclerosis.

    PubMed

    Wilensky, Robert L; Macphee, Colin H

    2009-10-01

    There is substantial data from over 50 000 patients that increased lipoprotein-associated phospholipase A2 (Lp-PLA2) mass or activity is associated with an increased risk of cardiac death, myocardial infarction, acute coronary syndromes and ischemic stroke. However, only recently have data emerged demonstrating a role of Lp-PLA2 in development of advanced coronary artery disease. Indeed, Lp-PLA2 may be an important link between lipid homeostasis and the vascular inflammatory response. Lp-PLA2, also known as platelet-activating factor acetylhydrolase, rapidly cleaves oxidized phosphatidylcholine molecules produced during the oxidation of LDL and atherogenic lipoprotein Lp(a), generating the soluble proinflammatory and proapoptotic lipid mediators, lyso-phosphatidylcholine and oxidized nonesterified fatty acids. These proinflammatory lipids play an important role in the development of atherosclerotic necrotic cores, the substrate for acute unstable coronary disease by recruiting and activating leukocytes/macrophages, inducing apoptosis and impairing the subsequent removal of dead cells. Selective inhibition of Lp-PLA2 reduces development of necrotic cores and may result in stabilization of atherosclerotic plaques. Recent data have shown that immune pathways play a major role in the development and progression of high-risk atherosclerosis, which leads to ischemic sudden death, myocardial infarction, acute coronary syndromes and ischemic strokes. Persistent and sustained macrophage apoptosis appears to play a major role in the resulting local inflammatory response in part by effects elicited by Lp-PLA2. Selective inhibition of Lp-PLA2 has been postulated to reduce necrotic core progression and the clinical sequelae of advanced, unstable atherosclerosis.

  1. Phospholipase A2-treated human high-density lipoprotein and cholesterol movements: exchange processes and lecithin: cholesterol acyltransferase reactivity.

    PubMed

    Chollet, F; Perret, B P; Chap, H; Douste-Blazy, L

    1986-02-12

    Human HDL3 (d 1.125-1.21 g/ml) were treated by an exogenous phospholipase A2 from Crotalus adamenteus in the presence of albumin. Phosphatidylcholine hydrolysis ranged between 30 and 90% and the reisolated particle was essentially devoid of lipolysis products. (1) An exchange of free cholesterol was recorded between radiolabelled erythrocytes at 5-10% haematocrit and HDL3 (0.6 mM total cholesterol) from 0 to 12-15 h. Isotopic equilibration was reached. Kinetic analysis of the data indicated a constant rate of free cholesterol exchange of 13.0 microM/h with a half-time of equilibration around 3 h. Very similar values of cholesterol exchange, specific radioactivities and kinetic parameters were measured when phospholipase-treated HDL replaced control HDL. (2) The lecithin: cholesterol acyltransferase reactivity of HDL3, containing different amounts of phosphatidylcholine, as achieved by various degrees of phospholipase A2 treatment, was measured using a crude preparation of lecithin: cholesterol acyltransferase (the d 1.21-1.25 g/ml plasma fraction). The rate of esterification was determined between 0 and 12 h. Following a 15-30% lipolysis, the lecithin: cholesterol acyltransferase reactivity of HDL3 was reduced about 30-40%, and then continued to decrease, though more slowly, as the phospholipid content was further lowered in the particle. (3) The addition of the lecithin: cholesterol acyltransferase preparation into an incubation medium made of labelled erythrocytes and HDL3 promoted a movement of radioactive cholesterol out of cells, above the values of exchange, and an accumulation of cholesteryl esters in HDL. This reflected a mass consumption of free cholesterol, from both the cellular and the lipoprotein compartments upon the lecithin: cholesterol acyltransferase action. As a consequence of a decreased reactivity, phospholipase-treated HDL (with 2/3 of phosphatidylcholine hydrolyzed) proved much less effective in the lecithin: cholesterol acyltransferase

  2. Human epidermis is a novel site of phospholipase B expression.

    PubMed

    Maury, Eric; Prévost, Marie Claude; Nauze, Michel; Redoulès, Daniel; Tarroux, Roger; Charvéron, Marie; Salles, Jean Pierre; Perret, Bertrand; Chap, Hugues; Gassama-Diagne, Ama

    2002-07-12

    Phospholipase B (PLB) is an enzyme that displays both phospholipase A(2) and lysophospholipase activities. Analysis of human epidermis homogenates indicated the presence of a 97 kDa PLB protein, as well as a phospholipase A(2) activity, both being enriched in the soluble fraction. Immunolabelling and in situ hybridization experiments showed that this enzyme is expressed in the different layers of epidermis with an accumulation at the dermo-epidermis junction. RT-PCR data indicated that PLB is specifically expressed in natural and reconstructed epidermis. By 3'-RACE-PCR and screening of human genome databases, we obtained a 3600 bp cDNA coding for human PLB highly homologous to already described intestinal brush border PLBs. These data led us to conclude that the soluble PLB corresponds to a proteolytic cleavage of the membrane anchored protein. Altogether, our results provide the first characterization of human PLB which should play an important role in epidermal barrier function.

  3. Emergence of a metalloproteinase / phospholipase A2 axis of systemic inflammation

    PubMed Central

    Fernandez-Patron, Carlos; Leung, Dickson

    2015-01-01

    We review select aspects of the biology of matrix metalloproteinases (MMPs) with a focus on the modulation of inflammatory responses by MMP-2. MMP-2 is a zinc- and calcium-dependent endoprotease with substrates including extracellular matrix proteins, vasoactive peptides and chemokines. Humans and mice with MMP-2 deficiency exhibit a predominantly inflammatory phenotype. Recent research shows that MMP-2 deficient mice display elevated activity of a secreted phospholipase A2 in the heart. Additionally, MMP-2 deficient mice exhibit abnormally high prostaglandin E2 levels in various organs (i.e., the heart, brain and liver), signs of inflammation and exacerbated lipopolysaccharide-induced fever. We briefly review the biology of sPLA2 enzymes to propose the existence of a heart-centric MMP-2/sPLA2 axis of systemic inflammation. Moreover, we postulate that PLA2 activation is induced by chemokines, whose ability to signal inflammation is regulated in a tissue-specific fashion by MMPs. Thus, genetic and pharmacologically induced MMP-deficiencies can be expected to perturb PLA2-mediated inflammatory mechanisms. PMID:26491703

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

    NASA Astrophysics Data System (ADS)

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

    1998-12-01

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

  5. Effect of intensive insulin treatment on plasma levels of lipoprotein-associated phospholipase A2 and secretory phospholipase A2 in patients with newly diagnosed type 2 diabetes.

    PubMed

    Lin, Xiu-Hong; Xu, Ming-Tong; Tang, Jv-Ying; Mai, Li-Fang; Wang, Xiao-Yi; Ren, Meng; Yan, Li

    2016-11-23

    China has the highest absolute disease burden of diabetes worldwide. For diabetic patients, diabetes-related vascular complications are major causes of morbidity and mortality. The roles of lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) and secretory phospholipase A 2 (sPLA 2 ) as inflammatory markers have been recently evaluated in the pathogenesis of both diabetes and atherosclerosis. We aimed to determine the mechanism through which patients with newly diagnosed type 2 diabetes gain long-term vascular benefit from intensive insulin therapy by evaluating the change in Lp-PLA 2 and sPLA 2 levels after early intensive insulin treatment and its relevance with insulin resistance and pancreatic β-cell function. In total, 90 patients with newly diagnosed type 2 diabetes mellitus were enrolled. All patients received continuous subcutaneous insulin infusion (CSII) for approximately 2 weeks. Intravenous glucose-tolerance test (IVGTT) and oral glucose-tolerance test (OGTT) were performed, and plasma concentrations of Lp-PLA 2 and sPLA 2 were measured before and after CSII. Levels of Lp-PLA 2 and sPLA 2 were significantly higher in diabetic patients with macroangiopathy than in those without (P < 0.05). After CSII, the sPLA 2 level decreased significantly in all diabetic patients (P < 0.05), while the Lp-PLA2 level changed only in those with macroangiopathy (P < 0.05). The area under the curve of insulin in IVGTT and OGTT, the acute insulin response (AIR 3-5 ), early phase of insulin secretion (ΔIns30/ΔG30), modified β-cell function index, and homeostatic model assessment for β-cell function (HOMA-β) increased after treatment even when adjusted for the influence of insulin resistance (IR; P < 0.001). The HOMA-IR was lower after treatment, and the three other indicators adopted to estimate insulin sensitivity (ISI ced , IAI, and QUICKI) were higher after treatment (P < 0.05). Correlation analysis showed that the decrease in the Lp-PLA 2 and s

  6. Discovery of AZD2716: A Novel Secreted Phospholipase A2 (sPLA2) Inhibitor for the Treatment of Coronary Artery Disease

    PubMed Central

    2016-01-01

    Expedited structure-based optimization of the initial fragment hit 1 led to the design of (R)-7 (AZD2716) a novel, potent secreted phospholipase A2 (sPLA2) inhibitor with excellent preclinical pharmacokinetic properties across species, clear in vivo efficacy, and minimized safety risk. Based on accumulated profiling data, (R)-7 was selected as a clinical candidate for the treatment of coronary artery disease. PMID:27774123

  7. Role of phospholipase A2 pathway in regulating activation of Bufo arenarum oocytes.

    PubMed

    Ajmat, M T; Bonilla, F; Hermosilla, P C; Zelarayán, L; Bühler, M I

    2013-08-01

    Transient increases in the concentration of cytosolic Ca(2+) are essential for triggering egg activation events. Increased Ca(2+) results from its rapid release from intracellular stores, mainly mediated by one or both intracellular calcium channels: the inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Several regulatory pathways that tailor the response of these channels to the specific cell type have been proposed. Among its many modulatory actions, calcium can serve as an activator of a cytosolic phospholipase A(2) (cPLA2), which releases arachidonic acid from phospholipids of the endoplasmic reticulum as well as from the nuclear envelope. Previous studies have suggested that arachidonic acid and/or its metabolites were able to modulate the activity of several ion channels. Based on these findings, we have studied the participation of the phospholipase A(2) (PLA(2)) pathway in the process of Bufo arenarum oocyte activation and the interrelation between any of its metabolites and the ion channels involved in the calcium release from the intracellular reservoirs at fertilization. We found that addition of both melittin, a potent PLA(2) activator, and arachidonic acid, the main PLA(2) reaction metabolite, was able to induce activation events in a bell-shaped manner. Differential regulation of IP3Rs and RyRs by arachidonic acid and its products could explain melittin and arachidonic acid behaviour in Bufo arenarum egg activation. The concerted action of arachidonic acid and/or its metabolites could provide controlled mobilization of calcium from intracellular reservoirs and useful tools for understanding calcium homeostasis in eggs that express both types of receptors.

  8. Fluorescent Phosphatidylinositol 4,5-Bisphosphate Derivatives with Modified 6-Hydroxy Group as Novel Substrates for Phospholipase C

    PubMed Central

    Wang, Xiaoyang; Barrett, Matthew; Sondek, John; Harden, T. Kendall; Zhang, Qisheng

    2013-01-01

    The capacity to monitor spatiotemporal activity of phospholipase C (PLC) isozymes with a PLC-selective sensor would dramatically enhance understanding of the physiological function and disease relevance of these signaling proteins. Previous structural and biochemical studies defined critical roles for several of the functional groups of the endogenous substrate of PLC isozymes, phosphatidylinositol 4,5-bisphosphate (PIP2), indicating that these sites cannot be readily modified without compromising interactions with the lipase active site. However, the role of the 6-hydroxy group of PIP2 for interaction and hydrolysis by PLC has not been explored, possibly due to challenges in synthesizing 6-hydroxy derivatives. Here, we describe an efficient route for the synthesis of novel, fluorescent PIP2 derivatives modified at the 6-hydroxy group. Two of these derivatives were used in assays of PLC activity in which the fluorescent PIP2 substrates were separated from their diacylglycerol products and reaction rates quantified by fluorescence. Both PIP2 analogues effectively function as substrates of PLC-δ1, and the KM and Vmax values obtained with one of these are similar to those observed with native PIP2 substrate. These results indicate that the 6-hydroxy group can be modified to develop functional substrates for PLC isozymes, thereby serving as the foundation for further development of PLC-selective sensors. PMID:22703043

  9. The Phospholipase A2 Activity of Peroxiredoxin 6.

    PubMed

    Fisher, Aron B

    2018-05-01

    Peroxiredoxin 6 (Prdx6) is a Ca2+-independent intracellular phospholipase A2 (called aiPLA2) that is localized to cytosol and acidic organelles (lysosomes and lysosomal-related organelles). Activity is minimal at cytosolic pH but is increased significantly at acidic pH, in the presence of oxidized phospholipid substrate, with protein oxidation, and with enzyme phosphorylation; maximal activity with phosphorylated aiPLA2 is ~2 μmol/min/mg protein. Prdx6 is a ″moonlighting″ protein that also expresses peroxidase and lysophosphatidylcholine acyl transferase activities.The active site for aiPLA2 activity is Ser32-H26-D140. Activity is inhibited by a serine ″protease″ inhibitor diethyl p-nitrophenyl phosphate (DENP) ,a transition state analogue 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol (MJ33),and two naturally occurring proteins, surfactant protein A (SP-A) and p67phox. aiPLA2 activity has important physiological roles in the turnover (degradation and synthesis) of lung surfactant phospholipids, in the repair of peroxidized cell membranes, and in the activation of NADPH oxidase (NOX2). The enzyme has been implicated in acute lung injury, carcinogenesis, neurodegenerative diseases, diabetes, male infertility, and sundry other conditions although its specific roles have not been well defined. Protein mutations and animal models are now available to further investigate the potentially important roles of Prdx6-aiPLA2 activity in normal and pathological physiology. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Class specific peptide inhibitors for secretory phospholipases A2.

    PubMed

    Mahalka, Ajay K; Kinnunen, Paavo K J

    2013-06-28

    Phospholipases A2 (PLA2) catalyze the hydrolytic cleavage of free fatty acids from the sn-2 OH-moiety of glycerophospholipids. These enzymes have a number of functions, from digestion to signaling and toxicity of several venoms. They have also been implicated in inflammation and are connected to diverse diseases, such as cancer, ischemia, atherosclerosis, and schizophrenia. Accordingly, there is a keen interest to develop selective inhibitors for therapeutic use. We recently proposed a novel mechanism for the control of PLA2 activity with highly active protofibrils of PLA2 existing transiently before conversion to inactive amyloid fibrils [19]. In keeping with the above mechanism several algorithms identified (85)KMYFNLI(91) and (17)AALSYGFYG(25) in bee venom (bv) and human lacrimal fluid (Lf) PLA2, respectively, as a regions potentially forming amyloid type aggregates. Interestingly, in keeping with the proposed role of these sequences in the control of the activity of these enzymes, preincubation of 2nM bvPLA2 with (85)KMYFNLI(91) caused complete inhibition of PLA2 activity while the scrambled control peptide YNFLIMK had no effect. Approximately 36% attenuation of the hydrolytic activity of LfPLA2 present in human lacrimal fluid was observed in the presence of 80nM (17)AALSYGFYG(25). Copyright © 2013 Elsevier Inc. All rights reserved.

  11. p67(phox) terminates the phospholipase A(2)-derived signal for activation of NADPH oxidase (NOX2).

    PubMed

    Krishnaiah, Saikumari Y; Dodia, Chandra; Feinstein, Sheldon I; Fisher, Aron B

    2013-05-01

    The phospholipase A2 (PLA2)activity of phosphorylated peroxiredoxin 6 (Prdx6) is required for activation of NADPH oxidase (NOX2). We investigated the interaction of Prdx6 with p67(phox) and its effect on NOX2 activity. With the use of specific antibodies, coimmunoprecipitation of p67(phox) and phosphorylated Prdx6 was demonstrated with lysates of mouse pulmonary microvascular endothelial cells (MPMVECs) that were stimulated with angiotensin II; the interaction of p67(phox) with nonphosphorylated Prdx6 was relatively weak. Association of p67(phox) and phosphoPrdx6 in intact MPMVECs after angiotensin II stimulation was demonstrated by proximity ligation assay and was abolished by U0126, a MAP kinase inhibitor. By isothermal titration calorimetry, p67(phox) bound strongly to phosphoPrdx6 but bound poorly to Prdx6; phosphorylated p67(phox) did not bind to either Prdx6 or phosphoPrdx6. PLA2 activity of recombinant phosphoPrdx6 was decreased by >98% in the presence of p67(phox); the calculated dissociation constant (Kd) of the p67(phox): phosphoPrdx6 complex was 65 nM. PLA2 activity (MJ33 sensitive) in cell lysates following angiotensin II treatment of MPMVECs was increased by 85% following knockdown of p67(phox) with siRNA. These data indicate that p67(phox) binds to phosphoPrdx6 and inhibits its PLA2 activity, an interaction that could function to terminate the PLA2-mediated NOX2 activation signal.-Krishnaiah, S. Y., Dodia, C., Feinstein, S. I., and Fisher, A. B. p67(phox) terminates the phospholipase A2-derived signal for activation of NADPH oxidase (NOX2).

  12. Citicoline decreases phospholipase A2 stimulation and hydroxyl radical generation in transient cerebral ischemia.

    PubMed

    Adibhatla, Rao Muralikrishna; Hatcher, James F

    2003-08-01

    Neuroprotection by citicoline (CDP-choline) in transient cerebral ischemia has been demonstrated previously. Citicoline has undergone several Phase III clinical trials for stroke, and is being evaluated for treatment of Alzheimer's and Parkinson's diseases. Phospholipid degradation and generation of reactive oxygen species (ROS) are major factors causing neuronal injury in CNS trauma and neurodegenerative diseases. Oxidative metabolism of arachidonic acid (released by the action of phospholipases) contributes to ROS generation. We examined the effect of citicoline on phospholipase A(2) (PLA(2)) activity in relation to the attenuation of hydroxyl radical (OH.) generation after transient forebrain ischemia of gerbil. PLA(2) activity (requires mM Ca(2+)) increased significantly (P < 0.05) in both membrane (50.2 +/- 2.2 pmol/min/mg protein compared to sham 35.9 +/- 3.2) and mitochondrial fractions (77.0 +/- 1.2 pmol/min/mg protein compared to sham 33.9 +/- 1.2) after cerebral ischemia and 2 hr reperfusion in gerbil, which was significantly attenuated (P < 0.01) by citicoline (membrane, 39.9. +/- 2.2 and mitochondria, 41.9 +/- 3.2 pmol/min/mg protein). In vitro, citicoline and its components cytidine and choline had no effect on PLA(2) activity, and thus citicoline as such is not a PLA(2) inhibitor. Ischemia/reperfusion resulted in significant OH. generation (P < 0.01) and citicoline significantly (P < 0.01) attenuated their formation (expressed as 2,3-dihydroxybenzoic acid/salicylate ratio; ischemia/24 hr reperfusion, 6.30 +/- 0.23; sham, 2.56 +/- 0.27; ischemia/24 hr reperfusion + citicoline, 4.85 +/- 0.35). These results suggest that citicoline affects PLA(2) stimulation and decreases OH. generation after transient cerebral ischemia. Copyright 2003 Wiley-Liss, Inc.

  13. SPLASH (PLA2IID), a novel member of phospholipase A2 family, is associated with lymphotoxin deficiency.

    PubMed

    Shakhov, A N; Rubtsov, A V; Lyakhov, I G; Tumanov, A V; Nedospasov, S A

    2000-02-01

    Lymphotoxin (LT) deficient mice have profound defects in the splenic microarchitecture associated with defective expression on certain gene products, including chemokines. By using subtraction cloning of splenic cDNA from wild-type and LT alpha or TNF/LT alpha double deficient mice we isolated a novel murine gene encoding a secretory type phospholipase A2, called SPLASH. The two major alternative transcripts of SPLASH gene are predominantly expressed in lymphoid tissues, such as spleen and lymph nodes. SPLASH maps to the distal part of chromosome 4, to which several cancer-related loci have been also mapped.

  14. Effects of the propeptide of group X secreted phospholipase A(2) on substrate specificity and interfacial activity on phospholipid monolayers.

    PubMed

    Point, Vanessa; Bénarouche, Anaïs; Jemel, Ikram; Parsiegla, Goetz; Lambeau, Gérard; Carrière, Frédéric; Cavalier, Jean-François

    2013-01-01

    Group X secreted phospholipase A(2) (GX sPLA(2)) plays important physiological roles in the gastrointestinal tract, in immune and sperm cells and is involved in several types of inflammatory diseases. It is secreted either as a mature enzyme or as a mixture of proenzyme (with a basic 11 amino acid propeptide) and mature enzyme. The role of the propeptide in the repression of sPLA(2) activity has been studied extensively using liposomes and micelles as model interfaces. These substrates are however not always suitable for detecting some fine tuning of lipolytic enzymes. In the present study, the monolayer technique is used to compare PLA(2) activity of recombinant mouse GX sPLA(2) (mGX) and its pro-form (PromGX) on monomolecular films of dilauroyl-phosphatidyl-ethanolamine (DLPE), -choline (DLPC) and -glycerol (DLPG). The PLA(2) activity and substrate specificity of mGX (PE ≈ PG > PC) were found to be surface pressure-dependent. mGX displayed a high activity on DLPE and DLPG but not on DLPC monolayers up to surface pressures corresponding to the lateral pressure of biological membranes (30-35 mN/m). Overall, the propeptide impaired the enzyme activity, particularly on DLPE whatever the surface pressure. However some conditions could be found where the propeptide had little effects on the repression of PLA(2) activity. In particular, both PromGX and mGX had similar activities on DLPG at a surface pressure of 30 mN/m. These findings show that PromGX can be potentially active depending on the presentation of the substrate (i.e., lipid packing) and one cannot exclude such an activity in a physiological context. A structural model of PromGX was built to investigate how the propeptide controls the activity of GX sPLA(2). This model shows that the propeptide is located within the interfacial binding site (i-face) and could disrupt both the interfacial binding of the enzyme and the access to the active site by steric hindrance. Copyright © 2012 Elsevier Masson SAS

  15. Trichomonas vaginalis: identification of soluble and membrane-associated phospholipase A1 and A2 activities with direct and indirect hemolytic effects.

    PubMed

    Vargas-Villarreal, Javier; Mata-Cárdenas, Benito David; Palacios-Corona, Rebeca; González-Salazar, Francisco; Cortes-Gutierrez, Elva I; Martínez-Rodríguez, Herminia G; Said-Fernández, Salvador

    2005-02-01

    A direct hemolytic activity, dependent on phospholipase A (PLA) activity, was located in the particulate subcellular fraction (P30) of Trichomonas vaginalis. We identified soluble direct and indirect hemolytic activities in the spent medium and soluble fraction (S30) of T. vaginalis strain GT-13. Spent medium showed the highest specific indirect hemolytic activity (SIHA) at pH 6.0 (91 indirect hemolytic units [HU]/mg/hr). Spent medium and P30, but not S30, showed direct hemolytic activity. PLA activity was protein dose dependent and time dependent. The highest PLA activity was observed at pH 6.0. All trichomonad preparations showed phospholipase A1 (PLA A1) and phospholipase A2 (PLA A2) activities. Indirect and direct hemolytic activity and PLA A1 and PLA A2 diminished at pH 6.0 and 8.0 with increasing concentrations of Rosenthal's inhibitor. The greatest effect was observed with 80 microM at pH 6.0 on the SIHA of S30 (83% reduction) and the lowest at pH 8.0, also on the SIHA of S30 (26% reduction). In conclusion, T. vaginalis contains particulate and soluble acidic, and alkaline direct and indirect hemolytic activities, which are partially dependent on alkaline or acidic PLA A1 and PLA A2 enzymes. These could be responsible for the contact-dependent and -independent hemolytic and cytolytic activities of T. vaginalis.

  16. Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity

    PubMed Central

    Anderson, Mark T.; Mitchell, Lindsay A.

    2017-01-01

    ABSTRACT Serratia marcescens causes health care-associated opportunistic infections that can be difficult to treat due to a high incidence of antibiotic resistance. One of the many secreted proteins of S. marcescens is the PhlA phospholipase enzyme. Genes involved in the production and secretion of PhlA were identified by screening a transposon insertion library for phospholipase-deficient mutants on phosphatidylcholine-containing medium. Mutations were identified in four genes (cyaA, crp, fliJ, and fliP) that are involved in the flagellum-dependent PhlA secretion pathway. An additional phospholipase-deficient isolate harbored a transposon insertion in the cysE gene encoding a predicted serine O-acetyltransferase required for cysteine biosynthesis. The cysE requirement for extracellular phospholipase activity was confirmed using a fluorogenic phospholipase substrate. Phospholipase activity was restored to the cysE mutant by the addition of exogenous l-cysteine or O-acetylserine to the culture medium and by genetic complementation. Additionally, phlA transcript levels were decreased 6-fold in bacteria lacking cysE and were restored with added cysteine, indicating a role for cysteine-dependent transcriptional regulation of S. marcescens phospholipase activity. S. marcescens cysE mutants also exhibited a defect in swarming motility that was correlated with reduced levels of flhD and fliA flagellar regulator gene transcription. Together, these findings suggest a model in which cysteine is required for the regulation of both extracellular phospholipase activity and surface motility in S. marcescens. IMPORTANCE Serratia marcescens is known to secrete multiple extracellular enzymes, but PhlA is unusual in that this protein is thought to be exported by the flagellar transport apparatus. In this study, we demonstrate that both extracellular phospholipase activity and flagellar function are dependent on the cysteine biosynthesis pathway. Furthermore, a disruption of cysteine

  17. The effects of two phospholipase A2 inhibitors on the neuromuscular blocking activities of homologous phospholipases A2 from the venom of Pseudechis australis, the Australian king brown snake.

    PubMed

    Fatehi, M; Rowan, E G; Harvey, A L

    1995-12-01

    Previous studies have shown that homologous phospholipases A2 (PLA2) (Pa-3, Pa-9C, Pa-10F and Pa-11) from the venom of the Australian king brown snake, Pseudechis australis, significantly reduce the resting membrane potentials and quantal contents of endplate potentials recorded from endplate regions of mouse triangularis sterni nerve-muscle preparations. It is not clear whether PLA2 activity is essential for their neuromuscular activities. Therefore, pharmacological studies were carried out to determine whether neuromuscular activity of the toxins changed after treatment with the phospholipase A2 inhibitors 7,7-dimethyl-eicosadienoic acid (DEDA) and manoalide. After incubation of the toxins with manoalide (120 nM), or DEDA (50 microM), no PLA2 activity against 1-stearoyl 2-[3H]arachidonoylglycerophosphocholine was detected. After incubation with manoalide and/or DEDA, the toxins did not depolarize muscle fibre membranes up to 60 min after administration. However, manoalide and DEDA had different influences on the inhibitory effect of these toxic enzymes on acetylcholine release from nerve terminals. Manoalide abolished the inhibitory effect of the toxins on evoked release of acetylcholine. In contrast, DEDA was not able to prevent the reduction of quantal content of endplate potentials induced by the toxins. This study provides evidence that the depolarizing action and the inhibitory effect on release of acetylcholine exerted by these toxic PLA2 from king brown snake are independent phenomena. The evidence for this conclusion was that inhibition of enzymatic activity with an arachidonic acid analogue (DEDA) abolished the depolarizing effect of the toxins but not the effects on the quantal release of acetylcholine from mouse motor nerve terminals. The data suggest that the depolarizing effect of these toxins is probably due to the enzymatic activity. Since manoalide interacts with lysine residues of PLA2 polypeptides, and, as shown here, manoalide prevented

  18. p67phox terminates the phospholipase A2-derived signal for activation of NADPH oxidase (NOX2)

    PubMed Central

    Krishnaiah, Saikumari Y.; Dodia, Chandra; Feinstein, Sheldon I.; Fisher, Aron B.

    2013-01-01

    The phospholipase A2 (PLA2)activity of phosphorylated peroxiredoxin 6 (Prdx6) is required for activation of NADPH oxidase (NOX2). We investigated the interaction of Prdx6 with p67phox and its effect on NOX2 activity. With the use of specific antibodies, coimmunoprecipitation of p67phox and phosphorylated Prdx6 was demonstrated with lysates of mouse pulmonary microvascular endothelial cells (MPMVECs) that were stimulated with angiotensin II; the interaction of p67phox with nonphosphorylated Prdx6 was relatively weak. Association of p67phox and phosphoPrdx6 in intact MPMVECs after angiotensin II stimulation was demonstrated by proximity ligation assay and was abolished by U0126, a MAP kinase inhibitor. By isothermal titration calorimetry, p67phox bound strongly to phosphoPrdx6 but bound poorly to Prdx6; phosphorylated p67phox did not bind to either Prdx6 or phosphoPrdx6. PLA2 activity of recombinant phosphoPrdx6 was decreased by >98% in the presence of p67phox; the calculated dissociation constant (Kd) of the p67phox: phosphoPrdx6 complex was 65 nM. PLA2 activity (MJ33 sensitive) in cell lysates following angiotensin II treatment of MPMVECs was increased by 85% following knockdown of p67phox with siRNA. These data indicate that p67phox binds to phosphoPrdx6 and inhibits its PLA2 activity, an interaction that could function to terminate the PLA2-mediated NOX2 activation signal.—Krishnaiah, S. Y., Dodia, C., Feinstein, S. I., and Fisher, A. B. p67phox terminates the phospholipase A2-derived signal for activation of NADPH oxidase (NOX2). PMID:23401562

  19. MVL-PLA2, a Snake Venom Phospholipase A2, Inhibits Angiogenesis through an Increase in Microtubule Dynamics and Disorganization of Focal Adhesions

    PubMed Central

    Bazaa, Amine; Pasquier, Eddy; Defilles, Céline; Limam, Ines; Kessentini-Zouari, Raoudha; Kallech-Ziri, Olfa; Battari, Assou El; Braguer, Diane; Ayeb, Mohamed El; Marrakchi, Naziha; Luis, José

    2010-01-01

    Integrins are essential protagonists of the complex multi-step process of angiogenesis that has now become a major target for the development of anticancer therapies. We recently reported and characterized that MVL-PLA2, a novel phospholipase A2 from Macrovipera lebetina venom, exhibited anti-integrin activity. In this study, we show that MVL-PLA2 also displays potent anti-angiogenic properties. This phospholipase A2 inhibited adhesion and migration of human microvascular-endothelial cells (HMEC-1) in a dose-dependent manner without being cytotoxic. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrated that MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both in vitro and in vivo. We have also found that the actin cytoskeleton and the distribution of αvβ3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. In order to further investigate the mechanism of action of this protein on endothelial cells, we analyzed the dynamic instability behavior of microtubules in living endothelial cells. Interestingly, we showed that MVL-PLA2 significantly increased microtubule dynamicity in HMEC-1 cells by 40%. We propose that the enhancement of microtubule dynamics may explain the alterations in the formation of focal adhesions, leading to inhibition of cell adhesion and migration. PMID:20405031

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

    PubMed

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

    2016-04-01

    Pterois russelli is a venomous fish belonging to scorpionidae family. Regarding to high significance value for tracing potential therapeutic molecules and special agents from venomous marine creatures, the present study was aimed to characterization of the Persian Gulf lionfish venom. Proteolytic, phospholipase, hemolytic, coagulation, edematogenic and dermonecrotic activities were determined for extracted venom. The LD50 of P. russelli venom was determined by intravenous injection in white Balb/c mice. Phospholipase A2 activity was recorded at 20 μg of total venom. Coagulation activity on human plasma was shown by Prothrombin Time (PT) and activated Partial Thromboplastin Time (APTT) assays and coagulation visualized after 7 and 14 s respectively for 60 μg of crude venom. LD50 was calculated as 10.5 mg/kg. SDS-PAGE revealed the presence of major and minor protein bands between 6 and 205 kDa. Different amounts of crude venom ranged from 1.87 to 30 μg showed proteolytic activity on casein. The highest edematic activity was detected at 20 μg. Our findings showed that the edematic activity was dose dependent and persisted for 48 h after injection. The crude venom did not induce dermonecrotic activity on rabbit skin and showed no hemolytic activity on human, mouse and rabbit erythrocytes. This is the first report for phospholipase A2 and coagulation activity in venomous fish and venomous marine animals respectively. Proteolytic activity of P. russelli venom is in accordance with the other genara of scorpionidae family. According to venom activity on intrinsic and extrinsic coagulation pathways, lionfish venom would be contained an interesting pharmaceutical agent. This study is pending to further characterization of phospholipase A2, coagulation, and protease activities and also in vivo activity on animal model of surface and internal bleeding. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2009-01-01

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

  2. Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity.

    PubMed

    Anderson, Mark T; Mitchell, Lindsay A; Mobley, Harry L T

    2017-08-15

    Serratia marcescens causes health care-associated opportunistic infections that can be difficult to treat due to a high incidence of antibiotic resistance. One of the many secreted proteins of S. marcescens is the PhlA phospholipase enzyme. Genes involved in the production and secretion of PhlA were identified by screening a transposon insertion library for phospholipase-deficient mutants on phosphatidylcholine-containing medium. Mutations were identified in four genes ( cyaA , crp , fliJ , and fliP ) that are involved in the flagellum-dependent PhlA secretion pathway. An additional phospholipase-deficient isolate harbored a transposon insertion in the cysE gene encoding a predicted serine O -acetyltransferase required for cysteine biosynthesis. The cysE requirement for extracellular phospholipase activity was confirmed using a fluorogenic phospholipase substrate. Phospholipase activity was restored to the cysE mutant by the addition of exogenous l-cysteine or O -acetylserine to the culture medium and by genetic complementation. Additionally, phlA transcript levels were decreased 6-fold in bacteria lacking cysE and were restored with added cysteine, indicating a role for cysteine-dependent transcriptional regulation of S. marcescens phospholipase activity. S. marcescens cysE mutants also exhibited a defect in swarming motility that was correlated with reduced levels of flhD and fliA flagellar regulator gene transcription. Together, these findings suggest a model in which cysteine is required for the regulation of both extracellular phospholipase activity and surface motility in S. marcescens IMPORTANCE Serratia marcescens is known to secrete multiple extracellular enzymes, but PhlA is unusual in that this protein is thought to be exported by the flagellar transport apparatus. In this study, we demonstrate that both extracellular phospholipase activity and flagellar function are dependent on the cysteine biosynthesis pathway. Furthermore, a disruption of cysteine

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

    PubMed

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

    2016-10-01

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

  4. Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A{sub 2}-induced degranulation in mast cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nishikawa, Hirofumi; Kitani, Seiichi, E-mail: drkitani@kaiyodai.ac.jp

    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 {beta}-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation andmore » cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G{sub M1}), di-sialoganglioside (G{sub D1a}) and tri-sialoganglioside (G{sub T1b}). In contrast, honeybee venom-derived phospholipase A{sub 2} induced the net degranulation directly without cytotoxicity, which was not inhibited by G{sub M1}, G{sub D1a} and G{sub T1b}. For analysis of distribution of G{alpha}{sub q} and G{alpha}{sub i} protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of G{alpha}{sub q} and G{alpha}{sub i} 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 A{sub 2}-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A{sub 2}-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.« less

  5. Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state.

    PubMed

    Gabriel, N E; Agman, N V; Roberts, M F

    1987-11-17

    Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.

  6. Phospholipase D1 increases Bcl-2 expression during neuronal differentiation of rat neural stem cells.

    PubMed

    Park, Shin-Young; Ma, Weina; Yoon, Sung Nyo; Kang, Min Jeong; Han, Joong-Soo

    2015-01-01

    We studied the possible role of phospholipase D1 (PLD1) in the neuronal differentiation, including neurite formation of neural stem cells. PLD1 protein and PLD activity increased during neuronal differentiation. Bcl-2 also increased. Downregulation of PLD1 by transfection with PLD1 siRNA or a dominant-negative form of PLD1 (DN-PLD1) inhibited both neurite outgrowth and Bcl-2 expression. PLD activity was dramatically reduced by a PLCγ (phospholipase Cγ) inhibitor (U73122), a Ca(2+)chelator (BAPTA-AM), and a PKCα (protein kinase Cα) inhibitor (RO320432). Furthermore, treatment with arachidonic acid (AA) which is generated by the action of PLA2 (phospholipase A2) on phosphatidic acid (a PLD1 product), increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, indicating that PLA2 is involved in the differentiation process resulting from PLD1 activation. PGE2 (prostaglandin E2), a cyclooxygenase product of AA, also increased during neuronal differentiation. Moreover, treatment with PGE2 increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, and this effect was inhibited by a PKA inhibitor (Rp-cAMP). As expected, inhibition of p38 MAPK resulted in loss of CREB activity, and when CREB activity was blocked with CREB siRNA, Bcl-2 production also decreased. We also showed that the EP4 receptor was required for the PKA/p38MAPK/CREB/Bcl-2 pathway. Taken together, these observations indicate that PLD1 is activated by PLCγ/PKCα signaling and stimulate Bcl-2 expression through PLA2/Cox2/EP4/PKA/p38MAPK/CREB during neuronal differentiation of rat neural stem cells.

  7. Antitumor effects of cationic synthetic peptides derived from Lys49 phospholipase A2 homologues of snake venoms.

    PubMed

    Araya, Cindy; Lomonte, Bruno

    2007-03-01

    The effects of two cationic synthetic peptides, derived from the C-terminal region of Lys49 phospholipase A2 homologues from snake venoms, upon various murine tumor cell lines (B16 melanoma, EMT6 mammary carcinoma, S-180 sarcoma, P3X myeloma, tEnd endothelial cells) were evaluated. The peptides are 13-mers derived from Agkistrodon piscivorus piscivorus Lys49 PLA2 (p-AppK: KKYKAYFKLKCKK) and Bothrops asper Lys49 myotoxin II (pEM-2[D]: KKWRWWLKALAKK), respectively, in the latter case with slight modifications and with all-D amino acids. All tumor cells tested were susceptible to the lytic action of the peptides. The susceptibility of tumor cell lines was not higher than that of C2C12 skeletal muscle myoblasts, utilized as a non-transformed cell line control. However, in a murine model of subcutaneous solid tumor growth of EMT6 mammary carcinoma, the intraperitoneal administration of pEM-2[D] caused a tumor mass reduction of 36% (p<0.05), which was of similar magnitude to that achieved by the administration of paclitaxel, an antitumor drug in clinical use. Thus, the C-terminal peptides of Lys49 phospholipase A2 homologues present antitumor effects that might be of interest in developing therapeutic strategies against cancer.

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

    PubMed

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

    2015-07-01

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

  9. Purification of a phospholipase A2 from Daboia russelii siamensis venom with anticancer effects

    PubMed Central

    Khunsap, Suchitra; Pakmanee, Narumol; Khow, Orawan; Chanhome, Lawan; Sitprija, Visith; Suntravat, Montamas; Lucena, Sara E; Perez, John C; Sánchez, Elda E

    2011-01-01

    Venom phospholipases A2 (PLA2) are associated with neurotoxic, myotoxic, cardiotoxic, platelet aggregation, and edema activities. A PLA2 (Drs-PLA2) was purified from Daboia russelii siamensis venom by a two-step purification procedure consisting of size-exclusion, followed by anion exchange high performance liquid chromatography (HPLC). The molecular weight of the Drs-PLA2 was 13,679Da, which was determined by MALDI-TOF mass spectrometry. Its N-terminal amino acid sequence was homologous to basic PLA2s of viperid snake venoms. The Drs-PLA2 had indirect hemolytic and anticoagulant activities, cytotoxic activity with a CC50 of 65.8nM, and inhibited SK-MEL-28 cell migration with an IC50 of 25.6nM. In addition, the Drs-PLA2 inhibited the colonization of B16F10 cells in lungs of BALB/c mice by ∼65%. PMID:22091349

  10. Effects of dexamethasone on palate mesenchymal cell phospholipase activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bulleit, R.F.; Zimmerman, E.F.

    1984-09-15

    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 (1more » 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.« less

  11. Ethanol causes desensitization of receptor-mediated phospholipase C activation in isolated hepatocytes.

    PubMed

    Higashi, K; Hoek, J B

    1991-02-05

    The effect of ethanol on receptor-mediated phospholipase C-linked signal transduction processes was investigated in isolated rat hepatocytes. Pretreatment of the cells with ethanol (6-300 mM) markedly inhibited a subsequent stimulation of phospholipase C by vasopressin, angiotensin II, or epidermal growth factor. By contrast, the effects of the alpha 1-adrenergic agonist phenylephrine and of glucagon were not affected by ethanol pretreatment. Ethanol inhibited the agonist-induced decrease in polyphosphoinositides, the formation of inositol phosphates, and the increase in cytosolic free Ca2+ levels, as detected with the intracellular Ca2+ indicator indo-1. The effects of ethanol were concentration dependent and were pronounced at low concentrations of agonists but were not significant at saturating levels. Pretreatment of the cells with the protein kinase C inhibitor H7 partly prevented the inhibition by ethanol of vasopressin-induced phospholipase C activation. By contrast, pretreatment of the cells with (Rp)-adenosine cyclic 3':5'-phosphorothioate [Rp)-cAMP-S), a competitive inhibitor of protein kinase A, potentiated the inhibitory effect of ethanol on the Ca2+ mobilization by vasopressin. (Rp)-cAMP-S similarly potentiated the inhibition of phospholipase C by the protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The kinase A inhibitor also made the Ca2+ mobilization by phenylephrine sensitive to ethanol, indicating that the formation of cAMP in the cells played a role in suppressing the sensitivity to ethanol. Pretreatment of the cells with ethanol enhanced the inhibitory effects of TPA on the vasopressin-induced phospholipase C activation at all concentrations of the hormone; however, these synergistic effects were prevented when TPA was added prior to ethanol, a condition that prevents the activation of phospholipase C by ethanol. The data indicate that ethanol causes desensitization of the receptor-mediated phospholipase C

  12. Modeling of substrate and inhibitor binding to phospholipase A2.

    PubMed

    Sessions, R B; Dauber-Osguthorpe, P; Campbell, M M; Osguthorpe, D J

    1992-09-01

    Molecular graphics and molecular mechanics techniques have been used to study the mode of ligand binding and mechanism of action of the enzyme phospholipase A2. A substrate-enzyme complex was constructed based on the crystal structure of the apoenzyme. The complex was minimized to relieve initial strain, and the structural and energetic features of the resultant complex analyzed in detail, at the molecular and residue level. The minimized complex was then used as a basis for examining the action of the enzyme on modified substrates, binding of inhibitors to the enzyme, and possible reaction intermediate complexes. The model is compatible with the suggested mechanism of hydrolysis and with experimental data about stereoselectivity, efficiency of hydrolysis of modified substrates, and inhibitor potency. In conclusion, the model can be used as a tool in evaluating new ligands as possible substrates and in the rational design of inhibitors, for the therapeutic treatment of diseases such as rheumatoid arthritis, atherosclerosis, and asthma.

  13. The group VIA calcium-independent phospholipase A2 and NFATc4 pathway mediates IL-1β-induced expression of chemokines CCL2 and CXCL10 in rat fibroblasts.

    PubMed

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

    2018-06-01

    Chemokines are secreted proteins that regulate cell migration and are involved in inflammatory and immune responses. Here, we sought to define the functional crosstalk between the lipid signaling and chemokine signaling. We obtained evidence that the induction of some chemokines is regulated by group VIA calcium-independent phospholipase A 2 β (iPLA 2 β) in IL-1β-stimulated rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with IL-1β elicited an increased release of chemotactic factor(s) for monocytic THP-1 cells into culture medium in a time-dependent manner. Inhibitor studies revealed that an intracellular PLA 2 inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF 3 ), but not the cyclooxygenase inhibitor indomethacin, attenuated the release of chemotactic factor(s). The chemotactic activity was inactivated by treatment with either heat or proteinase K, suggesting this chemotactic factor(s) is a proteinaceous factor(s). We purified the chemotactic factor(s) from the conditioned medium of IL-1β-stimulated 3Y1 cells using a heparin column and identified several chemokines, including CCL2 and CXCL10. The inducible expressions of CCL2 and CXCL10 were significantly attenuated by pretreatment with AACOCF 3 . Gene silencing using siRNA revealed that the inductions of CCL2 and CXCL10 were attenuated by iPLA 2 β knockdown. Additionally, the transcriptional activation of nuclear factor of activated T-cell proteins (NFATs), but not nuclear factor-κB, by IL-1β stimulation was markedly attenuated by the iPLA 2 inhibitor bromoenol lactone, and NFATc4 knockdown markedly attenuated the IL-1β-induced expression of both CCL2 and CXCL10. Collectively, these results indicated that iPLA 2 β plays roles in IL-1β-induced chemokine expression, in part via NFATc4 signaling. © 2018 Federation of European Biochemical Societies.

  14. Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells.

    PubMed

    Martin, T W

    1988-10-14

    The conversion of phosphatidylcholine (PC) to diacylglycerol (DAG) was studied in sonicated endothelial cells and in subcellular fractions in the presence of 0.05% Triton X-100 and 2 mM EDTA. DAG formation occurred predominantly in an organelle fraction that sedimented at 15,000 x g. In parallel reactions with exogenous 1-oleoyl-2-[3H]oleoyl-PC (sn-2-[3H]DOPC) and phosphatidyl[3H]choline ([choline-3H]PC), [3H]DAG was formed by a reaction pathway in which [3H]choline was the only product derived from [choline-3H]PC. [3H]Choline was not formed secondarily from [3H]glycerophosphocholine or [3H]phosphocholine. Small amounts of [3H]phosphatidate ([3H]PA) were isolated from reactions with sn-2-[3H]DOPC at short incubation times, and substantial PA phosphatase activity was demonstrated. These data, taken together, supported a phospholipase D-PA phosphatase pathway of DAG formation. Kinetic data established that the low ratio of [3H]PA/[3H]DAG formed in reactions with sn-2-[3H]DOPC was due to a 15-fold higher Vmax and 7-fold lower apparent Km of the PA phosphatase. The [3H]PA/[3H]DAG product ratio was increased by addition of unlabeled PA or by selective extraction of phospholipase D with Triton X-100. The characteristics of the phospholipase D indicated a unique enzyme. Activity was optimal in the presence of EDTA and was almost totally dependent upon Triton X-100. The pH profile displayed a peak at 7.0. Of particular significance was the stringent substrate specificity. Phosphatidylinositol was not hydrolyzed, and activities towards phosphatidylethanolamine and sphingomyelin were at most 30- to 50-fold lower than those towards PC. Phospholipase D and PA phosphatase were identified in a number of rat tissues and other cells. The highest activities of phospholipase D were present in lung and endothelial cells. Phospholipase D was partially purified from rat lung by Triton X-100 extraction and anion exchange chromatography. When linked with PA phosphatase, the phospholipase

  15. Interaction of human synovial phospholipase A2 with mixed lipid bilayers: a coarse-grain and all-atom molecular dynamics simulation study.

    PubMed

    Qin, Shan-Shan; Yu, Yang-Xin; Li, Qi-Kai; Yu, Zhi-Wu

    2013-02-26

    Human secreted phospholipase A2s have been shown to promote inflammation in mammals by catalyzing the first step of the arachidonic acid pathway by breaking down phospholipids, producing fatty acids, including arachidonic acid. They bind to the membrane water interface to access their phospholipid substrates from the membrane. Their binding modes on membrane surfaces are regulated by diverse factors, including membrane charge, fluidity, and heterogeneity. The influence of these factors on the binding modes of the enzymes is not well understood. Here we have studied several human synovial phospholipase A2 (hs-PLA2)/mixed bilayer systems through a combined coarse-grain and all-atom molecular dynamics simulation. It was found that hydrophobic residues Leu2, Val3, Ala18, Leu19, Phe23, Gly30, and Phe63 that form the edge of the entrance of the hydrophobic binding pocket in hs-PLA2 tend to penetrate into the hydrophobic area of lipid bilayers, and more than half of the total amino acid residues make contact with the lipid headgroups. Each enzyme molecule forms 19-38 hydrogen bonds with the bilayer to which it binds, most of which are with the phosphate groups. Analysis of the root-mean-square deviation (rmsd) shows that residues Val30-Thr40, Tyr66-Gln80, and Lys107-Arg118 have relatively large rmsds during all-atom molecular dynamics simulations, in accordance with the observation of an enlarged entrance region of the hydrophobic binding pocket. The amino acid sequences forming the entrance of the binding pocket prefer to interact with lipid molecules that are more fluid or negatively charged, and the opening of the binding pocket would be larger when the lipid components are more fluid.

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

    PubMed

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

    2016-08-22

    The venom of Micrurus lemniscatus, a coral snake of wide geographical distribution in South America, was fractionated by reverse-phase HPLC and the fractions screened for phospholipase A2 (PLA2) activity. The major component of the venom, a PLA2, here referred to as 'Lemnitoxin', was isolated and characterized biochemically and toxicologically. It induces myotoxicity upon intramuscular or intravenous injection into mice. The amino acid residues Arg15, Ala100, Asn108, and a hydrophobic residue at position 109, which are characteristic of myotoxic class I phospholipases A2, are present in Lemnitoxin. This PLA2 is antigenically related to M. nigrocinctus nigroxin, Notechis scutatus notexin, Pseudechis australis mulgotoxin, and Pseudonaja textilis textilotoxin, as demonstrated with monoclonal and polyclonal antibodies. Lemnitoxin is highly selective in its targeting of cells, being cytotoxic for differentiated myotubes in vitro and muscle fibers in vivo, but not for undifferentiated myoblasts or endothelial cells. Lemnitoxin is not lethal after intravenous injection at doses up to 2μg/g in mice, evidencing its lack of significant neurotoxicity. Lemnitoxin displays anticoagulant effect on human plasma and proinflammatory activity also, as it induces paw edema and mast cell degranulation. Thus, the results of this work demonstrate that Lemnitoxin is a potent myotoxic and proinflammatory class I PLA2. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

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

    2015-09-01

    The Micrurus genus is the American representative of Elapidae family. Micrurus spixii is endemic of South America and northern states of Brazil. Elapidic venoms contain neurotoxins that promote curare-mimetic neuromuscular blockage. In this study, biochemical and functional characterizations of M. spixii crude venom were performed and a new neurotoxic phospholipase A2 called MsPLA2-I was isolated. M. spixii crude venom caused severe swelling in the legs of tested mice and significant release of creatine kinase (CK) showing its myotoxic activity. Leishmanicidal activity against Leishmania amazonensis (IC50 1.24 μg/mL) was also observed, along with antiplasmodial activity against Plasmodium falciparum, which are unprecedented for Micrurus venoms. MsPLA2-I with a Mr 12,809.4 Da was isolated from the crude venom of M. spixii. The N-terminal sequencing of a fragment of 60 amino acids showed 80% similarity with another PLA2 from Micrurus altirostris. This toxin and the crude venom showed phospholipase activity. In a mouse phrenic nerve-diaphragm preparation, M. spixii venom and MsPLA2-I induced the blockage of both direct and indirect twitches. While the venom presented a pronounced myotoxic activity, MsPLA2-I expressed a summation of neurotoxic activity. The results of this study make M. spixii crude venom promising compounds in the exploration of molecules with microbicidal potential. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2017-10-01

    Phospholipases A 2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIβ and PLIγ. Phospholipases A 2 (PLA 2 s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107-116) may be responsible for PLA 2 inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Development of a highly efficient oil degumming process using a novel phosphatidylinositol-specific phospholipase C enzyme.

    PubMed

    Cerminati, Sebastián; Eberhardt, Florencia; Elena, Claudia E; Peirú, Salvador; Castelli, María E; Menzella, Hugo G

    2017-06-01

    Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14 g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

  20. Role of phospholipase A2 in cholesterol gallstone formation is associated with biliary phospholipid species selection at the site of hepatic excretion: indirect evidence.

    PubMed

    Hattori, Y; Tazuma, S; Yamashita, G; Ochi, H; Sunami, Y; Nishioka, T; Hyogo, H; Yasumiba, S; Kajihara, T; Nakai, K; Tsuboi, K; Asamoto, Y; Sakomoto, M; Kajiyama, G

    2000-07-01

    Phospholipase A2 plays a role in cholesterol gallstone development by hydrolyzing bile phospholipids into lysolecithin and free fatty acids. Lysolecithin and polyunsaturated free fatty acids are known to stimulate the synthesis and/or secretion of gallbladder mucin via a prostanoid pathway, leading to enhancing cholesterol crystal nucleation and growth, and therefore, the action of phospholipase A2 is associated, in part, with bile phospholipid fatty acid. To clarify this hypothesis, we evaluated the effect on bile lipid metastability in vitro of replacing phospholipids with lysolecithin and various free fatty acids. Supersaturated model biles were created with an identical composition (cholesterol saturation index, 1.8; egg yolk lecithin, 34 mM; taurocholate, 120 mM; cholesterol, 25 mM) except for 5%, 10%, or 20% replacement of egg yolk lecithin with a combination of palmitoyl-lysolecithin and a free fatty acid (palmitate, stearate, oleate, linoleate, or arachidonate), followed by time-sequentially monitoring of vesicles and cholesterol crystals using spectrophotometer and video-enhanced differential contrast microscopy. Replacement with hydrophilic fatty acids (linoleate and arachidonate) reduced vesicle formation and promoted cholesterol crystallization, whereas an enhanced cholesterol-holding capacity was evident after replacement with hydrophobic fatty acids (palmitate and stearate). These results indicate that the effect of phospholipase A2 on bile lithogenecity is modulated by the fatty acid species in bile phospholipids, and therefore, that the role of phospholipase A2 in cholesterol gallstone formation is dependent, in part, on biliary phospholipid species selection at the site of hepatic excretion.

  1. Genetic association between the phospholipase A2 gene and unipolar affective disorder: a multicentre case-control study.

    PubMed

    Papadimitriou, George N; Dikeos, Dimitris G; Souery, Daniel; Del-Favero, Jurgen; Massat, Isabelle; Avramopoulos, Dimitrios; Blairy, Sylvie; Cichon, Sven; Ivezic, Sladjana; Kaneva, Radka; Karadima, Georgia; Lilli, Roberta; Milanova, Vihra; Nöthen, Markus; Oruc, Lilijana; Rietschel, Marcella; Serretti, Alessandro; Van Broeckhoven, Christine; Stefanis, Costas N; Mendlewicz, Julien

    2003-12-01

    The co-segregation in one pedigree of bipolar affective disorder with Darier's disease whose gene is on chromosome 12q23-q24.1, and findings from linkage and association studies with the neighbouring gene of phospholipase A2 (PLA2) indicate that PLA2 may be considered as a candidate gene for affective disorders. All relevant genetic association studies, however, were conducted on bipolar patients. In the present study, the possible association between the PLA2 gene and unipolar affective disorder was examined on 321 unipolar patients and 604 controls (all personally interviewed), recruited from six countries (Belgium, Bulgaria, Croatia, Germany, Greece, and Italy) participating in the European Collaborative Project on Affective Disorders. After controlling for population group and gender, one of the eight alleles of the investigated marker (allele 7) was found to be more frequent among unipolar patients with more than three major depressive episodes than among controls (P<0.01); genotypic association was also observed, under the dominant model of genetic transmission (P<0.02). In addition, presence of allele 7 was correlated with a higher frequency of depressive episodes (P<0.02). These findings suggest that structural variations at the PLA2 gene or the chromosomal region around it may confer susceptibility for unipolar affective disorder.

  2. Effect of lipoprotein-associated phospholipase A2 inhibitor on insulin resistance in streptozotocin-induced diabetic pregnant rats.

    PubMed

    Wang, Guo-Hua; Jin, Jun; Sun, Li-Zhou

    2018-06-21

    This paper aims to investigate the influence of lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor, darapladib, on insulin resistance (IR) in streptozotocin (STZ)-induced diabetic pregnant rats. The rat models were divided into Control (normal pregnancy), STZ + saline (STZ-induced diabetic pregnant rats), STZ + Low-dose and STZ + High-dose darapladib (STZ-induced diabetic pregnant rats treated with low-/high-dose darapladib) groups. Pathological changes were observed by Hematoxylin-eosin (HE) and Immunohistochemistry staining. Lp-PLA2 levels were determined by enzyme-linked immunosorbent assay (ELISA). An automatic biochemical analyzer was used to measure the serum levels of biochemical indicators, and homeostatic model assessment for insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) were calculated. Western blot was applied to determine levels of inflammatory cytokines. Compared with Control group, rats in the STZ + saline group were significantly decreased in body weight, the number of embryo implantation, the number of insulin positive cells and pancreatic islet size as well as the islet endocrine cells, and high-density lipoprotein (HDL-C) level, but substantially increased in Lp-PLA2, low-density lipoprotein (LDL-C), fatty acids (FFA), serum total cholesterol (TC), triglyceride (TG) levels. Moreover, the increased fasting plasma glucose (FPG) and HOMA-IR and inflammatory cytokines but decreased fasting insulin (FINS) and ISI were also found in diabetic pregnant rats. On the contrary, rats in the darapladib-treated groups were just opposite to the STZ + saline group, and STZ + High-dose group improved better than STZ + Low-dose group. Thus, darapladib can improve lipid metabolism, and enhance insulin sensitivity of diabetic pregnant rats by regulating inflammatory cytokines.

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

    PubMed

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

    2014-11-07

    Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Crystallization and preliminary X-ray diffraction analysis of a novel Arg49 phospholipase A{sub 2} homologue from Zhaoermia mangshanensis venom

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Murakami, Mário T.; Center for Applied Toxinology, CAT-CEPID, São Paulo, SP; Advanced Center for Genomics and Proteomics, UNESP-State University of São Paulo, São José do Rio Preto 15054-000

    2007-07-01

    A single crystal of zhaoermiatoxin with maximum dimensions of 0.2 × 0.2 × 0.5 mm was used for X-ray diffraction data collection to a resolution of 2.05 Å using synchrotron radiation and the diffraction pattern was indexed in the hexagonal space group P6{sub 4}, with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 Å. Zhaoermiatoxin, an Arg49 phospholipase A{sub 2} homologue from Zhaoermia mangshanensis (formerly Trimeresurus mangshanensis, Ermia mangshanensis) venom is a novel member of the PLA{sub 2}-homologue family that possesses an arginine residue at position 49, probably arising from a secondary Lys49→Arg substitution that does notmore » alter the catalytic inactivity towards phospholipids. Like other Lys49 PLA{sub 2} homologues, zhaoermiatoxin induces oedema and strong myonecrosis without detectable PLA{sub 2} catalytic activity. A single crystal with maximum dimensions of 0.2 × 0.2 × 0.5 mm was used for X-ray diffraction data collection to a resolution of 2.05 Å using synchrotron radiation and the diffraction pattern was indexed in the hexagonal space group P6{sub 4}, with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 Å.« less

  5. Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins[S

    PubMed Central

    Subra, Caroline; Grand, David; Laulagnier, Karine; Stella, Alexandre; Lambeau, Gérard; Paillasse, Michael; De Medina, Philippe; Monsarrat, Bernard; Perret, Bertrand; Silvente-Poirot, Sandrine; Poirot, Marc; Record, Michel

    2010-01-01

    Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA2-IVA, the calcium-independent iPLA2-VIA, and the secreted sPLA2-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPγS triggered activation of phospholipase A2 (PLA2)and PLD2. A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandinJ2 (15-d PGJ2), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell. PMID:20424270

  6. Structural analysis of secretory phospholipase A2 from Clonorchis sinensis: therapeutic implications for hepatic fibrosis.

    PubMed

    Hariprasad, Gururao; Kaur, Punit; Srinivasan, Alagiri; Singh, Tej Pal; Kumar, Manoj

    2012-07-01

    Hepatic fibrosis is a common complication of the infection by the parasite, Clonorchis sinensis. There is a high incidence of this disease in the Asian countries with an increased risk of conversion to cancer. A secretory phospholipase A(2) (PLA(2)) enzyme from the parasite is implicated in the pathology. This is an attractive drug target in the light of extensive structural characterization of this class of enzyme. In this study, the structure of the enzyme was modeled based on its sequence homology to the group III bee venom PLA(2). On analysis, the overall structure essentially is comprised of three helices, two sets of β-wings and an elongated C-terminal extension. The structure is stabilized by four disulfide bonds. The structure is comprised of a calcium binding loop, active site and a substrate binding hydrophobic channel. The active site of the enzyme shows the classical features of PLA(2) with the participation of the three residues: histidine-aspartic acid-tyrosine in hydrogen bond formation. This is an interesting variation from the house keeping group III PLA(2) enzyme of human which has a histidine-aspartic acid and phenylalanine arrangement at the active site. This difference is therefore an important structural parameter that can be exploited to design specific inhibitor molecules against the pathogen PLA(2). Likewise, there are certain unique structural features in the hydrophobic channel and the putative membrane binding surface of the PLA(2) from Clonorchis sinensis that not only help understand the mechanism of action but also provide knowledge for a targeted therapy of liver fibrosis caused by the parasite.

  7. Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

    NASA Technical Reports Server (NTRS)

    Vandenburgh, H. H.; Shansky, J.; Karlisch, P.; Solerssi, R. L.

    1993-01-01

    Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E2 and F2 alpha which regulate protein turnover rates and muscle cell growth. These stretch-induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of 3H-arachidonic acid labelled phospholipids, releasing free 3H-arachidonic acid, the rate-limiting precursor of PG synthesis. Mechanical stimulation also increases 3H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-[2-3H]inositol labelled phospholipids. Phospholipase A2 (PLA2), phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch-induced increases in PG production, 3H-arachidonic acid labelled phospholipid breakdown, and 3H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitive) whereas the formation of inositol phosphates from myo-[2-3H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol-specific PLC.

  8. Possible regulation of cation-induced pinocytosis in Amoeba proteus by phospholipase A.

    PubMed

    Josefsson, J O; Arvidson, G; Cobbold, P

    1988-04-01

    We have studied the effects of exogenous phospholipids and compounds which are known to alter the activity of phospholipase A (PLA) on Ca2+-dependent, Na+-induced pinocytosis in Amoeba proteus. The PLA-inhibitors mepacrine, p-bromophenacyl bromide (pBPB) and Rosenthal's inhibitor depressed pinocytosis. Normal pinocytotic intensity was restored by the addition of Ca2+ or picomolar concentrations of lysolecithin. Very low concentrations of lysophospholipids and different molecular species of lecithins increased the capacity for pinocytosis in starved amoebae. The effect of the lecithins but not of the corresponding lysolecithins was abolished by PLA-inhibitors. Also, the restoration of the pinocytotic capacity of starved amoebae by melittin and mastoparan, which are known to stimulate PLA, was inhibited by mepacrine and pBPB. Isolated amoeba plasma membranes contain phospholipase A1 and A2 activity and the amoebae secrete a lipid (PRF, pinocytosis regulating factor) which has lysolecithin-like effects on pinocytosis. The enzyme activities and the release of PRF were markedly decreased by the PLA-inhibitors. Our observations support the hypothesis that PRF is a lysophospholipid that may constitute a signal for the formation of pinocytotic channels in the initial stages of pinocytosis. The phospholipase A activity of the amoeba must therefore be assigned an important role in the regulation of the Ca2+-dependent, cation-induced pinocytosis.

  9. Antiparasitic effects induced by polyclonal IgY antibodies anti-phospholipase A2 from Bothrops pauloensis venom.

    PubMed

    Borges, Isabela Pacheco; Silva, Mariana Ferreira; Santiago, Fernanda Maria; de Faria, Lucas Silva; Júnior, Álvaro Ferreira; da Silva, Rafaela José; Costa, Mônica Soares; de Freitas, Vitor; Yoneyama, Kelly Aparecida Geraldo; Ferro, Eloísa Amália Vieira; Lopes, Daiana Silva; Rodrigues, Renata Santos; de Melo Rodrigues, Veridiana

    2018-06-01

    Activities of phospholipases (PLAs) have been linked to pathogenesis in various microorganisms, and implicated in cell invasion and so the interest in these enzymes as potential targets that could contribute to the control of parasite survival and proliferation. Chicken eggs immunized with BnSP-7, a Lys49 phospholipase A 2 (PLA 2 ) homologue from Bothrops pauloensis snake venom, represent an excellent source of polyclonal antibodies with potential inhibitory activity on parasite PLA s. Herein, we report the production, characterization and anti-parasitic effect of IgY antibodies from egg yolks of hens immunized with BnSP-7. Produced antibodies presented increasing avidity and affinity for antigenic toxin epitopes throughout immunization, attaining a plateau after 4weeks. Pooled egg yolks-purified anti-BnSP-7 IgY antibodies were able to specifically recognize different PLA 2 s from Bothrops pauloensis and Bothrops jararacussu venom. Antibodies also neutralized BnSP-7 cytotoxic activity in C2C12 cells. Also, the antibodies recognized targets in Leishmania (Leishmania) amazonensis and Toxoplasma gondii extracts by ELISA and immunofluorescence assays. Anti-BnSP-7 IgY antibodies were cytotoxic to T. gondii tachyzoite and L. (L.) amazonensis promastigotes, and were able to decrease proliferation of both parasites treated before infection. These data suggest that the anti-BnSP-7 IgY is an important tool for discovering new parasite targets and blocking parasitic effects. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Contrasting respirable quartz and kaolin retention of lecithin surfactant and expression of membranolytic activity following phospholipase A2 digestion.

    PubMed

    Wallace, W E; Keane, M J; Mike, P S; Hill, C A; Vallyathan, V; Regad, E D

    1992-11-01

    Respirable-sized quartz, a well-established fibrogenic mineral dust, is compared with kaolin in erythrocyte hemolysis assays after treatment with saline dispersion of dipalmitoyl phosphatidylcholine, a primary phospholipid component of pulmonary surfactant. Both dusts are rendered inactive after treatment, but the membranolytic activity is partly to fully restored after treatment with phospholipase A2, an enzyme normally associated with cellular plasma membranes and lysosomes. Phospholipid-coated dusts were incubated for periods of 2-72 h at a series of applied enzyme concentrations, and the adsorbed lipid species and hemolytic activity were quantitated at each time for both dusts. Surfactant was lost more readily from quartz than from kaolin, with consequent more rapid restoration of mineral surface hemolytic activity for quartz. Interactions of surfactant and mineral surface functional groups responsible for the mineral-specific rate differences, and implications for determining the mineral surface bioavailability of silica and silicate dusts, are discussed.

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

    PubMed

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

    2007-11-30

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

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

    PubMed

    Ben Bacha, Abir; Abid, Islem

    2013-03-01

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

  13. Tilmicosin reduces lipopolysaccharide-stimulated bovine alveolar macrophage prostaglandin E(2) production via a mechanism involving phospholipases.

    PubMed

    Lakritz, Jeffrey; Tyler, Jeff W; Marsh, Antoinette E; Romesburg-Cockrell, Mary; Smith, Kathy; Holle, Julie M

    2002-01-01

    Tilmicosin is a potent antimicrobial with broad-spectrum activity against the bacterial agents involved in the bovine respiratory disease complex. Recent studies indicate that in addition to being bactericidal, tilmicosin is capable of modulating inflammation in the lung. A series of experiments were designed to determine whether tilmicosin alters alveolar macrophage-prostaglandin E(2) (PGE(2)) production induced by Escherichia coli (O55:B5) lipopolysaccharide (LPS). Twenty-two healthy Holstein bull calves were used to study the effects of LPS-induced PGE(2) production of alveolar macrophages after in vivo or in vitro treatment with tilmicosin. In Experiment 1, tilmicosin was given by subcutaneous injection (15 mg/kg) twice, 48 hours apart, to four calves; four control calves received no treatment. Twenty-four hours after the second treatment, alveolar macrophages were stimulated with LPS in vitro. In Experiment 2, alveolar macrophages from five untreated calves were harvested and treated in vitro with tilmicosin, followed by LPS stimulation. In Experiment 3, the ability of in vitro tilmicosin treatment to alter the expression of LPS-induced cyclooxygenase-2 (COX-2) mRNA was evaluated. In Experiments 4 and 5, secretory phospholipase A(2) activity was examined in untreated calves. Treatment of calves with tilmicosin resulted in reduced LPS-induced alveolar macrophage PGE(2) production. Similar reductions in PGE(2) by LPS-stimulated alveolar macrophages after in vitro tilmicosin treatment were noted. This in vitro tilmicosin treatment was not associated with reduction of the expression of LPS-induced COX-2. Alveolar macrophage phospholipase A(2) activity induced by LPS was significantly reduced by prior tilmicosin treatment in vitro. Tilmicosin (in vivo and in vitro) appears to reduce the PGE(2) eicosanoid response of LPS-stimulated alveolar macrophages by reducing the in vitro substrate availability without altering in vitro COX-2 mRNA expression.

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

    PubMed Central

    2011-01-01

    Background Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food. Results The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used for immunohistochimical and immunofluorescence analysis in order to determine the cellular location of mSDPLA2. Our results showed essentially that mSDPLA2 was detected inside in specific vesicles tentatively named (mSDPLA2+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion in the snail might occur in specific granules inside the digestive cells. Conclusion The cellular location of mSDPLA2 suggests that intracellular phospholipids digestion, like other food components digestion of snail diet, occurs in these digestive cells. The hepatopancreas of H. trunculus has been pointed out as the main region for digestion, absorption and storage of lipids. PMID:21631952

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

    PubMed

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

    2011-06-01

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

  16. Structure of a Premicellar Complex of Alkyl Sulfates with the Interfacial Binding Surfaces of 4 Subunits of Phospholipase A2

    PubMed Central

    Pan, Ying H.; Bahnson, Brian J.

    2010-01-01

    The properties of three discrete premicellar complexes (E1#, E2#, E3#) of pig pancreatic group-IB secreted phospholipase A2 (sPLA2) with monodisperse alkyl sulfates has been characterized [Berg, O. G., et al., Biochemistry 43, 7999–8013, 2004]. Here we have solved the 2.7 Å crystal structure of group-IB sPLA2 complexed with 12 molecules of octyl sulfate (C8S) in a form consistent with a tetrameric oligomeric that exists during the E1# phase of premicellar complexes. The alkyl tails of the C8S molecules are centered in the middle of the tetrameric cluster of sPLA2 subunits. Three of the four sPLA2 subunits also contain a C8S molecule in the active site pocket. The sulfate oxygen of a C8S ligand is complexed to the active site calcium in 3 of the 4 protein active sites. The interactions of the alkyl sulfate head group with Arg-6 and Lys-10, as well as the backbone amide of Met-20, are analogous to those observed in the previously solved sPLA2 crystal structures with bound phosphate and sulfate anions. The cluster of three anions found in the present structure is postulated to be the site for nucleating the binding of anionic amphiphiles to the interfacial surface of the protein, and therefore this binding interaction has implications for interfacial activation of the enzyme. PMID:20302975

  17. Structure of a premicellar complex of alkyl sulfates with the interfacial binding surfaces of four subunits of phospholipase A2.

    PubMed

    Pan, Ying H; Bahnson, Brian J

    2010-07-01

    The properties of three discrete premicellar complexes (E1#, E2#, E3#) of pig pancreatic group-IB secreted phospholipase A2 (sPLA2) with monodisperse alkyl sulfates have been characterized [Berg, O. G. et al., Biochemistry 43, 7999-8013, 2004]. Here we have solved the 2.7 A crystal structure of group-IB sPLA2 complexed with 12 molecules of octyl sulfate (C8S) in a form consistent with a tetrameric oligomeric that exists during the E1# phase of premicellar complexes. The alkyl tails of the C8S molecules are centered in the middle of the tetrameric cluster of sPLA2 subunits. Three of the four sPLA2 subunits also contain a C8S molecule in the active site pocket. The sulfate oxygen of a C8S ligand is complexed to the active site calcium in three of the four protein active sites. The interactions of the alkyl sulfate head group with Arg-6 and Lys-10, as well as the backbone amide of Met-20, are analogous to those observed in the previously solved sPLA2 crystal structures with bound phosphate and sulfate anions. The cluster of three anions found in the present structure is postulated to be the site for nucleating the binding of anionic amphiphiles to the interfacial surface of the protein, and therefore this binding interaction has implications for interfacial activation of the enzyme. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  18. Plasma phospholipase, γ-CEHC and antioxidant capacity in fibromyalgia.

    PubMed

    Fais, Antonella; Cacace, Enrico; Atzori, Luigi; Era, Benedetta; Ruggiero, Valeria

    2017-05-01

    Recent studies have suggested a possible role of high levels of plasma lysophosphocholines (lysoPCs) in fibromyalgia syndrome (FMS). The aim of this study was to evaluate the content of plasma phospholipases (e.g., Platelet Activating Factor Acetyl Hydrolase [PAF-AH], secretory Phospholipase A 2 [sPLA 2 ], Total Antioxidant Capacity [TAOC] and 2,7,8-trimethyl-2-(2-carboxyethyl)-6-hydroxy chroman [γ-CEHC]) in FMS patients and their association with clinical status and quality of life. Thirty-six females meeting the 2011 American College of Rheumatology criteria for the classification of FMS and thirty-four healthy females were enrolled for the study. Plasma enzyme levels were quantified using commercial enzyme-linked-immunosorbent-assay (ELISA). In order to assess the disease severity and the functional status of patients, the Fibromyalgia Impact Questionnarie (FIQ) was used. Higher levels of sPLA 2 and lower PAF-AH and γ-CEHC were observed in the plasma of FMS patients compared to the controls. A decrease in PAF-AH and TAOC levels were found in severe FMS (S-FMS) compared to mild/slight (MS-FMS) forms. The results of the study indicate a possible involvement of phospholipases and γ-CEHC in fibromyalgia syndrome. © 2015 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  19. [Molecular cloning and characterization in silico of phospholipase A(2) transcript isolated from Lachesis muta peruvian snake venom].

    PubMed

    Jimenez, Karim L; Zavaleta, Amparo I; Izaguirre, Victor; Yarleque, Armando; Inga, Rosio R

    2010-01-01

    Isolate and characterize in silico gene phospholipase A(2) (PLA(2)) isolated from Lachesis muta venom of the Peruvian Amazon. Technique RT-PCR from total RNA was using specific primers, the amplified DNA product was inserted into the pGEM vector for subsequent sequencing. By bioinformatic analysis identified an open reading frame of 414 nucleotides that encoded 138 amino acids including a signal peptide of 16 aminoacids, molecular weight and pI were 13,976 kDa and 5.66 respectively. The aminoacid sequence was called Lm-PLA(2)-Peru, contains an aspartate at position 49, this aminoacid in conjunction with other conserved residues such as Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 are important for enzymatic activity. The comparison with the amino acid sequence data banks showed of similarity between PLA(2) from Lachesis stenophrys (93%) and other PLA(2) snake venoms and over 80% of other sPLA(2) family Viperidae venoms. A phylogenetic analysis showed that Lm-PLA(2)-Peru grouped with other acidic [Asp(49)] sPLA(2) previously isolated from Bothriechis schlegelii venom showing 89 % nucleotide sequence identity. Finally, the computer modeling indicated that enzyme had the characteristic structure of sPLA(2) group II that consisted of three α-helices, a β-wing, a short helix and a calcium-binding loop. The nucleotide sequence corresponding to the first transcript of gene from PLA(2) cloned of Lachesis muta venom, snake from the Peruvian rainforest.

  20. Crystallization and preliminary X-ray diffraction analysis of a class II phospholipase D from Loxosceles intermedia venom

    PubMed Central

    Ullah, Anwar; de Giuseppe, Priscila Oliveira; Murakami, Mario Tyago; Trevisan-Silva, Dilza; Wille, Ana Carolina Martins; Chaves-Moreira, Daniele; Gremski, Luiza Helena; da Silveira, Rafael Bertoni; Sennf-Ribeiro, Andrea; Chaim, Olga Meiri; Veiga, Silvio Sanches; Arni, Raghuvir Krishnaswamy

    2011-01-01

    Phospholipases D are the major dermonecrotic component of Loxosceles venom and catalyze the hydrolysis of phospholipids, resulting in the formation of lipid mediators such as ceramide-1-phosphate and lysophosphatidic acid which can induce pathological and biological responses. Phospholipases D can be classified into two classes depending on their catalytic efficiency and the presence of an additional disulfide bridge. In this work, both wild-type and H12A-mutant forms of the class II phospholipase D from L. intermedia venom were crystallized. Wild-type and H12A-mutant crystals were grown under very similar conditions using PEG 200 as a precipitant and belonged to space group P1211, with unit-cell parameters a = 50.1, b = 49.5, c = 56.5 Å, β = 105.9°. Wild-type and H12A-mutant crystals diffracted to maximum resolutions of 1.95 and 1.60 Å, respectively. PMID:21301094

  1. A new inhibitor of synovial phospholipase A2 from fermentations of Penicillium sp. 62-92.

    PubMed

    Witter, L; Anke, T; Sterner, O

    1998-01-01

    Penidiamide, a new tripetide containing dehydrotryptamine, glycine and anthranilic acid linked together by two amide bonds, and oxindole were isolated from submerged cultures of Penicillium sp. 62-92. Both compounds preferentially inhibited human synovial phospholipase A2, penidiamide with an IC50 of 30 microM and oxindole of 380 microM. With the exception of U 937 cells (leukemia, human), no cytotoxic activities were detected against HL-60- (leukemia, human), HeLa S3- (epitheloid carcinoma, human), BHK 21- (kidney fibroblasts, hamster), and L1210-cells (leukemia, mouse). No antimicrobial activity was detected for oxindole, and only weak antibacterial activity for penidiamide. The structure of penidiamide was elucidated by spectroscopic methods.

  2. Antimycobacterial activity of lecithin-cholesterol liposomes in the presence of phospholipase A2.

    PubMed

    Kondo, E; Kanai, K

    1978-06-01

    Tubercle bacilli were preincubated with lecithin-cholesterol liposomes to be subsequently exposed to phospholipase A2. After further incubation in the environment of acidic buffer, viable units in the final mixture were enumerated by inoculating the serial dilutions of an aliquot onto Kirchner agar medium containing horse serum in 5%. Another aliquot was used for lipid analyses to confirm hydrolysis of lecithin. In addition to this bactericidal type of experiments, bacteriostatic tests were also conducted with Kirchner semi-solid agar medium, into which liposome-treated bacilli were inoculated with the enzyme at a time. Various natural and synthetic lecithins different in constituent fatty acids were employed. The results indicated that toxic fatty acids released from lecithin acted to kill the bacilli or to inhibit their growth.

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

    PubMed

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

    2012-05-01

    Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A(2). Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A(2). These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A(2), it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. 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

    PubMed Central

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

    2012-01-01

    Summary 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 (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. PMID:22266334

  5. Phospholipase D function in Saccharomyces cerevisiae.

    PubMed

    Mendonsa, Rima; Engebrecht, JoAnne

    2009-09-01

    Phosphatidylinositol 4,5-bisphosphate-regulated phosphatidylcholine-specific phospholipase D is conserved from yeast to man. The essential role of this enzyme in yeast is to mediate the fusion of Golgi and endosome-derived vesicles to generate the prospore membrane during the developmental program of sporulation, through the production of the fusogenic lipid phosphatidic acid. In addition to recruiting proteins required for fusion, phosphatidic acid is believed to lower the energy barrier to stimulate membrane curvature. During mitotic growth, phospholipase D activity is dispensable unless the major phosphatidylinositol/phosphatidylcholine transfer protein is absent; it also appears to play a nonessential role in the mating signal transduction pathway. The regulation of phospholipase D activity during both sporulation and mitotic growth is still not fully understood and awaits further characterization.

  6. Thyrotropin-induced hydrogen peroxide production in FRTL-5 thyroid cells is mediated not by adenosine 3',5'-monophosphate, but by Ca2+ signaling followed by phospholipase-A2 activation and potentiated by an adenosine derivative.

    PubMed

    Kimura, T; Okajima, F; Sho, K; Kobayashi, I; Kondo, Y

    1995-01-01

    The production of hydrogen peroxide (H2O2) as an essential process for iodide organification is a key reaction in TSH-induced thyroid hormone synthesis. Here we characterize the signal transduction pathway involved in TSH-induced H2O2 production in FRTL-5 thyroid cells. At higher than 1 nM TSH, N6-(L-2-phenylisopropyl)adenosine (PIA), an adenosine receptor agonist having, by itself, no influence on H2O2 generation, potentiated this TSH action, whereas the TSH increase and PIA addition reduced cAMP accumulation. RO 20-1724, a phosphodiesterase inhibitor, amplified the TSH-induced cAMP accumulation, but did not change H2O2 generation in the whole range of TSH used. Ca(2+)-mobilizing agonists, GTP and ATP, also induced H2O2 production without stimulating cAMP accumulation. Chelation of intracellular Ca2+ markedly inhibited the TSH action, but intracellular Ca2+ increases by either thapsigargin or ionomycin mimicking it. All of the findings show the participation of Ca2+, but not cAMP, in the action of TSH. Desensitization of protein kinase-C (PKC) did not influence the receptor-mediated H2O2 production, suggesting the reduced importance of PKC activation compared to Ca2+ signaling to the reaction. A rise in intracellular Ca2+ independent of receptor activation also induced H2O2 production as well as arachidonate release, and both were potentiated by PIA. In addition, inhibitors of phospholipase-A2 and the arachidonate metabolic pathway depressed H2O2 generation, suggesting the participation of an arachidonate cascade in the Ca(2+)-dependent H2O2 production. Lipoxygenase inhibitors depressed the Ca2+ action without influencing arachidonate release, suggesting the involvement of a lipoxygenase product(s) of arachidonate in the Ca(2+)-signaling mechanism. In conclusion, in FRTL-5 cells, TSH-induced H2O2 production is mediated not by cAMP, but by the phospholipase-C/Ca2+ cascade, possibly followed by the Ca(2+)-dependent phospholipase-A2/arachidonate cascade. PIA

  7. Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1.

    PubMed

    Taguchi, R; Ikezawa, H

    1987-10-01

    The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)

  8. The manifold phospholipases A of Legionella pneumophila - identification, export, regulation, and their link to bacterial virulence.

    PubMed

    Banerji, Sangeeta; Aurass, Philipp; Flieger, Antje

    2008-04-01

    The intracellular lung pathogen Legionella pneumophila expresses secreted and cell-associated phospholipase A (PLA) and lysophospholipase A (LPLA) activities belonging to at least three enzyme families. The first family consists of three secreted PLA and LPLA activities displaying the amino acid signature motif 'GDSL'; PlaA, PlaC and PlaD. The second group contains the cell-associated and very potent PLA/LPLA, PlaB. The third group, the patatin-like proteins, comprises 11 members. One patatin-like protein, PatA/VipD, shows LPLA and PLA activities and interferes with vesicular trafficking when expressed in yeast and therefore is possibly involved in the intracellular infection process. Likewise, members of the first two phospholipase families have roles in bacterial virulence because phospholipases are important virulence factors that have been shown to promote bacterial survival, spread and host cell modification/damage. The GDSL enzyme PlaA detoxifies cytolytic lysophospholipids, and PlaB shows contact-dependent haemolytic activity. PlaC acylates cholesterol, a lipid present in eukaryotic hosts but not in the bacterium. Many of the L. pneumophila PLAs are exported by the type II Lsp or the type IVB Dot/Icm secretion systems involved in virulence factor export. Moreover, the regulation of lipolytic activities depends on the transcriptional regulators LetA/S and RpoS, inducing the expression of virulence traits, and on posttranscriptional activators like the zinc metalloprotease ProA.

  9. Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury.

    PubMed

    Chao, Honglu; Liu, Yinlong; Lin, Chao; Xu, Xiupeng; Li, Zheng; Bao, Zhongyuan; Fan, Liang; Tao, Chao; Zhao, Lin; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

    2018-06-09

    Phospholipase A 2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A 2 (cPLA 2 )-related inflammatory responses after TBI. We found that cPLA 2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA 2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA 2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA 2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA 2 -related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA 2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA 2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA 2 -related inflammatory response from the PKC pathway. Copyright © 2018. Published by Elsevier B.V.

  10. [Lipase and phospholipase C from Staphylococcus aureus of different origin. I. Determination and occurrence (author's transl)].

    PubMed

    Berete, Y J; Schaeg, W; Brückler, J; Blobel, H

    1980-11-01

    Lipase and phospholipase C from Staphylococcus aureus of different origin were demonstrated qualitatively by agar diffusion on tributyrin- and lecithin agar. On test media with either 0,3% Na-azide or 0,3% KCN lipase-activity was not inhibited, phospholipase C, on the other hand, completely blocked (Table 1, Fig. 2). In this manner a tentative differentiation was possible between lipase and phospholipase C. For the quantitative determination of lipase the hydrolysis of p-nitrophenyl palmitate proved to be most useful (Fig. 1). S. aureus-cultures of human origin produced more often and more actively lipase and phospholipase C than those from cattle (Table 2).

  11. AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs

    PubMed Central

    El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

    2015-01-01

    Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs. PMID:26162096

  12. AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs.

    PubMed

    El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

    2015-01-01

    Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

  13. Petunia Phospholipase C1 Is Involved in Pollen Tube Growth[W

    PubMed Central

    Dowd, Peter E.; Coursol, Sylvie; Skirpan, Andrea L.; Kao, Teh-hui; Gilroy, Simon

    2006-01-01

    Although pollen tube growth is essential for plant fertilization and reproductive success, the regulators of the actin-related growth machinery and the cytosolic Ca2+ gradient thought to determine how these cells elongate remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators; however, the relevant phospholipase(s) have not been characterized. Therefore, we cloned cDNA for a pollen-expressed phosphatidylinositol 4,5-bisphosphate (PtdInsP2)–cleaving phospholipase C (PLC) from Petunia inflata, named Pet PLC1. Expressing a catalytically inactive form of Pet PLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton, and delocalization of growth at the tube tip. These phenotypes were suppressed by depolymerizing actin with low concentrations of latrunculin B, suggesting that a critical site of action of Pet PLC1 is in regulating actin structure at the growing tip. A green fluorescent protein (GFP) fusion to Pet PLC1 caused enrichment in regions of the apical plasma membrane not undergoing rapid expansion, whereas a GFP fusion to the PtdInsP2 binding domain of mammalian PLC δ1 caused enrichment in apical regions depleted in PLC. Thus, Pet PLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell. PMID:16648366

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

    PubMed

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

    2015-07-01

    Azithromycin is a member of macrolides, utilized in the treatment of infections. Independently, these antibiotics also possess anti-inflammatory and immunomodulatory properties. Phospholipase A2 isotypes, which are implicated in the pathophysiology of inflammatory lung disorders, are produced by alveolar macrophages and other lung cells during inflammatory response and can promote lung injury by destructing lung surfactant. The aim of the study was to investigate whether in lung cells azithromycin can inhibit secretory and cytosolic phospholipases A2, (sPLA2) and (cPLA2), respectively, which are induced by an inflammatory trigger. In this respect, we studied the lipopolysaccharide (LPS)-mediated production or secretion of sPLA2 and cPLA2 from A549 cells, a cancer bronchial epithelial cell line, and alveolar macrophages, isolated from bronchoalveolar lavage fluid of ARDS and control patients without cardiopulmonary disease or sepsis. Pre-treatment of cells with azithromycin caused a dose-dependent decrease in the LPS-induced sPLA2-IIA levels in A549 cells. This inhibition was rather due to reduced PLA2G2A mRNA expression and secretion of sPLA2-IIA protein levels, as observed by western blotting and indirect immunofluorescence by confocal microscopy, respectively, than to the inhibition of the enzymic activity per se. On the contrary, azithromycin had no effect on the LPS-induced production or secretion of sPLA2-IIA from alveolar macrophages. The levels of LPS-induced c-PLA2 were not significantly affected by azithromycin in either cell type. We conclude that azithromycin exerts anti-inflammatory properties on lung epithelial cells through the inhibition of both the expression and secretion of LPS-induced sPLA2-IIA, while it does not affect alveolar macrophages. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Negative correlation between phospholipase and esterase activity produced by Fusarium isolates.

    PubMed

    Ishida, K; Alviano, D S; Silva, B G; Guerra, C R; Costa, A S; Nucci, M; Alviano, C S; Rozental, S

    2012-05-01

    Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

  16. Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2.

    PubMed

    Sitkiewicz, Izabela; Nagiec, Michal J; Sumby, Paul; Butler, Stephanie D; Cywes-Bentley, Colette; Musser, James M

    2006-10-24

    The molecular basis of pathogen clone emergence is relatively poorly understood. Acquisition of a bacteriophage encoding a previously unknown secreted phospholipase A(2) (designated SlaA) has been implicated in the rapid emergence in the mid-1980s of a new hypervirulent clone of serotype M3 group A Streptococcus. Although several lines of circumstantial evidence suggest that SlaA is a virulence factor, this issue has not been addressed experimentally. We found that an isogenic DeltaslaA mutant strain was significantly impaired in ability to adhere to and kill human epithelial cells compared with the wild-type parental strain. The mutant strain was less virulent for mice than the wild-type strain, and immunization with purified SlaA significantly protected mice from invasive disease. Importantly, the mutant strain was significantly attenuated for colonization in a monkey model of pharyngitis. We conclude that transductional acquisition of the ability of a GAS strain to produce SlaA enhanced the spread and virulence of the serotype M3 precursor strain. Hence, these studies identified a crucial molecular event underlying the evolution, rapid emergence, and widespread dissemination of unusually severe human infections caused by a distinct bacterial clone.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  19. Requirement of GM2 ganglioside activator for phospholipase D activation

    PubMed Central

    Nakamura, Shun-ichi; Akisue, Toshihiro; Jinnai, Hitoshi; Hitomi, Tomohiro; Sarkar, Sukumar; Miwa, Noriko; Okada, Taro; Yoshida, Kimihisa; Kuroda, Shun’ichi; Kikkawa, Ushio; Nishizuka, Yasutomi

    1998-01-01

    Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by β-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792–815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown. PMID:9770472

  20. Membrane-induced Allosteric Control of Phospholipase C-β Isozymes*

    PubMed Central

    Charpentier, Thomas H.; Waldo, Gary L.; Barrett, Matthew O.; Huang, Weigang; Zhang, Qisheng; Harden, T. Kendall; Sondek, John

    2014-01-01

    All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-β (PLC-β) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-β isozymes are autoinhibited, and several proteins, including Gαq, Gβγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP2 that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gαq or Gβ1γ2 did not activate purified PLC-β3 under these conditions despite their robust capacity to activate PLC-β3 at membranes. In addition, mutants of PLC-β3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP2 in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-β isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-β isozymes. PMID:25193662

  1. Preliminary X-ray crystallographic studies of a tetrameric phospholipase A{sub 2} formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Marchi-Salvador, D. P.; Corrêa, L. C.; Salvador, G. H. M.

    2007-12-01

    Crotoxin B is a basic phospholipase A{sub 2} found in the venom of C. durissus terrificus and is one of the subunits that constitute crotoxin. Here, the crystallization, X-ray diffraction data collection and molecular-replacement solution of a novel tetrameric complex formed by two dimers of crotoxin B isoforms are presented. Crotoxin B is a basic phospholipase A{sub 2} found in the venom of Crotalus durissus terrificus and is one of the subunits that constitute crotoxin. This heterodimeric toxin, which is the main component of C. d. terrificus venom, is completed by an acidic, nontoxic and non-enzymatic component (crotoxin A) andmore » is involved in important envenomation effects, such as neurological disorders, myotoxicity and renal failure. Although crotoxin was first crystallized in 1938, no crystal structure is currently available for crotoxin, crotoxin A or crotoxin B. In this work, the crystallization, X-ray diffraction data collection to 2.28 Å resolution and molecular-replacement solution of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2) is presented.« less

  2. Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera.

    PubMed

    Machiah, Deepa K; Gowda, T Veerabasappa

    2006-06-01

    A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.

  3. Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.; Shansky, Janet; Karlisch, Patricia; Solerssi, Rosa Lopez

    1991-01-01

    Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins E2 and F2(alpha) which regulate protein turnover rates and muscle cell growth. Mechnical stimulation significantly increases the breakdown rate of (3)H-arachidonic acid labelled phospholipids, releasing free (3)H-arachidonic acid, and the rate-limiting precursor of prostaglandin synthesis. Mechanical stimulation also significantly increases (3)H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-2-(3)H inositol labelled phospholipids. Phospholipase A2, phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are activated by stretch. The lipase inhibitors bromophenacylbromide and RHC80267 together reduce stretch-induced prostaglandin production by 73-83 percent. The stretch-induced increases in prostaglandin production, (3)H-arachidonic acid labelled phospholipid breakdown, and (3)H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitive) whereas the formation of inositol phosphates from myo-2-(3)H inositol labelled phospholipids are dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and prostaglandins through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol-specific PLC.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

  5. Inhibition of untransformed prostaglandin H(2) production and stretch-induced contraction of rabbit pulmonary arteries by indoxam, a selective secretory phospholipase A(2) inhibitor.

    PubMed

    Tanabe, Yoshiyuki; Saito, Maki; Morikawa, Yuki; Kamataki, Akihisa; Sawai, Takashi; Hirose, Masamichi; Nakayama, Koichi

    2011-01-01

    Involvement of secretory phospholipase A(2) (sPLA(2)) in the stretch-induced production of untransformed prostaglandin H(2) (PGH(2)) in the endothelium of rabbit pulmonary arteries was investigated. The stretch-induced contraction was significantly inhibited by indoxam, a selective inhibitor for sPLA(2), and NS-398, a selective inhibitor for cyclooxygenase-2 (COX-2). Indoxam inhibited the RGD-sensitive-integrin-independent production of untransformed PGH(2), but did not affect the RGD-sensitive-integrin-dependent production of thromboxane A(2) (TXA(2)). These results suggest that the stretch-induced contraction and untransformed PGH(2) production was mediated by sPLA(2)-COX-2 pathway, making it a new possible target for pharmacological intervention of pulmonary artery contractility.

  6. Definition of the specific roles of lysolecithin and palmitic acid in altering the susceptibility of dipalmitoylphosphatidylcholine bilayers to phospholipase A2.

    PubMed

    Henshaw, J B; Olsen, C A; Farnbach, A R; Nielson, K H; Bell, J D

    1998-07-28

    Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperature near the main bilayer phase transition and calcium concentration modulate the effectiveness of the reaction products. The purpose of this study was to examine the individual contributions of lysolecithin and palmitic acid to the susceptibility of dipalmitoylphosphatidylcholine vesicles and to rationalize the effects of temperature and calcium. Various fluorescent probes (Prodan, Laurdan, pyrene-labeled fatty acid, and dansyl-labeled phospholipid) were used to assess changes in the ability of the reaction products to perturb the bilayer and to affect the interactions with the enzyme. Un-ionized palmitic acid decreased bilayer polarity and perturbed the membrane surface exposing some of the Prodan to bulk water. Lysolecithin increased bilayer polarity and the rate of dipolar relaxation in response to the excited states of Laurdan and Prodan. A combination of the individual contributions of each product was observed when palmitic acid and lysolecithin were present together at low calcium, and the effects of lysolecithin dominated at high calcium. Palmitic acid, but not lysolecithin, promoted the binding of phospholipase A2 to the bilayer surface in the absence of calcium. Lysolecithin reduced the ability of fatty acid to enhance binding apparently by altering the structure of fatty acid domains in the membrane. Furthermore, increased temperature and ionization of the fatty acid tended to cause segregation of bound phospholipase A2 into domains poor in phospholipid content which presumably impeded bilayer hydrolysis. In contrast, un-ionized palmitic acid and lysolecithin promoted hydrolysis by augmenting a step distal to the adsorption of enzyme to the bilayer. This kinetic response to lysolecithin was calcium-dependent. A

  7. Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

    PubMed Central

    Ishida, K.; Alviano, D.S.; Silva, B.G.; Guerra, C.R.; Costa, A.S.; Nucci, M.; Alviano, C.S.; Rozental, S.

    2012-01-01

    Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes. PMID:22415116

  8. Efficient heterologous expression, functional characterization and molecular modeling of annular seabream digestive phospholipase A2.

    PubMed

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

    2018-03-01

    Here we report the cDNA cloning of a phospholipase A 2 (PLA 2 ) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA 2 . In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA 2 are closer to avian PLA 2 group IB than to mammals' ones. In order to understand the structure-function relationships of these enzymes, we report here the recombinant expression in E.coli, the refolding and characterization of His-tagged annular seabream PLA 2 (AsPLA 2 ). A single Ni-affinity chromatography step was used to obtain a highly purified recombinant AsPLA 2 with a molecular mass of 15kDa as attested by gel electrophoresis and MALDI-TOF mass spectrometry data. The enzyme has a specific activity of 400U.mg -1 measured on phosphatidylcholine at pH 8.5 and 50°C. The enzyme high thermo-activity and thermo-stability make it a potential candidate in various biological applications. The 3D structure models of these enzymes were compared with structures of phylogenetically related pancreatic PLA 2 . By following these models and utilizing molecular dynamics simulations, the resistance of the AsPLA 2 at high temperatures was explained. Using the monomolecular film technique, AsPLA 2 was found to be active on various phospholipids spread at the air/water interface at a surface pressure between 12 and 25dyncm -1 . Interestingly, this enzyme was shown to be mostly active on dilauroyl-phosphatidylglycerol monolayers and this behavior was confirmed by molecular docking and dynamics simulations analysis. The discovery of a thermo-active new member of Sparidae PLA 2 , provides new insights on structure-activity relationships of fish PLA 2 . Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Reminiscence of phospholipase B in Penicillium notatum

    PubMed Central

    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, Fe3+, 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. Investigating interactions between phospholipase B-Like 2 and antibodies during Protein A chromatography.

    PubMed

    Tran, Benjamin; Grosskopf, Vanessa; Wang, Xiangdan; Yang, Jihong; Walker, Don; Yu, Christopher; McDonald, Paul

    2016-03-18

    Purification processes for therapeutic antibodies typically exploit multiple and orthogonal chromatography steps in order to remove impurities, such as host-cell proteins. While the majority of host-cell proteins are cleared through purification processes, individual host-cell proteins such as Phospholipase B-like 2 (PLBL2) are more challenging to remove and can persist into the final purification pool even after multiple chromatography steps. With packed-bed chromatography runs using host-cell protein ELISAs and mass spectrometry analysis, we demonstrated that different therapeutic antibodies interact to varying degrees with host-cell proteins in general, and PLBL2 specifically. We then used a high-throughput Protein A chromatography method to further examine the interaction between our antibodies and PLBL2. Our results showed that the co-elution of PLBL2 during Protein A chromatography is highly dependent on the individual antibody and PLBL2 concentration in the chromatographic load. Process parameters such as antibody resin load density and pre-elution wash conditions also influence the levels of PLBL2 in the Protein A eluate. Furthermore, using surface plasmon resonance, we demonstrated that there is a preference for PLBL2 to interact with IgG4 subclass antibodies compared to IgG1 antibodies. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2015-09-25

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

  12. Predominant Role of Cytosolic Phospholipase A2α in Dioxin-induced Neonatal Hydronephrosis in Mice

    PubMed Central

    Yoshioka, Wataru; Kawaguchi, Tatsuya; Fujisawa, Nozomi; Aida-Yasuoka, Keiko; Shimizu, Takao; Matsumura, Fumio; Tohyama, Chiharu

    2014-01-01

    Hydronephrosis is a common disease characterized by dilation of the renal pelvis and calices, resulting in loss of kidney function in the most severe cases. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces nonobstructive hydronephrosis in mouse neonates through upregulation of prostaglandin E2 (PGE2) synthesis pathway consisting of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) by a yet unknown mechanism. We here studied possible involvement of cytosolic phospholipase A2α (cPLA2α) in this mechanism. To this end, we used a cPLA2α-null mouse model and found that cPLA2α has a significant role in the upregulation of the PGE2 synthesis pathway through a noncanonical pathway of aryl hydrocarbon receptor. This study is the first to demonstrate the predominant role of cPLA2α in hydronephrosis. Elucidation of the pathway leading to the onset of hydronephrosis using the TCDD-exposed mouse model will deepen our understanding of the molecular basis of nonobstructive hydronephrosis in humans. PMID:24509627

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

    PubMed Central

    Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

    2010-01-01

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

  14. The chemical nature of the products obtained by the action of cabbage-leaf phospholipase D on lysolecithin: the structure of lysolecithin

    PubMed Central

    Long, C.; Odavić, R.; Sargent, Elizabeth J.

    1967-01-01

    1. Lysolecithin, prepared by the action of snake-venom phospholipase A on ovolecithin, when incubated with Savoy-cabbage phospholipase D, in the presence of Ca2+ ions, gave two degradation products (designated A and B) in the form of their calcium salts. 2. These calcium salts were separated quantitatively by solvent fractionation and converted into the corresponding sodium salts. 3. Substance B proved to be a lysophosphatidic acid of conventional structure (1-monoacyl-l-3-glycerophosphoric acid). When the phosphate group was removed by means of prostatic acid phosphomonoesterase, a 1-monoglyceride was formed quantitatively. Alkaline hydrolysis gave the theoretical yield of l-3-glycerophosphate. 4. Substance A, on the other hand, had all the properties expected for a cyclic phosphate of a 1-monoglyceride. It was unaffected by phosphomonoesterase. On alkaline hydrolysis, the acyl group was removed and ring opening of the presumed cyclic phosphate group gave an approximately equimolar mixture of 2- and l-3-glycerophosphates. 5. The structures of substances A and B confirm lysolecithin as 1-monoacyl-l-3-glycerylphosphorylcholine. PMID:4291559

  15. Phospholipases of Mineralization Competent Cells and Matrix Vesicles: Roles in Physiological and Pathological Mineralizations

    PubMed Central

    Mebarek, Saida; Abousalham, Abdelkarim; Magne, David; Do, Le Duy; Bandorowicz-Pikula, Joanna; Pikula, Slawomir; Buchet, René

    2013-01-01

    The present review aims to systematically and critically analyze the current knowledge on phospholipases and their role in physiological and pathological mineralization undertaken by mineralization competent cells. Cellular lipid metabolism plays an important role in biological mineralization. The physiological mechanisms of mineralization are likely to take place in tissues other than in bones and teeth under specific pathological conditions. For instance, vascular calcification in arteries of patients with renal failure, diabetes mellitus or atherosclerosis recapitulates the mechanisms of bone formation. Osteoporosis—a bone resorbing disease—and rheumatoid arthritis originating from the inflammation in the synovium are also affected by cellular lipid metabolism. The focus is on the lipid metabolism due to the effects of dietary lipids on bone health. These and other phenomena indicate that phospholipases may participate in bone remodelling as evidenced by their expression in smooth muscle cells, in bone forming osteoblasts, chondrocytes and in bone resorbing osteoclasts. Among various enzymes involved, phospholipases A1 or A2, phospholipase C, phospholipase D, autotaxin and sphingomyelinase are engaged in membrane lipid remodelling during early stages of mineralization and cell maturation in mineralization-competent cells. Numerous experimental evidences suggested that phospholipases exert their action at various stages of mineralization by affecting intracellular signaling and cell differentiation. The lipid metabolites—such as arachidonic acid, lysophospholipids, and sphingosine-1-phosphate are involved in cell signaling and inflammation reactions. Phospholipases are also important members of the cellular machinery engaged in matrix vesicle (MV) biogenesis and exocytosis. They may favour mineral formation inside MVs, may catalyse MV membrane breakdown necessary for the release of mineral deposits into extracellular matrix (ECM), or participate in

  16. Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

    PubMed

    Wu, Yinjuan; Li, Ye; Shang, Mei; Jian, Yu; Wang, Caiqin; Bardeesi, Adham Sameer A; Li, Zhaolei; Chen, Tingjin; Zhao, Lu; Zhou, Lina; He, Ai; Huang, Yan; Lv, Zhiyue; Yu, Xinbing; Li, Xuerong

    2017-03-16

    Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, it's necessary to express CssPLA2 in water-soluble form and determine whether the enzymatic activity of CssPLA2 or cell signalling pathways is involved in liver fibrosis caused by clonorchiasis. Balb/C mice were given an abdominal injection of MBP-CssPLA2. Liver sections with HE and Masson staining were observed to detect accumulation of collagen. Western blot of mouse liver was done to detect the activation of JNK signalling pathway. In vitro, HSCs were incubated with MBP-CssPLA2 to detect the activation of HSCs as well as the activation of JNK signalling pathway. The mutant of MBP-CssPLA2 without enzymatic activity was constructed and was also incubated with HSCs to check whether activation of the HSCs was related to the enzymatic activity of MBP-CssPLA2. The recombinant protein MBP-CssPLA2 was expressed soluble and of good enzymatic activity. A mutant of CssPLA2, without enzymatic activity, was also constructed. In vivo liver sections of Balb/C mice that were given an abdominal injection of 50 μg/ml MBP-CssPLA2 showed an obvious accumulation of collagen and a clear band of P-JNK1 could be seen by western blot of the liver tissue. In vitro, MBP-CssPLA2, as well as the mutant, was incubated with HSCs and it was proved that activation of HSCs was related to activation of the JNK signalling pathway instead of the enzymatic activity of MBP-CssPLA2. Activation of HSCs by CssPLA2 is related to the activation of the JNK signalling pathway instead of the enzymatic activity of CssPLA2. This finding

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

    2011-01-01

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

  19. Synthesis and activity of a novel diether phosphonoglycerol in phospholipase-resistant synthetic lipid:peptide lung surfactants†

    PubMed Central

    Schwan, Adrian L.; Singh, Suneel P.; Davy, Jason A.; Waring, Alan J.; Gordon, Larry M.; Walther, Frans J.; Wang, Zhengdong; Notter, Robert H.

    2012-01-01

    This paper reports the chemical synthesis and purification of a novel phospholipase-resistant C16:0, C16:1 diether phosphonoglycerol with structural analogy to ester-linked anionic phosphatidylglycerol (PG) in endogenous pulmonary surfactant. This diether phosphonoglycerol (PG 1) is studied for phospholipase A2 (PLA2) resistance and for surface activity in synthetic exogenous surfactants combined with Super Mini-B (S-MB) peptide and DEPN-8, a previously-reported diether phosphonolipid analog of dipalmitoyl phosphatidylcholine (DPPC, the major zwitterionic phospholipid in native lung surfactant). Activity experiments measured both adsorption and dynamic surface tension lowering due to the known importance of these surface behaviors in lung surfactant function in vivo. Synthetic surfactants containing 9 : 1 DEPN-8:PG 1 + 3% S-MB were resistant to degradation by PLA2 in chromatographic studies, while calf lung surfactant extract (CLSE, the substance of the bovine clinical surfactant Infasurf®) was significantly degraded by PLA2. The 9 : 1 DEPN-8:PG 1 + 3% S-MB mixture also had small but consistent increases in both adsorption and dynamic surface tension lowering ability compared to DEPN-8 + 3% S-MB. Consistent with these surface activity increases, molecular dynamics simulations using Protein Modeller, GROMACS force-field, and PyMOL showed that bilayers containing DPPC and palmitoyl-oleoyl-PC (POPC) as surrogates of DEPN-8 and PG 1 were penetrated to a greater extent by S-MB peptide than bilayers of DPPC alone. These results suggest that PG 1 or related anionic phosphono-PG analogs may have functional utility in phospholipase-resistant synthetic surfactants targeting forms of acute pulmonary injury where endogenous surfactant becomes dysfunctional due to phospholipase activity in the innate inflammatory response. PMID:22530092

  20. Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: A sup 31 P NMR study

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shashidhar, M.S.; Kuppe, A.; Volwerk, J.J.

    1990-09-04

    The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by {sup 31}P NMR. {sup 31}P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are {minus}0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. The authors also report the new and unexpected observation that themore » phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by {sup 31}P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B.cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. They propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.« less

  1. Intestinal Candida phospholipase is not elevated in patients with antibiotic-associated diarrhea.

    PubMed

    Krause, Robert; Haberl, Renate; Strempfl, Christina; Daxböck, Florian; Krejs, Günter J; Reisinger, Emil C; Wenisch, Christoph

    2002-01-01

    In order to assess the role of Candida-secreted phospholipase in antibiotic-associated diarrhea (AAD), 43 fecal Candida isolates from patients with AAD and from controls were tested on egg yolk agar for production of phospholipase. Phospholipase zones did not differ between the isolates from patients with AAD and from controls. The data indicate that the fungal virulence factor phospholipase may not be responsible for AAD in adults.

  2. Prostaglandin F(2alpha) stimulates tyrosine phosphorylation of phospholipase C-gamma1.

    PubMed

    Husain, Shahid; Jafri, Farahdiba

    2002-10-11

    In this study, we investigated the ability of prostaglandin F(2alpha) (PGF(2alpha)) to induce tyrosine phosphorylation of phospholipase C-gamma1 (PLC-gamma1) in cat iris sphincter smooth muscle (CISM) cells. PGF(2alpha)(1 microM) stimulated PLC-gamma1 tyrosine phosphorylation in a time- and dose-dependent manner with a maximum increase of 3-fold at 0.5min. The protein tyrosine kinase inhibitors, genistein, and tyrphostin A-25, blocked the stimulatory effects of PGF(2alpha), suggesting involvement of protein tyrosine kinase activity in the physiological actions of the PGF(2alpha). Furthermore, PGF(2alpha)-induced p42/p44 MAP kinase activation was also completely blocked by protein tyrosine kinase inhibitors. In summary, these findings show that PGF(2alpha) stimulates tyrosine phosphorylation of PLC-gamma1 in CISM cells and indicate that PGF(2alpha)-stimulated tyrosine phosphorylation is responsible for an early signal transduction event.

  3. Dimethyl ester of bilirubin exhibits anti-inflammatory activity through inhibition of secretory phospholipase A2, lipoxygenase and cyclooxygenase.

    PubMed

    Joshi, Vikram; Umashankara, M; Ramakrishnan, Chandrasekaran; Nanjaraj Urs, Ankanahalli N; Suvilesh, Kanve Nagaraj; Velmurugan, Devadasan; Rangappa, Kanchugarakoppal S; Vishwanath, Bannikuppe Sannanaik

    2016-05-15

    Overproduction of arachidonic acid (AA) mediated by secretory phospholipase A2 group IIA (sPLA2IIA) is a hallmark of many inflammatory disorders. AA is subsequently converted into pro-inflammatory eicosanoids through 5-lipoxygenase (5-LOX) and cyclooxygenase-1/2 (COX-1/2) activities. Hence, inhibition of sPLA2IIA, 5-LOX and COX-1/2 activities is critical in regulating inflammation. We have previously reported unconjugated bilirubin (UCB), an endogenous antioxidant, as sPLA2IIA inhibitor. However, lipophilic UCB gets conjugated in liver with glucuronic acid into hydrophilic conjugated bilirubin (CB). Since hydrophobicity is pre-requisite for sPLA2IIA inhibition, conjugation reduces the efficacy of UCB. In this regard, UCB was chemically modified and derivatives were evaluated for sPLA2IIA, 5-LOX and COX-1/2 inhibition. Among the derivatives, BD1 (dimethyl ester of bilirubin) exhibited ∼ 3 fold greater inhibitory potency towards sPLA2IIA compared to UCB. Both UCB and BD1 inhibited human 5-LOX and COX-2 activities; however only BD1 inhibited AA induced platelet aggregation. Molecular docking studies demonstrated BD1 as better inhibitor of aforesaid enzymes than UCB and other endogenous antioxidants. These data suggest that BD1 exhibits strong anti-inflammatory activity through inhibition of AA cascade enzymes which is of great therapeutic importance. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. The effect of ω-fatty acids on the expression of phospholipase A2 group 2A in human gastric cancer patients.

    PubMed

    Shariati, Mahboube; Aghaei, Mahmoud; Movahedian, Ahmad; Somi, Mohammad Hosein; Dolatkhah, Homayun; Aghazade, Ahmad Mirza

    2016-01-01

    Studies show that polyunsaturated fatty acids (PUFAs) may have an inhibitory role in carcinogenesis. It was previously shown that PLA2 group 2A (PLA2G2A) messenger RNA (mRNA) expression is associated with less frequent metastasis and longer survival in gastric adenocarcinoma. This study intends to investigate the effect of PUFAs on the expression of PLA2G2A in patients with gastric cancer. Thirty-four patients with gastric cancer (GC) were randomly divided into two groups. The first group received cisplatin medication. The second group received cisplatin medication and supplements of ω-fatty acids for three courses. The total RNA was extracted from the tissues and cDNA was synthesized. The gene expression of PLA2G2A was evaluated by the real-time polymerase chain reaction (PCR) method. To confirm the changes in gene expression, frozen section was utilized. The frozen tissue samples were sectioned and stained using the immunohistochemistry technique. After chemotherapy and chemotherapy plus supplement, the relative mean of PLA2G2A gene expression increased 1.5 ± 0.5-fold and 7.4 ± 2.6-fold, respectively ( P = 0.006). The relative mean of gene expression in patients who received cisplatin and ω-fatty acids supplement increased more significantly (7.5 ± 3.3-fold) than in patients who received only cisplatin ( P = 0.016). It was found that PUFAs increased the gene and protein expression of PLA2G2A in gastric cancer. Concerning the fact that studies reveal protective function of PLA2G2A in gastric cancer, it is suggested that increased expression of PLA2G2A is helpful. Furthermore, PUFAs can be considered as a useful therapeutic supplement for patients with gastric cancer.

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

    PubMed

    Romero, Paco; Gandía, Mónica; Alférez, Fernando

    2013-09-01

    The interplay between abscisic acid (ABA) and phospholipases A2 and D (PLA2 and PLD) in the response of citrus fruit to water stress was investigated during postharvest by using an ABA-deficient mutant from 'Navelate' orange named 'Pinalate'. Fruit from both varieties harvested at two different maturation stages (mature-green and full-mature) were subjected to prolonged water loss inducing stem-end rind breakdown (SERB) in full-mature fruit. Treatment with PLA2 inhibitor aristolochic acid (AT) and PLD inhibitor lysophosphatidylethanolamine (LPE) reduced the disorder in both varieties, suggesting that phospholipid metabolism is involved in citrus peel quality. Expression of CsPLDα and CsPLDβ, and CssPLA2α and CssPLA2β was studied by real-time RT-PCR during water stress and in response to ABA. CsPLDα expression increased in mature-green fruit from 'Navelate' but not in 'Pinalate' and ABA did not counteract this effect. ABA enhanced repression of CsPLDα in full-mature fruit. CsPLDβ gene expression decreased in mature-green 'Pinalate', remained unchanged in 'Navelate' and was induced in full-mature fruit from both varieties. CssPLA2α expression increased in mature-green fruit from both varieties whereas in full-mature fruit only increased in 'Navelate'. CssPLA2β expression increased in mature-green flavedo from both varieties, but in full-mature fruit remained steady in 'Navelate' and barely increased in 'Pinalate' fruit. ABA reduced expression in both after prolonged storage. Responsiveness to ABA increased with maturation. Our results show interplay between PLA2 and PLD and suggest that ABA action is upstream phospholipase activation. Response to ABA during water stress in citrus is regulated during fruit maturation and involves membrane phospholipid degradation. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  6. Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

    PubMed Central

    Bruntz, Ronald C.; Lindsley, Craig W.

    2014-01-01

    Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein–coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions. PMID:25244928

  7. Role of protein kinase C alpha and mitogen-activated protein kinases in endothelin-1-stimulation of cytosolic phospholipase A2 in iris sphincter smooth muscle.

    PubMed

    Abdel-Latif, A A; Husain, S; Yousufzai, S Y

    2000-11-01

    We have investigated the roles of protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) in the phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in endothelin-1- (ET-1) stimulated cat iris sphincter smooth muscle (CISM) cells. We found that in these cells both PKC and p38 MAP kinases play a critical role in ET-1-induced cPLA, phosphorylation and arachidonic acid (AA) release. Our findings indicate that stimulation of the endothelin-A- (ET(A)) receptor leads to: (1) activation of Gq protein which stimulates phospholipase C to hydrolyze the polyphosphoinositide PIP, into diacylglycerol (DAG) and inositol trisphosphate (IP3), the DAG may then activate PKC to phosphorylate and activate cPLA2; and (2) activation of Gi protein, which, through a series of kinases, leads to the stimulation of p38 MAPK and subsequently to phosphorylation and activation of cPLA2. The ability of the activated ET(A)-receptor, which is coupled to both Gq and Gi proteins, to recruit and activate this complex signal transduction mechanism remains to be clarified.

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2010-08-01

    Oxidative stress leads to drastic modifications of both the biophysical properties of biomembranes and their associated chemistry imparted upon the formation of oxidatively modified lipids. To this end, oxidized phospholipid derivatives bearing an aldehyde function, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) can covalently react with proteins that come into direct contact. Intriguingly, we observed PoxnoPC in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix to shorten and abolish the lag time in the action of phospholipase A2 (PLA2) on this composite substrate, with concomitant augmented decrement in pH, indicating more extensive hydrolysis, which was in keeping with enhanced 90 degrees light scattering. The latter was abolished by the aldehyde scavenger methoxyamine, thus suggesting the involvement of Schiff base. Enhanced hydrolysis of a fluorescent phospholipid analogue was seen for PLA2 preincubated with PoxnoPC. Mixing PLA2 with submicellar (22 microM) PoxnoPC caused a pronounced increase in Thioflavin T fluorescence, in keeping with the formation of amyloid-type fibers, which were seen also by electron microscopy. Copyright 2010 Elsevier B.V. All rights reserved.

  10. Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

    PubMed Central

    Stith, Bradley J.

    2015-01-01

    This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG. PMID:25748412

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

    PubMed

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

    2014-01-01

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

  12. Phosphatidic acid (PA)-preferring phospholipase A1 regulates mitochondrial dynamics.

    PubMed

    Baba, Takashi; Kashiwagi, Yuriko; Arimitsu, Nagisa; Kogure, Takeshi; Edo, Ayumi; Maruyama, Tomohiro; Nakao, Kazuki; Nakanishi, Hiroki; Kinoshita, Makoto; Frohman, Michael A; Yamamoto, Akitsugu; Tani, Katsuko

    2014-04-18

    Recent studies have suggested that phosphatidic acid (PA), a cone-shaped phospholipid that can generate negative curvature of lipid membranes, participates in mitochondrial fusion. However, precise mechanisms underling the production and consumption of PA on the mitochondrial surface are not fully understood. Phosphatidic acid-preferring phospholipase A1 (PA-PLA1)/DDHD1 is the first identified intracellular phospholipase A1 and preferentially hydrolyzes PA in vitro. Its cellular and physiological functions have not been elucidated. In this study, we show that PA-PLA1 regulates mitochondrial dynamics. PA-PLA1, when ectopically expressed in HeLa cells, induced mitochondrial fragmentation, whereas its depletion caused mitochondrial elongation. The effects of PA-PLA1 on mitochondrial morphology appear to counteract those of MitoPLD, a mitochondrion-localized phospholipase D that produces PA from cardiolipin. Consistent with high levels of expression of PA-PLA1 in testis, PA-PLA1 knock-out mice have a defect in sperm formation. In PA-PLA1-deficient sperm, the mitochondrial structure is disorganized, and an abnormal gap structure exists between the middle and principal pieces. A flagellum is bent at that position, leading to a loss of motility. Our results suggest a possible mechanism of PA regulation of the mitochondrial membrane and demonstrate an in vivo function of PA-PLA1 in the organization of mitochondria during spermiogenesis.

  13. Phospholipase A2 activation regulates cytotoxicity of methylmercury in vascular endothelial cells.

    PubMed

    Mazerik, Jessica N; Hagele, Thomas; Sherwani, Shariq; Ciapala, Valorie; Butler, Susan; Kuppusamy, M Lakshmi; Hunter, Melissa; Kuppusamy, Periannan; Marsh, Clay B; Parinandi, Narasimham L

    2007-01-01

    Mercury has been identified as a risk factor for cardiovascular disease among humans. Through diet, mainly fish consumption, humans are exposed to methylmercury, the biomethylated organic form of environmental mercury. As the endothelium is an important player in homeostasis of the cardiovascular system, here, the authors tested their hypothesis that methylmercury activates the lipid signaling enzyme phospholipase A(2) (PLA(2)) in vascular endothelial cells (ECs), causing upstream regulation of cytotoxicity. To test this hypothesis, the authors used bovine pulmonary artery ECs (BPAECs) cultured in monolayers, following labeling of their membrane phospholipids with [(3)H]arachidonic acid (AA). The cells were exposed to methylmercury chloride (MMC) and then the release of free AA (index of PLA(2) activity) and lactate dehydrogenase (LDH; index of cytotoxicity) were determined by liquid scintillation counting and spectrophotometry, respectively. MMC significantly activated PLA(2) in a dose-dependent (5 to 15 microM) and time-dependent (0 to 60 min) fashion. Sulfhydryl (thiol-protective) agents, calcium chelators, antioxidants, and PLA(2)-specific inhibitors attenuated the MMC-induced PLA(2) activation, suggesting the role of thiols, reactive oxygen species (ROS), and calcium in the activation of PLA(2) in BPAECs. MMC also induced the loss of thiols and increase of lipid peroxidation in BPAECs. MMC induced cytotoxicity in BPAECs as observed by the altered cell morphology and LDH leak, which was significantly attenuated by PLA(2) inhibitors. This study established that PLA(2) activation through thiols, calcium, and oxidative stress was associated with the cytotoxicity of MMC in BPAECs, drawing attention to the involvement of PLA(2) signaling in the methylmercury-induced vascular endothelial dysfunctions.

  14. Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer.

    PubMed

    Bruntz, Ronald C; Lindsley, Craig W; Brown, H Alex

    2014-10-01

    Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein-coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  15. Group 1B phospholipase A₂ inactivation suppresses atherosclerosis and metabolic diseases in LDL receptor-deficient mice.

    PubMed

    Hollie, Norris I; Konaniah, Eddy S; Goodin, Colleen; Hui, David Y

    2014-06-01

    Previous studies have shown that inactivation of the group 1B phospholipase A2 (Pla2g1b) suppresses diet-induced obesity, hyperglycemia, insulin resistance, and hyperlipidemia in C57BL/6 mice. A possible influence of Pla2g1b inactivation on atherosclerosis has not been addressed previously. The current study utilized LDL receptor-deficient (Ldlr(-/-)) mice with plasma lipid levels and distribution similar to hyperlipidemic human subjects as a preclinical animal model to test the effectiveness of Pla2g1b inactivation on atherosclerosis. The Pla2g1b(+/+)Ldlr(-/-) and Pla2g1b(-/-)Ldlr(-/-) mice were fed a low fat chow diet or a hypercaloric diet with 58.5 kcal% fat and 25 kcal% sucrose for 10 weeks. Minimal differences were observed between Pla2g1b(+/+)Ldlr(-/-) and Pla2g1b(-/-)Ldlr(-/-) mice when the animals were maintained on the low fat chow diet. However, when the animals were maintained on the hypercaloric diet, the Pla2g1(+/+)Ldlr(-/-) mice showed the expected body weight gain but the Pla2g1b(-/-)Ldlr(-/-) mice were resistant to diet-induced body weight gain. The Pla2g1b(-/-)Ldlr(-/-) mice also displayed lower fasting glucose, insulin, and plasma lipid levels compared to the Pla2g1b(+/+)Ldlr(-/-) mice, which displayed robust hyperglycemia, hyperinsulinemia, and hyperlipidemia in response to the hypercaloric diet. Importantly, atherosclerotic lesions in the aortic roots were also reduced 7-fold in the Pla2g1b(-/-)Ldlr(-/-) mice. The effectiveness of Pla2g1b inactivation to suppress diet-induced body weight gain and reduce diabetes and atherosclerosis in LDL receptor-deficient mice suggests that pharmacological inhibition of Pla2g1b may be a viable strategy to decrease diet-induced obesity and the risk of diabetes and atherosclerosis in humans. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. A molecular dynamics study of the three-dimensional model of human synovial fluid phospholipase A2--transition state mimic complexes.

    PubMed

    Hariprasad, V; Kulkarni, V M

    1996-01-01

    Different modes of binding of transition state mimics: amide, phosphonate and difluoro ketone, to human synovial fluid phospholipase A2 (HSF PLA2) are studies by molecular dynamics simulations computed in solvent. The results are analysed in the light of primary binding sites. Hydrogen bonding interaction plays an important role for amino acids such as Gly32, Val30, and Glu55, apart from the well known active site residues viz Asp48, Gly25, Gly29, Gly31, His27, His47, Lys62, Phe23, Asn114 and Tyr112. In addition, the hydrogen bonding interaction between Sn-1 tetrahedral phosphonate group of amide and difluoro ketone inhibitors and crystallographic water molecules (H2O 523, H2O 524 and H2O 401) seems to have a significant role. Many of the active site charged residues display considerable movement upon ligand binding. The structural effects of ligand binding were analyzed from RMS deviations of C alpha in the resulting energy-minimized average structures of the receptor-ligand complexes. The values of the RMS deviations differ among the HSF PLA2s, in a pattern that is not the same for the three complexes. This suggests that ligands with different pharmacological efficacies induce different types of conformational changes of the receptor. Our active-orientation model is, at least qualitatively, consistent with experimental data and should be useful for the rational design of more potent inhibitors.

  17. M-type phospholipase A2 receptor (PLA2R) glomerular staining in pediatric idiopathic membranous nephropathy.

    PubMed

    Kanda, Shoichiro; Horita, Shigeru; Yanagihara, Takeshi; Shimizu, Akira; Hattori, Motoshi

    2017-04-01

    Identifying M-type phospholipase A 2 receptor (PLA 2 R) is a landmark breakthrough for understanding adult idiopathic membranous nephropathy (iMN). However, potential roles for PLA 2 R in pediatric iMN have not been well investigated. A total of 34 pediatric iMN patients who underwent kidney biopsy between 1972 and 2015 were enrolled in this study. The study cohort consisted of 15 children aged from 3 to 9 years and 19 aged from 10 to 15 years. In all cases, secondary causes of MN, including infections, autoimmune diseases, and others, were ruled out. We examined PLA 2 R glomerular staining in stored, formalin-fixed, paraffin-embedded kidney biopsy samples. Kidney biopsy specimens obtained from an adult patient with iMN and an adult patient with lupus-associated MN were also examined to assess our PLA 2 R staining procedure. Granular staining of PLA 2 R along glomerular capillary loops was present in two patients: an 11-year-old girl and 12-year-old boy identified during a school urine screening test and who presented with mild proteinuria at the time of biopsy. Interestingly, the intensity of PLA 2 R glomerular staining in these patients was weaker than that of a PLA 2 R-positive adult iMN patient. There were no PLA 2 R-positive patients among our cohort of children younger than 10 years. This preliminary study suggests PLA 2 R may play a role in some adolescent and preteen iMN patients but may be less frequently associated with iMN during childhood.

  18. Pancreatic islets and insulinoma cells express a novel isoform of group VIA phospholipase A2 (iPLA2 beta) that participates in glucose-stimulated insulin secretion and is not produced by alternate splicing of the iPLA2 beta transcript.

    PubMed

    Ramanadham, Sasanka; Song, Haowei; Hsu, Fong-Fu; Zhang, Sheng; Crankshaw, Mark; Grant, Gregory A; Newgard, Christopher B; Bao, Shunzhong; Ma, Zhongmin; Turk, John

    2003-12-02

    Many cells express a group VIA 84 kDa phospholipase A(2) (iPLA(2)beta) that is sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate. Inhibition of iPLA(2)beta in pancreatic islets and insulinoma cells suppresses, and overexpression of iPLA(2)beta in INS-1 insulinoma cells amplifies, glucose-stimulated insulin secretion, suggesting that iPLA(2)beta participates in secretion. Western blotting analyses reveal that glucose-responsive 832/13 INS-1 cells express essentially no 84 kDa iPLA(2)beta-immunoreactive protein but predominantly express a previously unrecognized immunoreactive iPLA(2)beta protein in the 70 kDa region that is not generated by a mechanism of alternate splicing of the iPLA(2)beta transcript. To determine if the 70 kDa-immunoreactive protein is a short isoform of iPLA(2)beta, protein from the 70 kDa region was digested with trypsin and analyzed by mass spectrometry. Such analyses reveal several peptides with masses and amino acid sequences that exactly match iPLA(2)beta tryptic peptides. Peptide sequences identified in the 70 kDa tryptic digest include iPLA(2)beta residues 7-53, suggesting that the N-terminus is preserved. We also report here that the 832/13 INS-1 cells express iPLA(2)beta catalytic activity and that BEL inhibits secretagogue-stimulated insulin secretion from these cells but not the incorporation of arachidonic acid into membrane PC pools of these cells. These observations suggest that the catalytic iPLA(2)beta activity expressed in 832/13 INS-1 cells is attributable to a short isoform of iPLA(2)beta and that this isoform participates in insulin secretory but not in membrane phospholipid remodeling pathways. Further, the finding that pancreatic islets also express predominantly a 70 kDa iPLA(2)beta-immunoreactive protein suggests that a signal transduction role of iPLA(2)beta in the native beta-cell might be attributable to a 70 kDa isoform of iPLA(2)beta.

  19. Genetic Ablation of Calcium-independent Phospholipase A2γ Induces Glomerular Injury in Mice*

    PubMed Central

    Elimam, Hanan; Papillon, Joan; Kaufman, Daniel R.; Guillemette, Julie; Aoudjit, Lamine; Gross, Richard W.; Takano, Tomoko; Cybulsky, Andrey V.

    2016-01-01

    Glomerular visceral epithelial cells (podocytes) play a critical role in the maintenance of glomerular permselectivity. Podocyte injury, manifesting as proteinuria, is the cause of many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA2γ KO mice have demonstrated an important role for iPLA2γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA2γ KO mice to better understand the role of iPLA2γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA2γ did not cause detectable albuminuria; however, it resulted in mitochondrial structural abnormalities and enhanced autophagy in podocytes as well as loss of podocytes in aging KO mice. Moreover, after induction of anti-glomerular basement membrane nephritis in young mice, iPLA2γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA2γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA2γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria. PMID:27226532

  20. Yor022c protein is a phospholipase A{sub 1} that localizes to the mitochondrial matrix

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Urafuji, Kyosei; Arioka, Manabu

    In mammals, three types of intracellular phospholipase A{sub 1} (iPLA{sub 1}) enzymes have been characterized and are thought to be involved in various cellular processes such as phospholipid metabolism, organelle biogenesis, and membrane trafficking. In this study we analyzed the unique iPLA{sub 1}-like protein, Yor022c, in the budding yeast Saccharomyces cerevisiae. By the mass spectrometry analysis, we demonstrate that Yor022c is actually a phospholipase displaying sn-1-specific activity toward phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid, generating 2-acyl lysophospholipids. GFP-fused Yor022c co-stained with the mitochondrial dye MitoTracker, indicating that, unlike its mammalian counterparts, it is a mitochondrial protein. Further biochemical fractionation experiment combinedmore » with protease sensitivity assay showed that Yor022c localizes to the mitochondrial matrix. Thus Yor022c is the first PLA{sub 1} putatively involved in the maintenance of sn-1 acyl chains of phospholipids in the mitochondrial inner membrane. - Highlights: • Yeast Yor022c protein displays phospholipase A{sub 1} activity to various phospholipids. • Yor022c-GFP fusion protein localizes to mitochondria. • Biochemical fractionation showed that Yor022c localizes to the mitochondrial matrix.« less

  1. Ca2+-induced changes in the secondary structure of a 60 kDa phosphoinositide-specific phospholipase C from bovine brain cytosol.

    PubMed Central

    Herrero, C; Cornet, M E; Lopez, C; Barreno, P G; Municio, A M; Moscat, J

    1988-01-01

    The purification to homogeneity of a 60 kDa phosphoinositide-specific phospholipase C from bovine brain cytosol is reported here. This enzyme exhibits the same properties, in terms of response to Ca2+, as does the cytosolic activity in a variety of cell types. We show here that Ca2+ does not appear to modulate the binding of the enzyme to the substrate, but induces dramatic changes in its secondary structure. Therefore we suggest that a decrease in the alpha-helix content of this enzyme correlates with its ability to be activated by Ca2+. Images Fig. 1. PMID:2850798

  2. Phospholipase D1 modulates protein kinase C-epsilon in retinal pigment epithelium cells during inflammatory response.

    PubMed

    Tenconi, Paula E; Giusto, Norma M; Salvador, Gabriela A; Mateos, Melina V

    2016-12-01

    Inflammation is a key factor in the pathogenesis of several retinal diseases. In view of the essential role of the retinal pigment epithelium in visual function, elucidating the molecular mechanisms elicited by inflammation in this tissue could provide new insights for the treatment of retinal diseases. The aim of the present work was to study protein kinase C signaling and its modulation by phospholipases D in ARPE-19 cells exposed to lipopolysaccharide. This bacterial endotoxin induced protein kinase C-α/βII phosphorylation and protein kinase-ε translocation to the plasma membrane in ARPE-19 cells. Pre-incubation with selective phospholipase D inhibitors demonstrated that protein kinase C-α phosphorylation depends on phospholipase D1 and 2 while protein kinase C-ε activation depends only on phospholipase D1. The inhibition of α and β protein kinase C isoforms with Go 6976 did not modify the reduced mitochondrial function induced by lipopolysaccharide. On the contrary, the inhibition of protein kinase C-α, β and ε with Ro 31-8220 potentiated the decrease in mitochondrial function. Moreover, inhibition of protein kinase C-ε reduced Bcl-2 expression and Akt activation and increased Caspase-3 cleavage in cells treated or not with lipopolysaccharide. Our results demonstrate that through protein kinase C-ε regulation, phospholipase D1 protects retinal pigment epithelium cells from lipopolysaccharide-induced damage. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Structure-Guided Discovery of Novel, Potent, and Orally Bioavailable Inhibitors of Lipoprotein-Associated Phospholipase A2.

    PubMed

    Liu, Qiufeng; Huang, Fubao; Yuan, Xiaojing; Wang, Kai; Zou, Yi; Shen, Jianhua; Xu, Yechun

    2017-12-28

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a promising therapeutic target for atherosclerosis, Alzheimer's disease, and diabetic macular edema. Here we report the identification of novel sulfonamide scaffold Lp-PLA2 inhibitors derived from a relatively weak fragment. Similarity searching on this fragment followed by molecular docking leads to the discovery of a micromolar inhibitor with a 300-fold potency improvement. Subsequently, by the application of a structure-guided design strategy, a successful hit-to-lead optimization was achieved and a number of Lp-PLA2 inhibitors with single-digit nanomolar potency were obtained. After preliminary evaluation of the properties of drug-likeness in vitro and in vivo, compound 37 stands out from this congeneric series of inhibitors for good inhibitory activity and favorable oral bioavailability in male Sprague-Dawley rats, providing a quality candidate for further development. The present study thus clearly demonstrates the power and advantage of integrally employing fragment screening, crystal structures determination, virtual screening, and medicinal chemistry in an efficient lead discovery project, providing a good example for structure-based drug design.

  4. In vivo Detection of Phospholipase C by Enzyme-Activated Near-infrared Probes

    PubMed Central

    Mawn, Theresa M.; Popov, Anatoliy V.; Beardsley, Nancy J.; Stefflova, Klara; Milkevitch, Matthew; Zheng, Gang; Delikatny, E. James

    2011-01-01

    In this paper the characterization of the first near-infrared (NIR) phospholipase-activated molecular beacon is reported and its utility for in vivo cancer imaging is demonstrated. The probe consists of three elements: a phospholipid (PL) backbone to which the NIR fluorophore, pyropheophorbide a (Pyro), and the NIR Black Hole Quencher 3 (BHQ) were conjugated. Due to the close proximity of BHQ to Pyro, the Pyro-PtdEtn-BHQ probe is self-quenched until enzyme hydrolysis releases the fluorophore. The Pyro-PtdEtn-BHQ probe is highly specific to one isoform of phospholipase C, phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine. Incubation of Pyro-PtdEtn-BHQ in vitro with PC-PLC demonstrated a 150-fold increase in fluorescence that could be inhibited by the specific PC-PLC inhibitor tricyclodecan-9-yl xanthogenate (D609) with an IC50 of 34±8 µM. Since elevations in phosphocholine have been consistently observed by magnetic resonance spectroscopy in a wide array of cancer cells and solid tumors, we assessed the utility of Pyro-PtdEtn-BHQ as a probe for targeted tumor imaging. Injection of Pyro-PtdEtn-BHQ into mice bearing DU145 human prostate tumor xenografts followed by in vivo NIR imaging resulted in a 4-fold increase in tumor radiance over background and a 2 fold increase in the tumor:muscle ratio. Tumor fluorescence enhancement was inhibited with administration of D609. The ability to image PC-PLC activity in vivo provides a unique and sensitive method of monitoring one of the critical phospholipase signaling pathways activated in cancer, as well as the phospholipase activities that are altered in response to cancer treatment. PMID:22034913

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-12-01

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

  7. Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons.

    PubMed

    de Carvalho, Nathalia Delazeri; Garcia, Raphael CaioTamborelli; Ferreira, Adilson Kleber; Batista, Daniel Rodrigo; Cassola, Antonio Carlos; Maria, Durvanei; Lebrun, Ivo; Carneiro, Sylvia Mendes; Afeche, Solange Castro; Marcourakis, Tania; Sandoval, Maria Regina Lopes

    2014-03-13

    The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-9 toxins, an increase in free cytosolic Ca(2+) and a reduction in mitochondrial transmembrane potential (ΔΨm) became evident and occurred prior to the morphological changes and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed in the electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic Ca(2+) and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Plasma proteins in the acquired denture pellicle enhance substrate surface free energy and Candida albicans phospholipase and proteinase activities.

    PubMed

    Custodio, William; Silva, Wander J; Paes Leme, Adriana F; Cury, Jaime A; Del Bel Cury, Altair A

    2015-11-01

    The objective of the present study was to determine if blood plasma proteins could change the proteome of the acquired denture pellicle by label-free quantitative proteomics. As pellicle proteome modulates the interaction between substrates and Candida cells, we investigated its effect on the surface free energy (SFE) of the coated resin and on Candida albicans phospholipase and aspartyl proteinase activities. Poly(methylmethacrylate) discs were exposed to saliva (control) or saliva enriched with blood plasma (experimental group). The pellicle proteome was analyzed by mass spectrometry coupled with liquid chromatography. SFE was determined by acid-base technique. After biofilm formation, phospholipase and proteinase activities were determined accordingly to classic plate methods. Data were analyzed by two-way anova and Tukey test (P < 0.05). α-Amylase, cystatins, mucins, and host-immune system proteins were the main proteins identified in the control group. Fibrinogen and albumin were observed only in the experimental group. Coated discs of the experimental group presented an increased SFE (P < 0.05). For both enzymes tested, the experimental group showed higher proteolytic activity (P < 0.001). Blood plasma changes the proteome of the acquired denture pellicle, increasing surface free energy and the activity of Candida albicans phospholipase and aspartyl proteinase. © 2014 Wiley Publishing Asia Pty Ltd.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  11. Phospholipase A2 in experimental allergic bronchitis: a lesson from mouse and rat models.

    PubMed

    Mruwat, Rufayda; Yedgar, Saul; Lavon, Iris; Ariel, Amiram; Krimsky, Miron; Shoseyov, David

    2013-01-01

    Phospholipases A2 (PLA2) hydrolyzes phospholipids, initiating the production of inflammatory lipid mediators. We have previously shown that in rats, sPLA2 and cPLA2 play opposing roles in the pathophysiology of ovalbumin (OVA)-induced experimental allergic bronchitis (OVA-EAB), an asthma model: Upon disease induction sPLA2 expression and production of the broncho-constricting CysLTs are elevated, whereas cPLA2 expression and the broncho-dilating PGE2 production are suppressed. These were reversed upon disease amelioration by treatment with an sPLA2 inhibitor. However, studies in mice reported the involvement of both sPLA2 and cPLA2 in EAB induction. To examine the relevance of mouse and rat models to understanding asthma pathophysiology. OVA-EAB was induced in mice using the same methodology applied in rats. Disease and biochemical markers in mice were compared with those in rats. As in rats, EAB in mice was associated with increased mRNA of sPLA2, specifically sPLA2gX, in the lungs, and production of the broncho-constricting eicosanoids CysLTs, PGD2 and TBX2 in bronchoalveolar lavage (BAL). In contrast, EAB in mice was associated also with elevated cPLA2 mRNA and PGE2 production. Yet, treatment with an sPLA2 inhibitor ameliorated the EAB concomitantly with reverting the expression of both cPLA2 and sPLA2, and eicosanoid production. In both mice and rats sPLA2 is pivotal in OVA-induced EAB. Yet, amelioration of asthma markers in mouse models, and human tissues, was observed also upon cPLA2 inhibition. It is plausible that airway conditions, involving multiple cell types and organs, require the combined action of more than one, essential, PLA2s.

  12. GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Oberdisse, E.; Lapetina, E.G.

    1987-05-14

    Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with (/sup 3/H)inositol. Ca/sup 2 +/ has opposite effects on the formation of (/sup 3/H)inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of (/sup 3/H)inositol phosphates is inhibited by Ca/sup 2 +/, action of thrombin is stimulated by Ca/sup 2 +/. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma Smore » on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca/sup 2 +/ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca/sup 2 +/ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.« less

  13. Phospholipase PlaB is a new virulence factor of Legionella pneumophila.

    PubMed

    Schunder, Eva; Adam, Patrick; Higa, Futoshi; Remer, Katharina A; Lorenz, Udo; Bender, Jennifer; Schulz, Tino; Flieger, Antje; Steinert, Michael; Heuner, Klaus

    2010-06-01

    We previously identified Legionella pneumophila PlaB as the major cell-associated phospholipase A/lysophospholipase A with contact-dependent hemolytic activity. In this study, we further characterized this protein and found it to be involved in the virulence of L. pneumophila. PlaB was mainly expressed and active during exponential growth. Active PlaB was outer membrane-associated and at least in parts surface-exposed. Transport to the outer membrane was not dependent on the type I (T1SS), II (T2SS), IVB (T4BSS) or Tat secretion pathways. Furthermore, PlaB activity was not dependent on the presence of the macrophage infectivity potentiator (Mip) or the major secreted zinc metalloproteinase A (MspA). Despite the fact that PlaB is not essential for replication in protozoa or macrophage cell lines, we found that plaB mutants were impaired for replication in the lungs and dissemination to the spleen in the guinea pig infection model. Histological sections monitored less inflammation and destruction of the lung tissue after infection with the plaB mutants compared to L. pneumophila wild type. Taken together, PlaB is the first phospholipase A/lysophospholipase A with a confirmed role in the establishment of Legionnaires' disease. Copyright 2010 Elsevier GmbH. All rights reserved.

  14. Effect of phospholipase A treatment of low density lipoproteins on the dextran sulfate--lipoprotein interaction.

    PubMed

    Nishida, T

    1968-09-01

    The effect of phospholipase A on the interaction of low density lipoproteins of the S(f) 0-10 class with dextran sulfate was studied in phosphate buffer of pH 7.4, ionic strength 0.1, by chemical, spectrophotometric, and centrifugal methods. When low density lipoproteins that had been treated with phospholipase A were substituted for untreated lipoproteins, the amount of insoluble dextran sulfate-lipoprotein complex formed was greatly reduced. Hydrolysis of over 20% of the lecithin and phosphatidyl ethanolamine constituents of the lipoproteins prevented the formation of insoluble complex. However, even the lipoproteins in which almost all the phosphoglycerides were hydrolyzed produced soluble complex, which was converted to insoluble complex upon addition of magnesium sulfate. It is apparent that the lipoproteins altered extensively by treatment with phospholipase A retain many characteristic properties of native low density lipoproteins. Fatty acids, but not lysolecithin, released by the action of phospholipase A interfered with the formation of insoluble complex; this interference was due to association of the fatty acids with the lipoproteins. With increases in the concentration of the associated fatty acids, the amounts of magnesium ion required for the conversion of soluble complex to insoluble complex increased progressively. Charge interaction is evidently of paramount importance in the formation of sulfated polysaccharide-lipoprotein complexes.

  15. Multivalent Nanoparticle Networks Enable Point of Care Detection of Human Phospholipase-A2 in Serum

    PubMed Central

    Burnapp, Mark; Bentham, Andrew; Hillier, David; Zabron, Abigail; Khan, Shahid; Tyreman, Matthew; Stevens, Molly M.

    2017-01-01

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

  16. Pancreatic Islets and Insulinoma Cells Express a Novel Isoform of Group VIA Phospholipase A2 (iPLA2β) that Participates in Glucose-Stimulated Insulin Secretion and Is Not Produced by Alternate Splicing of the iPLA2β Transcript†

    PubMed Central

    Ramanadham, Sasanka; Song, Haowei; Hsu, Fong-Fu; Zhang, Sheng; Crankshaw, Mark; Grant, Gregory A.; Newgard, Christopher B.; Bao, Shunzhong; Ma, Zhongmin; Turk, John

    2013-01-01

    Many cells express a group VIA 84 kDa phospholipase A2 (iPLA2β) that is sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate. Inhibition of iPLA2β in pancreatic islets and insulinoma cells suppresses, and overexpression of iPLA2β in INS-1 insulinoma cells amplifies, glucose-stimulated insulin secretion, suggesting that iPLA2β participates in secretion. Western blotting analyses reveal that glucose-responsive 832/13 INS-1 cells express essentially no 84 kDa iPLA2β-immunoreactive protein but predominantly express a previously unrecognized immunoreactive iPLA2β protein in the 70 kDa region that is not generated by a mechanism of alternate splicing of the iPLA2β transcript. To determine if the 70 kDa-immunoreactive protein is a short isoform of iPLA2β, protein from the 70 kDa region was digested with trypsin and analyzed by mass spectrometry. Such analyses reveal several peptides with masses and amino acid sequences that exactly match iPLA2β tryptic peptides. Peptide sequences identified in the 70 kDa tryptic digest include iPLA2β residues 7–53, suggesting that the N-terminus is preserved. We also report here that the 832/13 INS-1 cells express iPLA2β catalytic activity and that BEL inhibits secretagogue-stimulated insulin secretion from these cells but not the incorporation of arachidonic acid into membrane PC pools of these cells. These observations suggest that the catalytic iPLA2β activity expressed in 832/13 INS-1 cells is attributable to a short isoform of iPLA2β and that this isoform participates in insulin secretory but not in membrane phospholipid remodeling pathways. Further, the finding that pancreatic islets also express predominantly a 70 kDa iPLA2β-immunoreactive protein suggests that a signal transduction role of iPLA2β in the native β-cell might be attributable to a 70 kDa isoform of iPLA2β. PMID:14636061

  17. Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications.

    PubMed

    Borrelli, Grazia M; Trono, Daniela

    2015-09-01

    Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes.

  18. Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications

    PubMed Central

    Borrelli, Grazia M.; Trono, Daniela

    2015-01-01

    Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes. PMID:26340621

  19. The kinetics of the phospholipase A2-catalyzed hydrolysis of Egg phosphatidylcholine in unilamellar vesicles. Product inhibition and its relief by serum albumin.

    PubMed

    Kupferberg, J P; Yokoyama, S; Kézdy, F J

    1981-06-25

    Only the lecithin in the outer leaflet (representing 70% of the total) of egg lecithin unilamellar vesicles is hydrolyzed by Crotalus atrox phospholipase A2. Hydrolyzed vesicles remain intact and impermeable to ionic solutes. The fatty acids produced in the hydrolysis remain on the vesicle and are only partially ionized at neutral pH due to electrostatic repulsions. About 40% of the lysolecithin product is desorbed from the vesicle. In the presence of a large excess of bovine serum albumin, the reaction is first order with respect to both the enzyme and the substrate. At 21 degrees C, pH 7.2, I = 0.16 M, and [Ca2+] = 7 mM, the second order rate constant is kex(2) = 1.5 X 10(6) M-1 s-1. In the absence of albumin, the reaction is inhibited competitively by both the monomeric (KIm = 4.5 X 10(-8) M) and micellar (nKIa = 3.7 X 10(-7) M) forms of lysolecithin ([critical micelle concentration] = 4.3 X 10(-6) M). Bovine serum albumin complexes two molecules of lysolecithin with a dissociation constant, Kb = 5 X 10(-8) M. With substoichiometric albumin, the reaction is biphasic, and, when the albumin is saturated with lysolecithin, the kinetics become similar to those observed in the absence of albumin. The action of phospholipase A2 shows that in unilamellar vesicles there is only one major lecithin conformation in the outer leaflet, or that all conformations are rapidly interconvertible.

  20. Calcium-independent phospholipase A2 participates in KCl-induced calcium sensitization of vascular smooth muscle.

    PubMed

    Ratz, Paul H; Miner, Amy S; Barbour, Suzanne E

    2009-07-01

    In vascular smooth muscle, KCl not only elevates intracellular free Ca(2+) ([Ca(2+)](i)), myosin light chain kinase activity and tension (T), but also can inhibit myosin light chain phosphatase activity by activation of rhoA kinase (ROCK), resulting in Ca(2+) sensitization (increased T/[Ca(2+)](i) ratio). Precisely how KCl causes ROCK-dependent Ca(2+) sensitization remains to be determined. Using Fura-2-loaded isometric rings of rabbit artery, we found that the Ca(2+)-independent phospholipase A(2) (iPLA(2)) inhibitor, bromoenol lactone (BEL), reduced the KCl-induced tonic but not early phasic phase of T and potentiated [Ca(2+)](i), reducing Ca(2+) sensitization. The PKC inhibitor, GF-109203X (> or =3 microM) and the pseudo-substrate inhibitor of PKCzeta produced a response similar to BEL. BEL reduced basal and KCl-stimulated myosin phosphatase phosphorylation. Whereas BEL and H-1152 produced strong inhibition of KCl-induced tonic T (approximately 50%), H-1152 did not induce additional inhibition of tissues already inhibited by BEL, suggesting that iPLA(2) links KCl stimulation with ROCK activation. The cPLA(2) inhibitor, pyrrolidine-1, inhibited KCl-induced tonic increases in [Ca(2+)](i) but not T, whereas the inhibitor of 20-HETE production, HET0016, acted like the ROCK inhibitor H-1152 by causing Ca(2+) desensitization. These data support a model in which iPLA(2) activity regulates Ca(2+) sensitivity.

  1. A Model for the Interfacial Kinetics of Phospholipase D Activity on Long-Chain Lipids

    DTIC Science & Technology

    2013-07-01

    we extend this model to account for the interaction between PLD and its reaction product, phosphatidic acid (PA), which is a long-chain lipid and... phosphatidic acid , and lecithin/ phosphatidic acid fixed monolayers: a Langmuit film balance study. J. Colloid Interface Sci. 79:319–338. 55. Morris, A...Dennis. 1988. Kinetic analysis of the Ca2þ-dependent, membrane-bound, macrophage phospholipase A2 and the effects of arachidonic acid . J. Biol. Chem. 263

  2. Interaction of Phospholipase A/Acyltransferase-3 with Pex19p

    PubMed Central

    Uyama, Toru; Kawai, Katsuhisa; Kono, Nozomu; Watanabe, Masahiro; Tsuboi, Kazuhito; Inoue, Tomohito; Araki, Nobukazu; Arai, Hiroyuki; Ueda, Natsuo

    2015-01-01

    Phospholipase A/acyltransferase (PLA/AT)-3 (also known as H-rev107 or AdPLA) was originally isolated as a tumor suppressor and was later shown to have phospholipase A1/A2 activity. We have also found that the overexpression of PLA/AT-3 in mammalian cells results in specific disappearance of peroxisomes. However, its molecular mechanism remained unclear. In the present study, we first established a HEK293 cell line, which stably expresses a fluorescent peroxisome marker protein (DsRed2-Peroxi) and expresses PLA/AT-3 in a tetracycline-dependent manner. The treatment with tetracycline, as expected, caused disappearance of peroxisomes within 24 h, as revealed by diffuse signals of DsRed2-Peroxi and a remarkable decrease in a peroxisomal membrane protein, PMP70. A time-dependent decrease in ether-type lipid levels was also seen. Because the activation of LC3, a marker of autophagy, was not observed, the involvement of autophagy was unlikely. Among various peroxins responsible for peroxisome biogenesis, Pex19p functions as a chaperone protein for the transportation of peroxisomal membrane proteins. Immunoprecipitation analysis showed that PLA/AT-3 binds to Pex19p through its N-terminal proline-rich and C-terminal hydrophobic domains. The protein level and enzyme activity of PLA/AT-3 were increased by its coexpression with Pex19p. Moreover, PLA/AT-3 inhibited the binding of Pex19 to peroxisomal membrane proteins, such as Pex3p and Pex11βp. A catalytically inactive point mutant of PLA/AT-3 could bind to Pex19p but did not inhibit the chaperone activity of Pex19p. Altogether, these results suggest a novel regulatory mechanism for peroxisome biogenesis through the interaction between Pex19p and PLA/AT-3. PMID:26018079

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

    PubMed Central

    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 venom phospholipase A2 fraction 38 (BF-38) was isolated by ion exchange chromatography, molecular weight was determined by mass spectrometry and its N terminal amino acid sequence was identified. Monospecific rabbit antiserum was raised against the PLA2 in presence of Freund complete adjuvant. The neutralization of PLA2 induced toxicities was done in in vitro and in in vivo models using synthetic herbal compounds, anti PLA2 rabbit antiserum and commercial polyvalent snake venom antiserum. Results: A toxic PLA2 (BF-38) was purified from the B. fasciatus venom by CM-cellulose and HPLC, of 13.17 kDa and a minor band of 7.3 kDa using ESI-MS. The 13.17 kDa PLA2 sequence was NLYQFKNMIQC. The 7.3 kDa toxin sequence was RKCLTKYSQDNES and was found to be <10 per cent w/w. Anti PLA2 rabbit antiserum produced faint precipitant band in immunogel diffusion and showed low titre value. The commercial polyvalent snake venom antiserum, anti PLA2 rabbit antiserum and the synthetic herbal compounds neutralized the PLA 2 induced toxicities at different intensities. Interpretation & conclusions: Our results suggested that synthetic herbal compound (BA) along with antiserum might provide effective protection against PLA2 induced toxicities of B. fasciatus venom. PMID:22885262

  4. Hyaluronidase, phospholipase A2 and protease inhibitory activity of plants used in traditional treatment of snakebite-induced tissue necrosis in Mali, DR Congo and South Africa.

    PubMed

    Molander, Marianne; Nielsen, Line; Søgaard, Søren; Staerk, Dan; Rønsted, Nina; Diallo, Drissa; Chifundera, Kusamba Zacharie; van Staden, Johannes; Jäger, Anna K

    2014-11-18

    Snakebite envenomation, every year, causes estimated 5-10,000 mortalities and results in more than 5-15,000 amputations in sub-Saharan Africa alone. Antiserum is not easily accessible in these regions or doctors are simply not available, thus more than 80% of all patients seek traditional practitioners as first-choice. Therefore it is important to investigate whether the plants used in traditional medicine systems contain compounds against the necrosis-inducing enzymes of snake venom. Extracts from traditionally used plants from DR Congo, Mali and South Africa were tested in hyaluronidase, phospholipase A2 and protease enzyme bioassays using Bitis arietans and Naja nigricollis as enzyme source. A total of 226 extracts from 94 different plant species from the three countries, Mali, Democratic Republic of Congo and South Africa were tested in phospholipase A2, proteases and hyaluronidase enzyme assays. Forty plant species showed more than 90% inhibition in one or more assay. Fabaceae, Anacardiaceae and Malvaceae were the families with the highest number of active species, and the active compounds were distributed in different plant parts depending on plant species. Polyphenols were removed in the search for specific enzyme inhibitors against hyaluronidase, phospholipase A2 or proteases from extracts with IC50 values below 100µg/ml. Water extracts of Pupalia lappacea, Combretum molle, Strychnos innocua and Grewia mollis and ethanol extract of Lannea acida and Bauhinia thonningii still showed IC50 values below 100µg/ml in either the hyaluronidase or protease bioassay after removal of polyphenols. As four of the active plants are widely distributed in the areas where the snake species Bitis arietans and Naja nigricollis occur a potential inhibitor of the necrotic enzymes is accessible for many people in sub-Saharan Africa. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

    Davidsen, Jesper; Jørgensen, Kent; Andresen, Thomas L; Mouritsen, Ole G

    2003-01-10

    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 PLA(2) 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 destabilization and drug release at the same time as the permeability of the target membrane is enhanced. Moreover, the proposed system can be made thermosensitive and offers a rational way for developing smart liposome-based drug delivery systems. This can be achieved by incorporating specific lipid-based proenhancers or prodestabilisers into the liposome carrier, which automatically becomes activated by PLA(2) only at the diseased target sites, such as inflamed or cancerous tissue.

  6. Cloning, Sequencing, and Role in Virulence of Two Phospholipases (A1 and C) from Mesophilic Aeromonas sp. Serogroup O:34

    PubMed Central

    Merino, Susana; Aguilar, Alicia; Nogueras, Maria Mercedes; Regue, Miguel; Swift, Simon; Tomás, Juan M.

    1999-01-01

    Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5α. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process. PMID:10417167

  7. Phospholipase A₂: the key to reversing long-term memory impairment in a gastropod model of aging.

    PubMed

    Watson, Shawn N; Wright, Natasha; Hermann, Petra M; Wildering, Willem C

    2013-02-01

    Memory failure associated with changes in neuronal circuit functions rather than cell death is a common feature of normal aging in diverse animal species. The (neuro)biological foundations of this phenomenon are not well understood although oxidative stress, particularly in the guise of lipid peroxidation, is suspected to play a key role. Using an invertebrate model system of age-associated memory impairment that supports direct correlation between behavioral deficits and changes in the underlying neural substrate, we show that inhibition of phospholipase A(2) (PLA(2)) abolishes both long-term memory (LTM) and neural defects observed in senescent subjects and subjects exposed to experimental oxidative stress. Using a combination of behavioral assessments and electrophysiological techniques, we provide evidence for a close link between lipid peroxidation, provocation of phospholipase A(2)-dependent free fatty acid release, decline of neuronal excitability, and age-related long-term memory impairments. This supports the view that these processes suspend rather than irreversibly extinguish the aging nervous system's intrinsic capacity for plasticity. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

    PubMed

    Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

    1999-03-01

    The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

  9. Ceruleotoxin: identification in the venom of Bungarus fasciatus, molecular properties and importance of phospholipase A2 activity for neurotoxicity.

    PubMed

    Bon, C; Saliou, B

    1983-01-01

    Ceruleotoxin is a potent neurotoxin which was originally purified from a batch of venom labelled Bungarus caeruleus, from the Pasteur Institute. Since NOBLE et al. have shown that this batch differs in its protein composition from that of B. caeruleus provided by Miami Serpentarium, we decided to clarify this point by comparing the composition of venoms from various Bungarus species of several origins. Although individual variations exist between samples of the same species, the venom from B. multicinctus, B. caeruleus and B. fasciatus possess characteristic protein compositions which allowed us to identify the batch used to purify ceruleotoxin as a B. fasciatus venom. We identified and purified ceruleotoxin from each of the five samples of B. fasciatus venoms tested. We failed to find this neurotoxin in either B. multicinctus or B. caeruleus venoms. Purified ceruleotoxin is a slightly basic protein with an isoelectric point of 7.4 which possesses a significant phospholipase A2 activity (200 mumoles lecithin hydrolyzed per min per mg) and a high lethal potency (i.v. LD50 in mice 0.03-0.07 mg/kg). It is composed of two identical subunits of 13,000 mol. wt. which resemble pancreas and snake venom phospholipases in their amino acid composition. Like crotoxin, ceruleotoxin irreversibly blocks the postsynaptic response of Torpedo and Electrophorus electroplaques to cholinergic agonists without preventing the binding of acetylcholine to its receptor. By hydrolyzing critical lipids of the postsynaptic membrane, it stabilizes the acetylcholine receptor - ionophore assembly in a desensitized state.

  10. Quercetin-induced downregulation of phospholipase D1 inhibits proliferation and invasion in U87 glioma cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Park, Mi Hee; Min, Do Sik, E-mail: minds@pusan.ac.kr

    Highlights: {yields} Quercetin, a bioactive flavonoid, suppresses expression and enzymatic activity of phospholipase D1. {yields} Quercetin abolishes NFkB-induced phospholipase D1 expression via inhibition of NFkB transactivation. {yields} Quercetin-induced suppression of phospholipase D1 inhibits invasion and proliferation of human glioma cells. -- Abstract: Phospholipase D (PLD) has been recognized as a regulator of cell proliferation and tumorigenesis, but little is known about the molecules regulating PLD expression. Thus, the identification of small molecules inhibiting PLD expression would be an important advance in PLD-mediated physiology. Quercetin, a ubiquitous bioactive flavonoid, is known to inhibit proliferation and induce apoptosis in a variety ofmore » cancer cells. In the present study, we examined the effect of quercetin on the expression of PLD in U87 glioma cells. Quercetin significantly suppressed the expression of PLD1 at the transcriptional level. Moreover, quercetin abolished the protein expression of PLD1 in a time and dose-dependent manner, as well as inhibited PLD activity. Quercetin suppressed NF{kappa}B-induced PLD1 expression via inhibition of NFkB transactivation. Furthermore, quercetin inhibited activation and invasion of metalloproteinase-2 (MMP-2), a key modulator of glioma cell invasion, induced by phosphatidic acid (PA), a product of PLD activity. Taken together these data demonstrate that quercetin abolishes PLD1 expression and subsequently inhibits invasion and proliferation of glioma cells.« less

  11. Activation of farnesoid X receptor promotes triglycerides lowering by suppressing phospholipase A2 G12B expression.

    PubMed

    Liu, Qingli; Yang, Meng; Fu, Xuekun; Liu, Renzhong; Sun, Caijun; Pan, Haobo; Wong, Chi-Wai; Guan, Min

    2016-11-15

    As a novel mediator of hepatic very low-density lipoproteins (VLDL) secretion, phospholipase A2 G12B (PLA2G12B) is transcriptionally regulated by hepatocyte nuclear factor-4 alpha (HNF-4α). Farnesoid X receptor (FXR) plays a critical role in maintaining bile acids and triglycerides (TG) homeostasis. Here we report that FXR regulates serum TG level in part through PLA2G12B. Activation of FXR by chenodeoxycholic acid (CDCA) or GW4064 significantly decreased PLA2G12B expression in HepG2 cells. PLA2G12B expression was transcriptionally repressed due to an FXR-mediated up-regulation of small heterodimer partner (SHP) which functionally suppresses HNF-4α activity. We found that hepatic PLA2G12B expression was suppressed and serum TG level reduced in high fat diet mice treated with CDCA. Concurrently, CDCA treatment lowered hepatic VLDL-TG secretion. Our data demonstrate that activation of FXR promotes TG lowering, not only by decreasing de novo lipogenesis but also reducing hepatic secretion of TG-rich VLDL particles in part through suppressing PLA2G12B expression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Extracellular UDP enhances P2X-mediated bladder smooth muscle contractility via P2Y6 activation of the phospholipase C/inositol trisphosphate pathway

    PubMed Central

    Yu, Weiqun; Sun, Xiaofeng; Robson, Simon C.; Hill, Warren G.

    2013-01-01

    Bladder dysfunction characterized by abnormal bladder smooth muscle (BSM) contractions is pivotal to the disease process in overactive bladder, urge incontinence, and spinal cord injury. Purinergic signaling comprises one key pathway in modulating BSM contractility, but molecular mechanisms remain unclear. Here we demonstrate, using myography, that activation of P2Y6 by either UDP or a specific agonist (MRS 2693) induced a sustained increase in BSM tone (up to 2 mN) in a concentration-dependent manner. Notably, activation of P2Y6 enhanced ATP-mediated BSM contractile force by up to 45%, indicating synergistic interactions between P2X and P2Y signaling. P2Y6-activated responses were abolished by phospholipase C (PLC) and inositol trisphosphate (IP3) receptor antagonists U73122 and xestospongin C, demonstrating involvement of the PLC/IP3 signal pathway. Mice null for Entpd1, an ectonucleotidase on BSM, demonstrated increased force generation on P2Y6 activation (150%). Thus, in vivo perturbations to purinergic signaling resulted in altered P2Y6 activity and bladder contractility. We conclude that UDP, acting on P2Y6, regulates BSM tone and in doing so selectively maximizes P2X1-mediated contraction forces. This novel neurotransmitter pathway may play an important role in urinary voiding disorders characterized by abnormal bladder motility.—Yu, W., Sun, X., Robson, S. C., Hill, W. G. Extracellular UDP enhances P2X-mediated bladder smooth muscle contractility via P2Y6 activation of the phospholipase C/inositol trisphosphate pathway. PMID:23362118

  13. Hydrolysis of Phosphatidylcholine-Isoprostanes (PtdCho-IP) by Peripheral Human Group IIA, V and X Secretory Phospholipases A2 (sPLA2).

    PubMed

    Kuksis, Arnis; Pruzanski, Waldemar

    2017-06-01

    Biologically active F- and E/D-type-prostane ring isomers (F 2 -IP and E 2 /D 2 -IP, respectively) are produced in situ by non-enzymatic peroxidation of arachidonic acid esterified to GroPCho (PtdCho-IP) and are universally distributed in tissue lipoproteins and cell membranes. Previous work has shown that platelet-activating factor acetylhydrolases (PAF-AH) are the main endogenous PLA 2 involved in degradation of PtdCho-IP. The present study shows that the PtdCho-IP are also subject to hydrolysis by group IIA, V and X secretory PLA 2 , which also have a wide peripheral tissue distribution. For this demonstration, we compared the LC/MS profiles of PtdCho-IP of auto-oxidized plasma lipoproteins after incubation for 1-4 h (37 °C) in the absence or presence of recombinant human sPLA 2 (1-2.5 µg/ml). In the absence of exogenously added sPLA 2 the total PtdCho-IP level after 4 h incubation reached 15.9, 21.6 and 8.7 nmol/mg protein of LDL, HDL and HDL 3 , respectively. In the presence of group V or group X sPLA 2 (2.5 µg/ml), the PtdCho-IP was completely hydrolyzed in 1 h, while in the presence of group IIA sPLA 2 (2.5 µg/ml) the hydrolysis was less than 25% in 4 h, although it was complete after 8-24 h incubation. This report provides the first demonstration that PtdCho-IP are readily hydrolyzed by group IIA, V and X sPLA 2 . A co-location of sPLA 2 and the substrates in various tissues has been recorded. Thus, the initiation of interaction and production of isoprostanes in situ are highly probable.

  14. Phospholipase and proteinase activities of Candida spp. isolates from vulvovaginitis in Iran.

    PubMed

    Shirkhani, S; Sepahvand, A; Mirzaee, M; Anbari, K

    2016-09-01

    This study aims to characterize phospholipase and proteinase activities of Candida isolates from 82 vulvovaginal candidiasis (VVC) and to study the relationship of these activities with vulvovaginitis. Totally 82 Candida isolates from vagina samples of VVC patients were randomly collected over the period between September and December 2014 from hospitalized patients at the general hospitals of Lorestan province, Iran. Isolates were previously identified by conventional mycological methods. The phospholipase and proteinase activities were evaluated by Egg yolk agar, Tween 80 opacity medium and agar plate methods. The most common Candida species was identified Candida albicans (n=34, 41.5%), followed by Candida famata (n=13, 15.8%), Candida tropicalis (n=11, 13.4%), and Candida parapsilosis (n=9, 11%). The most phospholipase activity was observed in Candida colliculosa (40%), followed by C. famata (38.5%), and Candida krusei (33.3%). The findings revealed that the correlation between phospholipase production by Candida spp. and the presence of VVC was not found to be statistically significant (P=0.91). All Candida spp. exhibited considerable proteinase activity; so that 100% of C. colliculosa, C. parapsilosis, Candida kefyr, and Candida intermedia isolates produced high proteinase activity with Pz 4+ scores. There was a significant correlation between proteinase production by Candida spp. and the presence of VVC (P=0.009). The obtained findings revealed that Candida spp. isolates may produce both virulence factors, phospholipase and proteinase. Although the phospholipase production was only observed in <40% of the isolates; however there was a significant association between proteinase production by Candida spp. and VVC. Copyright © 2016. Published by Elsevier Masson SAS.

  15. Native and recombinant phospholipases A2 of Scorpio maurus venom glands impair angiogenesis by targeting integrins α5β1 and αvβ3.

    PubMed

    Krayem, Najeh; Abdelkefi-Koubaa, Zaineb; Marrakchi, Naziha; Gargouri, Youssef; Luis, José

    2018-04-30

    We recently purified an heterodimeric phospholipase A 2 named Sm-PLGV from the venom glands of scorpion Scorpio maurus containing a Long chain, a penta-peptide insertion, which is cut out during the maturation, followed by a short chain. Three recombinant forms of Sm-PLGV were produced in Escherichia coli: rPLA 2 (+5) containing the full-length sequence including the penta-peptide insert, rPLA 2 (-5) a fused continuous chain of the Long and the short chains without the penta-peptide and the Long chain alone without the short one. In this study, we showed that Sm-PLGV, rPLA 2 (+5) and rPLA 2 (-5) displayed more potent anti-angiogenic properties than the recombinant Long chain and the short chain obtained by chemical synthesis. These phospholipases A 2 inhibited in a dose-dependent manner adhesion, migration and invasion of human microvascular endothelial cells through the alteration of α5β1 and αvβ3 integrins function. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrated that Sm-PLGV, rPLA 2 (+5) and rPLA 2 (-5) significantly inhibited both in vitro and in vivo angiogenesis. We also showed a clear dissociation of the anti-angiogenic effect of Sm-PLGV and its catalytic activity. This is the first study describing an anti-angiogenic effect for recombinant scorpion venom enzymes. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. The effect of centrally injected CDP-choline on respiratory system; involvement of phospholipase to thromboxane signaling pathway.

    PubMed

    Topuz, Bora B; Altinbas, Burcin; Yilmaz, Mustafa S; Saha, Sikha; Batten, Trevor F; Savci, Vahide; Yalcin, Murat

    2014-05-01

    CDP-choline is an endogenous metabolite in phosphatidylcholine biosynthesis. Exogenous administration of CDP-choline has been shown to affect brain metabolism and to exhibit cardiovascular, neuroendocrine neuroprotective actions. On the other hand, little is known regarding its respiratory actions and/or central mechanism of its respiratory effect. Therefore the current study was designed to investigate the possible effects of centrally injected CDP-choline on respiratory system and the mediation of the central cholinergic receptors and phospholipase to thromboxane signaling pathway on CDP-choline-induced respiratory effects in anaesthetized rats. Intracerebroventricularly (i.c.v.) administration of CDP-choline induced dose- and time-dependent increased respiratory rates, tidal volume and minute ventilation of male anaesthetized Spraque Dawley rats. İ.c.v. pretreatment with atropine failed to alter the hyperventilation responses to CDP-choline whereas mecamylamine, cholinergic nicotinic receptor antagonist, mepacrine, phospholipase A2 inhibitor, and neomycin phospholipase C inhibitor, blocked completely the hyperventilation induced by CDP-choline. In addition, central pretreatment with furegrelate, thromboxane A2 synthesis inhibitor, also partially blocked CDP-choline-evoked hyperventilation effects. These data show that centrally administered CDP-choline induces hyperventilation which is mediated by activation of central nicotinic receptors and phospholipase to thromboxane signaling pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells.

    PubMed

    Jiang, Feng; Waterfield, Nicholas R; Yang, Jian; Yang, Guowei; Jin, Qi

    2014-05-14

    Widely found in animal and plant-associated proteobacteria, type VI secretion systems (T6SSs) are potentially capable of facilitating diverse interactions with eukaryotes and/or other bacteria. Pseudomonas aeruginosa encodes three distinct T6SS haemolysin coregulated protein (Hcp) secretion islands (H1, H2, and H3-T6SS), each involved in different aspects of the bacterium's interaction with other organisms. Here we describe the characterization of a P. aeruginosa H3-T6SS-dependent phospholipase D effector, PldB, and its three tightly linked cognate immunity proteins. PldB targets the periplasm of prokaryotic cells and exerts an antibacterial activity. Surprisingly, PldB also facilitates intracellular invasion of host eukaryotic cells by activation of the PI3K/Akt pathway, revealing it to be a trans-kingdom effector. Our findings imply a potentially widespread T6SS-mediated mechanism, which deploys a single phospholipase effector to influence both prokaryotic cells and eukaryotic hosts. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Infrared neural stimulation induces intracellular Ca2+ release mediated by phospholipase C.

    PubMed

    Moreau, David; Lefort, Claire; Pas, Jolien; Bardet, Sylvia M; Leveque, Philippe; O'Connor, Rodney P

    2018-02-01

    The influence of infrared laser pulses on intracellular Ca 2+ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo-4 was used to measure Ca 2+ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo-4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca 2+ transient with similar dose-response characteristics in hippocampal neurons and glioblastoma cells (half-maximal effective energy density EC 50 of around 58 J.cm -2 ). For both type of cells, the source of the infrared-induced Ca 2+ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP 3 -induced Ca 2+ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP 3 cell signaling cascade has many potential applications including the development of optoceutical therapeutics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

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

    2015-10-07

    Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

  20. Inhibition of Cytosolic Phospholipase A2α Impairs an Early Step of Coronavirus Replication in Cell Culture.

    PubMed

    Müller, Christin; Hardt, Martin; Schwudke, Dominik; Neuman, Benjamin W; Pleschka, Stephan; Ziebuhr, John

    2018-02-15

    Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double-membrane vesicles (DMVs) and other membranous structures that are referred to as replicative organelles (ROs). The latter provide a structural scaffold for viral replication/transcription complexes (RTCs) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand RNA (+RNA) virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase A 2 α (cPLA 2 α) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLA 2 α activity, which produces lysophospholipids (LPLs) by cleaving at the sn -2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i) viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells, and (iii) this increase was diminished in the presence of the cPLA 2 α inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the Coronaviridae and Togaviridae families, while members of the Picornaviridae were not affected. Taken together, the study provides evidence that cPLA 2 α activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development. IMPORTANCE Examples of highly conserved RNA virus proteins that qualify as drug targets for broad

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

    PubMed

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

    2017-01-01

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

  2. Aberrant methylation of the M-type phospholipase A2 receptor gene in leukemic cells

    PubMed Central

    2012-01-01

    Background The M-type phospholipase A2 receptor (PLA2R1) plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS) or acute leukemia. Methods Sites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM) analysis was then carried out to quantify PLA2R1 methylation at 5`-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA. Results Expression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2´-deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% ± 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia. Conclusions The study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis. PMID:23217014

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

    PubMed

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

    2013-01-01

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

  4. Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells.

    PubMed

    Chang, Mei-Chi; Lin, Szu-I; Lin, Li-Deh; Chan, Chiu-Po; Lee, Ming-Shu; Wang, Tong-Mei; Jeng, Po-Yuan; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

    2016-04-01

    Prostaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation. Primary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate-activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2. PGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect. These results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  5. Hydrolysis of membrane phospholipids by phospholipases of rat liver lysosomes

    PubMed Central

    Richards, Donald E.; Irvine, Robin F.; Dawson, Rex M. C.

    1979-01-01

    (1) The hydrolysis of 32P- or myo-[2-3H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca2+ inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca2+ than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[3H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed. PMID:508301

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

    PubMed

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

    2016-04-12

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

  7. Specificity of Lipoprotein-Associated Phospholipase A2 Towards Oxidized Phosphatidylserines: LC-ESI-MS Characterization of Products and Computer Modeling of Interactions

    PubMed Central

    Tyurin, Vladimir A.; Yanamala, Naveena; Tyurina, Yulia Y.; Klein-Seetharaman, Judith; Macphee, Colin H.; Kagan, Valerian E.

    2013-01-01

    Ca2+ independent lipoprotein associated phospholipase A2 (Lp-PLA2) is a member of the phospholipase A2 superfamily with a distinguishing characteristic of high specificity for oxidatively modified sn-2 fatty acid residues in phospholipids which has been especially well characterized for peroxidized species of phosphatidylcholines (PC). The ability of Lp-PLA2 to hydrolyze peroxidized species of phosphatidylserine (PS) – acting as a recognition signal for clearance of apoptotic cells by professional phagocytes - as well as the products of the reaction have not been investigated. We performed LC-MS-ESI-based structural characterization of oxygenated/hydrolyzed molecular species of PS - containing linoleic acid in either sn-2 position (C18:0/C18:2) or in both sn-1 and sn-2 positions (C18:2/C18:2) - formed in cytochrome c/ H2O2 driven enzymatic oxidation reaction. Cytochrome c has been chosen as a catalyst of peroxidation reactions due to its likely involvement in PS oxidation in apoptotic cells. We found that Lp-PLA2 catalyzed the hydrolysis of both non-truncated and truncated (oxidatively fragmented) species of oxidized PS species albeit with different efficiencies and performed detailed characterization of the major reaction products – oxygenated derivatives of linoleic acid as well as non-oxygenated and oxygenated species of lyso-PS. Among linoleic acid products, derivatives oxygenated at the C9 position, including 9-hydroxyoctadecadienoic acid (9-HODE) – a potent ligand of G protein-coupled receptor G2A - were the most abundant. Computer modeling of interactions of Lp-PLA2 with different PS oxidized species indicated that they are able to bind in proximity (<5Å) to Ser273 and His351 of the catalytic triad. For 9-hydroxy- and 9-hydroperoxy- derivatives of oxidized PS, the sn-2 ester bond was positioned within the very close proximity (<3Å) from the Ser273 residue - a nucleophile directly attacking the sn-2 bond – thus favoring the hydrolysis reaction. We

  8. Listeria phospholipases subvert host autophagic defenses by stalling pre-autophagosomal structures

    PubMed Central

    Tattoli, Ivan; Sorbara, Matthew T; Yang, Chloe; Tooze, Sharon A; Philpott, Dana J; Girardin, Stephen E

    2013-01-01

    Listeria can escape host autophagy defense pathways through mechanisms that remain poorly understood. We show here that in epithelial cells, Listeriolysin (LLO)-dependent cytosolic escape of Listeria triggered a transient amino-acid starvation host response characterized by GCN2 phosphorylation, ATF3 induction and mTOR inhibition, the latter favouring a pro-autophagic cellular environment. Surprisingly, rapid recovery of mTOR signalling was neither sufficient nor necessary for Listeria avoidance of autophagic targeting. Instead, we observed that Listeria phospholipases PlcA and PlcB reduced autophagic flux and phosphatidylinositol 3-phosphate (PI3P) levels, causing pre-autophagosomal structure stalling and preventing efficient targeting of cytosolic bacteria. In co-infection experiments, wild-type Listeria protected PlcA/B-deficient bacteria from autophagy-mediated clearance. Thus, our results uncover a critical role for Listeria phospholipases C in the inhibition of autophagic flux, favouring bacterial escape from host autophagic defense. PMID:24162724

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

    PubMed

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

    2017-06-01

    Snake venom phospholipases A 2 (PLA 2 ) share high sequence identities and a conserved structural scaffold, but show important functional differences. Only a few PLA 2 s have been purified and characterized from coral snake (Micrurus spp.) venoms, and their role in envenomation remains largely unknown. In this report, we describe the isolation, sequencing and partial functional characterization of two Micrurus PLA 2 s: MmipPLA 2 from Micrurus mipartitus and MdumPLA 2 from Micrurus dumerilii, two species of clinical importance in Colombia. MmipPLA 2 consisted of 119 amino acid residues with a predicted pI of 8.4, whereas MdumPLA 2 consisted of 117 residues with a pI of 5.6. Both PLA 2 s showed the conserved 'group I' cysteine pattern and were enzymatically active, although MdumPLA 2 had higher activity. The two enzymes differed notably in their toxicity, with MmipPLA 2 being highly lethal to mice and mildly myotoxic, whereas MdumPLA 2 was not lethal (up to 3 μg/g body weight) but strongly myotoxic. MdumPLA 2 displayed higher anticoagulant activity than MmipPLA 2 in vitro and caused more sustained edema in the mouse footpad assay. Neither of these enzymes was cytolytic to cultured skeletal muscle C2C12 myotubes. Based on their structural differences, the two enzymes were placed in separate lineages in a partial phylogeny of Micrurus venom PLA 2 s and this classification agreed with their divergent biological activities. Overall, these findings highlight the structural and functional diversity of Micrurus venom PLA 2 s. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  10. Exploration of immunoglobulin transcriptomes from mice immunized with three-finger toxins and phospholipases A2 from the Central American coral snake, Micrurus nigrocinctus.

    PubMed

    Laustsen, Andreas H; Engmark, Mikael; Clouser, Christopher; Timberlake, Sonia; Vigneault, Francois; Gutiérrez, José María; Lomonte, Bruno

    2017-01-01

    Snakebite envenomings represent a neglected public health issue in many parts of the rural tropical world. Animal-derived antivenoms have existed for more than a hundred years and are effective in neutralizing snake venom toxins when timely administered. However, the low immunogenicity of many small but potent snake venom toxins represents a challenge for obtaining a balanced immune response against the medically relevant components of the venom. Here, we employ high-throughput sequencing of the immunoglobulin (Ig) transcriptome of mice immunized with a three-finger toxin and a phospholipase A 2 from the venom of the Central American coral snake, Micrurus nigrocinctus. Although exploratory in nature, our indicate results showed that only low frequencies of mRNA encoding IgG isotypes, the most relevant isotype for therapeutic purposes, were present in splenocytes of five mice immunized with 6 doses of the two types of toxins over 90 days. Furthermore, analysis of Ig heavy chain transcripts showed that no particular combination of variable (V) and joining (J) gene segments had been selected in the immunization process, as would be expected after a strong humoral immune response to a single antigen. Combined with the titration of toxin-specific antibodies in the sera of immunized mice, these data support the low immunogenicity of three-finger toxins and phospholipases A 2 found in M. nigrocinctus venoms, and highlight the need for future studies analyzing the complexity of antibody responses to toxins at the molecular level.

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

    PubMed Central

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2013-01-01

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

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

    PubMed

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

    2011-01-01

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

  13. SGR2, a Phospholipase-Like Protein, and ZIG/SGR4, a SNARE, Are Involved in the Shoot Gravitropism of Arabidopsis

    PubMed Central

    Kato, Takehide; Morita, Miyo Terao; Fukaki, Hidehiro; Yamauchi, Yoshiro; Uehara, Michiko; Niihama, Mitsuru; Tasaka, Masao

    2002-01-01

    In higher plants, the shoot and the root generally show negative and positive gravitropism, respectively. To elucidate the molecular mechanisms involved in gravitropism, we have isolated many shoot gravitropism mutants in Arabidopsis. The sgr2 and zig/sgr4 mutants exhibited abnormal gravitropism in both inflorescence stems and hypocotyls. These genes probably are involved in the early step(s) of the gravitropic response. The sgr2 mutants also had misshapen seed and seedlings, whereas the stem of the zig/sgr4 mutants elongated in a zigzag fashion. The SGR2 gene encodes a novel protein that may be part of a gene family represented by bovine phosphatidic acid–preferring phospholipase A1 containing a putative transmembrane domain. This gene family has been reported only in eukaryotes. The ZIG gene was found to encode AtVTI11, a protein that is homologous with yeast VTI1 and is involved in vesicle transport. Our observations suggest that the two genes may be involved in a vacuolar membrane system that affects shoot gravitropism. PMID:11826297

  14. Phospholipases A2 isolated from Micrurus lemniscatus coral snake venom: behavioral, electroencephalographic, and neuropathological aspects.

    PubMed

    Oliveira, D A; Harasawa, C; Seibert, C S; Casais e Silva, L L; Pimenta, D C; Lebrun, I; Sandoval, M R L

    2008-03-28

    The present study evaluated four phospholipase A2 (PLA2) (Mlx-8, Mlx-9, Mlx-11 and Mlx-12) isolated from Micrurus lemniscatus snake venom (Elapidae). The effects of intrahippocampal administration of these toxins have been determined on behavior, electroencephalography, and neuronal degeneration in rats. These four PLA2 toxins induced motor and EEG alterations in a dose-dependent manner. Behavioral convulsions were characterized by clonic movements and were often accompanied by EEG alterations. Mlx toxins were convulsive but weakly epileptogenic, since low rates of seizure discharges were observed in EEG records. Neuronal injury seemed to depend on the dose of the toxin used. The highest doses of toxins caused severe intoxication and death of some animals. The injury of hippocampal cells was characterized by massive neuronal loss and hippocampal gliosis. A high occurrence of compulsive scratching was observed. Moreover, the onset of seizures induced by Mlx toxins was markedly delayed. The similarities between the effects of Mlx PLA2s and those isolated from other Elapidae snakes venoms suggest that their toxicity are probably due to their specific binding to neuronal membranes and to the catalysis of phospholipid hydrolysis, producing lysophospholipids and fatty acids. These compounds likely disturb the membrane conformation, causing a marked increase in the release of neurotransmitters and concurrent inhibition of vesicle fission and recycling. These toxins can be a useful tool to investigate properties of endogenous secretory PLA2s and therefore may be important both to study mechanisms involved in neurotransmitter release at nerve terminals and to explain the convulsive properties of PLA2s toxins.

  15. Deinhibition of cardiac Na/sup +/-K/sup +/-ATPase after exposure to exogenous phospholipase A/sub 2/

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Colvin, R.A.

    1987-01-01

    After 2 h of exogenous phospholipase A/sub 2/ (PLA/sub 2/) exposure, membrane phospholipid decreased from 3.22 +/- 0.31 to 1.06 +/- 0.13 ..mu..mol/mg (33% of control). All classes of phospholipid, except sphingomyelin, were hydrolyzed, whereas total cholesterol content was unaffected. Increases in nonesterified fatty acids (NEFA) were reflected primarily in oleic (18:1), linoleic (18:2), and arachidonic (20:4). Na/sup +/-K/sup +/-adenosinetriphosphatase (ATPase) activity was inhibited to 29% of control by 2 h of PLA/sub 2/ treatment, and this inhibition was reversed (albeit, not completely after 5 min of PLA/sub 2/ treatment) by removal of the hydrolysis products with 0.1% bovine serummore » albumin (BSA). In contrast, the apparent binding capacity for (/sup 3/H)ouabain was not affected by PLA/sub 2/ treatment. Unmasking of latent (/sup 3/H)ouabain binding by alamethicin was utilized to estimate changes in the proportion of sealed vesicles present before and after PLA/sub 2/ treatment. PLA/sub 2/ treatment resulted in a time-dependent loss of sealed vesicles that paralleled the time course of phospholipid hydrolysis and was not reversed by washing with BSA. These studies demonstrate that cardiac Na/sup +/-K/sup +/-ATPase activity is inhibited by accumulation of endogenously produced lysophospholipids and NEFA. In contrast, loss of vesicle integrity may result from both accumulation of endogenously produced hydrolysis products and membrane phospholipid depletion.« less

  16. Inhibition of phospholipase C disrupts cytoskeletal organization and gravitropic growth in Arabidopsis roots.

    PubMed

    Andreeva, Zornitza; Barton, Deborah; Armour, William J; Li, Min Y; Liao, Li-Fen; McKellar, Heather L; Pethybridge, Kylie A; Marc, Jan

    2010-10-01

    The phospholipase protein superfamily plays an important role in hormonal signalling and cellular responses to environmental stimuli. There is also growing evidence for interactions between phospholipases and the cytoskeleton. In this report we used a pharmacological approach to investigate whether inhibiting a member of the phospholipase superfamily, phospholipase C (PLC), affects microtubules and actin microfilaments as well as root growth and morphology of Arabidopsis thaliana seedlings. Inhibiting PLC activity using the aminosteroid U73122 significantly inhibited root elongation and disrupted root morphology in a concentration-dependent manner, with the response being saturated at 5 μM, whereas the inactive analogue U73343 was ineffective. The primary root appeared to lose growth directionality accompanied by root waving and formation of curls. Immunolabelling of roots exposed to increasingly higher U73122 concentrations revealed that the normal transverse arrays of cortical microtubules in the elongation zone became progressively more disorganized or depolymerized, with the disorganization appearing within 1 h of incubation. Likewise, actin microfilament arrays also were disrupted. Inhibiting PLC using an alternative inhibitor, neomycin, caused similar disruptions to both cytoskeletal organization and root morphology. In seedlings gravistimulated by rotating the culture plates by 90°, both U73122 and neomycin disrupted the normal gravitropic growth of roots and etiolated hypocotyls. The effects of PLC inhibitors are therefore consistent with the notion that, as with phospholipases A and D, PLC likewise interacts with the cytoskeleton, alters growth morphology, and is involved in gravitropism.

  17. Cytosolic PhospholipaseA2 Inhibition with PLA-695 Radiosensitizes Tumors in Lung Cancer Animal Models

    PubMed Central

    Ferraro, Daniel J.; Kotipatruni, Rama P.; Bhave, Sandeep R.; Jaboin, Jerry J.; Hallahan, Dennis E.

    2013-01-01

    Lung cancer remains the leading cause of cancer deaths in the United States and the rest of the world. The advent of molecularly directed therapies holds promise for improvement in therapeutic efficacy. Cytosolic phospholipase A2 (cPLA2) is associated with tumor progression and radioresistance in mouse tumor models. Utilizing the cPLA2 specific inhibitor PLA-695, we determined if cPLA2 inhibition radiosensitizes non small cell lung cancer (NSCLC) cells and tumors. Treatment with PLA-695 attenuated radiation induced increases of phospho-ERK and phospho-Akt in endothelial cells. NSCLC cells (LLC and A549) co-cultured with endothelial cells (bEND3 and HUVEC) and pre-treated with PLA-695 showed radiosensitization. PLA-695 in combination with irradiation (IR) significantly reduced migration and proliferation in endothelial cells (HUVEC & bEND3) and induced cell death and attenuated invasion by tumor cells (LLC &A549). In a heterotopic tumor model, the combination of PLA-695 and radiation delayed growth in both LLC and A549 tumors. LLC and A549 tumors treated with a combination of PLA-695 and radiation displayed reduced tumor vasculature. In a dorsal skin fold model of LLC tumors, inhibition of cPLA2 in combination with radiation led to enhanced destruction of tumor blood vessels. The anti-angiogenic effects of PLA-695 and its enhancement of the efficacy of radiotherapy in mouse models of NSCLC suggest that clinical trials for its capacity to improve radiotherapy outcomes are warranted. PMID:23894523

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

    PubMed

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

    2015-06-01

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

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

    PubMed Central

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

    2015-01-01

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

  20. Uncarinic acids: phospholipase Cgamma1 inhibitors from hooks of Uncaria rhynchophylla.

    PubMed

    Lee, J S; Yang, M Y; Yeo, H; Kim, J; Lee, H S; Ahn, J S

    1999-05-17

    Bioactivity-guided fractionation of the CHCl3 extract from hooks of Uncaria rhynchophylla led to the isolation of two triterpene esters, namely uncarinic acids A (1) and B (2). Their structures were established by spectroscopic and chemical methods. These compounds inhibited phospholipase Cgamma1 with IC50 values of 35.66 and 44.55 microM, respectively.

  1. Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of the impact of secreted phospholipase A2 on microparticle assessment.

    PubMed

    Rousseau, Matthieu; Belleannee, Clemence; Duchez, Anne-Claire; Cloutier, Nathalie; Levesque, Tania; Jacques, Frederic; Perron, Jean; Nigrovic, Peter A; Dieude, Melanie; Hebert, Marie-Josee; Gelb, Michael H; Boilard, Eric

    2015-01-01

    Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

  2. Synthesis and Biological Activity of Phospholipase C-Resistant Analogues of Phosphatidylinositol 4, 5-bisphosphate

    PubMed Central

    Zhang, Honglu; Xu, Yong; Zhang, Zheng; Liman, Emily R.; Prestwich, Glenn D

    2008-01-01

    The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is an important regulator in cell physiology. Hydrolysis of PtdIns(4,5)P2 by phospholipase C (PLC) releases two second messengers, Ins(1,4,5)P3 and diacylglycerol. To dissect the effects of PtdIns(4,5)P2 from those resulting from PLC-generated signals, a metabolically-stabilized analogue of PtdIns(4,5)P2 was required. Two analogues were designed in which the scissile O-P bond was replaced with a C-P bond that could not be hydrolyzed by PLC activity. Herein we describe the asymmetric total synthesis of the first metabolically-stabilized, phospholipase C-resistant analogues of PtdIns(4,5)P2. The key transformation was a Pd(0)-catalyzed coupling of an H-phosphite with a vinyl bromide to form the desired C-P linkage. The phosphonate analogues of PtdIns(4,5)P2 were found to be effective in restoring the sensitivity of the TRPM4 channel to Ca2+ activation. PMID:16637624

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

    PubMed Central

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

    2013-01-01

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

  4. Eicosapentaenoic and docosahexaenoic acids have different effects on peripheral phospholipase A2 gene expressions in acute depressed patients.

    PubMed

    Su, Kuan-Pin; Yang, Hui-Ting; Chang, Jane Pei-Chen; Shih, Yin-Hua; Guu, Ta-Wei; Kumaran, Satyanarayanan Senthil; Gałecki, Piotr; Walczewska, Anna; Pariante, Carmine M

    2018-01-03

    Omega-3 polyunsaturated fatty acids (PUFAs) have been proven critical in the development and management of major depressive disorder (MDD) by a number of epidemiological, clinical and preclinical studies, but the molecular mechanisms underlying this therapeutic action are yet to be understood. Although eicosapentaenoic acid (EPA) seems to be the active component of omega-3 PUFAs' antidepressant effects, the biological research about the difference of specific genetic regulations between EPA and docosahexaenoic acid (DHA), the two main components of omega-3 PUFAs, is still lacking in human subjects. We conducted a 12-week randomized-controlled trial comparing the effects of EPA and DHA on gene expressions of phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX2), serotonin transporter (5HTT), and Tryptophan hydroxylase 2 (TPH-2) in 27 MDD patients. In addition, the erythrocyte PUFA compositions and the candidate gene expressions were also compared between these 27 MDD patients and 22 healthy controls. EPA was associated with a significant decrease in HAM-D scores (CI: -13 to -21, p<0.001) and significant increases in erythrocyte levels of EPA (CI: +1.0% to +2.9%, p=0.001) and DHA (CI: +2.9% to +5.6%, p=0.007). DHA treatment was associated with a significant decrease in HAM-D scores (CI: -6 to -14, p<0.001) and a significant increase in DHA levels (CI: +0.2% to +2.3%, p=0.047), but not of EPA levels. The cPLA2 gene expression levels were significantly increased in patients received EPA (1.9 folds, p=0.038), but not DHA (1.08 folds, p=0.92). There was a tendency for both EPA and DHA groups to decrease COX-2 gene expressions. The gene expressions of COX-2, cPLA2, TPH-2 and 5-HTT did not differ between MDD cases and healthy controls. EPA differentiates from DHA in clinical antidepressant efficacy and in upregulating cPLA2 gene regulations, which supports the clinical observation showing the superiority of EPA's antidepressant effects. ClinicalTrials.gov identifier: NCT

  5. Generation of N-Acylphosphatidylethanolamine by Members of the Phospholipase A/Acyltransferase (PLA/AT) Family*

    PubMed Central

    Uyama, Toru; Ikematsu, Natsuki; Inoue, Manami; Shinohara, Naoki; Jin, Xing-Hua; Tsuboi, Kazuhito; Tonai, Takeharu; Tokumura, Akira; Ueda, Natsuo

    2012-01-01

    Bioactive N-acylethanolamines (NAEs), including N-palmitoylethanolamine, N-oleoylethanolamine, and N-arachidonoylethanolamine (anandamide), are formed from membrane glycerophospholipids in animal tissues. The pathway is initiated by N-acylation of phosphatidylethanolamine to form N-acylphosphatidylethanolamine (NAPE). Despite the physiological importance of this reaction, the enzyme responsible, N-acyltransferase, remains molecularly uncharacterized. We recently demonstrated that all five members of the HRAS-like suppressor tumor family are phospholipid-metabolizing enzymes with N-acyltransferase activity and are renamed HRASLS1–5 as phospholipase A/acyltransferase (PLA/AT)-1–5. However, it was poorly understood whether these proteins were involved in the formation of NAPE in living cells. In the present studies, we first show that COS-7 cells transiently expressing recombinant PLA/AT-1, -2, -4, or -5, and HEK293 cells stably expressing PLA/AT-2 generated significant amounts of [14C]NAPE and [14C]NAE when cells were metabolically labeled with [14C]ethanolamine. Second, as analyzed by liquid chromatography-tandem mass spectrometry, the stable expression of PLA/AT-2 in cells remarkably increased endogenous levels of NAPEs and NAEs with various N-acyl species. Third, when NAPE-hydrolyzing phospholipase D was additionally expressed in PLA/AT-2-expressing cells, accumulating NAPE was efficiently converted to NAE. We also found that PLA/AT-2 was partly responsible for NAPE formation in HeLa cells that endogenously express PLA/AT-2. These results suggest that PLA/AT family proteins may produce NAPEs serving as precursors of bioactive NAEs in vivo. PMID:22825852

  6. Clinical features, course and prognosis of idiopathic membranous nephropathy depending on the presence of antibodies against M-type phospholipase A2 receptor.

    PubMed

    Jatem Escalante, Elías; Segarra Medrano, Alfons; Carnicer Cáceres, Clara; Martín-Gómez, M Adoración; Salcedo Allende, María Teresa; Ostos Roldan, Helena; Agraz Pamplona, Irene

    2015-01-01

    In membranous nephropathy, the presence of antibodies against M-type phospholipase A2 receptor is considered highly specific for idiopathic forms. However, no specific association to a particular clinical profile has been found for such antibodies. To assess potential differences in initial clinical profile, course and prognosis of idiopathic membranous nephropathy depending on the presence of anti-PLA2R antibodies. Eighty-five patients with idiopathic membranous nephropathy were included (55 anti-PLA2R-positive and 30 anti-PLA2R-negative). Clinical, biochemical and pathological variables were recorded at the time of diagnosis. Frequency of spontaneous remission, incidence of response to first-line therapy, frequency and number of recurrences, survival of renal function free from renal replacement therapy, survival of renal function free from chronic renal insufficiency and frequency of occurrence of malignant, infectious or autoimmune diseases during follow-up were recorded. At the time of diagnosis, anti-PLA2R-negative patients were significantly older and had a higher frequency of spontaneous remission. No differences were noted in the response to first-line treatment, frequency and number of recurrences, survival of renal function free from renal replacement therapy, or survival of renal function free from chronic renal insufficiency. Anti-PLA2R-negative patients with idiopathic membranous nephropathy were older and experienced spontaneous remission more often than anti-PLA2R-positive patients. No differences in terms of treatment response, recurrences, and final prognosis were observed between both groups of patients. Copyright © 2015 The Authors. Published by Elsevier España, S.L.U. All rights reserved.

  7. Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms.

    PubMed

    Estacion, M; Sinkins, W G; Schilling, W P

    2001-01-01

    Patch clamp and fura-2 fluorescence were employed to characterize receptor-mediated activation of recombinant Drosophila TrpL channels expressed in Sf9 insect cells. TrpL was activated by receptor stimulation and by exogenous application of diacylglycerol (DAG) or poly-unsaturated fatty acids (PUFAs). Activation of TrpL was blocked more than 70% by U73122, suggesting that the effect of these agents was dependent upon phospholipase C (PLC). In fura-2 assays, extracellular application of bacterial phosphatidylinositol (PI)-PLC or phosphatidylcholine (PC)-PLC caused a transient increase in TrpL channel activity, the magnitude of which was significantly less than that observed following receptor stimulation. TrpL channels were also activated in excised inside-out patches by cytoplasmic application of mammalian PLC-b2, bacterial PI-PLC and PC-PLC, but not by phospholipase D (PLD). The phospholipases had little or no effect when examined in either whole-cell or cell-attached configurations.TrpL activity was inhibited by addition of phosphatidylinositol-4,5-bisphosphate (PIP2) to excised inside-out membrane patches exhibiting spontaneous channel activity or to patches pre-activated by treatment with PLC. The effect was reversible, specific for PIP2, and was not observed with phosphatidylethanolamine (PE), PI, PC or phosphatidylserine (PS). However, antibodies against PIP2 consistently failed to activate TrpL in inside-out patches. It is concluded that both the hydrolysis of PIP2 and the generation of DAG are required to rapidly activate TrpL following receptor stimulation, or that some other PLC-dependent mechanism plays a crucial role in the activation process.

  8. Identification of the Elusive Mammalian Enzyme Phosphatidylcholine-Specific Phospholipase C

    DTIC Science & Technology

    2015-09-01

    progression of rheumatoid arthritis (RA). Thus, the main scopes of this proposal are: 1. to identify the PC-PLC gene and protein; and 2. to test PC-PLC...Phosphatidycholine-specific phospholipase C, lipopolisaccharide, oxidized lipoproteins, serum, rheumatoid arthritis , transcriptome sequencing, HUVECs...progression of rheumatoid arthritis (RA). The identification of these factors may ultimately provide alternative “druggable” targets for the treatment

  9. Role of Ca2+-independent phospholipase A2 and cytochrome P-450 in store-operated calcium entry in 3T6 fibroblasts.

    PubMed

    Martínez, Javier; Moreno, Juan J

    2005-09-01

    Store-operated calcium (SOC) channels and capacitative Ca2+ entry play a key role in cellular functions, but their mechanism of activation remains unclear. Here, we show that thapsigargin induces [3H] arachidonic acid (AA) release, 45Ca2+ influx and a subsequent enhancement of intracellular calcium concentration ([Ca2+]i. Thapsigargin-induced elevation of [Ca2+]i was inhibited by cytochrome P-450 inhibitors and by cytochrome P-450 epoxygenase inhibitor and was reverted by 11,12 EET addition. However, cyclooxygenase and lipoxygenase inhibitors have no effect. Moreover, we observed that four EETs were able to induce 45Ca2+ influx. Finally, we reported that the effect of 11,12 EET on 45Ca2+ influx was sensible to receptor-operated Ca2+ channel blockers (NiCl2, LaCl3) but not to voltage-dependent Ca2+ channel blocker as verapamil. Thus, AA released by Ca2+-independent phospholipase A2 and AA metabolism through cytochrome P-450 pathway may be crucial molecular determinant in thapsigargin activation of SOC channels and store-operated Ca2+ entry pathway in 3T6 fibroblasts. Moreover, EETs, the main cytochrome P-450 epoxygenase metabolites of AA, are involved in thapsigargin-stimulated Ca2+ influx. In summary, our results suggest that EETs are components of calcium influx factor(s).

  10. Phospholipase activity in rat liver mitochondria studied by the use of endogenous substrates.

    PubMed

    Bjornstad, P

    1966-09-01

    The hydrolysis of endogenous phosphatidyl ethanolamine and lecithin in rat liver mitochondria has been studied by using mitochondria from rats injected with ethanolamine-1,2-(14)C or choline-1,2-(14)C. A phospholipase A-like enzyme has been demonstrated, which catalyzes the hydrolysis of one fatty acid ester linkage in phosphatidyl ethanolamine and lecithin. Phosphatidyl ethanolamine is hydrolyzed in preference to lecithin and the main reaction products are free fatty acids and lysophosphatidyl ethanolamine. The further breakdown of lysophospholipids appears to be limited in mitochondria, which indicates that lysophospholipase activity is mainly located extramitochondrially. The enzymic system is greatly stimulated by calcium ions, and also slightly by magnesium ions, while EDTA inhibits it almost completely. These findings are discussed in relation to previous observations on the effect of calcium and of EDTA on the functions of mitochondria. The possible function of the mitochondrial phospholipase for the formation of phospholipids with special fatty acids at the alpha- and -position is discussed.

  11. Transient receptor potential channel M5 and phospholipaseC-beta2 colocalizing in zebrafish taste receptor cells.

    PubMed

    Yoshida, Yuki; Saitoh, Kana; Aihara, Yoshiko; Okada, Shinji; Misaka, Takumi; Abe, Keiko

    2007-10-08

    In mammals, transient receptor potential (TRP) channel M5 (TRPM5) is coexpressed with phospholipaseC-beta2 (PLC-beta2) in the taste receptor cells, and both PLC-beta2 and TRPM5 are essential elements in the signal transduction of sweet, bitter and umami stimuli. In this study, we identified the zebrafish homologue of TRPM5 (zfTRPM5) and examined its expression in the gustatory system by in-situ hybridization. Using a transgenic zebrafish line that expressed green fluorescent protein under the control of the PLC-beta2 promoter, we showed that zfTRPM5 is expressed in green fluorescent protein-labeled cells of the taste buds. These results demonstrate that zfTRPM5 and PLC-beta2 colocalize in zebrafish taste receptor cells, suggesting their crucial roles in taste signaling via the fish taste receptors.

  12. A Novel Phosphatidylinositol 4,5-Bisphosphate Binding Domain Mediates Plasma Membrane Localization of ExoU and Other Patatin-like Phospholipases*

    PubMed Central

    Tyson, Gregory H.; Halavaty, Andrei S.; Kim, Hyunjin; Geissler, Brett; Agard, Mallory; Satchell, Karla J.; Cho, Wonhwa; Anderson, Wayne F.; Hauser, Alan R.

    2015-01-01

    Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P2, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains. PMID:25505182

  13. Streptococcus pyogenes Phospholipase A2 Induces the Expression of Adhesion Molecules on Human Umbilical Vein Endothelial Cells and Aorta of Mice.

    PubMed

    Oda, Masataka; Domon, Hisanori; Kurosawa, Mie; Isono, Toshihito; Maekawa, Tomoki; Yamaguchi, Masaya; Kawabata, Shigetada; Terao, Yutaka

    2017-01-01

    The Streptococcus pyogenes phospholipase A 2 (SlaA) gene is highly conserved in the M3 serotype of group A S. pyogenes , which often involves hypervirulent clones. However, the role of SlaA in S. pyogenes pathogenesis is unclear. Herein, we report that SlaA induces the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) via the arachidonic acid signaling cascade. Notably, recombinant SlaA induced ICAM1 and VCAM1 expression in human umbilical vein endothelial cells (HUVECs), resulting in enhanced adhesion of human monocytic leukemia (THP-1) cells. However, C134A, a variant enzyme with no enzymatic activity, did not induce such events. In addition, culture supernatants from S. pyogenes SSI-1 enhanced the adhesion of THP-1 cells to HUVECs, but culture supernatants from the Δ slaA isogenic mutant strain had limited effects. Aspirin, a cyclooxygenase 2 inhibitor, prevented the adhesion of THP-1 cells to HUVECs and did not induce ICAM1 and VCAM1 expression in HUVECs treated with SlaA. However, zileuton, a 5-lipoxygenase inhibitor, did not exhibit such effects. Furthermore, pre-administration of aspirin in mice intravenously injected with SlaA attenuated the transcriptional abundance of ICAM1 and VCAM1 in the aorta. These results suggested that SlaA from S. pyogenes stimulates the expression of adhesion molecules in vascular endothelial cells. Thus, SlaA contributes to the inflammation of vascular endothelial cells upon S. pyogenes infection.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    1991-02-01

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

  15. Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight.

    PubMed

    Trujillo Viera, Jonathan; El-Merahbi, Rabih; Nieswandt, Bernhard; Stegner, David; Sumara, Grzegorz

    2016-01-01

    Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that Pld1-/- and Pld2-/- mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes.

  16. Association between polymorphisms in phospholipase A2 genes and the plasma triglyceride response to an n-3 PUFA supplementation: a clinical trial.

    PubMed

    Tremblay, Bénédicte L; Cormier, Hubert; Rudkowska, Iwona; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

    2015-02-21

    Fish oil-derived long-chain omega-3 (n-3) polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), reduce plasma triglyceride (TG) levels. Genetic factors such as single-nucleotide polymorphisms (SNPs) found in genes involved in metabolic pathways of n-3 PUFA could be responsible for well-recognized heterogeneity in plasma TG response to n-3 PUFA supplementation. Previous studies have shown that genes in the glycerophospholipid metabolism such as phospholipase A2 (PLA2) group II, IV, and VI, demonstrate changes in their expression levels in peripheral blood mononuclear cells (PBMCs) after n-3 PUFA supplementation. A total of 208 subjects consumed 3 g/day of n-3 PUFA for 6 weeks. Plasma lipids were measured before and after the supplementation period. Five SNPs in PLA2G2A, six in PLA2G2C, eight in PLA2G2D, six in PLA2G2F, 22 in PLA2G4A, five in PLA2G6, and nine in PLA2G7 were genotyped. The MIXED Procedure for repeated measures adjusted for age, sex, BMI, and energy intake was used in order to test whether the genotype, supplementation or interaction (genotype by supplementation) were associated with plasma TG levels. The n-3 PUFA supplementation had an independent effect on plasma TG levels. Genotype effects on plasma TG levels were observed for rs2301475 in PLA2G2C, rs818571 in PLA2G2F, and rs1569480 in PLA2G4A. Genotype x supplementation interaction effects on plasma TG levels were observed for rs1805018 in PLA2G7 as well as for rs10752979, rs10737277, rs7540602, and rs3820185 in PLA2G4A. These results suggest that, SNPs in PLA2 genes may influence plasma TG levels during a supplementation with n-3 PUFA. This trial was registered at clinicaltrials.gov as NCT01343342.

  17. Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C

    PubMed Central

    Seo, Jong Bae; Jung, Seung-Ryoung; Huang, Weigang; Zhang, Qisheng; Koh, Duk-Su

    2015-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically. PMID:26658739

  18. Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C.

    PubMed

    Seo, Jong Bae; Jung, Seung-Ryoung; Huang, Weigang; Zhang, Qisheng; Koh, Duk-Su

    2015-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically.

  19. [In vitro examination of the influence of lipase and amylase on dog's pancreas tissue incubated with endotoxins, phospholipase A2 or cytokines].

    PubMed

    Kerekes, László; Antal-Szalmás, Péter; Dezso, Balázs; Sipka, Sándor; Furka, Andrea; Mikó, Irén; Sápy, Péter

    2005-04-01

    Proinflammatory cytokines are elevated during acute pancreatitis. The endotoxins and Phospholipase A2 (PLA2) also have important role in acute pancreatitis. The aim of this study was to determine, what factors are responsible for the tissue damage in acute pancreatitis. The examinations were performed on fixed and frozen sections of healthy dog's pancreas tissue. Direct effects of endotoxins, PLA2, and proinflammatory cytokines together with pancreas enzymes were examined on pancreatic tissue. Pancreas enzymes themselves did not cause any change in the structure of pancreas. The common influence of endotoxins, PLA2 and pancreas enzymes was examined, and finally the effect of proinflammatory cytokines and enzymes was examined on pancreas tissue. Our results show, that besides enzymes many other factors are necessary to inflict tissue damage in acute pancreatitis, but for necrosis the presence of TNF alfa is a must.

  20. Inhibition of carrageenin-induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2.

    PubMed Central

    Landucci, E C; Antunes, E; Donato, J L; Faro, R; Hyslop, S; Marangoni, S; Oliveira, B; Cirino, G; de Nucci, G

    1995-01-01

    1. The effect of purified crotapotin, a non-toxic non-enzymatic chaperon protein normally complexed to a phospholipase A2 (PLA2) in South America rattlesnake venom, was studied in the acute inflammatory response induced by carrageenin (1 mg/paw), compound 48/80 (3 micrograms/paw) and 5-hydroxytryptamine (5-HT) (3 micrograms/paw) in the rat hind-paw. The effects of crotapotin on platelet aggregation, mast cell degranulation and eicosanoid release from guinea-pig isolated lung were also investigated. 2. Subplantar co-injection of crotapotin (1 and 10 micrograms/paw) with carrageenin or injection of crotapotin (10 micrograms/paw) into the contralateral paw significantly inhibited the carrageenin-induced oedema. This inhibition was also observed when crotapotin (10-30 micrograms/paw) was administered either intraperitoneally or orally. Subplantar injection of heated crotapotin (15 min at 60 degrees C) failed to inhibit carrageenin-induced oedema. Subplantar injection of crotapotin (10 micrograms/paw) also significantly inhibited the rat paw oedema induced by compound 48/80, but it did not affect 5-HT-induced oedema. 3. In adrenalectomized animals, subplantar injection of crotapotin markedly inhibited the oedema induced by carrageenin. The inhibitory effect of crotapotin was also observed in rats depleted of histamine and 5-HT stores. 4. Crotapotin (30 micrograms/paw) had no effect on either the histamine release induced by compound 48/80 in vitro or on the platelet aggregation induced by both arachidonic acid (1 nM) and platelet activating factor (1 microM) in human platelet-rich plasma. The platelet aggregation and thromboxane B2 (TXB2) release induced by thrombin (100 mu ml-1) in washed human platelets were also not affected by crotapotin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7537590

  1. Integrin-mediated human glioblastoma cells adhesion, migration and invasion by native and recombinant phospholipases of Scorpio maurus venom glands.

    PubMed

    Krayem, Najeh; Abdelkefi-Koubaa, Zaineb; Gargouri, Youssef; Luis, José

    2018-05-01

    Integrins are a large family of cell surface receptors mediating the interaction of cells with their microenvironment and they play an important role in glioma biology. In the present work, we reported the anti-tumor effect of Sm-PLGV a phospholipase A 2 from Tunisian scorpion venom glands-as well as its recombinant forms expressed in Escherichia coli-through interference with integrin receptor function in malignant glioma cells U87. These phospholipases inhibited in a dose dependent manner the adhesion, migration and invasion onto fibrinogen and fibronectin without any cytotoxicity. We showed that Sm-PLGV and its recombinant constructs blocked U87 migration by reducing their velocity and directional persistence. The inhibitory effect was related to a blockage of the integrins αvβ3 and α5β1 function. Inactivation of the enzymatic activity of Sm-PLGV by chemical modification with p-bromophenacyl bromide did not affect its anti-tumor properties, suggesting the presence of 'pharmacological sites' distinct from the catalytic site in scorpion venom phospholipases A 2 . Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Contribution of phospholipase C-beta3 phosphorylation to the rapid attenuation of opioid-activated phosphoinositide response.

    PubMed

    Strassheim, D; Law, P Y; Loh, H H

    1998-06-01

    Activation of the delta-opioid receptor in NG108-15 neuroblastoma X glioma hybrid cells results in a transient increase at the intracellular level of inositol-1,4,5-triphosphate [Ins(1,4,5)P3]. This time course in the transient increase in the Ins(1,4,5)P3 level is distinctly different from that observed in the homologous opioid receptor desensitization as measured by the inhibition of adenylyl cyclase activity. One probable mechanism for this rapid loss in Ins(1,4,5)P3 response is the feedback regulation of the phospholipase C activity. Regulation by protein phosphorylation was suggested by the observations that the opioid-mediated response was potentiated by calphostin C, an inhibitor of protein kinase C (PKC), and was abolished by either phorbol-12-myristate-13-acetate, a PKC activator, or calyculin A, a protein phosphatase1/2A inhibitor. The direct phosphorylation of phospholipase C was demonstrated by immunoprecipitation of PLC-beta3 from metabolically labeled NG108-15 cells challenged with the delta-selective agonist [D-Pen2, D-Pen5]enkephalin (DPDPE). A time- and DPDPE concentration-dependent and naloxone-reversible increase in the PLC-beta3 phosphorylation can be demonstrated. This PLC-beta3 phosphorylation was mainly due to PKC activation because pretreatment of NG108-15 cells with calphostin C could block the DPDPE effect. Activation of the PLC-beta3 by DPDPE was one of the prerequisites for agonist-mediated PLC-beta3 phosphorylation because the aminosteroid phospholipase C inhibitor U73122 could block the DPDPE effect. In addition to DPDPE, lysophosphatidic acid (LPA) stimulated the PLC-beta3 phosphorylation, but bradykinin did not. Furthermore, the LPA- and DPDPE-mediated PLC-beta3 phosphorylation was additive and was much less than that observed with phorbol-12-myristate-13-acetate. The effect of DPDPE was specific to PLC-beta3; the betagamma-insensitive phospholipase C-beta1 was not phosphorylated in the presence of either DPDPE or LPA. These results

  3. Regulation of DUOX by the Galphaq-phospholipase Cbeta-Ca2+ pathway in Drosophila gut immunity.

    PubMed

    Ha, Eun-Mi; Lee, Kyung-Ah; Park, Seon Hwa; Kim, Sung-Hee; Nam, Hyuck-Jin; Lee, Hyo-Young; Kang, Dongmin; Lee, Won-Jae

    2009-03-01

    All metazoan guts are in constant contact with diverse food-borne microorganisms. The signaling mechanisms by which the host regulates gut-microbe interactions, however, are not yet clear. Here, we show that phospholipase C-beta (PLCbeta) signaling modulates dual oxidase (DUOX) activity to produce microbicidal reactive oxygen species (ROS) essential for normal host survival. Gut-microbe contact rapidly activates PLCbeta through Galphaq, which in turn mobilizes intracellular Ca(2+) through inositol 1,4,5-trisphosphate generation for DUOX-dependent ROS production. PLCbeta mutant flies had a short life span due to the uncontrolled propagation of an essential nutritional microbe, Saccharomyces cerevisiae, in the gut. Gut-specific reintroduction of the PLCbeta restored efficient DUOX-dependent microbe-eliminating capacity and normal host survival. These results demonstrate that the Galphaq-PLCbeta-Ca(2+)-DUOX-ROS signaling pathway acts as a bona fide first line of defense that enables gut epithelia to dynamically control yeast during the Drosophila life cycle.

  4. Membrane Environment Exerts an Important Influence on Rac-Mediated Activation of Phospholipase2 ▿ †

    PubMed Central

    Everett, Katy L.; Buehler, Anja; Bunney, Tom D.; Margineanu, Anca; Baxendale, Rhona W.; Vatter, Petra; Retlich, Michael; Walliser, Claudia; Manning, Hugh B.; Neil, Mark A. A.; Dunsby, Christopher; French, Paul M. W.; Gierschik, Peter; Katan, Matilda

    2011-01-01

    We performed analyses of the molecular mechanisms involved in the regulation of phospholipase2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation. PMID:21245382

  5. Distinct enzymatic and cellular characteristics of two secretory phospholipases A2 in the filamentous fungus Aspergillus oryzae.

    PubMed

    Nakahama, Tomoyuki; Nakanishi, Yoshito; Viscomi, Arturo R; Takaya, Kohei; Kitamoto, Katsuhiko; Ottonello, Simone; Arioka, Manabu

    2010-04-01

    Microbial secretory phospholipases A(2) (sPLA(2)s) are among the last discovered and least known members of this functionally diverse family of enzymes. We analyzed here two sPLA(2)s, named sPlaA and sPlaB, of the filamentous ascomycete Aspergillus oryzae. sPlaA and sPlaB consist of 222 and 160 amino acids, respectively, and share the conserved Cys and catalytic His-Asp residues typical of microbial sPLA(2)s. Two sPLA(2)s differ in pH optimum, Ca(2+) requirement and expression profile. The splaA mRNA was strongly upregulated in response to carbon starvation, oxidative stress and during conidiation, while splaB was constitutively expressed at low levels and was weakly upregulated by heat shock. Experiments with sPLA(2) overexpressing strains demonstrated that two enzymes produce subtly different phospholipid composition variations and also differ in their subcellular localization: sPlaA is most abundant in hyphal tips and secreted to the medium, whereas sPlaB predominantly localizes to the ER-like intracellular compartment. Both sPLA(2) overexpressing strains were defective in conidiation, which was more pronounced for sPlaB overexpressors. Although no major morphological abnormality was detected in either DeltasplaA or DeltasplaB mutants, hyphal growth of DeltasplaB, but not that of DeltasplaA, displayed increased sensitivity to H(2)O(2) treatment. These data indicate that two A. oryzae sPLA(2) enzymes display distinct, presumably non-redundant, physiological functions.

  6. Desensitization of the growth hormone-induced Janus kinase 2 (Jak 2)/signal transducer and activator of transcription 5 (Stat5)-signaling pathway requires protein synthesis and phospholipase C.

    PubMed

    Fernández, L; Flores-Morales, A; Lahuna, O; Sliva, D; Norstedt, G; Haldosén, L A; Mode, A; Gustafsson, J A

    1998-04-01

    Signal transducers and activators of transcription (Stat) proteins are latent cytoplasmic transcription factors that are tyrosine phosphorylated by Janus kinases (Jak) in response to GH and other cytokines. GH activates Stat5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation. However, the mechanisms that turn off the GH-activated Jak2/Stat5 pathway are unknown. Continuous exposure to GH of BRL-4 cells, a rat hepatoma cell line stably transfected with rat GH receptor, induces a rapid but transient activation of Jak2 and Stat5. GH-induced Stat5 DNA-binding activity was detected after 2 min and reached a maximum at 10 min. Continued exposure to GH resulted in a desensitization characterized by 1) a rapid decrease in Stat5 DNA-binding activity. The rate of decrease of activity was rapid up to 1 h of GH treatment, and the remaining activity declined slowly thereafter. The activity of Stat5 present after 5 h is still higher than the control levels and almost 10-20% with respect to maximal activity at 10 min; and 2) the inability of further GH treatment to reinduce activation of Stat5. In contrast, with transient exposures of BRL-4 cells to GH, Stat5 DNA-binding activity could repeatedly be induced. GH-induced Jak2 and Stat5 activities were independent of ongoing protein synthesis. However, Jak2 tyrosine phosphorylation and Stat5 DNA-binding activity were prolonged for at least 4 h in the presence of cycloheximide, which suggests that the maintenance of desensitization requires ongoing protein synthesis. Furthermore, inhibition of protein synthesis potentiated GH-induced transcriptional activity in BRL-4 cells transiently transfected with SPIGLE1CAT, a reporter plasmid activated by Stat5. GH-induced Jak2 and Stat5 activation were not affected by D609 or mepacrine, both inhibitors of phospholipase C. However, in the presence of D609 and mepacrine, GH maintained prolonged Jak2 and Stat5 activation. Transactivation of SPIGLE1 by GH was

  7. Structural determinants of the hyperalgesic activity of myotoxic Lys49-phospholipase A2.

    PubMed

    Zambelli, Vanessa Olzon; Chioato, Lucimara; Gutierrez, Vanessa Pacciari; Ward, Richard John; Cury, Yara

    2017-01-01

    Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A 2 (Lys49-PLA 2 ) from the venom of Bothrops jararacussu, which despite of the lack of catalytic activity induces myotoxicity, inflammation and pain. The C-terminal region of the Lys49-PLA 2 s is important for these effects; however, the amino acid residues that determine hyperalgesia and edema are unknown. The aim of this study was to characterize the structural determinants for the Lys49-PLA 2 -induced nociception and inflammation. Scanning alanine mutagenesis in the active-site and C-terminal regions of BthTx-I has been used to study the structural determinants of toxin activities. The R118A mutant was employed as this substitution decreases PLA 2 myotoxicity. In addition, K115A and K116A mutants - which contribute to decrease cytotoxicity - and the K122A mutant - which decreases both myotoxicity and cytotoxicity - were also used. The H48Q mutant - which does not interfere with membrane damage or myotoxic activity - was used to evaluate if the PLA 2 catalytic site is relevant for the non-catalytic PLA 2 -induced pain and inflammation. Wistar male rats received intraplantar injections with mutant PLA 2 . Subsequently, hyperalgesia and edema were evaluated by the paw pressure test and by a plethysmometer. Native and recombinant BthTx-I were used as controls. Native and recombinant BthTx-I induced hyperalgesia and edema, which peaked at 2 h. The R118A mutant did not induce nociception or edema. The mutations K115A and K116A abolished hyperalgesia without interfering with edema. Finally, the K122A mutant did not induce hyperalgesia and presented a decreased inflammatory response. The results obtained with the BthTx-I mutants suggest, for the first time, that there are distinct residues responsible for the hyperalgesia and edema induced by BthTx-I. In addition, we also showed that cytolytic activity is essential for the hyperalgesic effect but not for edematogenic activity, corroborating previous data showing that

  8. Silencing of the tomato phosphatidylinositol-phospholipase C2 (SlPLC2) reduces plant susceptibility to Botrytis cinerea.

    PubMed

    Gonorazky, Gabriela; Guzzo, María Carla; Abd-El-Haliem, Ahmed M; Joosten, Matthieu H A J; Laxalt, Ana María

    2016-12-01

    The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated on pathogen attack. We have previously shown that the fungal elicitor xylanase induces a raise of SlPLC2 and SlPLC5 transcripts and that SlPLC2, but not SlPLC5, is required for xylanase-induced expression of defense-related genes. In this work we studied the role of SlPLC2 in the interaction between tomato and the necrotrophic fungus Botrytis cinerea. Inoculation of tomato leaves with B. cinerea increases SlPLC2 transcript levels. We knocked-down the expression of SlPLC2 by virus-induced gene silencing and plant defense responses were analyzed upon B. cinerea inoculation. SlPLC2 silenced plants developed smaller necrotic lesions concomitantly with less proliferation of the fungus. Silencing of SlPLC2 resulted as well in a reduced production of reactive oxygen species. Upon B. cinerea inoculation, transcript levels of the salicylic acid (SA)-defense pathway marker gene SlPR1a were diminished in SlPLC2 silenced plants compared to non-silenced infected plants, while transcripts of the jasmonic acid (JA)-defense gene markers Proteinase Inhibitor I and II (SlPI-I and SlPI-II) were increased. This implies that SlPLC2 participates in plant susceptibility to B. cinerea. © 2016 BSPP and John Wiley & Sons Ltd.

  9. Signal transfer in the plant plasma membrane: phospholipase A(2) is regulated via an inhibitory Gα protein and a cyclophilin.

    PubMed

    Heinze, Michael; Herre, Madeleine; Massalski, Carolin; Hermann, Isabella; Conrad, Udo; Roos, Werner

    2013-03-15

    The plasma membrane of the California poppy is known to harbour a PLA2 (phospholipase A2) that is associated with the Gα protein which facilitates its activation by a yeast glycoprotein, thereby eliciting the biosynthesis of phytoalexins. To understand the functional architecture of the protein complex, we titrated purified plasma membranes with the Gα protein (native or recombinant) and found that critical amounts of this subunit keep PLA2 in a low-activity state from which it is released either by elicitor plus GTP or by raising the Gα concentration, which probably causes oligomerization of Gα, as supported by FRET (fluorescence resonance energy transfer)-orientated fluorescence imaging and a semiquantitative split-ubiquitin assay. All effects of Gα were blocked by specific antibodies. A low-Gα mutant showed elevated PLA2 activity and lacked the GTP-dependent stimulation by elicitor, but regained this capability after pre-incubation with Gα. The inhibition by Gα and the GTP-dependent stimulation of PLA2 were diminished by inhibitors of peptidylprolyl cis-trans isomerases. A cyclophilin was identified by sequence in the plasma membrane and in immunoprecipitates with anti-Gα antibodies. We conclude that soluble and target-associated Gα interact at the plasma membrane to build complexes of varying architecture and signal amplification. Protein-folding activity is probably required to convey conformational transitions from Gα to its target PLA2.

  10. Disruption of the Phospholipase D Gene Attenuates the Virulence of Aspergillus fumigatus

    PubMed Central

    Li, Xianping; Gao, Meihua; Han, Xuelin; Tao, Sha; Zheng, Dongyu; Cheng, Ying; Yu, Rentao; Han, Gaige; Schmidt, Martina

    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 protein family in A. fumigatus, was confirmed to significantly decrease both intracellular and extracellular PLD activity of A. fumigatus. The pld gene disruption did not alter conidial morphological characteristics, germination, growth, and biofilm formation but significantly suppressed the internalization of A. fumigatus into A549 epithelial cells without affecting conidial adhesion to epithelial cells. Importantly, the suppressed internalization was fully rescued in the presence of 100 μM phosphatidic acid, the PLD product. Indeed, complementation of pld restored the PLD activity and internalization capacity of A. fumigatus. Phagocytosis of A. fumigatus conidia by J774 macrophages was not affected by the absence of the pld gene. Pretreatment of conidia with 1-butanol and a specific PLD inhibitor decreased the internalization of A. fumigatus into A549 epithelial cells but had no effect on phagocytosis by J774 macrophages. Finally, loss of the pld gene attenuated the virulence of A. fumigatus in mice immunosuppressed with hydrocortisone acetate but not with cyclophosphamide. These data suggest that PLD of A. fumigatus regulates its internalization into lung epithelial cells and may represent an important virulence factor for A. fumigatus infection. PMID:22083709

  11. The anti-inflammatory effects of Yunnan Baiyao are involved in regulation of the phospholipase A2/arachidonic acid metabolites pathways in acute inflammation rat model.

    PubMed

    Ren, Xiaobin; Zhang, Mingzhu; Chen, Lingxiang; Zhang, Wanli; Huang, Yu; Luo, Huazhen; Li, Ling; He, Hongbing

    2017-10-01

    The traditional Chinese medicine Yunnan Baiyao (YNB) has been reported to possess anti‑inflammatory properties, however its mechanism of action remains unclear. It was previously reported that YNB ameliorated depression of arachidonic acid (AA) levels in a rat model of collagen-induced arthritis. In the current study, the capacity of YNB to ameliorate inflammation was compared in carrageenan‑induced and AA‑induced acute inflammation of the rat paw with celecoxib and mizolastine, respectively (n=24 per group). The capacity of YNB to affect the phospholipase A2 (PLA2)/AA pathway (using reverse transcription‑quantitative polymerase chain reaction) and release of inflammatory lipid mediators (by ELISA) were investigated. Celecoxib ameliorated carrageenan‑induced paw edema, and mizolastine ameliorated AA‑induced rat paw edema. YNB alleviated paw edema and inhibited inflammatory cell infiltration in the two models. YNB inhibited production of 5‑LOX AA metabolite leukotriene B4 (LTB4), and suppressed expression of 5‑LOX, cytosolic PLA2 (cPLA2), 5‑LOX‑activating protein, and LTB4 receptor mRNA in the AA‑induced inflammation model (P<0.05). YNB Inhibited the production of the COX‑2 AA metabolite prostaglandin E2 (PGE2) and suppressed expression of COX‑2, cPLA2, PGE2 mRNA in the carrageenan‑induced inflammation mode (P<0.05). Taken together, the data suggest that modulation of COX and LOX pathways in AA metabolism represent a novel anti-inflammatory mechanism of YNB.

  12. Multiple roles of phosphoinositide-specific phospholipase C isozymes.

    PubMed

    Suh, Pann-Ghill; Park, Jae-Il; Manzoli, Lucia; Cocco, Lucio; Peak, Joanna C; Katan, Matilda; Fukami, Kiyoko; Kataoka, Tohru; Yun, Sanguk; Ryu, Sung Ho

    2008-06-30

    Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.

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

    PubMed Central

    Burnett, Bruce P.; Bitto, Alessandra; Altavilla, Domenica; Squadrito, Francesco; Levy, Robert M.; Pillai, Lakshmi

    2011-01-01

    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). PMID:21765617

  14. Flavocoxid inhibits phospholipase A2, peroxidase moieties of the cyclooxygenases (COX), and 5-lipoxygenase, modifies COX-2 gene expression, and acts as an antioxidant.

    PubMed

    Burnett, Bruce P; Bitto, Alessandra; Altavilla, Domenica; Squadrito, Francesco; Levy, Robert M; Pillai, Lakshmi

    2011-01-01

    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 (PLA(2)) (IC(50) = 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 (IC(50) = 12.3) and COX-2 (IC(50) = 11.3 μg/mL) peroxidase (PO) moieties as well as 5-lipoxygenase (5-LOX) (IC(50) = 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 (IC(50) = 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).

  15. Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress.

    PubMed

    Jarc, Eva; Kump, Ana; Malavašič, Petra; Eichmann, Thomas O; Zimmermann, Robert; Petan, Toni

    2018-03-01

    Cancer cells driven by the Ras oncogene scavenge unsaturated fatty acids (FAs) from their environment to counter nutrient stress. The human group X secreted phospholipase A 2 (hGX sPLA 2 ) releases FAs from membrane phospholipids, stimulates lipid droplet (LD) biogenesis in Ras-driven triple-negative breast cancer (TNBC) cells and enables their survival during starvation. Here we examined the role of LDs, induced by hGX sPLA 2 and unsaturated FAs, in protection of TNBC cells against nutrient stress. We found that hGX sPLA 2 releases a mixture of unsaturated FAs, including ω-3 and ω-6 polyunsaturated FAs (PUFAs), from TNBC cells. Starvation-induced breakdown of LDs induced by low micromolar concentrations of unsaturated FAs, including PUFAs, was associated with protection from cell death. Interestingly, adipose triglyceride lipase (ATGL) contributed to LD breakdown during starvation, but it was not required for the pro-survival effects of hGX sPLA 2 and unsaturated FAs. High micromolar concentrations of PUFAs, but not OA, induced oxidative stress-dependent cell death in TNBC cells. Inhibition of triacylglycerol (TAG) synthesis suppressed LD biogenesis and potentiated PUFA-induced cell damage. On the contrary, stimulation of LD biogenesis by hGX sPLA 2 and suppression of LD breakdown by ATGL depletion reduced PUFA-induced oxidative stress and cell death. Finally, lipidomic analyses revealed that sequestration of PUFAs in LDs by sPLA 2 -induced TAG remodelling and retention of PUFAs in LDs by inhibition of ATGL-mediated TAG lipolysis protect from PUFA lipotoxicity. LDs are thus antioxidant and pro-survival organelles that guard TNBC cells against nutrient and lipotoxic stress and emerge as attractive targets for novel therapeutic interventions. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Isolation and biochemical characterization of a γ-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum.

    PubMed

    Gimenes, Sarah Natalie Cirilo; Ferreira, Francis Barbosa; Silveira, Ana Carolina Portella; Rodrigues, Renata Santos; Yoneyama, Kelly Aparecida Geraldo; Izabel Dos Santos, Juliana; Fontes, Marcos Roberto de Mattos; de Campos Brites, Vera Lúcia; Santos, André Luiz Quagliatto; Borges, Márcia Helena; Lopes, Daiana Silva; Rodrigues, Veridiana M

    2014-04-01

    In the present work, we describe the isolation and partial structural and biochemical characterization of the first phospholipase A2 inhibitor (γPLI) from Crotalus durissus collilineatus (Cdc) snake serum. Initially, the Cdc serum was subjected to a Q-Sepharose ion exchange column, producing six peaks at 280 nm absorbance (Q1-Q6). Subsequently, Q4 fraction was submitted to affinity chromatography with immobilized PLA2 BnSP-7, a step that resulted in two fractions (NHS-1 and NHS-2). The latter contained the inhibitor, denominated γCdcPLI. The molecular mass of γCdcPLI, determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), was 22,340 Da. Partial sequences obtained by Edman degradation and by mass spectrometry (MALDI-TOF/TOF), showed similarity, as expected, to other related inhibitors. Circular dichroism (CD) analysis showed the presence of approximately 22% alpha helices and 29% beta sheets in the protein secondary structure. Additionally, CD studies also indicated no significant changes in the secondary structure of γCdcPLI when it is complexed to BpPLA2-TXI. On the other hand, dynamic light scattering (DLS) assays showed a temperature-dependent oligomerization behavior for this inhibitor. Biochemical analyses showed γCdcPLI was able to inhibit the enzymatic, cytotoxic and myotoxic activities of PLA2s. Structural and functional studies performed on this inhibitor may elucidate the action mechanisms of PLA2 inhibitors. In addition, we hope this study may contribute to investigating the potential use of these inhibitors for the treatment of snakebite or inflammatory diseases in which PLA2s may be involved. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. The effect of Danshen extract on lipoprotein-associated phospholipase A2 levels in patients with stable angina pectoris: study protocol for a randomized controlled trial - the DOLPHIN study.

    PubMed

    Chen, A-Di; Wang, Chun-Ling; Qin, Yang; Tian, Liang; Chen, Li-Bin; Yuan, Xiao-Ming; Ma, Lin-Xiu; Wang, Yu-Feng; Sun, Ji-Rong; Wang, Hao-Sen; Dai, Neng

    2017-12-20

    Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ), a biomarker of oxidation and inflammation, has been associated with increased coronary artery disease risk. To date, very few studies have examined the Chinese herbal drug Danshen or its extract on Lp-PLA 2 in patients with stable angina pectoris. In this study, we aim to investigate the effect of Danshen extract on Lp-PLA 2 level in patients with stable angina. This is a randomized, single-blind, placebo-controlled, adaptive clinical trial. A total of 156 patients meeting the eligibility criteria will be randomly assigned to either the Danshen extract (DanshenDuofensuanyan injection and Danshen drop spill) group or the placebo group in a 1:1 ratio. Participants will then undergo treatment with DanshenDuofensuanyan injection or placebo (glucose) during hospitalization, followed by open-label Danshen drop spill (30 pills/day) in Danshen extract group for 60 days after discharge. Because this is an adaptive trial, two interim analyses are prospectively planned. These will be performed after one-third and two-thirds of the patients, respectively, have completed the trial. On the basis of the results of these interim analyses, a data monitoring committee will determine how to modify aspects of the study without undermining the validity and integrity of the trial. The primary outcome measure is the serum level of Lp-PLA 2 in the Danshen extract group and the placebo group. The secondary outcomes include the proportion of patients who show a clinically significant change, which is defined as at least a 20-point improvement in angina frequency score on the Seattle Angina Questionnaire and the carotid intima-media thickness, which will be measured using ultrasound. Other secondary efficacy and safety outcomes will also be assessed. This study will provide evidence that Danshen extract is beneficial for stable angina and may establish a possible mechanism of Danshen treatment effects on cardiovascular disease. This

  18. Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain*

    PubMed Central

    Cai, Jingfei; Guo, Su; Lomasney, Jon W.; Roberts, Mary F.

    2013-01-01

    Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca2+ that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane. PMID:24235144

  19. Genetic Ablation of Calcium-independent Phospholipase A2γ Prevents Obesity and Insulin Resistance during High Fat Feeding by Mitochondrial Uncoupling and Increased Adipocyte Fatty Acid Oxidation*

    PubMed Central

    Mancuso, David J.; Sims, Harold F.; Yang, Kui; Kiebish, Michael A.; Su, Xiong; Jenkins, Christopher M.; Guan, Shaoping; Moon, Sung Ho; Pietka, Terri; Nassir, Fatiha; Schappe, Timothy; Moore, Kristin; Han, Xianlin; Abumrad, Nada A.; Gross, Richard W.

    2010-01-01

    Phospholipases are critical enzyme mediators participating in many aspects of cellular function through modulating the generation of lipid 2nd messengers, membrane physical properties, and cellular bioenergetics. Here, we demonstrate that mice null for calcium-independent phospholipase A2γ (iPLA2γ−/−) are completely resistant to high fat diet-induced weight gain, adipocyte hypertrophy, hyperinsulinemia, and insulin resistance, which occur in iPLA2γ+/+ mice after high fat feeding. Notably, iPLA2γ−/− mice were lean, demonstrated abdominal lipodystrophy, and remained insulin-sensitive despite having a marked impairment in glucose-stimulated insulin secretion after high fat feeding. Respirometry of adipocyte explants from iPLA2γ−/− mice identified increased rates of oxidation of multiple different substrates in comparison with adipocyte explants from wild-type littermates. Shotgun lipidomics of adipose tissue from wild-type mice demonstrated the anticipated 2-fold increase in triglyceride content after high fat feeding. In sharp contrast, the adipocyte triglyceride content was identical in iPLA2γ−/− mice fed either a standard diet or a high fat diet. Respirometry of skeletal muscle mitochondria from iPLA2γ−/− mice demonstrated marked decreases in state 3 respiration using multiple substrates whose metabolism was uncoupled from ATP production. Shotgun lipidomics of skeletal muscle revealed a decreased content of cardiolipin with an altered molecular species composition thereby identifying the mechanism underlying mitochondrial uncoupling in the iPLA2γ−/− mouse. Collectively, these results identify iPLA2γ as an obligatory upstream enzyme that is necessary for efficient electron transport chain coupling and energy production through its participation in the alterations of cellular bioenergetics that promote the development of the metabolic syndrome. PMID:20817734

  20. Phospholipase D2 Is Involved in the Formation of Golgi Tubules and ArfGAP1 Recruitment

    PubMed Central

    Martínez-Martínez, Narcisa; Martínez-Alonso, Emma; Ballesta, José; Martínez-Menárguez, José A.

    2014-01-01

    Lipids and lipid-modifying enzymes play a key role in the biogenesis, maintenance and fission of transport carriers in the secretory and endocytic pathways. In the present study we demonstrate that phosphatidic acid generated by phospholipase D2 (PLD2) is involved in the formation of Golgi tubules. The main evidence to support this is: 1) inhibitors of phosphatidic acid formation and PLD2 depletion inhibit the formation of tubules containing resident enzymes and regulators of intra-Golgi transport in a low temperature (15°C) model of Golgi tubulation but do not affect brefeldin A-induced tubules, 2) inhibition of PLD2 enzymatic activity and PLD2 depletion in cells cultured under physiological conditions (37°C) induce the formation of tubules specifically containing Golgi matrix proteins, and, 3) over-expression of PLD2 induces the formation of a tubular network. In addition, it was found that the generation of this lipid by the isoenzyme is necessary for ArfGAP1 recruitment to Golgi membranes. These results suggest that both proteins are involved in the molecular mechanisms which drive the formation of different types of Golgi tubules. PMID:25354038

  1. Isolation and characterization of a 66-kDa protein from rat liver plasma membrane with RhoA-stimulated phospholipase D activity.

    PubMed

    Dunkirk, Shawn G; Wallert, Mark A; Baumgartner, Matt L; Provost, Joseph J

    2002-02-01

    A 66-kDa molecular weight protein with phospholipase D activity was solubilized and partially purified from rat liver plasma membrane. The activity and regulation of this phospholipase D have been characterized. Immunoblot analyses indicated that the enzyme was distinct from hPLD1 and PLD2, but was recognized by an antibody to the 12 terminal amino acids of PLD1. PLD activity was stimulated by 1-100 microM Ca(2+) and Mg(2+) and displayed a pH optimum of 7.5. Activity was inhibited by both saturated and unsaturated fatty acids. This PLD was activated in an ATP-independent manner by the PKC isozymes alpha and betaII but not activated by other PKC isozymes. It was also stimulated by the small G-proteins RhoA and ARF. RhoA stimulated the greatest activation, followed by ARF and PKC(alpha). This enzyme was further activated in a synergistic manner when combinations of PKC(alpha) and RhoA or ARF were used. This enzyme displayed a greater response activation by RhoA than to activation by ARF. While a potential breakdown product of PLD1, activation by RhoA indicates that the PLD characterized here is distinct from the other PLDs cloned or isolated to date. Copyright 2002 Elsevier Science (USA).

  2. Phospholipase D2 Enhances Epidermal Growth Factor-Induced Akt Activation in EL4 Lymphoma Cells.

    PubMed

    Chahal, Manpreet S; Brauner, Daniel J; Meier, Kathryn E

    2010-07-02

    Phospholipase D2 (PLD2) generates phosphatidic acid through hydrolysis of phosphatidylcholine. PLD2 has been shown to play a role in enhancing tumorigenesis. The epidermal growth factor receptor (EGFR) can both activate and interact with PLD2. Murine lymphoma EL4 cells lacking endogenous PLD2 present a unique model to elucidate the role of PLD2 in signal transduction. In the current study, we investigated effects of PLD2 on EGF response. Western blotting and RT-PCR were used to establish that both parental cells and PLD2 transfectants express endogenous EGFR. Levels of EGFR protein are increased in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. EGF stimulates proliferation of EL4 cells transfected with active PLD2, but not parental cells or cells transfected with inactive PLD2. EGF-mediated proliferation in cells expressing active PLD2 is dependent on the activities of both the EGFR and the PI3K/Akt pathway, as demonstrated by studies using protein kinase inhibitors. EGF-induced invasion through a synthetic extracellular matrix is enhanced in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. Taken together, the data suggest that PLD2 acts in concert with EGFR to enhance mitogenesis and invasion in lymphoma cells.

  3. Phospholipase D2 Enhances Epidermal Growth Factor-Induced Akt Activation in EL4 Lymphoma Cells

    PubMed Central

    Chahal, Manpreet S.; Brauner, Daniel J.; Meier, Kathryn E.

    2010-01-01

    Phospholipase D2 (PLD2) generates phosphatidic acid through hydrolysis of phosphatidylcholine. PLD2 has been shown to play a role in enhancing tumorigenesis. The epidermal growth factor receptor (EGFR) can both activate and interact with PLD2. Murine lymphoma EL4 cells lacking endogenous PLD2 present a unique model to elucidate the role of PLD2 in signal transduction. In the current study, we investigated effects of PLD2 on EGF response. Western blotting and RT-PCR were used to establish that both parental cells and PLD2 transfectants express endogenous EGFR. Levels of EGFR protein are increased in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. EGF stimulates proliferation of EL4 cells transfected with active PLD2, but not parental cells or cells transfected with inactive PLD2. EGF-mediated proliferation in cells expressing active PLD2 is dependent on the activities of both the EGFR and the PI3K/Akt pathway, as demonstrated by studies using protein kinase inhibitors. EGF-induced invasion through a synthetic extracellular matrix is enhanced in cells expressing active PLD2, as compared to parental cells or cells expressing inactive PLD2. Taken together, the data suggest that PLD2 acts in concert with EGFR to enhance mitogenesis and invasion in lymphoma cells. PMID:27713341

  4. Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions.

    PubMed

    Häkkinen, T; Luoma, J S; Hiltunen, M O; Macphee, C H; Milliner, K J; Patel, L; Rice, S Q; Tew, D G; Karkola, K; Ylä-Herttuala, S

    1999-12-01

    We studied the expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA(2) activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA(2) mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase-polymerase chain reaction analysis indicated an increased expression of Lp-PLA(2) mRNA in human atherosclerotic lesions. In addition, approximately 6-fold higher Lp-PLA(2) activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA(2), which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA(2) activity could lead to antiatherogenic effects in the vessel wall.

  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

    PubMed Central

    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

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

    PubMed

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

    1999-04-13

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

  7. Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

    PubMed

    Sutto-Ortiz, Priscila; Camacho-Ruiz, María de Los Angeles; Kirchmayr, Manuel R; Camacho-Ruiz, Rosa María; Mateos-Díaz, Juan Carlos; Noiriel, Alexandre; Carrière, Frédéric; Abousalham, Abdelkarim; Rodríguez, Jorge A

    2017-01-01

    Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl- sn -glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel

  8. Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation

    PubMed Central

    Sutto-Ortiz, Priscila; Camacho-Ruiz, María de los Angeles; Kirchmayr, Manuel R.; Camacho-Ruiz, Rosa María; Mateos-Díaz, Juan Carlos; Noiriel, Alexandre; Carrière, Frédéric; Abousalham, Abdelkarim

    2017-01-01

    Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel

  9. Rac-mediated Stimulation of Phospholipase2 Amplifies B Cell Receptor-induced Calcium Signaling*♦

    PubMed Central

    Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu.; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I.; Nienhaus, G. Ulrich; Gierschik, Peter

    2015-01-01

    The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139

  10. The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

    PubMed

    Gurd, J W; Bissoon, N

    1997-08-01

    The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

  11. Phospholipase activity after β-endorphin exposure discriminates Malassezia strains isolated from healthy and seborrhoeic dermatitis skin.

    PubMed

    Vlachos, Ch; Gaitanis, G; Alexopoulos, E C; Papadopoulou, C; Bassukas, I D

    2013-12-01

    Phospholipase activity and its induction by β-endorphin have been associated with pathogenic Malassezia pachydermatis animal isolates. To evaluate Malassezia phosholipase activity in human isolates from seborrhoeic dermatitis (SD) and healthy controls before and after β-endorphin exposure. Eighty-four volunteers with or without SD (N = 41) were sampled. Isolated Malassezia strains were incubated in Dixon's medium with and without 100 nmol/L β-endorphin. Subsequently, phospholipase activity was assessed in egg-yolk agar and the results were compared employing Wilcoxon sign test for paired data, chi-squared test and multinomial logistic regression analysis. A total of 64 Malassezia strains were isolated. SD strains tended to have decreased phospholipase activity before (P = 0.057) and increased after exposure to β-endorphin (P = 0.061) compared to isolates from healthy skin. Phospholipase activity after β-endorphin exposure related to basal enzyme activity as a measure of per strain phospholipase inducibility by β-endorphin did not depend on Malassezia species (P = 0.652). However, this latter biochemical trait discriminates strains isolated from SD lesional and healthy skin (P = 0.036). β-endorphin exposure modifies the in vitro phosholipase activity in Malassezia species isolated from SD lesional skin. This is in accordance with emerging evidence that enhanced local lipase activity is involved in the pathogenesis of SD. © 2012 The Authors. Journal of the European Academy of Dermatology and Venereology © 2012 European Academy of Dermatology and Venereology.

  12. THE EFFECT OF X-IRRADIATION ON THE PHOSPHOLIPASE AND ANTIOXIDANT ACTIVITIES OF RAT INTESTINAL MUCOSA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ottolenghi, A.; Bernheim, F.

    1960-04-01

    The antioxidant effect of intestinal mucosa is the result of the liberation of free fatty acid from phospholipid by phospholipase. The fatty acid binds the iron and thus inhibits peroxidation of unsaturated lipids in the test system. The phospholipase and antioxidant activity of rat intestinal mucosa decreases markedly 24 hours postirradiation and to approximately the same extent. (auth)

  13. Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells.

    PubMed

    Yao, Mu; Xie, Chanlu; Kiang, Mei-Yee; Teng, Ying; Harman, David; Tiffen, Jessamy; Wang, Qian; Sved, Paul; Bao, Shisan; Witting, Paul; Holst, Jeff; Dong, Qihan

    2015-10-27

    Cell cycle re-entry of quiescent cancer cells has been proposed to be involved in cancer progression and recurrence. Cytosolic phospholipase A2α (cPLA2α) is an enzyme that hydrolyzes membrane glycerophospholipids to release arachidonic acid and lysophospholipids that are implicated in cancer cell proliferation. The aim of this study was to determine the role of cPLA2α in cell cycle re-entry of quiescent prostate cancer cells. When PC-3 and LNCaP cells were rendered to a quiescent state, the active form of cPLA2α with a phosphorylation at Ser505 was lower compared to their proliferating state. Conversely, the phospho-cPLA2α levels were resurgent during the induction of cell cycle re-entry. Pharmacological inhibition of cPLA2α with Efipladib upon induction of cell cycle re-entry inhibited the re-entry process, as manifested by refrained DNA synthesis, persistent high proportion of cells in G0/G1 and low percentage of cells in S and G2/M phases, together with a stagnant recovery of Ki-67 expression. Simultaneously, Efipladib prohibited the emergence of Skp2 while maintained p27 at a high level in the nuclear compartment during cell cycle re-entry. Inhibition of cPLA2α also prevented an accumulation of cyclin D1/CDK4, cyclin E/CDK2, phospho-pRb, pre-replicative complex proteins CDC6, MCM7, ORC6 and DNA synthesis-related protein PCNA during induction of cell cycle re-entry. Moreover, a pre-treatment of the prostate cancer cells with Efipladib during induction of cell cycle re-entry subsequently compromised their tumorigenic capacity in vivo. Hence, cPLA2α plays an important role in cell cycle re-entry by quiescent prostate cancer cells.

  14. Cytosolic phospholipase A2α is critical for angiotensin II-induced hypertension and associated cardiovascular pathophysiology.

    PubMed

    Khan, Nayaab S; Song, Chi Young; Jennings, Brett L; Estes, Anne M; Fang, Xiao R; Bonventre, Joseph V; Malik, Kafait U

    2015-04-01

    Angiotensin II activates cytosolic phospholipase A(2)α (cPLA2α) and releases arachidonic acid from tissue phospholipids, which mediate or modulate ≥1 cardiovascular effects of angiotensin II and has been implicated in hypertension. Because arachidonic acid release is the rate limiting step in eicosanoid production, cPLA2α might play a central role in the development of angiotensin II-induced hypertension. To test this hypothesis, we investigated the effect of angiotensin II infusion for 13 days by micro-osmotic pumps on systolic blood pressure and associated pathogenesis in wild type (cPLA2α(+/+)) and cPLA2α(-/-) mice. Angiotensin II-induced increase in systolic blood pressure in cPLA2α(+/+) mice was abolished in cPLA2α(-/-) mice; increased systolic blood pressure was also abolished by the arachidonic acid metabolism inhibitor, 5,8,11,14-eicosatetraynoic acid in cPLA2α(+/+) mice. Angiotensin II in cPLA2α(+/+) mice increased cardiac cPLA2 activity and urinary eicosanoid excretion, decreased cardiac output, caused cardiovascular remodeling with endothelial dysfunction, and increased vascular reactivity in cPLA2α(+/+) mice; these changes were diminished in cPLA2α(-/-) mice. Angiotensin II also increased cardiac infiltration of F4/80(+) macrophages and CD3(+) T lymphocytes, cardiovascular oxidative stress, expression of endoplasmic reticulum stress markers p58(IPK), and CHOP in cPLA2α(+/+) but not cPLA2α(-/-) mice. Angiotensin II increased cardiac activity of ERK1/2 and cSrc in cPLA2α(+/+) but not cPLA2α(-/-) mice. These data suggest that angiotensin II-induced hypertension and associated cardiovascular pathophysiological changes are mediated by cPLA2α activation, most likely through the release of arachidonic acid and generation of eicosanoids with predominant prohypertensive effects and activation of ≥1 signaling molecules, including ERK1/2 and cSrc. © 2015 American Heart Association, Inc.

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

    PubMed

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

    2015-05-01

    Secreted phospholipase A2 inhibitor (sPLA2i) has been reported to have an anti-inflammatory function by blocking the production of inflammatory mediators. Obesity is characterized by low-grade inflammation and oxidative stress. The aim of this study was to investigate the effects of dietary supplementation of sPLA2i on inflammation, oxidative stress and serum fatty acid profile in dogs. Seven obese and seven lean Beagle dogs were used in a 28-day double blind cross-over design. Dogs were fed a control diet without supplemental sPLA2i or an sPLA2i supplemented diet. The sPLA2i diet decreased plasma fibrinogen levels and increased the protein:fibrinogen ratio in obese dogs to levels similar to those of lean dogs fed the same diet. Obese dogs had a higher plasma concentration of the lipophilic vitamin A with potential antioxidative capacity and a lower ratio of retinol binding protein 4:vitamin A compared to lean dogs, independent of the diets. A higher proportion of myristic acid (C14:0) and a lower proportion of linoleic acid (C18:2n-6) were observed in the dogs fed with the sPLA2i diet compared to dogs fed with the control diet. Furthermore, a higher ratio of n-6 to n-3, a lower proportion of n-3 polyunsaturated fatty acids and lower omega-3 index were observed in obese compared to lean dogs. The results indicate that obese dogs are characterized by a more 'proinflammatory' serum fatty acid profile and that diet inclusion of sPLA2i may reduce inflammation and alter fatty acid profile. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Immunolocalization of a glycosylphosphatidylinositol-specific phospholipase D in mast cells found in normal tissue and neurofibromatosis lesions.

    PubMed

    Metz, C N; Thomas, P; Davitz, M A

    1992-06-01

    A large number of eukaryotic proteins have been shown to be anchored to the cell membrane by glycosylphosphatidylinositol (GPI). This glycolipid anchor can serve as a substrate for anchor-specific phospholipases that convert the GPI-anchored membrane proteins into soluble forms. Soluble forms of many GPI anchored proteins have been identified in vivo in connective tissue, plasma, and urine. The authors have discovered that mammalian plasma contains a GPI-specific phospholipase D (GPI-PLD). Because it recognizes a portion of the conserved glycan core structure, all GPI-anchored proteins are potential substrates. The authors report the development of a murine monoclonal antibody specific for one form of the human GPI-PLD and the immunohistochemical localization of this enzyme to mast cells.

  17. A myotoxic Lys49 phospholipase A2-homologue is the major component of the venom of Bothrops cotiara from Misiones, Argentina.

    PubMed

    de Roodt, Adolfo; Fernández, Julián; Solano, Daniela; Lomonte, Bruno

    2018-06-15

    Bothrops cotiara is a pitviper found in Southeastern Brazil and, scarcely, in the Misiones province of Argentina. In contrast to considerable information available on the venom of the Brazilian snake population, that of Misiones has received little attention. While exploring the chromatographic venom profile of Argentinean B. cotiara, a major protein peak was found which, according to a previous study, is not present in the venom of Brazilian origin. The corresponding protein was isolated by RP-HPLC, and characterized by electrophoresis, mass spectrometry, phospholipase A 2 (PLA 2 ) assay, and myotoxic activities. Representing nearly 15% of B. cotiara venom from Misiones, this protein was identified as a Lys49 PLA 2 homologue. In accordance with the characteristics of this toxin family, the protein induced myotoxicity in mice and was devoid of PLA 2 activity. Since previous work reported that no PLA 2 or PLA 2 -homologues occur in B. cotiara venom of Brazilian origin, the presence of an abundant Lys49 PLA 2 homologue in the venom from Misiones highlights a striking phenotypic variation in toxin expression within two populations of a single snake species inhabiting different geographic areas. The considerable proportion of B. cotiara Lys49 PLA 2 homologue myotoxin in the venom alerts that skeletal muscle necrosis might be a potentially relevant consequence of eventual envenomings by this species in Misiones. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Phospholipase A2 activity-dependent and -independent fusogenic activity of Naja nigricollis CMS-9 on zwitterionic and anionic phospholipid vesicles.

    PubMed

    Chiou, Yi-Ling; Chen, Ying-Jung; Lin, Shinne-Ren; Chang, Long-Sen

    2011-11-01

    CMS-9, a phospholipase A(2) (PLA(2)) from Naja nigricollis venom, induced the death of human breast cancer MCF-7 cells accompanied with the formation of cell clumps without clear boundaries between cells. Annexin V-FITC staining indicated that abundant phosphatidylserine appeared on the outer membrane of MCF-7 cell clumps, implying the possibility that CMS-9 may promote membrane fusion via anionic phospholipids. To validate this proposition, fusogenic activity of CMS-9 on vesicles composed of zwitterionic phospholipid alone or a combination of zwitterionic and anionic phospholipids was examined. Although CMS-9-induced fusion of zwitterionic phospholipid vesicles depended on PLA(2) activity, CMS-9-induced fusion of vesicles containing anionic phospholipids could occur without the involvement of PLA(2) activity. Membrane-damaging activity of CMS-9 was associated with its fusogenicity. Moreover, CMS-9 induced differently membrane leakage and membrane fusion of vesicles with different compositions. Membrane fluidity and binding capability with phospholipid vesicles were not related to the fusogenicity of CMS-9. However, membrane-bound conformation and mode of CMS-9 depended on phospholipid compositions. Collectively, our data suggest that PLA(2) activity-dependent and -independent fusogenicity of CMS-9 are closely related to its membrane-bound modes and targeted membrane compositions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Substance P Activates Ca2+-Permeable Nonselective Cation Channels through a Phosphatidylcholine-Specific Phospholipase C Signaling Pathway in nNOS-Expressing GABAergic Neurons in Visual Cortex.

    PubMed

    Endo, Toshiaki; Yanagawa, Yuchio; Komatsu, Yukio

    2016-02-01

    To understand the functions of the neocortex, it is essential to characterize the properties of neurons constituting cortical circuits. Here, we focused on a distinct group of GABAergic neurons that are defined by a specific colocalization of intense labeling for both neuronal nitric oxide synthase (nNOS) and substance P (SP) receptor [neurokinin 1 (NK1) receptors]. We investigated the mechanisms of the SP actions on these neurons in visual cortical slices obtained from young glutamate decarboxylase 67-green fluorescent protein knock-in mice. Bath application of SP induced a nonselective cation current leading to depolarization that was inhibited by the NK1 antagonists in nNOS-immunopositive neurons. Ruthenium red and La(3+), transient receptor potential (TRP) channel blockers, suppressed the SP-induced current. The SP-induced current was mediated by G proteins and suppressed by D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), but not by inhibitors of phosphatidylinositol-specific PLC, adenylate cyclase or Src tyrosine kinases. Ca(2+) imaging experiments under voltage clamp showed that SP induced a rise in intracellular Ca(2+) that was abolished by removal of extracellular Ca(2+) but not by depletion of intracellular Ca(2+) stores. These results suggest that SP regulates nNOS neurons by activating TRP-like Ca(2+)-permeable nonselective cation channels through a PC-PLC-dependent signaling pathway. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. [Phospholipase and proteinase production by Malassezia pachydermatis isolated in dogs with and without otitis].

    PubMed

    Ortiz, Gustavo; Martín, M Carmen; Carrillo-Muñoz, Alfonso J; Payá, M Jesús

    2013-01-01

    Malassezia pachydermatis is part of the skin microbiota of dogs and cats. M. pachydermatis has been associated with external otitis and seborrhoeic dermatitis, reported more often in dogs than in cats. When the physical, chemical or immunological mechanisms of the skin are altered, M. pachydermatis could act as a pathogen. Thus, several virulence factors, such as the ability to produce esterase, lipase, lipoxygenase, protease, chondroitin sulphatase, and hyaluronidase, have been studied. In the present study, we aim to identify the phospholipase activity measured at pH 6.3, and the proteinase activity measured at pH 6.3 and pH 6.8 (pH from ears of dogs with external otitis) of M. pachydermatis strains isolated from dogs with and without external otitis. The phospholipase activity was measured using a semi-quantitative method with egg yolk, and the proteinase activity with a semi-quantitative method using bovine serum albumin agar. The study was performed on 96 isolates of M. pachydermatis, 43 isolated from dogs without clinical symptoms of otitis, and 52 isolated from dogs with otitis. In our study, 75.8% of the isolates showed phospholipase activity at pH 6.3, and 81 and 97.9% of them showed proteinase activity measured at pH 6.3 and 6.8, respectively. A higher phospholipase activity was detected in strains isolated from dogs with otitis. The proteinase activity was increased at a pH of 6.8 (97.9%) in comparison to a pH of 6.3 (81%). Our results suggest that the phospholipase activity may play an important role in the invasion of host tissues in chronic canine otitis cases. The proteinase activity results obtained in this study suggest that a reduction in the pH of the treatment may improve its efficacy in the resolution of M. pachydermatis otitis. Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  1. The βγ-crystallin domain of Lysinibacillus sphaericus phosphatidylinositol phospholipase C plays a central role in protein stability.

    PubMed

    Cerminati, Sebastián; Paoletti, Luciana; Peirú, Salvador; Menzella, Hugo G; Castelli, María Eugenia

    2018-06-16

    βγ-crystallin has emerged as a superfamily of structurally homologous proteins with representatives across all domains of life. A major portion of this superfamily is constituted by microbial members. This superfamily has also been recognized as a novel group of Ca 2+ -binding proteins with a large diversity and variable properties in Ca 2+ binding and stability. We have recently described a new phosphatidylinositol phospholipase C from Lysinibacillus sphaericus (LS-PIPLC) which was shown to efficiently remove phosphatidylinositol from crude vegetable oil. Here, the role of the C-terminal βγ-crystallin domain of LS-PIPLC was analyzed in the context of the whole protein. A truncated protein in which the C-terminal βγ-crystallin domain was deleted (LS-PIPLC ΔCRY ) is catalytically as efficient as the full-length protein (LS-PIPLC). However, the thermal and chemical stability of LS-PIPLC ΔCRY are highly affected, demonstrating a stabilizing role for this domain. It is also shown that the presence of Ca 2+ increases the thermal and chemical stability of the protein both in aqueous media and in oil, making LS-PIPLC an excellent candidate for use in industrial soybean oil degumming.

  2. Plasma secretory phospholipase A2-IIa as a potential biomarker for lung cancer in patients with solitary pulmonary nodules

    PubMed Central

    2011-01-01

    Background Five-year survival for lung cancer has remained at 16% over last several decades largely due to the fact that over 50% of patients are diagnosed with locally-advanced or metastatic disease. Diagnosis at an earlier and potentially curable stage is crucial. Solitary pulmonary nodules (SPNs) are common, but the difficulty lies in the determination of which SPN is malignant. Currently, there is no convenient and reliable biomarker effective for early diagnosis. Secretory phospholipase A2-IIa (sPLA2-IIa) is secreted into the circulation by cancer cells and may allow for an early detection of lung cancer. Methods Plasma samples from healthy donors, patients with only benign SPN, and patients with lung cancer were analyzed. Expression of sPLA2-IIa protein in lung cancer tissues was also determined. Results We found that the levels of plasma sPLA2-IIa were significantly elevated in lung cancer patients. The receiver operating characteristic curve analysis, comparing lung cancer patients to patients with benign nodules, revealed an optimum cutoff value for plasma sPLA2-IIa of 2.4 ng/ml to predict an early stage cancer with 48% sensitivity and 86% specificity and up to 67% sensitivity for T2 stage lung cancer. Combined sPLA2-IIa, CEA, and Cyfra21.1 tests increased the sensitivity for lung cancer prediction. High level of plasma sPLA2-IIa was associated with a decreased overall cancer survival. sPLA2-IIa was overexpressed in almost all non-small cell lung cancer and in the majority of small cell lung cancer by immunohistochemistry analysis. Conclusion Our finding strongly suggests that plasma sPLA2-IIa is a potential lung biomarker to distinguish benign nodules from lung cancer and to aid lung cancer diagnosis in patients with SPNs. PMID:22151235

  3. Recombinant-phospholipase A2 production and architecture of inclusion bodies are affected by pH in Escherichia coli.

    PubMed

    Calcines-Cruz, Carlos; Olvera, Alejandro; Castro-Acosta, Ricardo M; Zavala, Guadalupe; Alagón, Alejandro; Trujillo-Roldán, Mauricio A; Valdez-Cruz, Norma A

    2018-03-01

    Aggregation of recombinant proteins into inclusion bodies (IBs) is the major drawback of heterologous expression in Escherichia coli. Here, we evaluated the effects of a pH shift after expression induction on recombinant phospholipase A2 production and its aggregation in IBs in E. coli Origami™, as compared to cultures with pH maintained at 7.5 or uncontrolled pH. Cultures shifted from 7.5 to pH 6.5 or 8.5 produced ∼15-25% less biomass as compared with those kept at 7.5 or without pH control. The cultures shifted to pH 8.5 showed a ∼50% higher yield of acetate per biomass, and the rPLA2 yield was improved 2.4-fold. Purified IBs formed at pH 8.5 containing ∼50% of rPLA2, were more susceptible to proteinase-K cleavage and bound less thioflavin-T, indicating lower amyloid content, with the concomitant enrichment of α-helical and random-coil secondary structures, as demonstrated by FTIR. Moreover, only one IB per cell was formed at pH 8.5; instead, more than two were observed under the other culture pH conditions. Nevertheless, under uncontrolled pH conditions, ∼300nm larger IBs were observed. Our work presents evidence of the usefulness of recombinant protein expression cultivated at pH 8.5 allowing the reduction of amyloid content in IBs. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Evidence for the cytotoxic effects of Mycobacterium tuberculosis phospholipase C towards macrophages.

    PubMed

    Bakala N'goma, J C; Schué, M; Carrière, F; Geerlof, A; Canaan, S

    2010-12-01

    Phospholipase Cs (PLCs) contribute importantly to the virulence and pathogenicity of several bacteria. It has been reported in previous studies that mutations in the four predicted plc genes of Mycobacterium tuberculosis inhibit the growth of these bacteria during the late phase of infection in mice. These enzymes have not yet been fully characterised, mainly because they are not easy to produce in large quantities. With a view to elucidating the role of all Mycobacterium tuberculosis phospholipase Cs (PLC-A, PLC-B, PLC-C and PLC-D), a large amount of active, soluble recombinant PLCs, were expressed and purified using Mycobacterium smegmatis as expression system. These enzymes showed different pH activity profiles. PLC-C was found to be the most active of the four recombinant PLCs under acidic conditions. All the enzymes tested induced cytotoxic effects on mouse macrophage RAW 264.7 cell lines, via direct or indirect enzymatic hydrolysis of cell membrane phospholipids. These results open new prospects for characterising biochemical and structural features of Mycobacterium tuberculosis PLCs, which might lead to the identification of novel anti-tuberculosis drug targets. All mycobacterial phospholipase Cs can now be studied in order to determine their role in the virulence and pathogenicity of bacteria of this kind. 2010 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-07-01

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

  6. Hyperforin induces Ca(2+)-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A(2) activation through select phospholipid interactions.

    PubMed

    Hoffmann, Marika; Lopez, Jakob J; Pergola, Carlo; Feisst, Christian; Pawelczik, Sven; Jakobsson, Per-Johan; Sorg, Bernd L; Glaubitz, Clemens; Steinhilber, Dieter; Werz, Oliver

    2010-04-01

    Here, we investigated the modulation of cytosolic phospholipase A(2) (cPLA(2))-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10microM) versus unstimulated cells, which was blocked by cPLA(2)alpha-inhibition, and induced translocation of cPLA(2) to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca(2+) by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA(2) at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA(2) phosphorylation. However, neither AA release nor translocation of cPLA(2) was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA(2) in the presence of Ca(2+). However, when Ca(2+) was omitted, hyperforin caused a prominent increase in cPLA(2) activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional (1)H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA(2) activation by enabling its access towards select lipid membranes independent of Ca(2+) ions. Such Ca(2+)- and phosphorylation-independent mechanism of cPLA(2) activation may apply also to other membrane-interfering molecules. 2010 Elsevier B.V. All rights reserved.

  7. Structure of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-peptide with phospholipase A2 from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution.

    PubMed

    Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

    2014-03-10

    Alzheimer's disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD's neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer's Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ-Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD.

  8. Mechanisms involved in hemoglobin-mediated oxidation of lipids in washed fish muscle and inhibitory effects of phospholipase A2.

    PubMed

    Tatiyaborworntham, Nantawat; Richards, Mark P

    2018-05-01

    Hemoglobin (Hb) is a lipid oxidation promoter in fish muscle. Phospholipase A2 (PLA2; EC 3.1.1.4) is linked to an increased resistance to lipid oxidation of frozen-thawed cod fillets via an unknown mechanism. The present study aimed to investigate the mechanism of Hb-mediated lipid oxidation with a focus on ferryl Hb and methemoglobin (metHb), the pro-oxidative Hb species, and to examine how porcine pancreatic PLA2 inhibits Hb-mediated lipid oxidation in washed cod muscle (WCM). Lipid hydroperoxides (LOOHs) and thiobarbituric acid reactive substances (TBARS) were measured as primary and secondary lipid oxidation products, respectively. The formation of metHb and ferryl Hb was also monitored. Ferryl Hb and metHb formed during the Hb-mediated lipid oxidation. PLA2 inhibited the formation of LOOHs and TBARS and suppressed the formation of metHb and ferryl Hb. WCM was pre-oxidized by hemin to increase the amount of LOOHs. PLA2 promoted the depletion of LOOHs in the pre-oxidized WCM with limited TBARS formation at the expense of the heme moiety of Hb. The results of the present study suggest that ferryl Hb may play a role in Hb-mediated lipid oxidation and that PLA2 from pig pancreas may work together with Hb as a novel antioxidant with an ability to remove pre-formed LOOHs from a lipid substrate. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  9. Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals.

    PubMed Central

    Diaz-Meco, M T; Dominguez, I; Sanz, L; Municio, M M; Berra, E; Cornet, M E; Garcia de Herreros, A; Johansen, T; Moscat, J

    1992-01-01

    Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has been established that the mechanism whereby TGF-beta 1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-beta 1 action. In the study reported here, we demonstrate that TGF-beta 1 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-beta 1. Images PMID:1309592

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

    PubMed

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

    2017-12-01

    Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A 2 (PLA 2 ), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB 4 production, cytosolic PLA 2 s activity, myeloperoxidase (MPO) and superoxide anion (O 2 - ) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB 4 , but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O 2 - release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O 2 - . However, it induces LTB 4 and IL-8 production. These data show the influence of CNF on PBMCs' function by inducing TNF-α and LTB 4 production, and on neutrophils, by stimulating chemotaxis and LTB 4 production, via cytosolic PLA 2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Phospholipase Dζ Enhances Diacylglycerol Flux into Triacylglycerol

    DOE PAGES

    Yang, Wenyu; Wang, Geliang; Li, Jia; ...

    2017-03-21

    Plant seeds are the primary source of triacylglycerols (TAG) for food, feed, fuel, and industrial applications. As TAG is produced from diacylglycerol (DAG), successful engineering strategies to enhance TAG levels have focused on the conversion of DAG to TAG. However, the production of TAG can be limited by flux through the enzymatic reactions that supply DAG. In this study, two Arabidopsis phospholipase Dζ genes (AtPLDζ 1 and AtPLDζ 2) were coexpressed in Camelina sativa to test whether the conversion of phosphatidylcholine to DAG impacts TAG levels in seeds. The resulting transgenic plants produced 2% to 3% more TAG as amore » component of total seed biomass and had increased 18:3 and 20:1 fatty acid levels relative to wild type. Increased DAG and decreased PC levels were examined through the kinetics of lipid assembly by [ 14C]acetate and [ 14C]glycerol incorporation into glycerolipids. [ 14C]acetate was rapidly incorporated into TAG in both wild-type and overexpression lines, indicating a significant flux of nascent and elongated acyl-CoAs into the sn-3 position of TAG. Stereochemical analysis revealed that newly synthesized fatty acids were preferentially incorporated into the sn-2 position of PC, but the sn-1 position of de novo DAG and indicated similar rates of nascent acyl groups into the Kennedy pathway and acyl editing. [ 14C]glycerol studies demonstrated PC-derived DAG is the major source of DAG for TAG synthesis in both tissues. The results emphasize that the interconversions of DAG and PC pools can impact oil production and composition.« less

  12. Phospholipase Dζ Enhances Diacylglycerol Flux into Triacylglycerol

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Wenyu; Wang, Geliang; Li, Jia

    Plant seeds are the primary source of triacylglycerols (TAG) for food, feed, fuel, and industrial applications. As TAG is produced from diacylglycerol (DAG), successful engineering strategies to enhance TAG levels have focused on the conversion of DAG to TAG. However, the production of TAG can be limited by flux through the enzymatic reactions that supply DAG. In this study, two Arabidopsis phospholipase Dζ genes (AtPLDζ 1 and AtPLDζ 2) were coexpressed in Camelina sativa to test whether the conversion of phosphatidylcholine to DAG impacts TAG levels in seeds. The resulting transgenic plants produced 2% to 3% more TAG as amore » component of total seed biomass and had increased 18:3 and 20:1 fatty acid levels relative to wild type. Increased DAG and decreased PC levels were examined through the kinetics of lipid assembly by [ 14C]acetate and [ 14C]glycerol incorporation into glycerolipids. [ 14C]acetate was rapidly incorporated into TAG in both wild-type and overexpression lines, indicating a significant flux of nascent and elongated acyl-CoAs into the sn-3 position of TAG. Stereochemical analysis revealed that newly synthesized fatty acids were preferentially incorporated into the sn-2 position of PC, but the sn-1 position of de novo DAG and indicated similar rates of nascent acyl groups into the Kennedy pathway and acyl editing. [ 14C]glycerol studies demonstrated PC-derived DAG is the major source of DAG for TAG synthesis in both tissues. The results emphasize that the interconversions of DAG and PC pools can impact oil production and composition.« less

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  15. Secretory phospholipase A{sub 2}-mediated progression of hepatotoxicity initiated by acetaminophen is exacerbated in the absence of hepatic COX-2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bhave, Vishakha S.; Donthamsetty, Shashikiran; Latendresse, John R.

    2011-03-15

    We have previously reported that among the other death proteins, hepatic secretory phospholipase A{sub 2} (sPLA{sub 2}) is a leading mediator of progression of liver injury initiated by CCl{sub 4} in rats. The aim of our present study was to test the hypothesis that increased hepatic sPLA{sub 2} 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 highermore » susceptibility of COX-2 KO mice to sPLA{sub 2}-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 {sup 14}C-APAP in the livers of KO mice. Hepatic sPLA{sub 2} activity and plasma TNF-{alpha} were significantly higher after APAP administration in the KO mice. This was accompanied by a corresponding fall in hepatic PGE{sub 2} and lower compensatory liver regeneration and repair ({sup 3}H-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 sPLA{sub 2}-mediated progression of liver injury. These findings are consistent with the destructive role of sPLA{sub 2} 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.« less

  16. Phospholipase C as a potential target for cardioprotection during oxidative stress.

    PubMed

    Tappia, Paramjit S; Asemu, Girma; Rodriguez-Leyva, Delfin

    2010-03-01

    Cardiac dysfunction due to ischemia-reperfusion (I/R) is associated with marked changes in membrane function and subsequent Ca2+-handling abnormalities in cardiomyocytes. The membrane abnormalities in hearts subjected to I/R arise primarily from oxidative stress as a consequence of increased formation of reactive oxygen species and other oxidants, as well as reduced antioxidant defenses. Little is known, however, about the nature and mechanisms of the sarcolemmal membrane changes with respect to phospholipase C (PLC)-related signaling events. In addition, the mechanisms involved in protection of the postischemic myocardium and in ischemic preconditioning with respect to PLC function need to be established. Accordingly, this article reviews the historical and current information on PLC-mediated signal transduction mechanisms in I/R, as well as outlining future directions that should be addressed. Such information will extend our knowledge of ischemic heart disease and help improve its therapy.

  17. β-Endorphin enhances the phospholipase activity of the dandruff causing fungi Malassezia globosa and Malassezia restricta.

    PubMed

    Honnavar, Prasanna; Chakrabarti, Arunaloke; Prasad, Ghandam S; Singh, Pankaj; Dogra, Sunil; Rudramurthy, Shivaprakash M

    2017-02-01

    β-Endorphin is known to stimulate phospholipase production by Malassezia pachydermatis during canine dermatoses. The role of β-endorphin in Malassezia infection in humans is not well studied. The present study compares the influence of β-endorphin on Malassezia globosa and Malassezia restricta isolated from patients with seborrhoeic dermatitis/dandruff (SD/D) and healthy controls. Malassezia isolates (five each of the two species from patients and healthy controls) were grown on modified Dixon's agar with or without 100 nmol/L β-endorphin. Phospholipase activity was quantified based on its ability to hydrolyze L-α-phosphatidylcholine dimyristoyl (phospholipid substrate). Free fatty acid was measured by a colorimetry method. In isolates from patients, the phospholipase activity significantly increased after exposure to β-endorphin (M. globosa, P = .04; M. restricta, P = .001), which did not occur in isolates from healthy controls. Moreover, after β-endorphin exposure the patient isolates had significantly higher (P = .0004) phospholipase activity compared to the healthy control isolates. The results suggest that isolates of M. globosa and M. restricta from patients may differ from those of healthy humans. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Characterization of secretory phospholipase A₂ with phospholipase A₁ activity in tobacco, Nicotiana tabacum (L.).

    PubMed

    Fujikawa, Yukichi; Fujikawa, Ritsuko; Iijima, Noriaki; Esaka, Muneharu

    2012-03-01

    A cDNA encoding protein with homology to plant secretory phospholipase A₂ (sPLA₂), denoted as Nt1 PLA₂, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA₂ has 12 cysteines, Ca²⁺ binding loop and catalytic site domain that are commonly conserved in plant sPLA₂s. The recombinant Nt1 PLA₂ was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA₂ could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²⁺ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA₂ mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.

  19. Rosmarinic acid, a new snake venom phospholipase A2 inhibitor from Cordia verbenacea (Boraginaceae): antiserum action potentiation and molecular interaction.

    PubMed

    Ticli, Fábio K; Hage, Lorane I S; Cambraia, Rafael S; Pereira, Paulo S; Magro, Angelo J; Fontes, Marcos R M; Stábeli, Rodrigo G; Giglio, José R; França, Suzelei C; Soares, Andreimar M; Sampaio, Suely V

    2005-09-01

    Many plants are used in traditional medicine as active agents against various effects induced by snakebite. The methanolic extract from Cordia verbenacea (Cv) significantly inhibited paw edema induced by Bothrops jararacussu snake venom and by its main basic phospholipase A2 homologs, namely bothropstoxins I and II (BthTXs). The active component was isolated by chromatography on Sephadex LH-20 and by RP-HPLC on a C18 column and identified as rosmarinic acid (Cv-RA). Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid [2-O-cafeoil-3-(3,4-di-hydroxy-phenyl)-R-lactic acid]. This is the first report of RA in the species C. verbenacea ('baleeira', 'whaler') and of its anti-inflammatory and antimyotoxic properties against snake venoms and isolated toxins. RA inhibited the edema and myotoxic activity induced by the basic PLA2s BthTX-I and BthTX-II. It was, however, less efficient to inhibit the PLA2 activity of BthTX-II and, still less, the PLA2 and edema-inducing activities of the acidic isoform BthA-I-PLA2 from the same venom, showing therefore a higher inhibitory activity upon basic PLA2s. RA also inhibited most of the myotoxic and partially the edema-inducing effects of both basic PLA2s, thus reinforcing the idea of dissociation between the catalytic and pharmacological domains. The pure compound potentiated the ability of the commercial equine polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA2s in experimental models. CD data presented here suggest that, after binding, no significant conformation changes occur either in the Cv-RA or in the target PLA2. A possible model for the interaction of rosmarinic acid with Lys49-PLA2 BthTX-I is proposed.

  20. Investigation of Specific Substitutions in Virulence Genes Characterizing Phenotypic Groups of Low-Virulence Field Strains of Listeria monocytogenes

    PubMed Central

    Roche, S. M.; Gracieux, P.; Milohanic, E.; Albert, I.; Virlogeux-Payant, I.; Témoin, S.; Grépinet, O.; Kerouanton, A.; Jacquet, C.; Cossart, P.; Velge, P.

    2005-01-01

    Several models have shown that virulence varies from one strain of Listeria monocytogenes to another, but little is known about the cause of low virulence. Twenty-six field L. monocytogenes strains were shown to be of low virulence in a plaque-forming assay and in a subcutaneous inoculation test in mice. Using the results of cell infection assays and phospholipase activities, the low-virulence strains were assigned to one of four groups by cluster analysis and then virulence-related genes were sequenced. Group I included 11 strains that did not enter cells and had no phospholipase activity. These strains exhibited a mutated PrfA; eight strains had a single amino acid substitution, PrfAK220T, and the other three had a truncated PrfA, PrfAΔ174-237. These genetic modifications could explain the low virulence of group I strains, since mutated PrfA proteins were inactive. Group II and III strains entered cells but did not form plaques. Group II strains had low phosphatidylcholine phospholipase C activity, whereas group III strains had low phosphatidylinositol phospholipase C activity. Several substitutions were observed for five out of six group III strains in the plcA gene and for one out of three group II strains in the plcB gene. Group IV strains poorly colonized spleens of mice and were practically indistinguishable from fully virulent strains on the basis of the above-mentioned in vitro criteria. These results demonstrate a relationship between the phenotypic classification and the genotypic modifications for at least group I and III strains and suggest a common evolution of these strains within a group. PMID:16204519

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

    PubMed

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

    2017-10-01

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

  2. Involvement of Arabidopsis thaliana phospholipase Dzeta2 in root hydrotropism through the suppression of root gravitropism.

    PubMed

    Taniguchi, Yukimi Y; Taniguchi, Masatoshi; Tsuge, Tomohiko; Oka, Atsuhiro; Aoyama, Takashi

    2010-01-01

    Root hydrotropism is the phenomenon of directional root growth toward moisture under water-deficient conditions. Although physiological and genetic studies have revealed the involvement of the root cap in the sensing of moisture gradients, and those of auxin and abscisic acid (ABA) in the signal transduction for asymmetric root elongation, the overall mechanism of root hydrotropism is still unclear. We found that the promoter activity of the Arabidopsis phospholipase Dzeta2 gene (PLDzeta2) was localized to epidermal cells in the distal root elongation zone and lateral root cap cells adjacent to them, and that exogenous ABA enhanced the activity and extended its area to the entire root cap. Although pldzeta2 mutant root caps did not exhibit a morphological phenotype in either the absence or presence of exogenous ABA, the inhibitory effect of ABA on gravitropism, which was significant in wild-type roots, was not observed in pldzeta2 mutant roots. In root hydrotropism experiments, pldzeta2 mutations significantly retarded or disturbed root hydrotropic responses. A drought condition similar to that used in a hydrotropism experiment enhanced the PLDzeta2 promoter activity in the root cap, as did exogenous ABA. These results suggest that PLDzeta2 responds to drought through ABA signaling in the root cap and accelerates root hydrotropism through the suppression of root gravitropism.

  3. The postantifungal effect and phospholipase production of oral Candida albicans from smokers, diabetics, asthmatics, denture wearers and healthy individuals following brief exposure to subtherapeutic concentrations of chlorhexidine gluconate.

    PubMed

    Ellepola, Arjuna N B; Joseph, Bobby K; Khan, Z U

    2014-09-01

    Candida albicans is the major aetiological agent of oral candidosis and one of its important virulent factors is the production of extracellular phospholipases, which can be modulated by subtherapeutic concentrations of antifungal agents thus decreasing their pathogenicity. Hence, considering that chlorhexidine gluconate (CG) is a common antimicrobial mouthwash used in dentistry and that its concentration in the mouth reaches subtherapeutic levels during dosage intervals due to the diluent effect of saliva and cleansing effect of the oral musculature, the postantifungal effect (PAFE) and the phospholipase production of oral C. albicans following brief exposure to subtherapeutic concentrations of CG was studied. Fifty C. albicans planktonic oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to three subtherapeutic concentrations of CG (0.005%, 0.0025% and 0.00125%) for 1 h. Isolates unexposed to CG was the control group. Thereafter the antiseptic was removed and the PAFE and phospholipase production was determined by a turbidometric method and a plate assay using an egg yolk agar medium respectively. Mean PAFE (hours) of 50 oral isolates of C. albicans following 1-h exposure to 0.005%, 0.0025% and 0.00125% CG was 6.97, 1.85 and 0.62 respectively. The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 21.68, 18.20 and 14.04% following exposure to 0.005%, 0.0025% and 0.00125% CG respectively. Brief exposure of C. albicans isolates to subtherapeutic concentrations of CG would wield an antifungal effect by suppressing growth and phospholipase production, thereby quelling its pathogenicity. © 2014 Blackwell Verlag GmbH.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  6. Substance P receptor desensitization requires receptor activation but not phospholipase C

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sugiya, Hiroshi; Putney, J.W. Jr.

    1988-08-01

    Previous studies have shown that exposure of parotid acinar cells to substance P at 37{degree}C results in activation of phospholipase C, formation of ({sup 3}H)inositol 1,4,5-trisphosphate (IP{sub 3}), and persistent desensitization of the substance P response. In cells treated with antimycin in medium containing glucose, ATP was decreased to {approximately}20% of control values, IP{sub 3} formation was completely inhibited, but desensitization was unaffected. When cells were treated with antimycin in the absence of glucose, cellular ATP was decreased to {approximately}5% of control values, and both IP{sub 3} formation and desensitization were blocked. A series of substance P-related peptides increased themore » formation of ({sup 3}H)IP{sub 3} and induced desensitization of the substance P response with a similar rank order of potencies. The substance P antagonist, (D-Pro{sup 2}, D-Try{sup 7,9})-substance P, inhibited substance P-induced IP{sub 3} formation and desensitization but did not induce desensitization. These results suggest that the desensitization of substance P-induced IP{sub 3} formation requires agonist activation of a P-type substance P receptor, and that one or more cellular ATP-dependent processes are required for this reaction. However, activation of phospholipase C and the generation of inositol phosphates does not seem to be a prerequisite for desensitization.« less

  7. Mineral fiber-mediated activation of phosphoinositide-specific phospholipase c in human bronchoalveolar carcinoma-derived alveolar epithelial A549 cells.

    PubMed

    Loreto, Carla; Carnazza, Maria Luisa; Cardile, Venera; Libra, Massimo; Lombardo, Laura; Malaponte, Grazia; Martinez, Giuseppina; Musumeci, Giuseppe; Papa, Veronica; Cocco, Lucio

    2009-02-01

    Given the role of phosphoinositide-specific phospholipase C (PLC) isozymes in the control of cell growth and differentiation we were prompted to analyze the expression of some of these PLC in human bronchoalveolar carcinoma-derived alveolar epithelial A549 cells. The effects of several fluoro-edenite fibers were compared with those of tremolite, a member of the calcic amphibole group of asbestos that originates from Calabria (Italy), and crocidolite, that, due to its high toxicity, is one of the most studied asbestos amphiboles. Our data show an increased expression of both PLC beta1 and PLC gamma1 in A549 cells treated with asbestos-like fibers, hinting at a role of PLC signalling in those cancerous cells.

  8. Unexpected inverse relationship between impaired glucose metabolism and lipoprotein-associated phospholipase A2 activity in patients with stable vascular disease.

    PubMed

    Mayer, Otto; Seidlerová, Jitka; Filipovský, Jan; Timoracká, Katarina; Bruthans, Jan; Vaněk, Jiří; Cerná, Lenka; Wohlfahrt, Peter; Renata, Cífková; Trefil, Ladislav

    2014-07-01

    Elevated lipoprotein-associated phospholipase A2 activity (aLp-PLA2) is associated with increased risk of cardiovascular events. In patients with stable atherovascular disease, we aimed to investigate whether impaired glucose metabolism might be associated with higher risk of elevated aLp-PLA2. We conducted a cross-sectional study in 825 stable patients after acute coronary syndrome, coronary revascularization or after first ischemic stroke (Czech part of EUROASPIRE III surveys). We measured aLp-PLA2 using diaDexus commercial kit. In multiple step-wise regression analysis, the aLp-PLA2 was significantly positively associated with male gender, current smoking, LDL cholesterol and metabolic syndrome and negatively with statin treatment, body mass index and LDL/apoB ratio. After adjustment for these confounders, we observed an inverse relationship between aLp-PLA2 and fasting glycemia [β coefficient -2.18 (p<0.0001)] or glycated hemoglobin A1c (HbA1c) [β coefficient -5.89 (p<0.0001)]. Moreover, we found a positive association between aLp-PLA2 and pancreatic β cell function [β coefficient +0.10 (p<0.0001)], but not with an insulin sensitivity. In present study, we cannot confirm any additive risk of impaired glucose metabolism in terms of increased activity of Lp-PLA2. On the contrary, presence of inadequately controlled diabetes mellitus was independently associated with lower risk of elevated aLp-PLA2 . Copyright © 2014 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

  9. Preliminary X-ray crystallographic studies of BthTX-II, a myotoxic Asp49-phospholipase A{sub 2} with low catalytic activity from Bothrops jararacussu venom

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Corrêa, L. C.; Marchi-Salvador, D. P.; Cintra, A. C. O.

    2006-08-01

    A myotoxic Asp49-PLA{sub 2} with low catalytic activity from B. jararacussu (BthTX-II) was crystallized in the monoclinic crystal system; a complete X-ray diffraction data set was collected and a molecular-replacement solution was obtained. The oligomeric structure of BthTX-II resembles those of the Asp49-PLA{sub 2} PrTX-III and all bothropic Lys49-PLA{sub 2}s. For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A{sub 2} (Asp49-PLA{sub 2}) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resemblesmore » the myotoxin Asp49-PLA{sub 2} PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA{sub 2}s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA{sub 2} from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA{sub 2}s.« less

  10. Bruton's tyrosine kinase regulates B cell antigen receptor-mediated JNK1 response through Rac1 and phospholipase C-gamma2 activation.

    PubMed

    Inabe, Kazunori; Miyawaki, Toshio; Longnecker, Richard; Matsukura, Hiroyoshi; Tsukada, Satoshi; Kurosaki, Tomohiro

    2002-03-13

    Bruton's tyrosine kinase (Btk) is essential for B cell development and B cell antigen receptor (BCR) function. Recent studies have shown that Btk plays an important role in BCR-mediated c-Jun NH(2)-terminal kinase (JNK) 1 activation; however, the mechanism by which Btk participates in the JNK1 response remains elusive. Here we show that the BCR-mediated Rac1 activation is significantly inhibited by loss of Btk, while this Rac1 activation is not affected by loss of phospholipase C-gamma2 (PLC-gamma2). Since PLC-gamma2 is also required for BCR-mediated JNK1 response, our results suggest that Btk regulates Rac1 pathway as well as PLC-gamma2 pathway, both of which contribute to the BCR-mediated JNK1 response.

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

    PubMed

    Verlotta, Angelo; Trono, Daniela

    2014-09-01

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

  12. Phospholipase C δ4 regulates cold sensitivity in mice.

    PubMed

    Yudin, Yevgen; Lutz, Brianna; Tao, Yuan-Xiang; Rohacs, Tibor

    2016-07-01

    The cold- and menthol-activated transient receptor potential melastatin 8 (TRPM8) channels are thought to be regulated by phospholipase C (PLC), but neither the specific PLC isoform nor the in vivo relevance of this regulation has been established. Here we identify PLCδ4 as the key PLC isoform involved in regulation of TRPM8 channels in vivo. We show that in small PLCδ4(-/-) TRPM8-positive dorsal root ganglion neurons cold, menthol and WS-12, a selective TRPM8 agonist, evoked significantly larger currents than in wild-type neurons, and action potential frequencies induced by menthol or by current injections were also higher in PLCδ4(-/-) neurons. PLCδ4(-/-) mice showed increased behavioural responses to evaporative cooling, and this effect was inhibited by a TRPM8 antagonist; behavioural responses to heat and mechanical stimuli were not altered. We provide evidence for the involvement of a specific PLC isoform in the regulation of cold sensitivity in mice by regulating TRPM8 activity. The transient receptor potential melastatin 8 (TRPM8) ion channel is a major sensor of environmental low temperatures. Ca(2+) -induced activation of phospholipase C (PLC) has been implied in the regulation of TRPM8 channels during menthol- and cold-induced desensitization in vitro. Here we identify PLCδ4 as the key PLC isoform involved in regulation of TRPM8 in sensory dorsal root ganglion (DRG) neurons. We identified two TRPM8-positive neuronal subpopulations, based on their cell body size. Most TRPM8-positive small neurons also responded to capsaicin, and had significantly larger menthol-induced inward current densities than medium-large cells, most of which did not respond to capsaicin. Small, but not medium-large, PLCδ4(-/-) neurons showed significantly larger currents induced by cold, menthol or WS-12, a specific TRPM8 agonist, compared to wild-type (WT) neurons, but TRPM8 protein levels were not different between the two groups. In current-clamp experiments small neurons

  13. Specific inhibition of Xenorhabdus hominickii, an entomopathogenic bacterium, against different types of host insect phospholipase A2.

    PubMed

    Sadekuzzaman, Md; Kim, Yonggyun

    2017-10-01

    Phospholipase A 2 (PLA 2 ) hydrolyzes ester bond of phospholipids at the sn-2 position to release free fatty acid and lysophospholipids. Some PLA 2 s preferentially release arachidonic acid which is subsequently oxygenated into eicosanoids to mediate immune responses in insects. Xenorhabdus hominickii is an entomopathogenic bacterium that can suppress insect immunity by inhibiting PLA 2 activity. However, little is known about target PLA 2 types inhibited by X. hominickii. Therefore, the objective of this study was to determine PLA 2 types in the host insect, Spodoptera exigua using specific inhibitors. All developmental stages of S. exigua possessed significant PLA 2 activities, with late larval stages showing relatively higher PLA 2 activities. In different larval tissues, hemocytes had higher PLA 2 activities than fat body, gut, or epidermis. Various developmental and tissue extracts exhibited differential susceptibilities to three different PLA 2 inhibitors. Late larva-to-adult stages were highly susceptible to all three different types of PLA 2 inhibitors. In contrast, extracts from egg and young larval stages were not susceptible to secretory PLA 2 (sPLA 2 ) or calcium-independent cellular PLA 2 (iPLA 2 ) inhibitors, although they were susceptible to a calcium-dependent cellular PLA 2 (cPLA 2 ) inhibitor in a dose-dependent manner. Different tissues of fifth instars exhibited variation in susceptibility to inhibitors, with epidermal tissue being sensitive to cPLA 2 inhibitor only while other tissues were sensitive to all three types of inhibitors. Bacterial challenge with heat-killed X. hominickii significantly increased PLA 2 activity. However, live bacteria suppressed the induction of PLA 2 activity. An organic extract of X. hominickii-culture broth inhibited the susceptibility of S. exigua to sPLA 2 - and iPLA 2 - specific inhibitors, but not to cPLA 2 -specific inhibitor. Oxindole, a component of the organic extract, exhibited an inhibitory pattern

  14. Human soluble phospholipase A2 receptor is an inhibitor of the integrin-mediated cell migratory response to collagen-I.

    PubMed

    Watanabe, Kazunori; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-Ei; Kugiyama, Kiyotaka

    2018-05-23

    Murine membrane-bound phospholipase A 2 receptor 1 (PLA 2 R) is shed and released into plasma in a soluble form that retains all of the extracellular domains. Relatively little is known about human PLA 2 R. This study examined whether human soluble PLA 2 R may have biological functions and whether soluble PLA 2 R may exist in human plasma. Here, we showed that human recombinant soluble PLA 2 R (rsPLA 2 R) bound to collagen-I and inhibited interaction of collagen-I with the extracellular domain of integrin β1 on the cell surface of HEK293 cells. As a result, rsPLA 2 R suppressed integrin β1-mediated migratory responses of HEK293 cells to collagen-I in Boyden chamber experiments. Inhibition of phosphorylation of FAK Tyr397 was also observed. Similar results were obtained with experiments using soluble PLA 2 R released from HEK293 cells transfected with a construct encoding human soluble PLA 2 R. rsPLA 2 R lacking the fibronectin-like type II (FNII) domain had no inhibitory effects on cell responses to collagen-I, suggesting an important role of the FNII domain in the interaction of rsPLA 2 R with collagen-I. In addition, rsPLA 2 R suppressed the migratory response to collagen-IV and binding of collagen-IV to the cell surface of human podocytes that endogenously express membrane-bound full-length PLA 2 R. Immunoprecipitation and Western blotting showed the existence of immuno-reactive PLA 2 R in human plasma. In conclusion, human recombinant soluble PLA 2 R inhibits integrin β1-mediated cell responses to collagens. Further studies are warranted to elucidate whether immuno-reactive PLA 2 R in human plasma has the same properties as rsPLA 2 R.

  15. Expression, purification, refolding, and enzymatic characterization of two secretory phospholipases A₂ from Neurospora crassa.

    PubMed

    Takayanagi, Ayumi; Miyakawa, Takuya; Asano, Atsuko; Ohtsuka, Jun; Tanokura, Masaru; Arioka, Manabu

    2015-11-01

    Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of sn-2 linkage in the glycerophospholipid, thereby releasing fatty acid and 1-acyl lysophospholipid. Among sPLA2s from various organisms and tissues, group XIV fungal/bacterial sPLA2s are relatively less characterized compared to their mammalian counterparts. Here we report cloning, recombinant expression, refolding, and enzymatic characterization of two sPLA2s, NCU06650 and NCU09423, from the filamentous fungus Neurospora crassa. The hexahistidine-tagged putative mature region of both proteins was expressed in Escherichia coli. Inclusion bodies were solubilized using a high hydrostatic pressure refolding technique. NCU06650 was solubilized without any additives at alkaline pH, and the addition of arginine or non-detergent sulfobetain (NDSB) significantly improved the process at acidic pH. In contrast, NCU09423 was solubilized only when NDSB was added at alkaline pH. Both enzymes displayed a Ca(2+)-dependent lipolytic activity toward E. coli membrane. Mass spectrometry analysis using the synthetic phospholipids as substrates demonstrated that both enzymes preferentially cleaved the sn-2 ester linkage of substrates and generated 1-acyl lysophospholipids, demonstrating that they are bona fide PLA2. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-04-01

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

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

    PubMed

    Zambelli, Vanessa O; Picolo, Gisele; Fernandes, Carlos A H; Fontes, Marcos R M; Cury, Yara

    2017-12-19

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

  18. Protective effects of TES trioleate, an inhibitor of phospholipase A2, on reactive oxygen species and UVA-induced cell damage.

    PubMed

    Park, Soo Nam; Kim, Moon Jin; Ha, Ji Hoon; Lee, Nan Hee; Park, Jino; Lee, Jiwon; Kim, Dukha; Yoon, Chulsoo

    2016-11-01

    2-[Tris(oleoyloxymethyl)methylamino]-1-ethane sulfonic acid (TES trioleate) is an inhibitor of phospholipase A 2 (PLA2), which hydrolyzes cell membrane phospholipids to produce arachidonic acid (AA) and lysophospholipids (LysoPLs). Here, we investigated the protective effects of TES trioleate on cell damage caused by ultraviolet A (UVA) light and reactive oxygen species (ROS). Pre-incubation with 250-1000μM TES trioleate resulted in concentration-dependent protection from UVA-induced damage in HaCaT cells. Additionally, 25-1000μM TES trioleate provided protection against H 2 O 2 in a concentration-dependent manner. In human erythrocytes treated with 1 O 2 , 10-100μM TES trioleate showed concentration-dependent protective effects, similar to but stronger than the controls, 4-BPB and lipophilic antioxidant (+)-α-tocopherol at 100μM. TES trioleate did not have detectable radical scavenging activity. Moreover, compared with (+)-α-tocopherol and rutin, TES trioleate showed low ROS scavenging activity. Thus, although TES trioleate showed cell protective effects against UVA, H 2 O 2 , and 1 O 2 -induced damages, these effects were not caused by the scavenging ability of the radical or ROS. Finally, pretreatment of HaCaT cells and human erythrocytes with l-α-lysophosphatidylcholine produced by PLA2 promoted increased cell damage at low concentrations. Thus, the protective effects of TES trioleate on cellular damage by UVA and ROS may be associated with inhibition of PLA2-dependent cell damage rather than ROS scavenging. Copyright © 2016. Published by Elsevier B.V.

  19. Endothelial Dysfunction in Children With Obstructive Sleep Apnea Is Associated With Elevated Lipoprotein-Associated Phospholipase A2 Plasma Activity Levels.

    PubMed

    Kheirandish-Gozal, Leila; Philby, Mona F; Qiao, Zhuanghong; Khalyfa, Abdelnaby; Gozal, David

    2017-02-09

    Obstructive sleep apnea (OSA) is a highly prevalent condition, especially in obese children, and has been associated with increased risk for endothelial dysfunction and dislipidemia, which are precursors of atherosclerosis. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is recognized as an independent risk factor for cardiovascular risk and atheromatous plaque activity. We hypothesized that Lp-PLA2 levels would be elevated in children with OSA, particularly among obese children who also manifest evidence of endothelial dysfunction. One hundred sixty children (mean age 7.1±2.3 years), either nonobese with (n=40) and without OSA (n=40) or obese with (n=40) and without OSA (n=40) underwent overnight polysomnographic and postocclusive reperfusion evaluation and a fasting blood draw the morning after the sleep study. In addition to lipid profile, Lp-PLA2 plasma activity was assessed using a commercial kit. Obese children and OSA children had significantly elevated plasma Lp-PLA2 activity levels compared to controls. Furthermore, when both obesity and OSA were concurrently present or when endothelial function was present, Lp-PLA2 activity was higher. Treatment of OSA by adenotonsillectomy resulted in reductions of Lp-PLA2 activity (n=37; P <0.001). Lp-PLA2 plasma activity is increased in pediatric OSA and obesity, particularly when endothelial dysfunction is present, and exhibits decreases on OSA treatment. The short-term and long-term significance of these findings in relation to cardiovascular risk remain undefined. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  20. A ternary metal binding site in the C2 domain of phosphoinositide-specific phospholipase C-delta1.

    PubMed

    Essen, L O; Perisic, O; Lynch, D E; Katan, M; Williams, R L

    1997-03-11

    We have determined the crystal structures of complexes of phosphoinositide-specific phospholipase C-delta1 from rat with calcium, barium, and lanthanum at 2.5-2.6 A resolution. Binding of these metal ions is observed in the active site of the catalytic TIM barrel and in the calcium binding region (CBR) of the C2 domain. The C2 domain of PLC-delta1 is a circularly permuted topological variant (P-variant) of the synaptotagmin I C2A domain (S-variant). On the basis of sequence analysis, we propose that both the S-variant and P-variant topologies are present among other C2 domains. Multiple adjacent binding sites in the C2 domain were observed for calcium and the other metal/enzyme complexes. The maximum number of binding sites observed was for the calcium analogue lanthanum. This complex shows an array-like binding of three lanthanum ions (sites I-III) in a crevice on one end of the C2 beta-sandwich. Residues involved in metal binding are contained in three loops, CBR1, CBR2, and CBR3. Sites I and II are maintained in the calcium and barium complexes, whereas sites II and III coincide with a binary calcium binding site in the C2A domain of synaptotagmin I. Several conformers for CBR1 are observed. The conformation of CBR1 does not appear to be strictly dependent on metal binding; however, metal binding may stabilize certain conformers. No significant structural changes are observed for CBR2 or CBR3. The surface of this ternary binding site provides a cluster of freely accessible liganding positions for putative phospholipid ligands of the C2 domain. It may be that the ternary metal binding site is also a feature of calcium-dependent phospholipid binding in solution. A ternary metal binding site might be a conserved feature among C2 domains that contain the critical calcium ligands in their CBR's. The high cooperativity of calcium-mediated lipid binding by C2 domains described previously is explained by this novel type of calcium binding site.

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

    PubMed

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

    2011-12-01

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

  2. Identification of a novel phospholipase D with high transphosphatidylation activity and its application in synthesis of phosphatidylserine and DHA-phosphatidylserine.

    PubMed

    Mao, Xiangzhao; Liu, Qianqian; Qiu, Yongqian; Fan, Xiaoqin; Han, Qingqing; Liu, Yanjun; Zhang, Lujia; Xue, Changhu

    2017-05-10

    Phosphatidylserine (PS) and docosahexaenoic acid-phosphatidylserine (DHA-PS) have significant nutritional and biological functions, which are extensively used in functional food industries. Phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) or DHA-PC with l-serine, is an effective method for PS and DHA-PS preparation. However, because of the hydrolysis activity of PLD, PC and DHA-PC would be converted to the undesirable byproduct, phosphatidic acid (PA) and DHA-PA. In this study, a novel phospholipase D (PLD a2 ) was firstly cloned from Acinetobacter radioresistens a2 with high transphosphatidylation activity and no hydrolysis activity. In the PLD-catalyzed synthesis process (12h), both the transphosphatidylation conversion rate and selectivity of PS and DHA-PS were about 100%, which is the only PLD enzyme reported with this superiority up till now. In comparison with the majority of other known PLDs, PLD a2 exerted the highest activity at neutral pH, and it was stable from pH 4.0 to pH 9.0. In addition, PLD a2 had excellent thermal stability, with an optimum reaction temperature of 40°C and keeping more than 80% activity from 20°C to 60°C. The high catalytic selectivity mechanism of PLD a2 was explained by utilizing homology modeling, two-step docking, and binding energy and conformation analysis. PLD a2 ensured a stable supply of the biocatalyst with its most preponderant transphosphatidylation activity and PS selectivity, and had great potential in phospholipids industrial production. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Myxococcus CsgA, Drosophila Sniffer, and human HSD10 are cardiolipin phospholipases

    PubMed Central

    Boynton, Tye O'Hara; Shimkets, Lawrence Joseph

    2015-01-01

    Myxococcus xanthus development requires CsgA, a member of the short-chain alcohol dehydrogenase (SCAD) family of proteins. We show that CsgA and SocA, a protein that can replace CsgA function in vivo, oxidize the 2′-OH glycerol moiety on cardiolipin and phosphatidylglycerol to produce diacylglycerol (DAG), dihydroxyacetone, and orthophosphate. A lipid extract enriched in DAGs from wild-type cells initiates development and lipid body production in a csgA mutant to bypass the mutational block. This novel phospholipase C-like reaction is widespread. SCADs that prevent neurodegenerative disorders, such as Drosophila Sniffer and human HSD10, oxidize cardiolipin with similar kinetic parameters. HSD10 exhibits a strong preference for cardiolipin with oxidized fatty acids. This activity is inhibited in the presence of the amyloid β peptide. Three HSD10 variants associated with neurodegenerative disorders are inactive with cardiolipin. We suggest that HSD10 protects humans from reactive oxygen species by removing damaged cardiolipin before it induces apoptosis. PMID:26338420

  4. Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A/sub 2/

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gamache, D.A.; Kornberg, L.J.; Bartolf, M.

    1986-05-01

    Incubation of cardiac sarcoplasmic reticulum with xanthine oxidase alone at pH 7.0 resulted in a loss of lipid phosphorus that was potentiated by the addition of xanthine. Using autoclaved E.coli with 1-/sup 14/C-oleate in the 2-acyl position of membrane phospholipids, the authors demonstrate that many, but not all, commercial preparations of xanthine oxidase contain significant phospholipase A/sub 2/ (PLA/sub 2/) activity (64.3-545.6 nmols/min/mg). The PLA/sub 2/ was maximally active in the neutral-alkaline pH range, was Ca/sup 2 +/-dependent, and was unaffected by the addition of xanthine. PLA/sub 2/ activity was totally inhibited by 1mM EDTA whereas radical production by optimalmore » concentrations of xanthine/xanthine oxidase (X/XO) was unaffected by EDTA. Chromatographically purified xanthine oxidase (Sigma Grade III) contained high levels of PLA/sub 2/ activity (64.3 nmols/min/mg) compared to endogenous levels of neutral-active, Ca/sup 2 +/-dependent PLA/sub 2/ measured in various tissue homogenates (less than or equal to 0.5 nmols/ min/mg). Because X/XO mixtures are used extensively to study oxygen free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular PLA/sub 2/ may have influenced previously published reports, and such studies should be interpreted cautiously.« less

  5. Crystallization and preliminary X-ray diffraction analysis of a Lys49-phospholipase A2 complexed with caffeic acid, a molecule with inhibitory properties against snake venoms

    PubMed Central

    Shimabuku, Patrícia S.; Fernandes, Carlos A. H.; Magro, Angelo J.; Costa, Tássia R.; Soares, Andreimar M.; Fontes, Marcos R. M.

    2011-01-01

    Phospholipases A2 (PLA2s) are one of the main components of bothropic venoms; in addition to their phospholipid hydrolysis action, they are involved in a wide spectrum of pharmacological activities, including neurotoxicity, myo­toxicity and cardiotoxicity. Caffeic acid is an inhibitor that is present in several plants and is employed for the treatment of ophidian envenomations in the folk medicine of many developing countries; as bothropic snake bites are not efficiently neutralized by conventional serum therapy, it may be useful as an antivenom. In this work, the cocrystallization and preliminary X-ray diffraction analysis of the Lys49-PLA2 piratoxin I from Bothrops pirajai venom in the presence of the inhibitor caffeic acid (CA) are reported. The crystals diffracted X-rays to 1.65 Å resolution and the structure was solved by molecular-replacement techniques. The electron-density map unambiguously indicated the presence of three CA molecules that interact with the C-terminus of the protein. This is the first time a ligand has been observed bound to this region and is in agreement with various experiments previously reported in the literature. PMID:21301098

  6. Correlations between endotoxin, interferon-gamma, biopterin and serum phospholipase A2-activities during lethal gram negative sepsis in rats.

    PubMed

    Hunsicker, A; Kullich, W; Weissenhofer, W; Lorenz, D; Petermann, J; Rokos, H; Schwesinger, G

    1997-05-01

    To establish a standardised reproducible animal model of intraperitoneal sepsis, and to investigate early immunoserological responses to find a mediator-based system for evaluation and grading of diffuse peritonitis in patients Prospective experimental study 4 Teaching hospitals, Germany and Austria 42 LEW. 1W rats, 12 of which acted as controls Gram negative sepsis was induced by intraperitoneal injection of 6 ml of a mixture of Escherichia coli (K1:H+) 10(10) organisms/ml, autogenous haemoglobin 2.9 ml (haemoglobin concentration 3%), 0.9% sodium chloride 3 ml, and suspension 0.1 ml. Control rats were given physiological saline 6 ml alone. Concentrations of endotoxin, interferon gamma (IFN-gamma), and biopterin, and serum phospholipase A2 (PLA2) activity. There were significant differences between the septic and control rats in concentrations of endotoxin (EU/ml) (median (interquartile range) 21.85 (2.02-159.5) compared with 0, p < 0.0001; IFN-gamma (pg/ml) 1263.0 (271.0-7575.0) compared with 101.0 (89.0-141.0), p < 0.0001; biopterin (nmol/L) 111.0 (66.4-156.3) compared with 53.7 (38.3-67.6), p < 0.001; and PLA2 (U/L) 163.0 (125.8-209.0) compared with 112.5 (88.5-126.5) p < 0.01. Measurements of concentrations of endotoxin, IFN-gamma, pteridines, and PLA2 activity may well be adequate markers for early recognition of sepsis, and perhaps for grading it during the first 6 hours after induction. The allow a clear distinction to be made between septic and non-septic disorders in 87% of cases.

  7. Refining the Value of Secretory Phospholipase A2 as a Predictor of Acute Chest Syndrome in Sickle Cell Disease: Results of a Feasibility Study (PROACTIVE)

    PubMed Central

    Styles, Lori; Wager, Carrie G.; Labotka, Richard J.; Smith-Whitley, Kim; Thompson, Alexis A.; Lane, Peter A.; McMahon, Lillian E.C; Miller, Robin; Roseff, Susan; Iyer, Rathi; Hsu, Lewis L.; Castro, Oswaldo; Ataga, Kenneth; Onyekwere, Onyinye; Okam, Maureen; Bellevue, Rita; Miller, Scott T.

    2012-01-01

    Acute chest syndrome (ACS) is defined as fever, respiratory symptoms and a new pulmonary infiltrate in an individual with sickle cell disease (SCD). Nearly half of ACS episodes occur in SCD patients already hospitalized, potentially permitting pre-emptive therapy in high-risk patients. Simple transfusion of red blood cells may abort ACS if given to patients hospitalized for pain who develop fever and elevated levels of secretory phospholipase A2 (sPLA2). In a feasibility study (PROACTIVE; ClinicalTrials.gov NCT00951808), patients hospitalized for pain who developed fever and elevated sPLA2 were eligible for randomization to transfusion or observation; all others were enrolled in an observational arm. Of 237 enrolled, only 10 were randomized; one of the four to receive transfusion had delayed treatment. Of 233 subjects receiving standard care, 22 developed ACS. A threshold level of sPLA2 ≥ 48 ng/ml gave optimal sensitivity (73%), specificity (71%) and accuracy (71%), but a positive predictive value of only 24%. The predictive value of sPLA2 was improved in adults and patients with chest or back pain, lower haemoglobin concentration and higher white blood cell counts; and those receiving less than two-thirds maintenance fluids. The hurdles identified in PROACTIVE should facilitate design of a larger, definitive, phase 3 randomized controlled trial. PMID:22463614

  8. Phosphatidylserine-specific phospholipase A1 (PS-PLA1) expression in colorectal cancer correlates with tumor invasion and hematogenous metastasis.

    PubMed

    Iida, Yuuki; Sunami, Eiji; Yamashita, Hiroharu; Hiyoshi, Masaya; Ishihara, Soichiro; Yamaguchi, Hironori; Inoue, Asuka; Makide, Kumiko; Tsuno, Nelson H; Aoki, Junken; Kitayama, Joji; Watanabe, Toshiaki

    2015-03-01

    The function of phosphatidylserine-specific phospholipase A1 (PS-PLA1), a phospholipase that acts specifically on phosphatidylserine and produces lysophosphatidylserine, a lysophospholipid mediator, has not been fully elucidated. We evaluated the role of PS-PLA1 in oncogenesis and metastasis of colorectal cancer (CRC). Specimens from 85 patients with CRC were immunostained with a monoclonal antibody against PS-PLA1. The correlation between PS-PLA1 expression and the clinicopathological variables was analyzed. Tumor depth and hematogenous metastasis independently positively correlated with PS-PLA1 expression. High PS-PLA1 expression was associated with shorter disease-free survival, although it was not an independent predictive factor. PS-PLA1 expression in CRC is associated with tumor invasion and metastasis. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  9. Evaluation of Expression of Lipases and Phospholipases of Malassezia restricta in Patients with Seborrheic Dermatitis

    PubMed Central

    Lee, Yang Won; Lee, Shin Yung; Lee, Younghoon

    2013-01-01

    Background Malassezia species (spp.) are cutaneous opportunistic pathogens and associated with various dermatological diseases including seborrheic dermatitis, dandruff and atopic dermatitis. Almost all Malassezia spp. are obligatorily lipid-dependent, which might be caused by lack of the myristic acid synthesis. Recent genome analysis of M. restricta and M. globosa suggested that the absence of a gene encoding fatty acid synthesis might be compensated by abundant genes encoding hydrolases, which produce fatty acids, and that lipases and phospholipases may play a role in virulence of the fungus. Objective The current study aimed to investigate the contribution of lipases and phospholipases in virulence of the M. restricta as being the most frequently isolated Malassezia spp. from the human skin. Methods Swap samples of two different body sites of at least 18 patients with seborrheic dermatitis were obtained and in vivo expression of lipases and phospholipases of M. restricta was analyzed by the gene specific two-step nested RT-PCR. Results The results of the current study suggest that majority of the patients display expression of lipase RES_0242. Conclusion These data imply a possible role of lipase in the host environment to produce free fatty acids for the fungus. PMID:24003273

  10. Evaluation of Expression of Lipases and Phospholipases of Malassezia restricta in Patients with Seborrheic Dermatitis.

    PubMed

    Lee, Yang Won; Lee, Shin Yung; Lee, Younghoon; Jung, Won Hee

    2013-08-01

    Malassezia species (spp.) are cutaneous opportunistic pathogens and associated with various dermatological diseases including seborrheic dermatitis, dandruff and atopic dermatitis. Almost all Malassezia spp. are obligatorily lipid-dependent, which might be caused by lack of the myristic acid synthesis. Recent genome analysis of M. restricta and M. globosa suggested that the absence of a gene encoding fatty acid synthesis might be compensated by abundant genes encoding hydrolases, which produce fatty acids, and that lipases and phospholipases may play a role in virulence of the fungus. The current study aimed to investigate the contribution of lipases and phospholipases in virulence of the M. restricta as being the most frequently isolated Malassezia spp. from the human skin. Swap samples of two different body sites of at least 18 patients with seborrheic dermatitis were obtained and in vivo expression of lipases and phospholipases of M. restricta was analyzed by the gene specific two-step nested RT-PCR. The results of the current study suggest that majority of the patients display expression of lipase RES_0242. These data imply a possible role of lipase in the host environment to produce free fatty acids for the fungus.

  11. Eastern coral snake Micrurus fulvius venom toxicity in mice is mainly determined by neurotoxic phospholipases A2.

    PubMed

    Vergara, Irene; Pedraza-Escalona, Martha; Paniagua, Dayanira; Restano-Cassulini, Rita; Zamudio, Fernando; Batista, Cesar V F; Possani, Lourival D; Alagón, Alejandro

    2014-06-13

    Here we show for the first time that the venom from an elapid (Micrurus fulvius) contains three finger toxin (3FTxs) peptides with low toxicity but high content of lethal phospholipases A2 (PLA2). The intravenous venom LD50 in mice was 0.3μg/g. Fractionation on a C18 column yielded 22 fractions; in terms of abundance, 58.3% of them were components of 13-14kDa and 24.9% were molecules of 6-7kDa. Two fractions with PLA2 activity represented 33.4% of the whole venom and were the most lethal fractions. Fractions with low molecular mass (<7000Da) partially and reversibly blocked the nicotinic acetylcholine receptor (nAChR), with the exception of one that blocked it completely. The fraction that blocked 100% contained two protein species whose dose-response was determined; the IC50s were 13±1 and 9.5±0.3nM. Despite the apparent effect on nAChR none of the low molecular mass fractions were lethal in mice, at concentrations of 1μg/g. From 2D-PAGE and LC-MS/MS, we identified fourteen species of PLA2, four protein species of C-type lectin, three zinc metalloproteinases, one phosphodiesterase and one 3FTx. The N-terminal amino acid sequence of fractions with biological interest was obtained. In contrast with coral snake venoms from South America, M. fulvius has minor amounts of low molecular mass components, but high content of PLA2, which is responsible for the venom lethality of this species. The results reported here contribute to better understanding of envenomation development and to improve antivenom design and production. These findings break from the paradigm that neurotoxicity caused by Micrurus venoms is mainly attributable to 3FTx neurotoxins and encourage future studies on Micrurus evolution and venom specialization. This article is part of a Special Issue entitled Non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Elevation of oleate-activated phospholipase D activity during thymic atrophy

    PubMed Central

    Lee, Youngkyun; Song, Soo-Mee; Park, Heung Soon; Kim, Sungyeol; Koh, Eun-Hee; Choi, Myung Sun; Choi, Myung-Un

    2002-01-01

    Various phospholipases are thought to be associated with the in vitro apoptosis of thymocytes. In the present study, the in vivo phospholipase D (PLD) activity of rat thymus was studied after whole-body X-irradiation or injection of dexamethasone (DEX). Using exogenous [14C]dipalmitoyl phosphatidylcholine (PC) as the substrate, an elevation of oleate-activated PLD activity was observed during thymic atrophy. The activity increases were sevenfold at 48 hr after 5-Gy irradiation and fourfold at 72 hr after injection of 5 mg/kg DEX. The elevation of PLD activity appeared to parallel extensive thymus shrinkage. An increased level of thymic phosphatidic acid (PA), the presumed physiological product of PLD action on PC, was also detected. By comparing the acyl chains of PA with those of other phospholipids, PA appeared to originate from PC. To assess the role of PLD during thymic atrophy, thymocytes and stromal cells were isolated. Although thymocytes themselves exhibited significant PLD activation, the major elevation in PLD activity (greater than fourfold) was found in isolated stromal cells. PLD was also activated during in vitro phagocytosis of apoptotic thymocytes by the macrophage-like cell line P388D1. This in vitro phagocytosis was significantly inhibited by PLD action blockers, such as 2,3-diphosphoglycerate and 1-butanol. These observations strongly suggest that the alteration of oleate-activated PLD activity is part of an in vivo event in the progression of thymic atrophy, including phagocytic clearance of apoptotic thymocytes. PMID:12460188

  13. Analysis of the Active-Site Mechanism of Tyrosyl-DNA Phosphodiesterase I: A Member of the Phospholipase D Superfamily

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gajewski, Stefan; Comeaux, Evan Q.; Jafari, Nauzanene

    2012-03-15

    Tyrosyl-DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily that hydrolyzes 3'-phospho-DNA adducts via two conserved catalytic histidines - one acting as the lead nucleophile and the second acting as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease spinocerebellar ataxia with axonal neuropathy (SCAN1). We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics, and theoretical chemistry. The structures of wild-type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observedmore » in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts the access of nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitutions with Asn, Gln, Leu, Ala, Ser, and Thr all result in severely compromised enzymes and DNA topoisomerase I-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate that suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pK{sub a} of this histidine is crucially dependent on the second histidine and on the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.« less

  14. Analysis of the active site mechanism of Tyrosyl-DNA phosphodiesterase I: a member of the phospholipase D superfamily

    PubMed Central

    Gajewski, Stefan; Comeaux, Evan Q.; Jafari, Nauzanene; Bharatham, Nagakumar; Bashford, Donald; White, Stephen W.; van Waardenburg, Robert C.A.M.

    2011-01-01

    Tyrosyl DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily and hydrolyzes 3′phospho-DNA adducts via two conserved catalytic histidines, one acting as the lead nucleophile and the second as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease SCAN1. We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics and theoretical chemistry. The structures of wild type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts access of a nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitution with Asn, Gln, Leu, Ala, Ser and Thr all result in severely compromised enzymes and Top1-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate which suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pKa of this histidine is crucially dependent upon the second histidine and the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily. PMID:22155078

  15. Optimization of the degumming process for camellia oil by the use of phospholipase C in pilot-scale system.

    PubMed

    Jiang, Xiaofei; Chang, Ming; Jin, Qingzhe; Wang, Xingguo

    2015-06-01

    In present study, phospholipase C (PLC) was applied in camellia oil degumming and the response surface method (RSM) was used to determine the optimum degumming conditions (reaction time, reaction temperature and enzyme dosage) for this enzyme. The optimum conditions for the minimum residual phosphorus content (15.14 mg/kg) and maximum yield of camellia oil (98.2 %) were obtained at reaction temperature 53 ºC, reaction time 2.2 h, PLC dosage 400 mg/kg and pH 5.4. The application of phospholipase A (PLA) - assisted degumming process could further reduce the residual phosphorus content of camellia oil (6.84 mg/kg) to make the oil suitable for physical refining while maintaining the maximal oil yield (98.2 %). These results indicate that PLC degumming process in combination with PLA treatment can be a commercially viable alternative for traditional degumming process. Study on the quality changes of degummed oils showed that the oxidative stability of camellia oil was slightly deceased after the enzymatic treatment, thus more attention should be paid to the oxidative stability in the further application.

  16. A phenome-wide association study of a lipoprotein-associated phospholipase A2 loss-of-function variant in 90 000 Chinese adults

    PubMed Central

    Millwood, Iona Y; Bennett, Derrick A; Walters, Robin G; Clarke, Robert; Waterworth, Dawn; Johnson, Toby; Chen, Yiping; Yang, Ling; Guo, Yu; Bian, Zheng; Hacker, Alex; Yeo, Astrid; Parish, Sarah; Hill, Michael R; Chissoe, Stephanie; Peto, Richard; Cardon, Lon; Collins, Rory; Li, Liming; Chen, Zhengming

    2016-01-01

    Background: Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been implicated in development of atherosclerosis; however, recent randomized trials of Lp-PLA2 inhibition reported no beneficial effects on vascular diseases. In East Asians, a loss-of-function variant in the PLA2G7 gene can be used to assess the effects of genetically determined lower Lp-PLA2. Methods: PLA2G7 V279F (rs76863441) was genotyped in 91 428 individuals randomly selected from the China Kadoorie Biobank of 0.5 M participants recruited in 2004–08 from 10 regions of China, with 7 years’ follow-up. Linear regression was used to assess effects of V279F on baseline traits. Logistic regression was conducted for a range of vascular and non-vascular diseases, including 41 ICD-10 coded disease categories. Results: PLA2G7 V279F frequency was 5% overall (range 3–7% by region), and 9691 (11%) participants had at least one loss-of-function variant. V279F was not associated with baseline blood pressure, adiposity, blood glucose or lung function. V279F was not associated with major vascular events [7141 events; odds ratio (OR) = 0.98 per F variant, 95% confidence interval (CI) 0.90-1.06] or other vascular outcomes, including major coronary events (922 events; 0.96, 0.79-1.18) and stroke (5967 events; 1.00, 0.92-1.09). Individuals with V279F had lower risks of diabetes (7031 events; 0.91, 0.84-0.98) and asthma (182 events; 0.53, 0.28-0.98), but there was no association after adjustment for multiple testing. Conclusions: Lifelong lower Lp-PLA2 activity was not associated with major risks of vascular or non-vascular diseases in Chinese adults. Using functional genetic variants in large-scale prospective studies with linkage to a range of health outcomes is a valuable approach to inform drug development and repositioning. PMID:27301456

  17. Molecular docking and in vitro studies of soap nut trypsin inhibitor (SNTI) against phospholipase A2 isoforms in therapeutic intervention of inflammatory diseases.

    PubMed

    Sirisha, Gandreddi V D; Vijaya Rachel, K; Zaveri, Kunal; Yarla, Nagendra Sastry; Kiranmayi, P; Ganash, Magdah; Alkreathy, Huda Mohammad; Rajeh, Nisreen; Ashraf, Ghulam Md

    2018-07-15

    Therapeutic value of allelochemicals in inflammatory disorders and the potential drug targets need to be elucidated to alleviate tissue and vascular injury. Natural anti-inflammatory agents are known to cause minimal adverse effects. Presence of different secondary metabolites (allelochemicals), protease inhibitors like soap nut trypsin inhibitor (SNTI) from Sapindus trifoliatus and allied compounds from natural sources cannot be blithely ignored as natural therapeutics. In the present study, SNTI, a prospective protease inhibitor isolated from the seeds of Sapindus trifoliatus were subjected to docking against three isoforms of Phospholipase A 2 (PLA 2 ) molecules of the inflammatory pathways which are localized in the membrane, cytosol and pancreas. Eleven ligand molecules were selected from Sapindus trifoliatus and docked against membrane, cytosolic and pancreatic PLA 2 . Cytosolic PLA 2 showed a strong inhibition by Kampferol, a secondary metabolite from seed endosperm of Sapindus trifoliatus. SNTI showed best interaction with membrane PLA 2 in both in silico as well as in in vitro studies. SNTI showed IC 50 value of 29.02 μM in in vitro assay. Docking interaction profiles and in vitro studies validate selected molecules from Sapindus trifoliatus as immunomodulators and can mollify inflammatory responses. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Different phospholipase-C-coupled receptors differentially regulate capacitative and non-capacitative Ca2+ entry in A7r5 cells

    PubMed Central

    Moneer, Zahid; Pino, Irene; Taylor, Emily J. A.; Broad, Lisa M.; Liu, Yingjie; Tovey, Stephen C.; Staali, Leila; Taylor, Colin W.

    2005-01-01

    Several receptors, including those for AVP (Arg8-vasopressin) and 5-HT (5-hydroxytryptamine), share an ability to stimulate PLC (phospholipase C) and so production of IP3 (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol) in A7r5 vascular smooth muscle cells. Our previous analysis of the effects of AVP on Ca2+ entry [Moneer, Dyer and Taylor (2003) Biochem. J. 370, 439–448] showed that arachidonic acid released from DAG stimulated NO synthase. NO then stimulated an NCCE (non-capacitative Ca2+ entry) pathway, and, via cGMP and protein kinase G, it inhibited CCE (capacitative Ca2+ entry). This reciprocal regulation ensured that, in the presence of AVP, all Ca2+ entry occurred via NCCE to be followed by a transient activation of CCE only when AVP was removed [Moneer and Taylor (2002) Biochem. J. 362, 13–21]. We confirm that, in the presence of AVP, all Ca2+ entry occurs via NCCE, but 5-HT, despite activating PLC and evoking release of Ca2+ from intracellular stores, stimulates Ca2+ entry only via CCE. We conclude that two PLC-coupled receptors differentially regulate CCE and NCCE. We also address evidence that, in some A7r5 cells lines, AVP fails either to stimulate NCCE or inhibit CCE [Brueggemann, Markun, Barakat, Chen and Byron (2005) Biochem. J. 388, 237–244]. Quantitative PCR analysis suggests that these cells predominantly express TRPC1 (transient receptor potential canonical 1), whereas cells in which AVP reciprocally regulates CCE and NCCE express a greater variety of TRPC subtypes (TRPC1=6>2>3). PMID:15918794

  19. Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets.

    PubMed Central

    Wonerow, Peter; Obergfell, Achim; Wilde, Jonathan I; Bobe, Régis; Asazuma, Naoki; Brdicka, Tomás; Leo, Albrecht; Schraven, Burkhart; Horejsí, Václav; Shattil, Sanford J; Watson, Steve P

    2002-01-01

    The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis. PMID:12049640

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

    PubMed

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

    2017-12-14

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

  1. Phospholipase PLA2G7, associated with aggressive prostate cancer, promotes prostate cancer cell migration and invasion and is inhibited by statins

    PubMed Central

    Vainio, Paula; Lehtinen, Laura; Mirtti, Tuomas; Hilvo, Mika; Seppänen-Laakso, Tuulikki; Virtanen, Johannes; Sankila, Anna; Nordling, Stig; Lundin, Johan; Rannikko, Antti; Orešič, Matej; Kallioniemi, Olli; Iljin, Kristiina

    2011-01-01

    Prostate cancer is the second leading cause of cancer mortality in men in developed countries. Due to the heterogeneous nature of the disease, design of novel personalized treatments is required to achieve efficient therapeutic responses. We have recently identified phospholipase 2 group VII (PLA2G7) as a potential drug target especially in ERG oncogene positive prostate cancers. Here, the expression profile of PLA2G7 was studied in 1137 prostate cancer and 409 adjacent non-malignant prostate tissues using immunohistochemistry to validate its biomarker potential and putative association with disease progression. In order to reveal the molecular alterations induced by PLA2G7 impairment, lipidomic and gene expression profiling was performed in response to PLA2G7 silencing in cultured prostate cancer cells. Moreover, the antineoplastic effect of statins combined with PLA2G7 impairment was studied in prostate cancer cells to evaluate the potential of repositioning of in vivo compatible drugs developed for other indications towards anti-cancer purposes. The results indicated that PLA2G7 is a cancer-selective biomarker in 50% of prostate cancers and associates with aggressive disease. The alterations induced by PLA2G7 silencing highlighted the potential of PLA2G7 inhibition as an anti-proliferative, pro-apoptotic and anti-migratorial therapeutic approach in prostate cancer. Moreover, the anti-proliferative effect of PLA2G7 silencing was potentiated by lipid-lowering statins in prostate cancer cells. Taken together, our results support the potential of PLA2G7 as a biomarker and a drug target in prostate cancer and present a rationale for combining PLA2G7 inhibition with the use of statins in prostate cancer management. PMID:22202492

  2. Kidney Transplantation in a Patient Lacking Cytosolic Phospholipase A2 Proves Renal Origins of Urinary PGI-M and TX-M.

    PubMed

    Mitchell, Jane A; Knowles, Rebecca B; Kirkby, Nicholas S; Reed, Daniel M; Edin, Matthew L; White, William E; Chan, Melissa V; Longhurst, Hilary; Yaqoob, Magdi M; Milne, Ginger L; Zeldin, Darryl C; Warner, Timothy D

    2018-02-16

    The balance between vascular prostacyclin, which is antithrombotic, and platelet thromboxane A 2 , which is prothrombotic, is fundamental to cardiovascular health. Prostacyclin and thromboxane A 2 are formed after the concerted actions of cPLA 2 α (cytosolic phospholipase A 2 ) and COX (cyclooxygenase). Urinary 2,3-dinor-6-keto-PGF 1α (PGI-M) and 11-dehydro-TXB 2 (TX-M) have been taken as biomarkers of prostacyclin and thromboxane A 2 formation within the circulation and used to explain COX biology and patient phenotypes, despite concerns that urinary PGI-M and TX-M originate in the kidney. We report data from a remarkable patient carrying an extremely rare genetic mutation in cPLA 2 α, causing almost complete loss of prostacyclin and thromboxane A 2 , who was transplanted with a normal kidney resulting in an experimental scenario of whole-body cPLA 2 α knockout, kidney-specific knockin. By studying this patient, we can determine definitively the contribution of the kidney to the productions of PGI-M and TX-M and test their validity as markers of prostacyclin and thromboxane A 2 in the circulation. Metabolites were measured using liquid chromatography-tandem mass spectrometry. Endothelial cells were grown from blood progenitors. Before kidney transplantation, the patient's endothelial cells and platelets released negligible levels of prostacyclin (measured as 6-keto-prostaglandin F 1α ) and thromboxane A 2 (measured as TXB 2 ), respectively. Likewise, the urinary levels of PGI-M and TX-M were very low. After transplantation and the establishment of normal renal function, the levels of PGI-M and TX-M in the patient's urine rose to within normal ranges, whereas endothelial production of prostacyclin and platelet production of thromboxane A 2 remained negligible. These data show that PGI-M and TX-M can be derived exclusively from the kidney without contribution from prostacyclin made by endothelial cells or thromboxane A 2 by platelets in the general circulation

  3. Identification of a new phospholipase D in Carica papaya latex.

    PubMed

    Abdelkafi, Slim; Abousalham, Abdelkarim; Fendri, Imen; Ogata, Hiroyuki; Barouh, Nathalie; Fouquet, Benjamin; Scheirlinckx, Frantz; Villeneuve, Pierre; Carrière, Frédéric

    2012-05-15

    Phospholipase D (PLD) is a lipolytic enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. It catalyzes the hydrolysis and transphosphatidylation of glycerophospholipids at the terminal phosphodiester bond. The presence of a PLD in the latex of Carica papaya (CpPLD1) was demonstrated by transphosphatidylation of phosphatidylcholine (PtdCho) in the presence of 2% ethanol. Although the protein could not be purified to homogeneity due to its presence in high molecular mass aggregates, a protein band was separated by SDS-PAGE after SDS/chloroform-methanol/TCA-acetone extraction of the latex insoluble fraction. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by micro-LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (723 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 2424 bp encoding a protein of 808 amino acid residues, with a theoretical molecular mass of 92.05 kDa. From sequence analysis, CpPLD1 was identified as a PLD belonging to the plant phosphatidylcholine phosphatidohydrolase family. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. A Cell-Permeable Phospholipase C[gamma]1-Binding Peptide Transduces Neurons and Impairs Long-Term Spatial Memory

    ERIC Educational Resources Information Center

    Blum, Sonja; Dash, Pramod K.

    2004-01-01

    Growth factor-mediated signaling has emerged as an essential component of memory formation. In this study, we used a phospholipase C gamma 1 (PLC[gamma]1) binding, cell-penetrating peptide to sequester PLC[gamma]1 away from its target, the phosphotyrosine residues within the activated growth factor receptor. Peptides appear to transduce neurons…

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

    PubMed

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

    2014-10-01

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

  6. Plant phospholipase C family: Regulation and functional role in lipid signaling.

    PubMed

    Singh, Amarjeet; Bhatnagar, Nikita; Pandey, Amita; Pandey, Girdhar K

    2015-08-01

    Phospholipase C (PLC), a major membrane phospholipid hydrolyzing enzyme generates signaling messengers such as diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) in animals, and their phosphorylated forms such as phosphatidic acid (PA) and inositol hexakisphosphate (IP6) are thought to regulate various cellular processes in plants. Based on substrate specificity, plant PLC family is sub-divided into phosphatidylinositol-PLC (PI-PLC) and phosphatidylcholine-PLC (PC-PLC) groups. The activity of plant PLCs is regulated by various factors and the major ones include, Ca(2+) concentration, phospholipid substrate, post-translational modifications and interacting proteins. Most of the PLC members have been localized at the plasma membrane, suited for their function of membrane lipid hydrolysis. Several PLC members have been implicated in various cellular processes and signaling networks, triggered in response to a number of environmental cues and developmental events in different plant species, which makes them potential candidates for genetically engineering the crop plants for stress tolerance and enhancing the crop productivity. In this review article, we are focusing mainly on the plant PLC signaling and regulation, potential cellular and physiological role in different abiotic and biotic stresses, nutrient deficiency, growth and development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Myxococcus CsgA, Drosophila Sniffer, and human HSD10 are cardiolipin phospholipases.

    PubMed

    Boynton, Tye O'Hara; Shimkets, Lawrence Joseph

    2015-09-15

    Myxococcus xanthus development requires CsgA, a member of the short-chain alcohol dehydrogenase (SCAD) family of proteins. We show that CsgA and SocA, a protein that can replace CsgA function in vivo, oxidize the 2'-OH glycerol moiety on cardiolipin and phosphatidylglycerol to produce diacylglycerol (DAG), dihydroxyacetone, and orthophosphate. A lipid extract enriched in DAGs from wild-type cells initiates development and lipid body production in a csgA mutant to bypass the mutational block. This novel phospholipase C-like reaction is widespread. SCADs that prevent neurodegenerative disorders, such as Drosophila Sniffer and human HSD10, oxidize cardiolipin with similar kinetic parameters. HSD10 exhibits a strong preference for cardiolipin with oxidized fatty acids. This activity is inhibited in the presence of the amyloid β peptide. Three HSD10 variants associated with neurodegenerative disorders are inactive with cardiolipin. We suggest that HSD10 protects humans from reactive oxygen species by removing damaged cardiolipin before it induces apoptosis. © 2015 Boynton and Shimkets; Published by Cold Spring Harbor Laboratory Press.

  8. Phospholipase C-β in Immune Cells

    PubMed Central

    Kawakami, Toshiaki; Xiao, Wenbin

    2013-01-01

    Great progress has recently been made in structural and functional research of phospholipase C (PLC)-β. We now understand how PLC-β isoforms (β1-β4) are activated by GTP-bound Gαq downstream of G protein-coupled receptors. Numerous studies indicate that PLC-βs participate in the differentiation and activation of immune cells that control both the innate and adaptive immune systems. The PLC-β3 isoform also interplays with tyrosine kinase-based signaling pathways, to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1, with which PLC-β3 and Stat5 form a multi-molecular signaling platform, named SPS complex. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation. PMID:23981313

  9. Effect of perzinfotel and a proprietary phospholipase A(2) inhibitor on kinetic gait and subjective lameness scores in dogs with sodium urate-induced synovitis.

    PubMed

    Budsberg, Steven C; Torres, Bryan T; Zwijnenberg, Raphael J; Eppler, C Mark; Clark, James D; Cathcart, Curtis J; Reynolds, Lisa R; Al-Nadaf, Sami

    2011-06-01

    To investigate the ability of perzinfotel (an N-methyl-d-aspartate receptor antagonist) and a proprietary phospholipase A(2) (PLA(2)) inhibitor to attenuate lameness in dogs with sodium urate (SU)-induced synovitis. 8 adult dogs. A blinded 4-way crossover study was performed. Dogs received perzinfotel (10 mg/kg), a proprietary PLA(2) inhibitor (10 mg/kg), carprofen (4.4 mg/kg; positive control treatment), or no treatment (negative control treatment). On the fourth day after initiation of treatment, synovitis was induced via intra-articular injection of SU 1 hour before administration of the last treatment dose. Ground reaction forces were measured and clinical lameness evaluations were performed before (baseline [time 0]) and 2, 4, 6, 8, 12, and 25 hours after SU injection. There was a 21-day washout period between subsequent treatments. Data were analyzed via repeated-measures ANOVAs. Peak vertical force (PVF) and vertical impulse (VI) values for negative control and perzinfotel treatments were significantly lower at 2 and 4 hours, compared with baseline values. Values for PVF and VI for the PLA(2) inhibitor and positive control treatments did not differ from baseline values at any time points. Between-treatment comparisons revealed significantly higher PVF and VI values for the positive control treatment than for the negative control and perzinfotel treatments at 2 and 4 hours. Values for VI were higher for PLA(2) inhibitor treatment than for negative control treatment at 2 hours. Perzinfotel did not significantly alter SU-induced lameness. The proprietary PLA(2) inhibitor attenuated lameness but not as completely as did carprofen.

  10. Mitochondria-localized phospholipase A2, AoPlaA, in Aspergillus oryzae displays phosphatidylethanolamine-specific activity and is involved in the maintenance of mitochondrial phospholipid composition.

    PubMed

    Kotani, Shohei; Izawa, Sho; Komai, Noriyuki; Takayanagi, Ayumi; Arioka, Manabu

    2016-11-01

    In mammals, cytosolic phospholipases A 2 (cPLA 2 s) play important physiological roles by releasing arachidonic acid, a precursor for bioactive lipid mediators, from the biological membranes. In contrast, fungal cPLA 2 -like proteins are much less characterized and their roles have remained elusive. AoPlaA is a cPLA 2 -like protein in the filamentous fungus Aspergillus oryzae which, unlike mammalian cPLA 2 , localizes to mitochondria. In this study, we investigated the biochemical and physiological functions of AoPlaA. Recombinant AoPlaA produced in E. coli displayed Ca 2+ -independent lipolytic activity. Mass spectrometry analysis demonstrated that AoPlaA displayed PLA 2 activity to phosphatidylethanolamine (PE), but not to other phospholipids, and generated 1-acylated lysoPE. Catalytic site mutants of AoPlaA displayed almost no or largely reduced activity to PE. Consistent with PE-specific activity of AoPlaA, AoplaA-overexpressing strain showed decreased PE content in the mitochondrial fraction. In contrast, AoplaA-disruption strain displayed increased content of cardiolipin. AoplaA-overexpressing strain, but not its counterparts overexpressing the catalytic site mutants, exhibited retarded growth at low temperature, possibly because of the impairment of the mitochondrial function caused by excess degradation of PE. These results suggest that AoPlaA is a novel PE-specific PLA 2 that plays a regulatory role in the maintenance of mitochondrial phospholipid composition. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Regulation and physiological functions of mammalian phospholipase C.

    PubMed

    Nakamura, Yoshikazu; Fukami, Kiyoko

    2017-04-01

    Phospholipase C (PLC) is a key enzyme in phosphoinositide metabolism. PLC hydrolyses phosphatidylinositol 4,5-bis-phosphate to generate two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol, that generate diverse cellular responses. PLC is activated by various signalling molecules, including Ca2+, heterometric G proteins, small G proteins, and receptor/non-receptor tyrosine kinases. In addition to their enzymatic activity, some PLC subtypes also function as a guanine nucleotide exchange factor, GTPase-activating protein, and adaptor protein, independent of their lipase activity. There are 13 PLC isozymes in mammals, and they are categorized into six classes based on structure. Generation and analysis of genetically modified mice has revealed the unexpectedly diverse physiological functions of PLC isozymes. Although all PLC isozymes catalyze the same reaction, each PLC isozyme has unique physiological functions. This review focuses on the regulation and physiological functions of PLCs. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  12. Tissue-specific expression of the gene coding for human Clara cell 10-kD protein, a phospholipase A2-inhibitory protein.

    PubMed Central

    Peri, A; Cordella-Miele, E; Miele, L; Mukherjee, A B

    1993-01-01

    Clara cell 10-kD protein (cc10kD), a secretory phospholipase A2 inhibitor, is suggested to be the human counterpart of rabbit uteroglobin (UG). Because cc10kD is expressed constitutively at a very high level in the human respiratory epithelium, the 5' region of its gene may be useful in achieving organ-specific expression of recombinant DNA in gene therapy of diseases such as cystic fibrosis. However, it is important to establish the tissue-specific expression of this gene before designing gene transfer experiments. Since the UG gene in the rabbit is expressed in many other organs besides the lung and the endometrium, we investigated the organ and tissue specificity of human cc10kD gene expression using polymerase chain reaction, nucleotide sequence analysis, immunofluorescence, and Northern blotting. Our results indicate that, in addition to the lung, cc10kD is expressed in several nonrespiratory organs, with a distribution pattern very similar, if not identical, to that of UG in the rabbit. These results underscore the necessity for more detailed analyses of the 5' region of the human cc10kD gene before its usefulness in gene therapy could be fully assessed. These data also suggest that cc10kD and UG may have similar physiological function(s). Images PMID:8227325

  13. Pharmacologic inhibition of phospholipase C in the brain attenuates early memory formation in the honeybee (Apis mellifera L.)

    PubMed Central

    Iino, Shiori; Kubo, Takeo

    2018-01-01

    ABSTRACT Although the molecular mechanisms involved in learning and memory in insects have been studied intensively, the intracellular signaling mechanisms involved in early memory formation are not fully understood. We previously demonstrated that phospholipase C epsilon (PLCe), whose product is involved in calcium signaling, is almost selectively expressed in the mushroom bodies, a brain structure important for learning and memory in the honeybee. Here, we pharmacologically examined the role of phospholipase C (PLC) in learning and memory in the honeybee. First, we identified four genes for PLC subtypes in the honeybee genome database. Quantitative reverse transcription-polymerase chain reaction revealed that, among these four genes, three, including PLCe, were expressed higher in the brain than in sensory organs in worker honeybees, suggesting their main roles in the brain. Edelfosine and neomycin, pan-PLC inhibitors, significantly decreased PLC activities in homogenates of the brain tissues. These drugs injected into the head of foragers significantly attenuated memory acquisition in comparison with the control groups, whereas memory retention was not affected. These findings suggest that PLC in the brain is involved in early memory formation in the honeybee. To our knowledge, this is the first report of a role for PLC in learning and memory in an insect. PMID:29330349

  14. Families of phosphoinositide-specific phospholipase C: structure and function.

    PubMed

    Katan, M

    1998-12-08

    A large number of extracellular signals stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). PI-PLC isozymes have been found in a broad spectrum of organisms and although they have common catalytic properties, their regulation involves different signalling pathways. A number of recent studies provided an insight into domain organisation of PI-PLC isozymes and contributed towards better understanding of the structural basis for catalysis, cellular localisation and molecular changes that could underlie the process of their activation.

  15. Exclusion of phospholipases (PLs)-producing bacteria in raw milk flushed with nitrogen gas (N(2)).

    PubMed

    Munsch-Alatossava, Patricia; Gursoy, Oguz; Alatossava, Tapani

    2010-01-01

    Prolonged cold storage of raw milks favors the growth of psychrotrophs, which produce heat-resistant exoenzymes of considerable spoilage potential; the bacterial proteases and lipases affect raw milk quality; among them phospholipases (PLs) may target the milk fat globule. More importantly, bacterial PLs are key virulence factors for numerous species. Two studies examined the use of nitrogen (N(2)) gas and examined its effect on psychrotrophs, proteases and lipase producers when the milk was stored in closed vessels; however, the effect on PLs producers is unknown. Here we show that by considering an open system the PLs producers were sooner or later excluded in raw milk (whereas the PLs producers in the non-treated controls culminated at 10(8)CFU/ml), by effective gas treatments that bring oxygen (O(2)) levels in milk lower than 0.1ppm. No increase of the PLs producers among the anaerobes was noticed during the course of the experiments. In the experiments performed at 6.0 degrees C, the delay after which the PLs producers were no longer detectable seemed independent of the initial level of PLs producers in raw milk (lower than 10(3)CFU/ml). We anticipate that flushing pure N(2) gas in raw milk tanks, considered as open systems, along the cold chain of raw milk storage and transportation, may be an additional technique to control psychrotrophs, and may also constitute an interesting perspective for limiting their spoilage and pathogenic potential in food materials in general.

  16. Genotype × Adiposity Interaction Linkage Analyses Reveal a Locus on Chromosome 1 for Lipoprotein-Associated Phospholipase A2, a Marker of Inflammation and Oxidative Stress

    PubMed Central

    Diego, Vincent P.; Rainwater, David L.; Wang, Xing-Li; Cole, Shelley A.; Curran, Joanne E.; Johnson, Matthew P.; Jowett, Jeremy B. M.; Dyer, Thomas D.; Williams, Jeff T.; Moses, Eric K.; Comuzzie, Anthony G.; MacCluer, Jean W.; Mahaney, Michael C.; Blangero, John

    2007-01-01

    Because obesity leads to a state of chronic, low-grade inflammation and oxidative stress, we hypothesized that the contribution of genes to variation in a biomarker of these two processes may be influenced by the degree of adiposity. We tested this hypothesis using samples from the San Antonio Family Heart Study that were assayed for activity of lipoprotein-associated phospholipase A2 (Lp-PLA2), a marker of inflammation and oxidative stress. Using an approach to model discrete genotype×environment (G×E) interaction, we assigned individuals to one of two discrete diagnostic states (or “adiposity environments”): nonobese or obese, according to criteria suggested by the World Health Organization. We found a genomewide maximum LOD of 3.39 at 153 cM on chromosome 1 for Lp-PLA2. Significant G×E interaction for Lp-PLA2 at the genomewide maximum (P=1.16×10-4) was also found. Microarray gene-expression data were analyzed within the 1-LOD interval of the linkage signal on chromosome 1. We found two transcripts—namely, for Fc gamma receptor IIA and heat-shock protein (70 kDa)—that were significantly associated with Lp-PLA2 (P<.001 for both) and showed evidence of cis-regulation with nominal LOD scores of 2.75 and 13.82, respectively. It would seem that there is a significant genetic response to the adiposity environment in this marker of inflammation and oxidative stress. Additionally, we conclude that G×E interaction analyses can improve our ability to identify and localize quantitative-trait loci. PMID:17160904

  17. Melatonin Promotes Brain-Derived Neurotrophic Factor (BDNF) Expression and Anti-Apoptotic Effects in Neonatal Hemolytic Hyperbilirubinemia via a Phospholipase (PLC)-Mediated Mechanism

    PubMed Central

    Luo, Yong; Peng, Mei; Wei, Hong

    2017-01-01

    Background Melatonin therapy shows positive effects on neuroprotective factor brain-derived neurotrophic factor (BDNF) expression and neuronal apoptosis in neonatal hemolytic hyperbilirubinemia. We hypothesized that melatonin promotes BDNF expression and anti-apoptotic effects in neonatal hemolytic hyperbilirubinemia through a phospholipase (PLC)-mediated mechanism. Material/Methods A phenylhydrazine hydrochloride (PHZ)-induced neonatal hemolytic hyperbilirubinemia model was constructed in neonatal rats. Four experimental groupsa control group (n=30), a PHZ group (n=30), a PHZ + melatonin group (n=30), and a PHZ + melatonin+U73122 (a PLC inhibitor) group (n=30) – were constructed. Trunk blood was assayed for serum hemoglobin, hematocrit, total and direct bilirubin, BDNF, S100B, and tau protein levels. Brain tissue levels of neuronal apoptosis, BDNF expression, PLC activity, IP3 content, phospho- and total Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV) expression, and phospho- and total cAMP response element binding protein (CREB) expression were also assayed. Results PHZ-induced hemolytic hyperbilirubinemia was validated by significantly decreased serum hemoglobin and hematocrit as well as significantly increased total and direct serum bilirubin (p<0.05). Neonatal bilirubin-induced neurotoxicity was validated by significantly decreased serum BDNF, brain BDNF, and serum S100B, along with significantly increased serum tau protein (p<0.05). PHZ-induced hemolytic hyperbilirubinemia significantly decreased serum BDNF, brain BDNF, and PLC/IP3/Ca2+ pathway activation while increasing neuronal apoptosis levels (p<0.05), all of which were partially rescued by melatonin therapy (p<0.05). Pre-treatment with the PLC inhibitor U73122 largely abolished the positive effects of melatonin on PLC/IP3/Ca2+ pathway activation, downstream BDNF levels, and neuronal apoptosis (p<0.05). Conclusions Promotion of BDNF expression and anti-apoptotic effects in neonatal

  18. Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.

    PubMed Central

    Benistant, C; Rubin, R

    1990-01-01

    Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization. Images p495-a PMID:2117442

  19. Triterpene derivatives as inhibitors of protein involved in the inflammatory process: molecules interfering with phospholipase A2, cycloxygenase, and lipoxygenase.

    PubMed

    Braca, Alessandra; Dal Piaz, Fabrizio; Marzocco, Stefania; Autore, Giuseppina; Vassallo, Antonio; De Tommasi, Nunziatina

    2011-03-01

    Over the past years, there was an explosion in the knowledge of the protein target and molecular mechanism associated with various disease types and in the new research of drugs of natural origin. The key idea is to evaluate bioactive natural products interacting with protein domains of different genetic origin but structurally preserved to develop libraries of compounds biologically validated and selected from an evolutionistic point of view. Compared with synthetic compounds, natural products have a major number of unused scaffolds and not comparable to the libraries of synthetic compounds, and could represent a promising starting points for the discovery of new bioactive compounds. Many natural products are reported to interact with proteins involved in serious diseases, such as inflammation and cancer. Recently various chemical classes of plant secondary metabolites have emerged as potential therapeutic compounds in several inflammatory diseases. Owing to the findings that triterpenoids, a common class of plant secondary metabolites, have anti-inflammatory and anti-cancer effects on humans, the interest in their potential application in human health and disease is increasing. The present review describes anti-inflammatory triterpenes derivatives from plant and fungi reported during the last two decades in order to provide an account of this field of investigation, sorting compounds according to their targets, phospholipase A(2) (PLA(2)), cycloxygenase (COX), and lipoxygenase (LOX). The attempt is also being made to enumerate the possible leads for further synthetic and drug discovery program development.

  20. Phospholipase C-β in immune cells.

    PubMed

    Kawakami, Toshiaki; Xiao, Wenbin

    2013-09-01

    Great progress has recently been made in structural and functional research of phospholipase C (PLC)-β. We now understand how PLC-β isoforms (β1-β4) are activated by GTP-bound Gαq downstream of G protein-coupled receptors. Numerous studies indicate that PLC-βs participate in the differentiation and activation of immune cells that control both the innate and adaptive immune systems. The PLC-β3 isoform also interplays with tyrosine kinase-based signaling pathways, to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1, with which PLC-β3 and Stat5 form a multi-molecular signaling platform, named SPS complex. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Substrate Efflux Propensity Is the Key Determinant of Ca2+-independent Phospholipase A-β (iPLAβ)-mediated Glycerophospholipid Hydrolysis*

    PubMed Central

    Batchu, Krishna Chaithanya; Hokynar, Kati; Jeltsch, Michael; Mattonet, Kenny; Somerharju, Pentti

    2015-01-01

    The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca2+-independent PLA-β (iPLAβ) in particular has been implicated in this essential process. However, the regulation of this enzyme, which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory (nonhomeostatic) PLAs. To study whether this is the case with iPLAβ as well, a mass spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation, and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn-1 and sn-2 acyl-binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLAβ-mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated. PMID:25713085

  2. Phosphoinositide-specific phospholipase C in oat roots: association with the actin cytoskeleton.

    PubMed

    Huang, Chiung-Hua; Crain, Richard C

    2009-10-01

    Phosphoinositide-specific phospholipase C (PI-PLC) activities are involved in mediating plant cell responses to environmental stimuli. Two variants of PI-PLC have been partially purified from the roots of oat seedlings; one cytosolic and one particulate. Although the cytosolic enzyme was significantly purified, the activity still co-migrated with a number of other proteins on heparin HPLC and also on size-exclusion chromatography. The partially purified PI-PLC was tested by Western blotting, and we found that actin and actin-binding proteins, profilin and tropomyosin, co-purified with cytosolic phospholipase C. After a non-ionic detergent (Triton X-100) treatment, PI-PLC activities still remained with the actin cytoskeleton. The effects of phalloidin and F-buffer confirmed this association; these conditions, which favor actin polymerization, decreased the release of PI-PLC from the cytoskeleton. The treatments of latrunculin and G-buffer, the conditions that favor actin depolymerization, increased the release of PI-PLC from the cytoskeleton. These results suggest that oat PI-PLC associates with the actin cytoskeleton.

  3. Draft Genome Sequence of Caenibacillus caldisaponilyticus B157T, a Thermophilic and Phospholipase-Producing Bacterium Isolated from Acidulocompost

    PubMed Central

    Tsujimoto, Yoshiyuki; Saito, Ryo; Sahara, Takehiko; Kimura, Nobutada; Tsuruoka, Naoki; Shigeri, Yasushi

    2017-01-01

    ABSTRACT Caenibacillus caldisaponilyticus B157T (= NBRC 111400T = DSM 101100T), in the family Sporolactobacillaceae, was isolated from acidulocompost as a thermophilic and phospholipid-degrading bacterium. Here, we report the 3.36-Mb draft genome sequence, with a G+C content of 51.8%, to provide the genetic information coding for phospholipases. PMID:28360164

  4. The Phospholipase C Isozymes and Their Regulation

    PubMed Central

    Gresset, Aurelie; Sondek, John

    2013-01-01

    The physiological effects of many extracellular neurotransmitters, hormones, growth factors, and other stimuli are mediated by receptor-promoted activation of phospholipase C (PLC) and consequential activation of inositol lipid signaling pathways. These signaling responses include the classically described conversion of phosphatidylinositol(4,5)P2 to the Ca2+-mobilizing second messenger inositol(1,4,5)P3 and the protein kinase C-activating second messenger diacylglycerol as well as alterations in membrane association or activity of many proteins that harbor phosphoinositide binding domains. The 13 mammalian PLCs elaborate a minimal catalytic core typified by PLC-δ to confer multiple modes of regulation of lipase activity. PLC-β isozymes are activated by Gαq- and Gβγ-subunits of heterotrimeric G proteins, and activation of PLC-γ isozymes occurs through phosphorylation promoted by receptor and non-receptor tyrosine kinases. PLC-ε and certain members of the PLC-β and PLC-γ subclasses of isozymes are activated by direct binding of small G proteins of the Ras, Rho, and Rac subfamilies of GTPases. Recent high resolution three dimensional structures together with biochemical studies have illustrated that the X/Y linker region of the catalytic core mediates autoinhibition of most if not all PLC isozymes. Activation occurs as a consequence of removal of this autoinhibition. PMID:22403074

  5. Costimulation of AMPA and metabotropic glutamate receptors underlies phospholipase C activation by glutamate in hippocampus.

    PubMed

    Kim, Hye-Hyun; Lee, Kyu-Hee; Lee, Doyun; Han, Young-Eun; Lee, Suk-Ho; Sohn, Jong-Woo; Ho, Won-Kyung

    2015-04-22

    Glutamate, a major neurotransmitter in the brain, activates ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs, respectively). The two types of glutamate receptors interact with each other, as exemplified by the modulation of iGluRs by mGluRs. However, the other way of interaction (i.e., modulation of mGluRs by iGluRs) has not received much attention. In this study, we found that group I mGluR-specific agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) alone is not sufficient to activate phospholipase C (PLC) in rat hippocampus, while glutamate robustly activates PLC. These results suggested that additional mechanisms provided by iGluRs are involved in group I mGluR-mediated PLC activation. A series of experiments demonstrated that glutamate-induced PLC activation is mediated by mGluR5 and is facilitated by local Ca(2+) signals that are induced by AMPA-mediated depolarization and L-type Ca(2+) channel activation. Finally, we found that PLC and L-type Ca(2+) channels are involved in hippocampal mGluR-dependent long-term depression (mGluR-LTD) induced by paired-pulse low-frequency stimulation, but not in DHPG-induced chemical LTD. Together, we propose that AMPA receptors initiate Ca(2+) influx via the L-type Ca(2+) channels that facilitate mGluR5-PLC signaling cascades, which underlie mGluR-LTD in rat hippocampus. Copyright © 2015 the authors 0270-6474/15/356401-12$15.00/0.

  6. Differential routes of Ca2+ influx in Swiss 3T3 fibroblasts in response to receptor stimulation.

    PubMed Central

    Miyakawa, T; Kojima, M; Ui, M

    1998-01-01

    Ca2+ influx into cells in response to stimulation of various receptors was studied with Swiss 3T3 fibroblasts. The mechanisms involved were found to be so diverse that they were classified into four groups, Type I to IV. Type-I influx occurred, via pertussis toxin-susceptible G-proteins, immediately after internal Ca2+ mobilization by bradykinin, thrombin, endothelin, vasopressin or angiotensin II. Type-II influx induced by bombesin differed from Type I in its insusceptibility to pertussis toxin treatment. Ca2+ influx induced by prostaglandin E1, referred to as Type-III influx, was unique in that phospholipase C was apparently not activated without extracellular Ca2+, strongly suggesting that the Ca2+ influx preceded and was responsible for InsP3 generation and internal Ca2+ mobilization. More Ca2+ entered the cells more slowly via the Type-IV route opened by platelet-derived and other growth factors. These types of Ca2+ influx could be differentiated by their different susceptibilities to protein kinase C maximally activated by 1 h of exposure of cells to PMA, which inhibited phospholipase Cbeta coupled to receptors involved in Type-I and -II influx but did not inhibit growth-factor-receptor-coupled phospholipase Cgamma. Type-I and -II Ca2+ influxes, together with store-operated influx induced by thapsigargin, were not directly inhibited by exposure of cells to PMA, but Type-III and -IV influxes were completely inhibited. In addition, stimulation of receptors involved in Type-I and -IV Ca2+ influx, but not Type-II and -III influx, led to phospholipase A2 activation in the presence of extracellular Ca2+. Inhibition of Type-I and -IV Ca2+ influxes by their respective inhibitors, diltiazem and nifedipine, resulted in abolition of phospholipase A2 activation induced by the respective receptor agonists, in agreement with the notion that Ca2+ influx via these routes is responsible for receptor-mediated phospholipase A2 activation. PMID:9405282

  7. Differential routes of Ca2+ influx in Swiss 3T3 fibroblasts in response to receptor stimulation.

    PubMed

    Miyakawa, T; Kojima, M; Ui, M

    1998-01-01

    Ca2+ influx into cells in response to stimulation of various receptors was studied with Swiss 3T3 fibroblasts. The mechanisms involved were found to be so diverse that they were classified into four groups, Type I to IV. Type-I influx occurred, via pertussis toxin-susceptible G-proteins, immediately after internal Ca2+ mobilization by bradykinin, thrombin, endothelin, vasopressin or angiotensin II. Type-II influx induced by bombesin differed from Type I in its insusceptibility to pertussis toxin treatment. Ca2+ influx induced by prostaglandin E1, referred to as Type-III influx, was unique in that phospholipase C was apparently not activated without extracellular Ca2+, strongly suggesting that the Ca2+ influx preceded and was responsible for InsP3 generation and internal Ca2+ mobilization. More Ca2+ entered the cells more slowly via the Type-IV route opened by platelet-derived and other growth factors. These types of Ca2+ influx could be differentiated by their different susceptibilities to protein kinase C maximally activated by 1 h of exposure of cells to PMA, which inhibited phospholipase Cbeta coupled to receptors involved in Type-I and -II influx but did not inhibit growth-factor-receptor-coupled phospholipase Cgamma. Type-I and -II Ca2+ influxes, together with store-operated influx induced by thapsigargin, were not directly inhibited by exposure of cells to PMA, but Type-III and -IV influxes were completely inhibited. In addition, stimulation of receptors involved in Type-I and -IV Ca2+ influx, but not Type-II and -III influx, led to phospholipase A2 activation in the presence of extracellular Ca2+. Inhibition of Type-I and -IV Ca2+ influxes by their respective inhibitors, diltiazem and nifedipine, resulted in abolition of phospholipase A2 activation induced by the respective receptor agonists, in agreement with the notion that Ca2+ influx via these routes is responsible for receptor-mediated phospholipase A2 activation.

  8. Role of protein kinase C alpha in endothelin-1 stimulation of cytosolic phospholipase A2 and arachidonic acid release in cultured cat iris sphincter smooth muscle cells.

    PubMed

    Husain, S; Abdel-Latif, A A

    1998-05-20

    We have investigated the role and mechanism of protein kinase C (PKC) isoforms in endothelin-1 (ET-1)-induced arachidonic acid (AA) release in cat iris sphincter smooth muscle (CISM) cells. ET-1 increased AA release in a concentration (EC50=8 nM) and time-dependent (t1/2=1.2 min) manner. Cytosolic phospholipase A2 (cPLA2), but not phospholipase C (PLC), is involved in the liberation of AA in the stimulated cells. This conclusion is supported by the findings that ET-1-induced AA release is inhibited by AACOCF3, quinacrine and manoalide, PLA2 inhibitors, but not by U-73122, a PLC inhibitor, or by RHC-80267, a diacylglycerol lipase inhibitor. A role for PKC in ET-1-induced AA release is supported by the findings that the phorbol ester, PDBu, increased AA release by 96%, that prolonged treatment of the cells with PDBu resulted in the selective down regulation of PKCalpha and the complete inhibition of ET-1-induced AA release, and that pretreatment of the cells with staurosporine or RO 31-8220, PKC inhibitors, blocked the ET-1-induced AA release. Gö-6976, a compound that inhibits PKCalpha and beta specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC50 value of 8 nM. Thymeatoxin (0.1 microM), a specific activator of PKCalpha, beta, and gamma induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKCalpha, but not PKCbeta, from cytosol to the particulate fraction. These results suggest that PKCalpha plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA2, p42mapk and p44mapk in the CISM cells. The data presented are consistent with a role for PKCalpha, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA2 activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA2 phosphorylation and cPLA2 activity, but had no inhibitory

  9. Trifluorothymidine resistance is associated with decreased thymidine kinase and equilibrative nucleoside transporter expression or increased secretory phospholipase A2.

    PubMed

    Temmink, Olaf H; Bijnsdorp, Irene V; Prins, Henk-Jan; Losekoot, Nienke; Adema, Auke D; Smid, Kees; Honeywell, Richard J; Ylstra, Bauke; Eijk, Paul P; Fukushima, Masakazu; Peters, Godefridus J

    2010-04-01

    Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance to TFT in H630 colon cancer cells using two different schedules and to analyze the resistance mechanism. Cells were exposed either continuously or intermittently to TFT, resulting in H630-cTFT and H630-4TFT, respectively. Cells were analyzed for cross-resistance, cell cycle, protein expression, and activity of thymidine phosphorylase (TP), thymidine kinase (TK), thymidylate synthase (TS), equilibrative nucleoside transporter (hENT), gene expression (microarray), and genomic alterations. Both cell lines were cross-resistant to 2'-deoxy-5-fluorouridine (>170-fold). Exposure to IC(75)-TFT increased the S/G(2)-M phase of H630 cells, whereas in the resistant variants, no change was observed. The two main target enzymes TS and TP remained unchanged in both TFT-resistant variants. In H630-4TFT cells, TK protein expression and activity were decreased, resulting in less activated TFT and was most likely the mechanism of TFT resistance. In H630-cTFT cells, hENT mRNA expression was decreased 2- to 3-fold, resulting in a 5- to 10-fold decreased TFT-nucleotide accumulation. Surprisingly, microarray-mRNA analysis revealed a strong increase of secretory phospholipase-A2 (sPLA2; 47-fold), which was also found by reverse transcription-PCR (RT-PCR; 211-fold). sPLA2 inhibition reversed TFT resistance partially. H630-cTFT had many chromosomal aberrations, but the exact role of sPLA2 in TFT resistance remains unclear. Altogether, resistance induction to TFT can lead to different mechanisms of resistance, including decreased TK protein expression and enzyme activity, decreased hENT expression, as well as (phospho)lipid metabolism. Mol Cancer Ther; 9(4); 1047-57. (c)2010 AACR.

  10. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis.

    PubMed

    Kumar, Dharmendra; Banerjee, Tuhina; Pratap, Chandra Bhan; Tilak, Ragini

    2015-04-15

    Since the emergence of pathogenic non-albicans Candida species, a number of new isolates have been added to the list. One such unusual species is Candida auris (C. auris), recently isolated and studied in few reports. In this study, a case of vulvovaginitis caused by Candida auris incidentally identified by molecular methods using internal transcribed spacer polymerase chain reaction (ITS PCR) is described. Antifungal susceptibility testing revealed the isolate to be resistant to itraconazole (MIC ≥ 2 µg/ml) and expressed important virulence factors including phospholipase, proteinase and hemolysin activity. The patient was successfully treated with oral fluconazole and did not have any invasive fungemia. Very few cases of this emerging pathogen have been reported. However, its isolation from clinical specimens reveals the significance of non-albicans candida species over C. albicans and the diversity of Candida spp causing infections.

  11. Cellular choline and glycine betaine pools impact osmoprotection and phospholipase C production in Pseudomonas aeruginosa.

    PubMed

    Fitzsimmons, Liam F; Hampel, Ken J; Wargo, Matthew J

    2012-09-01

    Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P. aeruginosa. We used (13)C nuclear magnetic resonance ((13)C-NMR) to demonstrate that P. aeruginosa maintains both choline and glycine betaine pools under a variety of conditions, in contrast to the transient glycine betaine pool reported for most bacteria. We were able to experimentally manipulate the choline and glycine betaine pools by overexpression of the cognate catabolic genes. Depletion of either the choline or glycine betaine pool reduced phospholipase production, a result unexpected for choline depletion. Depletion of the glycine betaine pool, but not the choline pool, inhibited growth under conditions of high salt with glucose as the primary carbon source. Depletion of the choline pool inhibited growth under high-salt conditions with choline as the sole carbon source, suggesting a role for the choline pool under these conditions. Here we have described the presence of a choline pool in P. aeruginosa and other pseudomonads that, with the glycine betaine pool, regulates osmoprotection and phospholipase production and impacts growth under high-salt conditions. These findings suggest that the levels of both pools are actively maintained and that perturbation of either pool impacts P. aeruginosa physiology.

  12. Cellular Choline and Glycine Betaine Pools Impact Osmoprotection and Phospholipase C Production in Pseudomonas aeruginosa

    PubMed Central

    Fitzsimmons, Liam F.; Hampel, Ken J.

    2012-01-01

    Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P. aeruginosa. We used 13C nuclear magnetic resonance (13C-NMR) to demonstrate that P. aeruginosa maintains both choline and glycine betaine pools under a variety of conditions, in contrast to the transient glycine betaine pool reported for most bacteria. We were able to experimentally manipulate the choline and glycine betaine pools by overexpression of the cognate catabolic genes. Depletion of either the choline or glycine betaine pool reduced phospholipase production, a result unexpected for choline depletion. Depletion of the glycine betaine pool, but not the choline pool, inhibited growth under conditions of high salt with glucose as the primary carbon source. Depletion of the choline pool inhibited growth under high-salt conditions with choline as the sole carbon source, suggesting a role for the choline pool under these conditions. Here we have described the presence of a choline pool in P. aeruginosa and other pseudomonads that, with the glycine betaine pool, regulates osmoprotection and phospholipase production and impacts growth under high-salt conditions. These findings suggest that the levels of both pools are actively maintained and that perturbation of either pool impacts P. aeruginosa physiology. PMID:22753069

  13. Contributions of PIP(2)-specific-phospholipase C and free salicylic acid to heat acclimation-induced thermotolerance in pea leaves.

    PubMed

    Liu, Hong-Tao; Huang, Wei-Dong; Pan, Qiu-Hong; Weng, Fang-Hua; Zhan, Ji-Cheng; Liu, Yan; Wan, Si-Bao; Liu, Yan-Yan

    2006-03-01

    The relationship between the accumulation in endogenous free salicylic acid (SA) induced by heat acclimation (37 degrees C) and the activity of PIP(2)-phospholipase C (PIP(2)-PLC; EC 3.1.4.3) in the plasma membrane fraction was investigated in pea (Pisum sativum L.) leaves. We focused our attention on the hypothesis that positive SA signals induced by heat acclimation may be relayed by PIP(2)-PLC. Heat acclimation induced an abrupt elevation of free SA preceding the activation of PLC toward PIP(2). Immunoblotting indicated a molecular mass with 66.5kDa PLC plays key role in the development of thermotolerance in pea leaves. In addition, some characterizations of PLC toward PIP(2) isolated from pea leaves with two-phase purification containing calcium concentration, pH and a protein concentration were also studied. Neomycin sulfate, a well-known PIP(2)-PLC inhibitor, was employed to access the involvement of PIP(2)-PLC in the acquisition of heat acclimation induced-thermotolerance. We were able to identify a PIP(2)-PLC, which was similar to a conventional PIP(2)-PLC in higher plants, from pea leaves suggesting that PIP(2)-PLC was involved in the signal pathway that leads to the acquisition of heat acclimation induced-thermotolerance. On the basis of these results, we conclude that the involvement of free SA may function as the upstream event in the stimulation of PIP(2)-PLC in response to heat acclimation treatment.

  14. Thrombin Promotes Sustained Signaling and Inflammatory Gene Expression through the CDC25 and Ras-associating Domains of Phospholipase Cϵ*

    PubMed Central

    Dusaban, Stephanie S.; Kunkel, Maya T.; Smrcka, Alan V.; Brown, Joan Heller

    2015-01-01

    Phospholipase C-epsilon (PLCϵ) plays a critical role in G-protein-coupled receptor-mediated inflammation. In addition to its ability to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, PLCϵ, unlike the other phospholipase C family members, is activated in a sustained manner. We hypothesized that the ability of PLCϵ to function as a guanine nucleotide exchange factor (GEF) for Rap1 supports sustained downstream signaling via feedback of Rap1 to the enzyme Ras-associating (RA2) domain. Using gene deletion and adenoviral rescue, we demonstrate that both the GEF (CDC25 homology domain) and RA2 domains of PLCϵ are required for long term protein kinase D (PKD) activation and subsequent induction of inflammatory genes. PLCϵ localization is largely intracellular and its compartmentalization could contribute to its sustained activation. Here we show that localization of PLCϵ to the Golgi is required for activation of PKD in this compartment as well as for subsequent induction of inflammatory genes. These data provide a molecular mechanism by which PLCϵ mediates sustained signaling and by which astrocytes mediate pathophysiological inflammatory responses. PMID:26350460

  15. Plasma glycosylphosphatidylinositol-specific phospholipase D predicts the change in insulin sensitivity in response to a low-fat but not a low-carbohydrate diet in obese women.

    PubMed

    Gray, Dona L; O'Brien, Kevin D; D'Alessio, David A; Brehm, Bonnie J; Deeg, Mark A

    2008-04-01

    Although circulating glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), a minor high-density lipoprotein-associated protein, is elevated in patients with insulin resistance or high triglycerides, no information is available on the effect of weight loss or changes in insulin sensitivity on circulating GPI-PLD levels. The objective of the study was to determine the effect of weight loss and changes in insulin sensitivity on plasma GPI-PLD levels. Forty-two nondiabetic obese women were included in the study, which involved a 3-month dietary intervention randomizing patients to a low-fat or a low-carbohydrate diet. The study's main outcome measures were plasma GPI-PLD levels and insulin sensitivity as estimated by the homeostasis model assessment. The very low carbohydrate diet group lost more weight after 3 months (-7.6 +/- 3.2 vs -4.2 +/- 3.5 kg, P < .01), although the decrease in insulin resistance was similar between groups. Weight loss with either diet did not alter plasma GPI-PLD levels. However, baseline GPI-PLD levels correlated with the change in insulin sensitivity in response to the low-fat diet, whereas baseline insulin sensitivity correlated with the change in insulin sensitivity in response to the low-carbohydrate diet. Plasma GPI-PLD may serve as a clinical tool to determine the effect of a low-fat diet on insulin sensitivity.

  16. Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes.

    PubMed

    Bonilla, Julia B; Cid, M Belén; Contreras, F-Xabier; Goñi, Félix M; Martín-Lomas, Manuel

    2006-02-01

    The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs.

  17. Iron-Regulated Phospholipase C Activity Contributes to the Cytolytic Activity and Virulence of Acinetobacter baumannii

    PubMed Central

    Fiester, Steven E.; Schmidt, Robert E.; Beckett, Amber C.; Ticak, Tomislav; Carrier, Mary V.; Ghosh, Rajarshi; Ohneck, Emily J.; Metz, Maeva L.; Sellin Jeffries, Marlo K.; Actis, Luis A.

    2016-01-01

    Acinetobacter baumannii is an opportunistic Gram-negative pathogen that causes a wide range of infections including pneumonia, septicemia, necrotizing fasciitis and severe wound and urinary tract infections. Analysis of A. baumannii representative strains grown in Chelex 100-treated medium for hemolytic activity demonstrated that this pathogen is increasingly hemolytic to sheep, human and horse erythrocytes, which interestingly contain increasing amounts of phosphatidylcholine in their membranes. Bioinformatic, genetic and functional analyses of 19 A. baumannii isolates showed that the genomes of each strain contained two phosphatidylcholine-specific phospholipase C (PC-PLC) genes, which were named plc1 and plc2. Accordingly, all of these strains were significantly hemolytic to horse erythrocytes and their culture supernatants tested positive for PC-PLC activity. Further analyses showed that the transcriptional expression of plc1 and plc2 and the production of phospholipase and thus hemolytic activity increased when bacteria were cultured under iron-chelation as compared to iron-rich conditions. Testing of the A. baumannii ATCC 19606T plc1::aph-FRT and plc2::aph isogenic insertion derivatives showed that these mutants had a significantly reduced PC-PLC activity as compared to the parental strain, while testing of plc1::ermAM/plc2::aph demonstrated that this double PC-PLC isogenic mutant expressed significantly reduced cytolytic and hemolytic activity. Interestingly, only plc1 was shown to contribute significantly to A. baumannii virulence using the Galleria mellonella infection model. Taken together, our data demonstrate that both PLC1 and PLC2, which have diverged from a common ancestor, play a concerted role in hemolytic and cytolytic activities; although PLC1 seems to play a more critical role in the virulence of A. baumannii when tested in an invertebrate model. These activities would provide access to intracellular iron stores this pathogen could use during

  18. Regulation of Transient Receptor Potential channels by the phospholipase C pathway

    PubMed Central

    Rohacs, Tibor

    2013-01-01

    Transient Receptor Potential (TRP) channels were discovered while analyzing visual mutants in drosophila. The protein encoded by the transient receptor potential (trp) gene is a Ca2+ permeable cation channel activated downstream of the phospholipase C (PLC) pathway. While searching for homologues in other organisms, a surprisingly large number of mammalian TRP channels were cloned. The regulation of TRP channels is quite diverse, but many of them are either activated downstream of the PLC pathway, or modulated by it. This review will summarize the current knowledge on regulation of TRP channels by the PLC pathway, with special focus on TRPC-s, which can be considered as effectors of the PLC pathway, and the heat and capsaicin sensitive TRPV1, which is modulated by the PLC pathway in a complex manner. PMID:23916247

  19. Impairment and restoration of response to TSH in dog thyroid slices after treatment with phospholipase A and lubrol PX.

    PubMed

    Yamashita, K; Oka, H; Kaneko, T; Ogata, E

    1976-01-01

    Incubation of dog thyroid slices with phospholipase A (10-40 U/Ml) or Lubrol PX (0.08-0.4%) caused a diminution in the subsequent TSH effect on the tissue cyclic AMP level and glucose oxidation. The same treatment had no effect on the basal level of these parameters. When the phospholipase A or Lubrol PX-treated slices were rinsed intensively with a Krebs-Ringer bicarbonate buffer and then incubated at 37degreesC in the same buffer for a further 1 to 3 hours, responsiveness to TSH recovered progressively reaching almost completely that of the control slices. Again, these procedures were without any significant effect on the responsiveness of the control slices. The above results together with those reported previously suggest strongly that phospholipids are an essential component of the plasma membrane system by which TSH stimulates adenylate cyclase activity. In addition, these essential lipids in the membrane appear to be renewed rather efficiently in this tissue, thus securing the functional integrity of the thyroid in the face of various deleterious situations.

  20. Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells.

    PubMed

    Gründling, Angelika; Gonzalez, Mark D; Higgins, Darren E

    2003-11-01

    In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in

  1. Phorbol ester inhibits arginine vasopressin activation of phospholipase C and promotes contraction of, and prostaglandin production by, cultured mesangial cells.

    PubMed Central

    Troyer, D A; Gonzalez, O F; Douglas, J G; Kreisberg, J I

    1988-01-01

    We have previously shown that arginine vasopressin (AVP) causes a rapid (5-10 min) contractile response in cultured mesangial cells plated onto slippery substrata such as poly(hydroxyethyl methacrylate)-coated dishes. This contraction is associated with an increase in the levels of inositol trisphosphate (InsP3), diacylglycerol and prostaglandin E2 (PGE2). We now report that agents which are known to activate protein kinase C, i.e. phorbol 12-myristate 13-acetate (PMA) and oleolylacetylglycerol (OAG), also contract mesangial cells; however, the contractile response is slow to develop (15-30 min). The inactive phorbol ester, 4 alpha -phorbol 12,13-didecanoate, did not elicit contraction. PMA and OAG did not increase InsP3 release in mesangial cells. However, pretreatment of mesangial cells with PMA inhibited the formation of InsP3. This inhibition could not be explained by a reduction in AVP binding since PMA treatment did not influence the number or affinity of [3H]AVP binding sites in intact cells. PMA alone stimulated PGE2 production in mesangial cells to a degree similar to AVP. Contrary to what was seen with InsP3, pretreatment of cells with PMA before AVP had an additive effect on arachidonic acid release and PGE2 production. Thus, there is an apparent dissociation of phospholipase C activity from that of phospholipase A2. Images Fig. 1. Fig. 2. PMID:3046605

  2. Neutralizing properties of Musa paradisiaca L. (Musaceae) juice on phospholipase A2, myotoxic, hemorrhagic and lethal activities of crotalidae venoms.

    PubMed

    Borges, M H; Alves, D L F; Raslan, D S; Piló-Veloso, D; Rodrigues, V M; Homsi-Brandeburgo, M I; de Lima, M E

    2005-04-08

    The use of plants as medicine has been referred to since ancient peoples, perhaps as early as Neanderthal man. Plants are a source of many biologically active products and nowadays they are of great interest to the pharmaceutical industry. The study of how people of different culture use plants in particular ways has led to the discovery of important new medicines. In this work, we verify the possible activity of Musa paradisiaca L. (Musaceae) against the toxicity of snake venoms. Musa paradisiaca, an important source of food in the world, has also been reported to be popularly used as an anti-venom. Interaction of Musa paradisiaca extract (MsE) with snake venom proteins has been examined in this study. Phospholipase A2 (PLA2), myotoxic and hemorrhagic activities, including lethality in mice, induced by crotalidae venoms were significantly inhibited when different amounts of MsE were mixed with these venoms before assays. On the other hand, mice that received MsE and venoms without previous mixture or by separated routes were not protected against venom toxicity. Partial chemical characterization of MsE showed the presence of polyphenols and tannins and they are known to non-specifically inactivate proteins. We suggest that these compounds can be responsible for the in vitro inhibition of the toxic effects of snake venoms. In conclusion, according to our results, using mice as experimental model, MsE does not show protection against the toxic effects of snake venoms in vivo, but if was very effective when the experiments were done in vitro.

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

    PubMed Central

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

    2016-01-01

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

  4. Bombesin and muscarinic receptor activation in rat pancreas generate cyclic inositol monophosphate: possible involvement of different phospholipase C isoenzymes.

    PubMed

    Sekar, M C; Sambandam, V; McDonald, J M

    1993-05-14

    Phospholipase C isoenzymes can generate different proportions of cyclic and non-cyclic inositol phosphates. Stimulation of [3H]-inositol labeled pancreatic minilobules by buffer, bombesin, neuromedin B or carbachol in presence of 10 mM lithium, followed by separation of inositol phosphates, yielded the following results for cyclic inositol monophosphate (cIP) [DPM/mg protein; Mean +/- SEM (n)]: control [21 +/- 6, (9)]; bombesin [145 +/- 24, (12)]; neuromedin B (99 +/- 22 (9)] and carbachol [512 +/- 60, (12)]. The generation of cIP and IP were significantly correlated [r2 = 0.72 (p < 0.05)] following carbachol activation, while no significant correlation was obtained following bombesin receptor activation by either bombesin or neuromedin B. Presence of zinc (100 microM) in the final incubation medium failed to amplify the bombesin-stimulated cIP accumulation. Based on our studies we postulate that different phospholipase C isoenzymes may be activated following muscarinic and bombesin receptor stimulation in pancrea.

  5. Patatin-related phospholipase A, pPLAIIIα, modulates the longitudinal growth of vegetative tissues and seeds in rice

    PubMed Central

    Liu, Guangmeng; Zhang, Ke; Ai, Jun; Deng, Xianjun; Hong, Yueyun; wang, Xuemin

    2015-01-01

    Patatin-related phospholipase A (pPLA) hydrolyses glycerolipids to produce fatty acids and lysoglycerolipids. The Oryza sativa genome has 21 putative pPLAs that are grouped into five subfamilies. Overexpression of OspPLAIIIα resulted in a dwarf phenotype with decreased length of rice stems, roots, leaves, seeds, panicles, and seeds, whereas OspPLAIIIα-knockout plants had longer panicles and seeds. OspPLAIIIα-overexpressing plants were less sensitive than wild-type and knockout plants to gibberellin-promoted seedling elongation. OspPLAIIIα overexpression and knockout had an opposite effect on the expression of the growth repressor SLENDER1 in the gibberellin signalling process. OspPLAIIIα-overexpressing plants had decreased mechanical strength and cellulose content, but exhibited increases in the expression of several cellulose synthase genes. These results indicate that OspPLAIIIα plays a role in rice vegetative and reproductive growth and that the constitutive, high activity of OspPLAIIIα suppresses cell elongation. The decreased gibberellin response in overexpressing plants is probably a result of the decreased ability to make cellulose for anisotropic cell expansion. PMID:26290597

  6. Phosphorylation of Nephrin Triggers Ca2+ Signaling by Recruitment and Activation of Phospholipase C-{gamma}1.

    PubMed

    Harita, Yutaka; Kurihara, Hidetake; Kosako, Hidetaka; Tezuka, Tohru; Sekine, Takashi; Igarashi, Takashi; Ohsawa, Ikuroh; Ohta, Shigeo; Hattori, Seisuke

    2009-03-27

    A specialized intercellular junction between podocytes, known as the slit diaphragm (SD), forms the essential structural frame-work for glomerular filtration in the kidney. In addition, mounting evidence demonstrates that the SD also plays a crucial role as a signaling platform in physiological and pathological states. Nephrin, the major component of the SD, is tyrosine-phosphorylated by a Src family tyrosine kinase, Fyn, in developing or injured podocytes, recruiting Nck to Nephrin via its Src homology 2 domain to regulate dynamic actin remodeling. Dysregulated Ca(2+) homeostasis has also been implicated in podocyte damage, but the mechanism of how podocytes respond to injury is largely unknown. Here we have identified phospholipase C-gamma1 (PLC-gamma1) as a novel phospho-Nephrin-binding protein. When HEK293T cells expressing a chimeric protein consisting of CD8 and Nephrin cytoplasmic domain (CD) were treated with anti-CD8 and anti-mouse antibodies, clustering of Nephrin and phosphorylation of Nephrin-CD were induced. Upon this clustering, PLC-gamma1 was bound to phosphorylated Nephrin Tyr-1204, which induced translocation of PLC-gamma1 from cytoplasm to the CD8/Nephrin cluster on the plasma membrane. The recruitment of PLC-gamma1 to Nephrin activated PLC-gamma1, as detected by phosphorylation of PLC-gamma1 Tyr-783 and increase in inositol 1,4,5-trisphosphate level. We also found that Nephrin Tyr-1204 phosphorylation triggers the Ca(2+) response in a PLC-gamma1-dependent fashion. Furthermore, PLC-gamma1 is significantly phosphorylated in injured podocytes in vivo. Given the profound effect of PLC-gamma in diverse cellular functions, regulation of the Ca(2+) signaling by Nephrin may be important in modulating the glomerular filtration barrier function.

  7. Recruitment of a phospholipase C/sphingomyelinase into non-lamellar lipid droplets during hydrolysis of lipid bilayers.

    PubMed

    Ibarguren, Maitane; Sot, Jesús; Montes, L Ruth; Vasil, Adriana I; Vasil, Michael L; Goñi, Félix M; Alonso, Alicia

    2013-01-01

    When giant unilamellar vesicles (GUVs) composed of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and cholesterol are treated with PlcHR(2), a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, the initial stages of lipid hydrolysis do not cause large changes in vesicle morphology (Ibarguren et al., 2011). However, when hydrolysis progresses confocal fluorescence microscopy reveals the formation of lipid aggregates, whose morphology is not compatible with that of bilayers. Smaller vesicles or droplets can also be seen inside the GUV. Our studies indicate that these aggregates or droplets are enriched in the non-lamellar lipid ceramide, an end-product of PlcHR(2) reaction. Moreover, the aggregates/droplets appear enriched in the hydrolytic enzyme PlcHR(2). At a final stage GUVs containing the enzyme-enriched droplets disintegrate and vanish from the microscope field. The observed non-lamellar enzyme-rich structures may be related to intermediates in the process of aggregation and fusion although the experimental design prevents vesicle free diffusion in the aqueous medium, thus actual aggregation or fusion cannot be observed. 2012 Elsevier Ireland Ltd. All rights reserved

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

    PubMed Central

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

    2014-01-01

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

  9. Peroxiredoxin 6 homodimerization and heterodimerization with glutathione S-transferase pi are required for its peroxidase but not phospholipase A2 activity.

    PubMed

    Zhou, Suiping; Sorokina, Elena M; Harper, Sandra; Li, Haitao; Ralat, Luis; Dodia, Chandra; Speicher, David W; Feinstein, Sheldon I; Fisher, Aron B

    2016-05-01

    Peroxiredoxin 6 (Prdx6) is a unique 1-Cys member of the peroxiredoxin family with both GSH peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of Prdx6 mediated by its heterodimerization with GSH S-transferase π (πGST) is required for its peroxidatic catalytic cycle. Recombinant human Prdx6 crystallizes as a homodimer and sedimentation equilibrium analysis confirmed that this protein exists as a high affinity dimer in solution. Based on measurement of molecular mass, dimeric Prdx6 that was oxidized to the sulfenic acid formed a sulfenylamide during storage. After examination of the dimer interface in the crystal structure, we postulated that the hydrophobic amino acids L145 and L148 play an important role in homodimerization of Prdx6 as well as in its heterodimerization with πGST. Oxidation of Prdx6 also was required for its heterodimerization. Sedimentation equilibrium analysis and the Duolink proximity ligation assay following mutation of the L145 and L148 residues of Prdx6 to Glu indicated greatly decreased dimerization propensity reflecting the loss of hydrophobic interactions between the protein monomers. Peroxidase activity was markedly reduced by mutation at either of the Leu sites and was essentially abolished by the double mutation, while PLA2 activity was unaffected. Decreased peroxidase activity following mutation of the interfacial leucines presumably is mediated via impaired heterodimerization of Prdx6 with πGST that is required for reduction and re-activation of the oxidized enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes.

    PubMed Central

    Geoffroy, C; Raveneau, J; Beretti, J L; Lecroisey, A; Vazquez-Boland, J A; Alouf, J E; Berche, P

    1991-01-01

    We purified and characterized an extracellular phospholipase produced by Listeria monocytogenes. This enzyme was separated as a homogeneous protein of 29 kDa by chromatography on DEAE-52 cellulose and Bio-Gel P100 columns. It is a zinc-dependent phospholipase C (PLC) that is mainly active at pH 6 to 7 and expresses lecithinase activity and a weaker sphingomyelinase activity. The exoenzyme also hydrolyzed phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin but not phosphatidylinositol. It was distinct from the 36-kDa phosphatidylinositol PLC produced by L. monocytogenes and from the L. ivanovii sphingomyelinase. The pure protein expressed a weak, calcium-independent hemolytic activity and was not toxic in mice. Western immunoblot analysis using a rabbit immune serum raised against the enzyme showed that all virulent strains of L. monocytogenes tested produced in the culture supernatant a 29-kDa PLC. In contrast, no proteins antigenically related to the 29-kDa PLC were detected in supernatants of L. ivanovii, L. seeligeri, L. innocua, or L. welshimeri. The role in virulence of the 29-kDa PLC specifically produced by L. monocytogenes remains to be established. Images PMID:1904842

  11. Lipoprotein-associated phospholipase A2 mass and activity in children with heterozygous familial hypercholesterolemia and unaffected siblings: effect of pravastatin.

    PubMed

    Ryu, Sung Kee; Hutten, Barbara A; Vissers, Maud N; Wiegman, Albert; Kastelein, John J P; Tsimikas, Sotirios

    2011-01-01

    Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is an independent risk factor of cardiovascular disease and a target of treatment. Lp-PLA(2) levels in children have not been previously reported. The effect of statin therapy on Lp-PLA(2) mass and activity in children with familial hypercholesterolemia (FH) is also not known. Lp-PLA(2) mass and activity levels were measured at baseline and after 2 years in 178 children with FH randomized to pravastatin or placebo and in 78 unaffected and untreated siblings. At the end of the randomized period, all FH children were then placed on pravastatin for an additional 2 years, and Lp-PLA(2) mass and activity levels were correlated with changes in carotid intima-media thickness during 4 years of follow-up. Baseline levels of Lp-PLA(2) mass and activity were significantly greater in children with FH compared with unaffected siblings (mass: 240.3 ± 41.6 vs 222.1 ± 36.5 ng/mL, P = .002; activity: 205.7 ± 41.6 vs 124.3±23.0 nmol/min/mL, P < .0001). In the randomized FH cohort, after 2 years treatment, Lp-PLA(2) mass (217.8 ± 35.0 vs 231.5 ± 34.8 ng/mL, P = .001) and activity (178.8 ± 37.3 vs 206.2 ± 33.5 nmol/min/mL, P < .0001) were significantly reduced by pravastatin compared with placebo. Change in Lp-PLA(2) activity was related to change in low-density lipoprotein cholesterol (pravastatin: r = 0.53, P < .0001, placebo: r = 0.23, P < .001) but change in Lp-PLA(2) mass was not related to change in low-density lipoprotein cholesterol. Baseline levels of Lp-PLA(2) mass and activity were not significantly associated with carotid intima-media thickness at baseline or at 4 years. Lp-PLA(2) mass and activity are significantly elevated in children with heterozygous FH compared with unaffected siblings and are significantly reduced by pravastatin therapy. Copyright © 2011 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  12. Enzymatic hydrolysis of 1-monoacyl-SN-glycerol-3-phosphoryl-choline (1-lysolecithin) by phospholipases from peanut seeds.

    PubMed

    Strauss, H; Leibovitz-Ben Gershon, Z; Heller, M

    1976-06-01

    Hydrolysis of 1-lysolecithin (1-acyl glycerophosphorylcholine [1-acyl GPC]) by preparations of phospholipase D from peanut seeds was investigated. 1-Lysolecithin was hydrolyzed at a much slower rate than phosphatidylcholine (lecithin). Although Ca+2 ions are required for the cleavage of lecithin by the enzyme, their effect on the hydrolysis of lysolecithin depended upon the concentration of the substrate: at 0.2 mM 1-lysolecithin, Ca+2 ions increased the reaction rates, whereas at concentrations of the substrate lower than 0.1 mM, Ca+2 ions were inhibitory. A broad pH activity curve between 5 and 8 was obtained with higher rates in the alkaline range, both in the absence and presence of Ca+2 ions. The increased hydrolysis of lysolecithin due to Ca+2 was noticed over the entire pH range. Upon storage of the enzyme solutions at 4 C, decreased rates of hydrolysis of lecithin were observed, with t 1/2 values of ca. 50 and 100 days depending on the purity of the preparation. During the same period, no reduction occurred in the activity of these preparations on lysolecithin as substrate. The effects of Ca+2 ions and the analysis of the products of 1-acyl GPC cleavage by the enzyme preparations revealed the presence of more than one enzyme and the formation of the following compounds: lysophosphatidic acids (1 acyl glycerophosphoric acids), free fatty acids, glycerophosphorylcholine, and choline. The possible pathways leading to the degradation of lysolecithin and the formation of these products include reactions catalyzed by lysophospholipase A1 (lysophosphatidylcholine 1-acyl hydrolase, E.C. 3.1.1.5) and a phosphodiesterase (L-3-glycerylphosphorylcholine glycerophosphohydrolase, E.C.3.1.4.2), in addition to phospholipase D (phosphatidyl-choline phosphatidohydrolase, E.C. 3.1.4.4).

  13. Mammalian phospholipase D: activation by ammonium sulfate and nucleotides.

    PubMed Central

    Nakamura, S; Shimooku, K; Akisue, T; Jinnai, H; Hitomi, T; Kiyohara, Y; Ogino, C; Yoshida, K; Nishizuka, Y

    1995-01-01

    Phospholipase D (PLD) associated with the rat kidney membrane was activated by guanine 5'-[gamma-thio]triphosphate and a cytosol fraction that contained ADP-ribosylation factor. When assayed by measuring the phosphatidyl transfer reaction to ethanol with exogenously added radioactive phosphatidylcholine as substrate, the PLD required a high concentration (1.6 M) of ammonium sulfate to exhibit high enzymatic activity. Other salts examined were far less effective or practically inactive, and this dramatic action of ammonium sulfate is not simply due to such high ionic strength. Addition of ATP but not of nonhydrolyzable ATP analogue adenosine 5'-[beta, gamma-imido]diphosphate further enhanced the PLD activation approximately equal to 2- to 3-fold. This enhancement by ATP needed cytosol, implying a role of protein phosphorylation. A survey of PLD activity in rat tissues revealed that, unlike in previous observations reported thus far, PLD was most abundant in membrane fractions of kidney, spleen, and liver in this order, and the enzymatic activity in brain and lung was low. PMID:8618893

  14. A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery.

    PubMed

    Flaugnatti, Nicolas; Le, Thi Thu Hang; Canaan, Stéphane; Aschtgen, Marie-Stéphanie; Nguyen, Van Son; Blangy, Stéphanie; Kellenberger, Christine; Roussel, Alain; Cambillau, Christian; Cascales, Eric; Journet, Laure

    2016-03-01

    The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins. © 2015 John Wiley & Sons Ltd.

  15. Intravascular hemolysis induced by the venom of the Eastern coral snake, Micrurus fulvius, in a mouse model: identification of directly hemolytic phospholipases A2.

    PubMed

    Arce-Bejarano, Ruth; Lomonte, Bruno; Gutiérrez, José María

    2014-11-01

    Intravascular hemolysis has been described in envenomings by the Eastern coral snake, Micrurus fulvius, in dogs. An experimental model of intravascular hemolysis was developed in mice after intravenous (i.v.) injection of M. fulvius venom. Within one hr, there was prominent hemolysis, associated with a drastic drop in hematocrit, morphological alterations of erythrocytes, hemoglobinemia, and hemoglobinuria. Hemoglobin was identified in urine by mass spectrometry. Histological sections of kidney revealed abundant hyaline casts, probably corresponding to hemoglobin. This effect was abrogated by p-bromophenacyl bromide, indicating that it is caused by phospholipases A2 (PLA2). A monospecific anti-Micrurus nigrocinctus antivenom neutralized hemolytic activity in vivo. When tested in vitro with erythrocytes of various species, a clear difference in susceptibility was observed. Mouse and dog erythrocytes showed the highest susceptibility, whereas human and rabbit erythrocytes were not affected at the experimental conditions tested. The higher susceptibility of dog and mouse erythrocytes correlates with a high ratio of phosphatidylcholine/sphingomyelin in erythrocyte plasma membrane. When mouse erythrocytes were subjected to mechanical stress, after incubation with venom, hemolysis increased significantly, suggesting that both phospholipid hydrolysis by PLA2s and mechanical stress associated with rheological factors are likely to contribute to cell lysis in vivo. Several PLA2s isolated from this venom reproduced the hemolytic effect, and the complete amino acid sequence of one of them (fraction 17), which also induces myotoxicity, is reported. Since very few PLA2s inducing intravascular hemolysis have been described from snake venoms, this enzyme is a valuable tool to identify the structural determinants of hemolytic activity. The mouse model described in this study may be useful to explore the pathophysiology of intravascular hemolysis. Copyright © 2014 Elsevier Ltd

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

    PubMed

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

    2018-03-01

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

  17. The galactolipase activity of Fusarium solani (phospho)lipase.

    PubMed

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

  18. VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones✰

    PubMed Central

    Filippone, Claudia; Zhi, Ning; Wong, Susan; Lu, Jun; Kajigaya, Sachiko; Gallinella, Giorgio; Kakkola, Laura; Venermo, Maria S Söderlund; Young, Neal S.; Brown, Kevin E.

    2008-01-01

    Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus. PMID:18252260

  19. VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones.

    PubMed

    Filippone, Claudia; Zhi, Ning; Wong, Susan; Lu, Jun; Kajigaya, Sachiko; Gallinella, Giorgio; Kakkola, Laura; Söderlund-Venermo, Maria; Young, Neal S; Brown, Kevin E

    2008-05-10

    Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus.

  20. Phosphatidylcholine-specific phospholipase C inhibition reduces HER2-overexpression, cell proliferation and in vivo tumor growth in a highly tumorigenic ovarian cancer model

    PubMed Central

    Spadaro, Francesca; Abalsamo, Laura; Pisanu, Maria Elena; Ricci, Alessandro; Cecchetti, Serena; Altabella, Luisa; Buoncervello, Maria; Lozneanu, Ludmila; Bagnoli, Marina; Ramoni, Carlo; Canevari, Silvana; Mezzanzanica, Delia

    2017-01-01

    Antagonizing the oncogenic effects of human epidermal growth factor receptor 2 (HER2) with current anti-HER2 agents has not yet yielded major progress in the treatment of advanced HER2-positive epithelial ovarian cancer (EOC). Using preclinical models to explore alternative molecular mechanisms affecting HER2 overexpression and oncogenicity may lead to new strategies for EOC patient treatment. We previously reported that phosphatidylcholine-specific phospholipase C (PC-PLC) exerts a pivotal role in regulating HER2 overexpression in breast cancer cells. The present study, conducted on two human HER2-overexpressing EOC cell lines - SKOV3 and its in vivo-passaged SKOV3.ip cell variant characterized by enhanced in vivo tumorigenicity - and on SKOV3.ip xenografts implanted in SCID mice, showed: a) about 2-fold higher PC-PLC and HER2 protein expression levels in SKOV3.ip compared to SKOV3 cells; b) physical association of PC-PLC with HER2 in non-raft domains; c) HER2 internalization and ca. 50% reduction of HER2 mRNA and protein expression levels in SKOV3.ip cells exposed to the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609); d) differential effects of D609 and trastuzumab on HER2 protein expression and cell proliferation; e) decreased in vivo tumor growth in SKOV3.ip xenografts during in vivo treatment with D609; f) potential use of in vivo magnetic resonance spectroscopy (MRS) and imaging (MRI) parameters as biomarkers of EOC response to PC-PLC inhibition. Overall, these findings support the view that PC-PLC inhibition may represent an effective means to target the tumorigenic effects of HER2 overexpression in EOC and that in vivo MR approaches can efficiently monitor its effects. PMID:28903399

  1. The Pleckstrin Homology Domain of Phospholipase Cβ Transmits Enzymatic Activation through Modulation of Membrane - Domain Orientation§

    PubMed Central

    Drin, Guillaume; Douguet, Dominique; Scarlata, Suzanne

    2008-01-01

    Phospholipase C-beta (PLCβ) enzymes are activated by Gαq and Gβγ subunits and catalyze the hydrolysis of the minor membrane lipid phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). Activation of PLCβ2 by Gβγ subunits has been shown to be conferred through its N-terminal pleckstrin homology (PH) domain although the underlying mechanism is unclear. Also unclear are observations that the extent of Gβγ activation differs on different membrane surfaces. In this study, we have identified a unique region of the PH domain of PLCβ2 domain (residues 71-88) which, when added to the enzyme as a peptide, causes enzyme activation similar to Gβγ subunits. This PH domain segment interacts strongly with membranes composed of lipid mixtures but not those containing lipids with electrically neutral zwitterionic head groups. Moreso, addition of this segment perturbs interaction of the catalytic domain, but not the PH domain, with membrane surfaces. We monitored the orientation of the PH and catalytic domains of PLC by intermolecular fluorescence resonance energy transfer (FRET) using the Gβγ activatable mutant, PLCβ2/δ1(C193S). We find an increase in FRET with binding to membranes with mixed lipids but not to those containing only lipids with electrically neutral head groups. These results suggest that enzymatic activation can be conferred through optimal association of the PHβ71-88 region to specific membrane surfaces. These studies allow us to understand the basis of variations of Gβγ activation on different membrane surfaces. PMID:16669615

  2. A novel synthetic peptide inspired on Lys49 phospholipase A2 from Crotalus oreganus abyssus snake venom active against multidrug-resistant clinical isolates.

    PubMed

    Almeida, José R; Mendes, Bruno; Lancellotti, Marcelo; Marangoni, Sergio; Vale, Nuno; Passos, Óscar; Ramos, Maria J; Fernandes, Pedro A; Gomes, Paula; Da Silva, Saulo L

    2018-04-10

    Currently, the evolving and complex mechanisms of bacterial resistance to conventional antibiotics are increasing, while alternative medicines are drying up, which urges the need to discover novel agents able to kill antibiotic-resistant bacteria. Lys49 phospholipase A 2 s (PLA 2 s) from snake venoms are multifunctional toxins able to induce a huge variety of therapeutic effects and consequently serve as templates for new drug leads. Hence, the present study was aimed at the synthesis of oligopeptides mimicking regions of the antibacterial Lys49 PLA 2 toxin (CoaTx-II), recently isolated from Crotalus oreganus abyssus snake venom, to identify small peptides able to reproduce the therapeutic action of the toxin. Five peptides, representing major regions of interest within CoaTx-II, were synthesized and screened for their antibacterial properties. The 13-mer peptide pC-CoaTxII, corresponding to residues 115-129 of CoaTx-II, was able to reproduce the promising bactericidal effect of the toxin against multi-resistant clinical isolates. Peptide pC-CoaTxII is mainly composed by positively charged and hydrophobic amino acids, a typical trait in most antimicrobial peptides, and presented no defined secondary structure in aqueous environment. The physicochemical properties of pC-CoaTxII are favorable towards a strong interaction with anionic lipid membranes as those in bacteria. Additional in silico studies suggest formation of a water channel across the membrane upon peptide insertion, eventually leading to bacterial cell disruption and death. Overall, our findings confirm the valuable potential of snake venom toxins towards design and synthesis of novel antimicrobials, thus representing key insights towards development of alternative efficient antimicrobials to fight bacterial resistance to current antibiotics. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

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

    PubMed

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

    2018-05-01

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

  4. Phosphatidylinositol-specific phospholipase C activity in Lactobacillus rhamnosus with capacity to translocate.

    PubMed

    Rodriguez, A V; Baigorí, M D; Alvarez, S; Castro, G R; Oliver, G

    2001-10-16

    Phosphatidylinositol-specific phospholipase C (PI-PLC) activity was investigated in 25 different lactic acid bacteria (LAB) strains belonging to the genera Lactobacillus, Weisella, and Enterococcus. PI-PLC activity was detected in 44% of the strains studied in culture medium without carbon source. From the PI-PLC positive strains, Lactobacillus rhamnosus ATCC 7469 was selected for translocation studies. Healthy mice were orally administered with a daily dose of 2.0 x 10(9) of viable L. rhamnosus suspension. Viable bacteria were detected in liver and spleen of mice fed with LAB for 7 days. Bacterial colonies isolated from liver were biochemically characterized, and further subjected to randomly amplified polymorphic DNA. Amplification patterns of five strains displayed identical profiles to L. rhamnosus. PI-PLC activity was determined in the strains recovered from liver.

  5. Phospholipase D2 Mediates Survival Signaling through Direct Regulation of Akt in Glioblastoma Cells*♦

    PubMed Central

    Bruntz, Ronald C.; Taylor, Harry E.; Lindsley, Craig W.; Brown, H. Alex

    2014-01-01

    The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipase D (PLD) is a novel regulator of Akt in GBM. Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase. PMID:24257753

  6. Chemical studies on damages of Escherichea coli by the immune bactericidal reaction. II. Release of phosphatidylethanolamine from a phospholipase A-deficient mutant of E. coli during the immune bactericidal reaction.

    PubMed

    Inoue, K; Yano, K; Amano, T

    1974-12-01

    When an antibody-sensitized, phospholipase A-deficient mutant of Escherichia coli B/SM was treated with complement in the absence of lysozyme, bacterial phosphatidylethanolamine (PE) was liberated into the lipid fraction of the surrounding medium, but only traces of its degradation products were found in this fraction. Therefore, most of the degradation of bacterial PE to FFA and LPE observed in the usual immune bactericidal reaction (Inoue et al., 1974) must be the result of the action of bacterial phospholipase A which is activated or becomes accessible to its substrate on formation of lesions by complement. The mechanism of complement-mediated formation of membrane lesions is discussed on the basis of these results.

  7. Genetic interactions between a phospholipase A2 and the Rim101 pathway components in S. cerevisiae reveal a role for this pathway in response to changes in membrane composition and shape

    PubMed Central

    Mattiazzi, M.; Jambhekar, A.; Kaferle, P.; DeRisi, J. L.; Križaj, I.

    2010-01-01

    Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A2 (PLA2s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA2 in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA2 activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA2 activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them. Electronic supplementary material The online version of this article (doi:10.1007/s00438-010-0533-8) contains supplementary material, which is available to authorized users. PMID:20379744

  8. The presence of a protein activator of sarcolemmal polyphosphoinositide phospholipase C in cardiac cytosol

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Quist, E.E.; Kriewaldt, S.D.; Powell, P.B.

    1989-01-01

    To study polyphosphoinositide phospholipase (PL) C, isolated sarcolemmal membranes were preincubated with Mg({sup 32}P)-ATP to label phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-diphosphate (PIP{sub 2}). After washing, PLC activity was determined by measuring the release of {sup 32}P-labeled inositol diphosphate (IP{sub 2}) and/or inositol trisphospate (IP{sub 3}) from membrane PIP and PIP{sub 2} during incubation at 25{degree}C and pH 7.4. Increasing concentrations of Ca{sup 2+} (0-100 {mu}M) increased IP{sub 2} by 100% over the 0 Ca{sup 2+} control levels. Ca{sup 2+} dependent PLC hydrolyzed both PIP and PIP{sub 2} with apparent D{sub A}'s of approximately 0.5 and 70 {mu}M. Addition ofmore » dialyzed cytosol further increased IP{sub 2} release by 250% without affecting the K{sub A}'s for Ca{sup 2+} activation. The cytosolic activator was partially purified by DEAE Sephacel chromatography was heat labile and sensitive to trypsin pretreatment identifying it as a protein. In contrast, 10 mM NaF increased the Ca{sup 2+} affinity for PLC 2-fold. These results show that cardiac sarcolemma possess a membrane bound Ca{sup 2+} dependent PLC activity which is regulated by a cytosolic protein activator and a G protein. The cytosolic activator would potentially amplify the amount of sarcolemmal polyphosphoinositides hydrolyzed by PLC in response to muscarinic receptor activation by acetylcholine. In addition, activation of PLC by NaF or other G protein activators could result from increasing the Ca{sup 2+} affinity of PLC to physiological intracellular Ca{sup 2+} levels.« less

  9. Nuclear diacylglycerol lipase-α in rat brain cortical neurons: evidence of 2-arachidonoylglycerol production in concert with phospholipase C-β activity.

    PubMed

    García del Caño, Gontzal; Aretxabala, Xabier; González-Burguera, Imanol; Montaña, Mario; López de Jesús, Maider; Barrondo, Sergio; Barrio, Ramón J; Sampedro, Carmen; Goicolea, M Arantzazu; Sallés, Joan

    2015-03-01

    In this report, we describe the localization of diacylglycerol lipase-α (DAGLα) in nuclei from adult cortical neurons, as assessed by double-immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double-labeling assays using the anti-DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα-signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC-35- and NeuN-signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C-β (PLCβ) family, PLCβ1, PLCβ2, and PLCβ4 exhibited the same distribution with respect to chromatin, lamin B1, SC-35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of 2-arachidonoylglycerol (2-AG) by liquid chromatography and mass spectrometry (LC-MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for 2-AG production, and its PLCβ/DAGLα-dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for 2-AG locally produced within the neuronal nucleus. © 2014 International Society for Neurochemistry.

  10. Patatin-related phospholipase A, pPLAIIIα, modulates the longitudinal growth of vegetative tissues and seeds in rice.

    PubMed

    Liu, Guangmeng; Zhang, Ke; Ai, Jun; Deng, Xianjun; Hong, Yueyun; Wang, Xuemin

    2015-11-01

    Patatin-related phospholipase A (pPLA) hydrolyses glycerolipids to produce fatty acids and lysoglycerolipids. The Oryza sativa genome has 21 putative pPLAs that are grouped into five subfamilies. Overexpression of OspPLAIIIα resulted in a dwarf phenotype with decreased length of rice stems, roots, leaves, seeds, panicles, and seeds, whereas OspPLAIIIα-knockout plants had longer panicles and seeds. OspPLAIIIα-overexpressing plants were less sensitive than wild-type and knockout plants to gibberellin-promoted seedling elongation. OspPLAIIIα overexpression and knockout had an opposite effect on the expression of the growth repressor SLENDER1 in the gibberellin signalling process. OspPLAIIIα-overexpressing plants had decreased mechanical strength and cellulose content, but exhibited increases in the expression of several cellulose synthase genes. These results indicate that OspPLAIIIα plays a role in rice vegetative and reproductive growth and that the constitutive, high activity of OspPLAIIIα suppresses cell elongation. The decreased gibberellin response in overexpressing plants is probably a result of the decreased ability to make cellulose for anisotropic cell expansion. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  11. The receptor tyrosine kinase MerTK activates phospholipase C γ2 during recognition of apoptotic thymocytes by murine macrophages

    PubMed Central

    Todt, Jill C.; Hu, Bin; Curtis, Jeffrey L.

    2008-01-01

    Apoptotic leukocytes must be cleared efficiently by macrophages (Mø). Apoptotic cell phagocytosis by Mø requires the receptor tyrosine kinase (RTK) MerTK (also known as c-Mer and Tyro12), the phosphatidylserine receptor (PS-R), and the classical protein kinase C (PKC) isoform βII, which translocates to Mø membrane and cytoskeletal fractions in a PS-R-dependent fashion. How these molecules cooperate to induce phagocytosis is unknown. Because the phosphatidylinositol-specific phospholipase (PI-PLC) PLC γ2 is downstream of RTKs in some cell types and can activate classical PKCs, we hypothesized that MerTK signals via PLC γ2. To test this hypothesis, we examined the interaction of MerTK and PLC γ2 in resident murine PMø and in the murine Mø cell line J774A.1 (J774) following exposure to apoptotic thymocytes. We found that, as with PMø, J774 phagocytosis of apoptotic thymocytes was inhibited by antibody against MerTK. Western blotting and immunoprecipitation showed that exposure to apoptotic cells produced three time-dependent changes in PMø and J774: (1) tyrosine phosphorylation of MerTK; (2) association of PLC γ2 with MerTK; and (3) tyrosine phosphorylation of PLC γ2. Phosphorylation of PLC γ2 and its association with MerTK was also induced by cross-linking MerTK using antibody. A PI-PLC appears to be required for phagocytosis of apoptotic cells because the PI-PLC inhibitor Et-18-OCH3 and the PLC inhibitor U73122, but not the inactive control U73343, blocked phagocytosis without impairing adhesion. On apoptotic cell adhesion to Mø, MerTK signals at least in part via PLC γ2. PMID:14704368

  12. Modulation of intracellular Ca(2+) via alpha(1B)-adrenoreceptor signaling molecules, G alpha(h) (transglutaminase II) and phospholipase C-delta 1.

    PubMed

    Kang, Sung Koo; Kim, Dae Kyong; Damron, Derek S; Baek, Kwang Jin; Im, Mie-Jae

    2002-04-26

    We characterized the alpha(1B)-adrenoreceptor (alpha(1B)-AR)-mediated intracellular Ca(2+) signaling involving G alpha(h) (transglutaminase II, TGII) and phospholipase C (PLC)-delta 1 using DDT1-MF2 cell. Expression of wild-type TGII and a TGII mutant lacking transglutaminase activity resulted in significant increases in a rapid peak and a sustained level of intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to activation of the alpha(1B)-AR. Expression of a TGII mutant lacking the interaction with the receptor or PLC-delta 1 substantially reduced both the peak and sustained levels of [Ca(2+)](i). Expression of TGII mutants lacking the interaction with PLC-delta 1 resulted in a reduced capacitative Ca(2+) entry. Reduced expression of PLC-delta 1 displayed a transient elevation of [Ca(2+)](i) and a reduction in capacitative Ca(2+) entry. Expression of the C2-domain of PLC-delta 1, which contains the TGII interaction site, resulted in reduction of the alpha(1B)-AR-evoked peak increase in [Ca(2+)](i), while the sustained elevation in [Ca(2+)](i) and capacitative Ca(2+) entry remained unchanged. These findings demonstrate that stimulation of PLC-delta 1 via coupling of the alpha(1B)-AR with TGII evokes both Ca(2+) release and capacitative Ca(2+) entry and that capacitative Ca(2+) entry is mediated by the interaction of TGII with PLC-delta 1.

  13. Genetic Ablation of Calcium-independent Phospholipase A2γ (iPLA2γ) Attenuates Calcium-induced Opening of the Mitochondrial Permeability Transition Pore and Resultant Cytochrome c Release*

    PubMed Central

    Moon, Sung Ho; Jenkins, Christopher M.; Kiebish, Michael A.; Sims, Harold F.; Mancuso, David J.; Gross, Richard W.

    2012-01-01

    Herein, we demonstrate that calcium-independent phospholipase A2γ (iPLA2γ) is a critical mechanistic participant in the calcium-induced opening of the mitochondrial permeability transition pore (mPTP). Liver mitochondria from iPLA2γ−/− mice were markedly resistant to calcium-induced swelling in the presence or absence of phosphate in comparison with wild-type littermates. Furthermore, the iPLA2γ enantioselective inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one ((R)-BEL) was markedly more potent than (S)-BEL in inhibiting mPTP opening in mitochondria from wild-type liver in comparison with hepatic mitochondria from iPLA2γ−/− mice. Intriguingly, low micromolar concentrations of long chain fatty acyl-CoAs and the non-hydrolyzable thioether analog of palmitoyl-CoA markedly accelerated Ca2+-induced mPTP opening in liver mitochondria from wild-type mice. The addition of l-carnitine enabled the metabolic channeling of acyl-CoA through carnitine palmitoyltransferases (CPT-1/2) and attenuated the palmitoyl-CoA-mediated amplification of calcium-induced mPTP opening. In contrast, mitochondria from iPLA2γ−/− mice were insensitive to fatty acyl-CoA-mediated augmentation of calcium-induced mPTP opening. Moreover, mitochondria from iPLA2γ−/− mouse liver were resistant to Ca2+/t-butyl hydroperoxide-induced mPTP opening in comparison with wild-type littermates. In support of these findings, cytochrome c release from iPLA2γ−/− mitochondria was dramatically decreased in response to calcium in the presence or absence of either t-butyl hydroperoxide or phenylarsine oxide in comparison with wild-type littermates. Collectively, these results identify iPLA2γ as an important mechanistic component of the mPTP, define its downstream products as potent regulators of mPTP opening, and demonstrate the integrated roles of mitochondrial bioenergetics and lipidomic flux in modulating mPTP opening promoting the activation of necrotic and

  14. Recent research progress with phospholipase C from Bacillus cereus.

    PubMed

    Lyu, Yan; Ye, Lidan; Xu, Jun; Yang, Xiaohong; Chen, Weiwei; Yu, Hongwei

    2016-01-01

    Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce phosphate monoesters and diacylglycerol. It has many applications in the enzymatic degumming of plant oils. PLC Bc , a bacterial PLC from Bacillus cereus, is an optimal choice for this activity in terms of its wide substrate spectrum, high activity, and approved safety. Unfortunately, its large-scale production and reliable high-throughput screening of PLC Bc remain challenging. Herein, we summarize the research progress regarding PLC Bc with emphasis on the screening methods, expression systems, catalytic mechanisms and inhibitor of PLC Bc . This review hopefully will inspire new achievements in related areas, to promote the sustainable development of PLC Bc and its application.

  15. Investigation of the Brain Biodistribution of the Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Inhibitor [18F]GSK2647544 in Healthy Male Subjects.

    PubMed

    Huiban, Mickael; Coello, Christopher; Wu, Kai; Xu, Yanmei; Lewis, Yvonne; Brown, Andrew P; Buraglio, Mauro; Guan, Chenbing; Shabbir, Shaila; Fong, Regan; Passchier, Jan; Rabiner, Eugenii A; Lockhart, Andrew

    2017-02-01

    GSK2647544 is a potent and specific inhibitor of lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ), which was in development as a potential treatment for Alzheimer's disease (AD). In order to refine therapeutic dose predictions and confirm brain penetration, a radiolabelled form of the inhibitor, [ 18 F]GSK2647544, was manufactured for use in a positron emission tomography (PET) biodistribution study. [ 18 F]GSK2647544 was produced using a novel, copper iodide (Cu(I)) mediated, [ 18 F]trifluoromethylation methodology. Healthy male subjects (n = 4, age range 34-42) received an oral dose of unlabelled GSK2647544 (100 mg) and after 2 h an intravenous (iv) injection of [ 18 F]GSK2647544 (average injected activity and mass were 106 ± 47 MBq and 179 ± 55 μg, respectively) followed by dynamic PET scans for 120 min. Defined regions of interest (ROI) throughout the brain were used to obtain regional time-activity curves (TACs) and compartmental modelling analysis used to estimate the primary outcome measure, whole brain volume of distribution (V T ). Secondary PK and safety endpoints were also recorded. PET dynamic data were successfully obtained from all four subjects and there were no clinically significant variations of the safety endpoints. Inspection of the TACs indicated a relatively homogenous uptake of [ 18 F]GSK2647544 across all the ROIs examined. The mean whole brain V T was 0.56 (95 % CI, 0.41-0.72). Secondary PK parameters, C max (geometric mean) and T max (median), were 354 ng/ml and 1.4 h, respectively. Metabolism of GSK2647544 was relatively consistent across subjects, with 20-40 % of the parent compound [ 18 F]GSK2647544 present after 120 min. The study provides evidence that GSK2647544 is able to cross the blood brain barrier in healthy male subjects leading to a measurable brain exposure. The administered doses of GSK2647544 were well tolerated. Exploratory modelling suggested that a twice-daily dose of 102 mg, at steady state

  16. Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

    PubMed Central

    Cruchaga, Carlos; Benitez, Bruno A.; Cai, Yefei; Guerreiro, Rita; Harari, Oscar; Norton, Joanne; Budde, John; Bertelsen, Sarah; Jeng, Amanda T.; Cooper, Breanna; Skorupa, Tara; Carrell, David; Levitch, Denise; Hsu, Simon; Choi, Jiyoon; Ryten, Mina; Sassi, Celeste; Bras, Jose; Gibbs, Raphael J.; Hernandez, Dena G.; Lupton, Michelle K.; Powell, John; Forabosco, Paola; Ridge, Perry G.; Corcoran, Christopher D.; Tschanz, JoAnn T.; Norton, Maria C.; Munger, Ronald G.; Schmutz, Cameron; Leary, Maegan; Demirci, F. Yesim; Bamne, Mikhil N.; Wang, Xingbin; Lopez, Oscar L.; Ganguli, Mary; Medway, Christopher; Turton, James; Lord, Jenny; Braae, Anne; Barber, Imelda; Brown, Kristelle; Pastor, Pau; Lorenzo-Betancor, Oswaldo; Brkanac, Zoran; Scott, Erick; Topol, Eric; Morgan, Kevin; Rogaeva, Ekaterina; Singleton, Andy; Hardy, John; Kamboh, M. Ilyas; George-Hyslop, Peter St; Cairns, Nigel; Morris, John C.; Kauwe, John S.K.; Goate, Alison M.

    2014-01-01

    Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD)1,2. These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low frequency coding variants with large effects on LOAD risk, we performed whole exome-sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large case-control datasets. A rare variant in PLD3 (phospholipase-D family, member 3, rs145999145; V232M) segregated with disease status in two independent families and doubled risk for AD in seven independent case-control series (V232M meta-analysis; OR= 2.10, CI=1.47-2.99; p= 2.93×10-5, 11,354 cases and controls of European-descent). Gene-based burden analyses in 4,387 cases and controls of European-descent and 302 African American cases and controls, with complete sequence data for PLD3, indicate that several variants in this gene increase risk for AD in both populations (EA: OR= 2.75, CI=2.05-3.68; p=1.44×10-11, AA: OR= 5.48, CI=1.77-16.92; p=1.40×10-3). PLD3 is highly expressed in brain regions vulnerable to AD pathology, including hippocampus and cortex, and is expressed at lower levels in neurons from AD brains compared to control brains (p=8.10×10-10). Over-expression of PLD3 leads to a significant decrease in intracellular APP and extracellular Aβ42 and Aβ40, while knock-down of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a two-fold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may be used to identify rare variants with large effects on risk for disease or other complex traits. PMID

  17. Patatin-like phospholipase domain-containing protein 3 (PNPLA3): A potential role in the association between liver disease and bipolar disorder.

    PubMed

    Kenneson, Aileen; Funderburk, Jennifer S

    2017-02-01

    Due to the increased prevalence of liver disease in patients with bipolar disorder, we examined the potential role of the patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant among individuals with bipolar disorder and those with no mood disorder. We used the National Health and Nutrition Examination Survey (NHANES) database (aged 15-39 years) to identify a group of individuals with a bipolar diagnosis and a control group of individuals with no mood disorder. A total of 1931 individuals were randomly selected, one from each family containing information on the PNPLA3 genotype to be used in the analysis. Analyses revealed individuals with the recessive variant genotype (MM) had an adjusted odds ratio for bipolar disorder of about 4.6 compared to individuals with either IM or II genotypes of the PNPLA3 variant. Limitations of this study include the use of a lay-administered survey in for diagnosis of bipolar disorder in NHANES. The association between the PNPLA3 variant and bipolar disorder may help guide further work on medication effectiveness, treatment options, prevention approaches, and understanding potential medication side effects among specific subgroups of individuals with the MM genotype. Published by Elsevier B.V.

  18. Inhibition of phospholipase cgamma1 and cancer cell proliferation by triterpene esters from Uncaria rhynchophylla.

    PubMed

    Lee, J S; Kim, J; Kim, B Y; Lee, H S; Ahn, J S; Chang, Y S

    2000-06-01

    Investigation of the hooks of Uncaria rhynchophylla resulted in isolation of six phospholipase Cgamma1 (PLCgamma1) inhibitors (1-6). The structures of these compounds were elucidated as pentacyclic triterpene esters by spectroscopic and chemical analysis. Three of them, namely uncarinic acids C (1), D (2), and E (3), are newly reported as natural products. All the compounds showed dose-dependent inhibitory activities against PLCgamma1 in vitro with IC(50) values of 9.5-44.6 microM and inhibited the proliferation of human cancer cells with IC(50) values of 0.5-6.5 microg/mL.

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

    PubMed

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

    2014-04-01

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

  20. Calmodulin is a phospholipase C-beta interacting protein.

    PubMed

    McCullar, Jennifer S; Larsen, Shana A; Millimaki, Ryan A; Filtz, Theresa M

    2003-09-05

    Phospholipase C-beta 3 (PLC beta 3) is an important effector enzyme in G protein-coupled signaling pathways. Activation of PLC beta 3 by G alpha and G beta gamma subunits has been fairly well characterized, but little is known about other protein interactions that may also regulate PLC beta 3 function. A yeast two-hybrid screen of a mouse brain cDNA library with the amino terminus of PLC beta 3 has yielded potential PLC beta 3 interacting proteins including calmodulin (CaM). Physical interaction between CaM and PLC beta 3 is supported by a positive secondary screen in yeast and the identification of a CaM binding site in the amino terminus of PLC beta 3. Co-precipitation of in vitro translated and transcribed amino- and carboxyl-terminal PLC beta 3 revealed CaM binding at a putative amino-terminal binding site. Direct physical interaction of PLC beta 3 and PLC beta 1 isoforms with CaM is supported by pull-down of both isoenzymes with CaM-Sepharose beads from 1321N1 cell lysates. CaM inhibitors reduced M1-muscarinic receptor stimulation of inositol phospholipid hydrolysis in 1321N1 astrocytoma cells consistent with a physiologic role for CaM in modulation of PLC beta activity. There was no effect of CaM kinase II inhibitors, KN-93 and KN-62, on M1-muscarinic receptor stimulation of inositol phosphate hydrolysis, consistent with a direct interaction between PLC beta isoforms and CaM.

  1. Attenuation of cysteamine-induced duodenal ulcer with Cochinchina momordica seed extract through inhibiting cytoplasmic phospholipase A2/5-lipoxygenase and activating γ-glutamylcysteine synthetase.

    PubMed

    Choi, Ki-Seok; Kim, Eun-Hee; Hong, Hua; Ock, Chan Young; Lee, Jeong Sang; Kim, Joo-Hyun; Hahm, Ki-Baik

    2012-04-01

    Cysteamine is a reducing aminothiol used for inducing duodenal ulcer through mechanisms of oxidative stress related to thiol-derived H(2)O(2) reaction. Cochinchina momordica saponins have been suggested to be protective against various gastric diseases based on their cytoprotective and anti-inflammatory mechanisms. This study was aimed to document the preventive effects of Cochinchina momordica seed extract against cysteamine-induced duodenal ulcer as well as the elucidation of its pharmacological mechanisms. Cochinchina momordica seed extract (50, 100, 200 mg/kg) was administrated intragastrically before cysteamine administration, after which the incidence of the duodenal ulcer, ulcer size, serum gastrin level, and the ratio of reduced glutathione (GSH)/oxidized glutathione disulfide (GSSG) as well as biochemical and molecular measurements of cytoplasmic phospholipase A(2) (cPLA(2)), cyclooxygenase-2 (COX-2), 5-lipoxygenase and the expression of proinflammatory genes including IL-1β, IL-6, COX-2 were measured in rat model. Additional experiments of electron spin resonance measurement and the changes of glutathione were performed. Cochinchina momordica seed extract effectively prevented cysteamine-induced duodenal ulcer in a dose-dependent manner as reflected with significant decreases in either duodenal ulcerogenesis or perforation accompanied with significantly decreased in serum gastrin in addition to inflammatory mediators including cPLA(2), COX-2, and 5-lipoxygenase. Cochinchina momordica seed extract induced the expression of γ-glutamylcysteine synthetase (γ-GCS)-related glutathione synthesis as well as significantly reduced the expression of cPLA(2). Cochinchina momordica seed extract preserved reduced glutathione through increased expressions of γ-GCS. Cochinchina momordica seed extracts exerted significantly protective effect against cysteamine-induced duodenal ulcer by either cPLA2 inhibition or glutathione preservation. © 2012 Journal of

  2. Conjugated polyelectrolyte based real-time fluorescence assay for phospholipase C.

    PubMed

    Liu, Yan; Ogawa, Katsu; Schanze, Kirk S

    2008-01-01

    A fluorescence turnoff assay for phospholipase C (PLC) from Clostridium perfringens is developed based on the reversible interaction between the natural substrate, phosphatidylcholine, and a fluorescent, water-soluble conjugated polyelectrolyte (CPE). The fluorescence intensity of the CPE in water is increased substantially by the addition of the phospholipid due to the formation of a CPE-lipid complex. Incubation of the CPE-lipid complex with the enzyme PLC causes the fluorescence intensity to decrease (turnoff sensor); the response arises due to PLC-catalyzed hydrolysis of the phosphatidylcholine, which effectively disrupts the CPE-lipid complex. The PLC assay operates with phospholipid substrate concentrations in the micromolar range, and the analytical detection limit for PLC is <1 nM. The optimized assay provides a convenient, rapid, and real-time sensor for PLC activity. The real-time fluorescence intensity from the CPE can be converted to substrate concentration by using an ex situ calibration curve, allowing PLC-catalyzed reaction rates and kinetic parameters to be determined. PLC activation by Ca2+ and inhibition by EDTA and fluoride ion are demonstrated using the optimized sensor.

  3. An electrophysiological study on the effects of Pa-1G (a phospholipase A(2)) from the venom of king brown snake, Pseudechis australis, on neuromuscular function.

    PubMed

    Fatehi, M; Rowan, E G; Harvey, A L

    2002-01-01

    The effects of Pa-1G, a phospholipase A(2) (PLA(2)) from the venom of the Australian king brown snake (Pseudechis australis) were determined on the release of acetylcholine, muscle resting membrane potential and motor nerve terminal action potential at mouse neuromuscular junction. Intracellular recording from endplate regions of mouse triangularis sterni nerve-muscle preparations revealed that Pa-1G (800 nM) significantly reduced the amplitude of endplate potentials within 10 min exposure. The quantal content of endplate potentials was decreased to 58+/-6% of control after 30 min exposure to 800 nM Pa-1G. The toxin also caused a partial depolarisation of mouse muscle fibres within 60 min exposure. Extracellular recording of action potentials at motor nerve terminals showed that Pa-1G reduced the waveforms associated with both sodium and potassium conductances. To investigate whether this was a direct or indirect effect of the toxin on these ionic currents, whole cell patch clamp experiments were performed using human neuroblastoma (SK-N-SH) cells and B82 mouse fibroblasts stably transfected with rKv1.2. Patch clamp recording experiments confirmed that potassium currents sensitive to alpha-dendrotoxin recorded from B82 cells and sodium currents in SK-N-SH cells were not affected by the toxin. Since neither facilitation of acetylcholine release at mouse neuromuscular junction nor depression of potassium currents in B82 cells has been observed, the apparent blockade of potassium currents at mouse motor nerve endings induced by the toxin is unlikely to be due to a selective block of potassium channels.

  4. Intravascular hemolysis induced by phospholipases A2 from the venom of the Eastern coral snake, Micrurus fulvius: Functional profiles of hemolytic and non-hemolytic isoforms.

    PubMed

    Fernández, María Laura; Quartino, Pablo Yunes; Arce-Bejarano, Ruth; Fernández, Julián; Camacho, Luis F; Gutiérrez, José María; Kuemmel, Daniel; Fidelio, Gerardo; Lomonte, Bruno

    2018-04-01

    A unique feature of the venom of Micrurus fulvius (Eastern coral snake) is its ability to induce severe intravascular hemolysis in particular species, such as dogs or mice. This effect was previously shown to be induced by distinct phospholipase A 2 (PLA 2 ) isoforms which cause direct hemolysis in vitro, an uncommon finding for such enzymes. The functional profiles of PLA 2 -17, a direct hemolytic enzyme, and PLA 2 -12, a co-existing venom isoform lacking such effect, were compared. The enzymes differed not only in their ability to cause intravascular hemolysis: PLA 2 -17 additionally displayed lethal, myotoxic, and anticoagulant actions, whereas PLA 2 -12 lacked these effects. PLA 2 -12 was much more active in hydrolyzing a monodisperse synthetic substrate than PLA 2 -17, but the catalytic activity of latter was notably higher on a micellar substrate, or towards pure phospholipid artificial monolayers under controlled lateral pressures. Interestingly, PLA 2 -17 could hydrolyze substrate at a pressure of 20 mN m -1 , in contrast to PLA 2 -12 or the non-toxic pancreatic PLA 2 . This suggests important differences in the monolayer penetrating power, which could be related to differences in toxicity. Comparative examination of primary structures and predicted three-dimensional folding of PLA 2 -12 and PLA 2 -17, revealed that differences concentrate in their N-terminal and central regions, leading to variations of the surface properties at the membrane interacting interface. PLA 2 -17 presents a less basic interfacial surface than PLA 2 -12, but more bulky aromatic residues, which could be associated to its higher membrane-penetrating strength. Altogether, these structural and functional comparative observations suggest that the ability of PLA 2 s to penetrate substrate interfaces could be a major determinant of toxicity, perhaps more important than protein surface charge. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2014-01-01

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

  6. Structural analysis of phospholipase A2 from functional perspective. 1. Functionally relevant solution structure and roles of the hydrogen-bonding network.

    PubMed

    Yuan, C; Byeon, I J; Li, Y; Tsai, M D

    1999-03-09

    Bovine pancreatic phospholipase A2 (PLA2), a small (13.8 kDa) Ca2+-dependent lipolytic enzyme, is rich in functional and structural character. In an effort to examine its detailed structure-function relationship, we determined its solution structure by multidimensional nuclear magnetic resonance (NMR) spectroscopy at a functionally relevant pH. An ensemble of 20 structures generated has an average root-mean-square deviation (RMSD) of 0.62 +/- 0.08 A for backbone (N, Calpha, C) atoms and 0.98 +/- 0.09 A for all heavy atoms. The overall structure shows several notable differences from the crystal structure: the first three residues at the N-terminus, the calcium-binding loop (Y25-T36), and the surface loop (V63-N72) appear to be flexible; the alpha-helical conformation of helix B (E17-F22) is absent; helix D appears to be shorter (D59-V63 instead of D59-D66); and the hydrogen-bonding network is less defined. These differences were analyzed in relation to the function of PLA2. We then further examined the H-bonding network, because its functional role or even its existence in solution has been in dispute recently. Our results show that part of the H-bonding network (the portion away from N-terminus) clearly exists in solution, as evidenced by direct observation (at 11.1 ppm) of a strong H-bond between Y73 and D99 and an implicated interaction between D99 and H48. Analyses of a series of mutants indicated that the existence of the Y73.D99 H-bond correlates directly with the conformational stability of the mutant. Loss of this H-bond results in a loss of 2-3 kcal/mol in the conformational stability of PLA2. The unequivocal identification and demonstration of the structural importance of a specific hydrogen bond, and the magnitude of its contribution to conformational stability, are uncommon to the best of our knowledge. Our results also suggest that, while the D99.H48 catalytic diad is the key catalytic machinery of PLA2, it also helps to maintain conformational

  7. Efficient Extracellular Expression of Phospholipase D in Escherichia Coli with an Optimized Signal Peptide

    NASA Astrophysics Data System (ADS)

    Yang, Leyun; Xu, Yu; Chen, Yong; Ying, Hanjie

    2018-01-01

    New secretion vectors containing the synthetic signal sequence (OmpA’) was constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli Phospholipase D structural gene (Accession number:NC_018658) fused to various signal sequence were expressed from the Lac promoter in E. coli Rosetta strains by induction with 0.4mM IPTG at 28°C for 48h. SDS-PaGe analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of Phospholipase D (PLD) not only to the medium but also remained in periplasm of E. coli with OmpA’ signal sequence at the N-terminus of PLD. Thus the study on the effects of various surfactants on PLD extracellular production in Escherichia coli in shake flasks revealed that optimal PLD extracellular production could be achieved by adding 0.4% Triton X-100 into the medium. The maximal extracellular PLD production and extracellular enzyme activity were 0.23mg ml-1 and 16U ml-1, respectively. These results demonstrate the possibility of efficient secretory production of recombinant PLD in E. coli should be a potential industrial applications.

  8. Effects of active and inactive phospholipase D2 on signal transduction, adhesion, migration, invasion, and metastasis in EL4 lymphoma cells.

    PubMed

    Knoepp, Stewart M; Chahal, Manpreet S; Xie, Yuhuan; Zhang, Zhihong; Brauner, Daniel J; Hallman, Mark A; Robinson, Stephanie A; Han, Shujie; Imai, Masaki; Tomlinson, Stephen; Meier, Kathryn E

    2008-09-01

    The phosphatidylcholine-using phospholipase D (PLD) isoform PLD2 is widely expressed in mammalian cells and is activated in response to a variety of promitogenic agonists. In this study, active and inactive hemagglutinin-tagged human PLD2 (HA-PLD2) constructs were stably expressed in an EL4 cell line lacking detectable endogenous PLD1 or PLD2. The overall goal of the study was to examine the roles of PLD2 in cellular signal transduction and cell phenotype. HA-PLD2 confers PLD activity that is activated by phorbol ester, ionomycin, and okadaic acid. Proliferation and Erk activation are unchanged in cells transfected with active PLD2; proliferation rate is decreased in cells expressing inactive PLD2. Basal tyrosine phosphorylation of focal adhesion kinase (FAK) is increased in cells expressing active PLD2, as is phosphorylation of Akt; inactive PLD2 has no effect. Expression of active PLD2 is associated with increased spreading and elongation of cells on tissue culture plastic, whereas inactive PLD2 inhibits cell spreading. Inactive PLD2 also inhibits cell adhesion, migration, and serum-induced invasion. Cells expressing active PLD2 form metastases in syngeneic mice, as do the parental cells; cells expressing inactive PLD2 form fewer metastases than parental cells. In summary, active PLD2 enhances FAK phosphorylation, Akt activation, and cell invasion in EL4 lymphoma cells, whereas inactive PLD2 exerts inhibitory effects on adhesion, migration, invasion, and tumor formation. Overall, expression of active PLD2 enhances processes favorable to lymphoma cell metastasis, whereas expression of inactive PLD2 inhibits metastasis.

  9. Kinetic study of sunflower phospholipase Dα: interactions with micellar substrate, detergents and metals.

    PubMed

    Abdelkafi, Slim; Abousalham, Abdelkarim

    2011-07-01

    Phospholipase Dα (PLDα) purified from six-day post-germinated sunflower seeds was inactive in vitro on bilamellar substrates. It was fully active on mixed micelles made with phospholipids and a mixture of Triton-X100 and SDS at equal concentrations. It had an absolute need for divalent ions and calcium ions at millimolar concentration were the most efficient. Calcium had two effects. Firstly, using the fluorescent probe 2-p-toluidinylnaphtalene-6-sulfonate, we showed that the enzyme was able to bind calcium with a dissociation constant of 40-50 mM. This high value is probably due to the modification of the C2 domain which lacks some coordination residues allowing the binding of the metal. Secondly, using turbidity measurements, we showed that the metal ions interact with the SDS contained in the mixed micelles thus leading to an aggregated form of the substrate which is more easily hydrolyzed by PLDα. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  10. Transgenic labeling of higher order neuronal circuits linked to phospholipase C-β2-expressing taste bud cells in medaka fish.

    PubMed

    Ieki, Takashi; Okada, Shinji; Aihara, Yoshiko; Ohmoto, Makoto; Abe, Keiko; Yasuoka, Akihito; Misaka, Takumi

    2013-06-01

    The sense of taste plays a pivotal role in the food-selecting behaviors of vertebrates. We have shown that the fish ortholog of the phospholipase C gene (plc-β2) is expressed in a subpopulation of taste bud cells that transmit taste stimuli to the central nervous system to evoke favorable and aversive behaviors. We generated transgenic medaka expressing wheat germ agglutinin (WGA) under the control of a regulatory region of the medaka plc-β2 gene to analyze the neuronal circuit connected to these sensory cells. Immunohistochemical analysis of the transgenic fish 12 days post fertilization revealed that the WGA protein was transferred to cranial sensory ganglia and several nuclei in the hindbrain. WGA signals were also detected in the secondary gustatory nucleus in the hindbrain of 3-month-old transgenic fish. WGA signals were observed in several diencephalic and telencephalic regions in 9-month-old transgenic fish. The age-dependent increase in the labeled brain regions strongly suggests that labeling occurred at taste bud cells and progressively extended to cranial nerves and neurons in the central nervous system. These data are the first to demonstrate the tracing of higher order gustatory neuronal circuitry that is associated with a specific subpopulation of taste bud cells. These results provide insight into the basic neuronal architecture of gustatory information processing that is common among vertebrates. Copyright © 2012 Wiley Periodicals, Inc.

  11. Production of a recombinant phospholipase A2 in Escherichia coli using resonant acoustic mixing that improves oxygen transfer in shake flasks.

    PubMed

    Valdez-Cruz, Norma A; Reynoso-Cereceda, Greta I; Pérez-Rodriguez, Saumel; Restrepo-Pineda, Sara; González-Santana, Jesus; Olvera, Alejandro; Zavala, Guadalupe; Alagón, Alejandro; Trujillo-Roldán, Mauricio A

    2017-07-25

    Shake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom. Comparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (k L a ≈ 80 h -1 ) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and β-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response. In moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in

  12. Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.

    PubMed Central

    Lehto, M T; Sharom, F J

    1998-01-01

    Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion

  13. Regulation of activity in vitro and in vivo of three phospholipases B from Saccharomyces cerevisiae

    PubMed Central

    2004-01-01

    The genome of the yeast, Saccharomyces cerevisiae, contains three highly similar genes coding for phospholipases B/lysophospholipases. These enzymes behave differently with respect to substrate preferences in vitro and relative contributions to phospholipid catabolism in vivo [Merkel, Fido, Mayr, Prüger, Raab, Zandonella, Kohlwein and Paltauf (1999) J. Biol. Chem. 274, 28121–28127]. It is shown in the present study that, in vitro, pH markedly affects the substrate preference of Plb1p and Plb2p, but not of Plb3p. At the pH optimum of 2.5–3.5, the order of substrate preference of Plb1p and Plb2p is PtdSer (phosphatidylserine)>PtdIns>PtdCho (phosphatidylcholine>PtdEtn (phosphatidylethanolamine). At pH values of 5 and above, the substrate preferences change to PtdCho=PtdEtn for Plb1p and PtdSer=PtdEtn for Plb2p. Accordingly, with cultured cells the ratio of PtdIns/PtdCho breakdown, as reflected in the ratio of GroPIns (glycerophosphoinositol)/GroPCho (glycerophosphocholine) released into the culture medium, is inversely related to the pH of the growth medium. This effect is ascribed to the pH response of Plb1p, because Plb2p does not contribute to the degradation of PtdIns and PtdCho in vivo. Bivalent and tervalent cations activate phospholipases B at pH 5.5, but are inhibitory at pH 2.5. Al3+ at a concentration of 20 mM increases Plb1p activity in vitro by 8-fold and leads to a 9-fold increase in GroPCho release by whole cells. In vivo, cycloheximide strongly inhibits the breakdown of PtdIns, and to a lesser extent PtdCho. However, Al3+-stimulated GroPCho release is almost completely inhibited by cycloheximide. Deletion of PLB3 leads to increased sensitivity to toxic Al3+. Addition of SDS or melittin to cultured cells leads to a significant increase in phospholipid degradation, which is insensitive to inhibition by cycloheximide. Deletion mutants defective in the PLB1 gene are significantly more resistant to SDS than are wild-type cells. PMID:15588231

  14. Phospholipase C-ε links Epac2 activation to the potentiation of glucose-stimulated insulin secretion from mouse islets of Langerhans

    PubMed Central

    Dzhura, Igor; Chepurny, Oleg G; Leech, Colin A; Roe, Michael W; Dzhura, Elvira; Xu, Xin; Lu, Youming; Schwede, Frank; Genieser, Hans-G; Smrcka, Alan V

    2011-01-01

    Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is potentiated by cAMP-elevating agents, such as the incretin hormone glucagon-like peptide-1 (GLP-1) and cAMP exerts its insulin secretagogue action by activating both protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor designated as Epac2. Although prior studies of mouse islets demonstrated that Epac2 acts via Rap1 GTPase to potentiate GSIS, it is not understood which downstream targets of Rap1 promote the exocytosis of insulin. Here, we measured insulin secretion stimulated by a cAMP analog that is a selective activator of Epac proteins in order to demonstrate that a Rap1-regulated phospholipase C-epsilon (PLC-ε) links Epac2 activation to the potentiation of GSIS. Our analysis demonstrates that the Epac activator 8-pCPT-2′-O-Me-cAMP-AM potentiates GSIS from the islets of wild-type (WT) mice, whereas it has a greatly reduced insulin secretagogue action in the islets of Epac2 (−/−) and PLC-ε (−/−) knockout (KO) mice. Importantly, the insulin secretagogue action of 8-pCPT-2′-O-Me-cAMP-AM in WT mouse islets cannot be explained by an unexpected action of this cAMP analog to activate PKA, as verified through the use of a FRET-based A-kinase activity reporter (AKAR3) that reports PKA activation. Since the KO of PLC-ε disrupts the ability of 8-pCPT-2′-O-Me-cAMP-AM to potentiate GSIS, while also disrupting its ability to stimulate an increase of β-cell [Ca2+]i, the available evidence indicates that it is a Rap1-regulated PLC-ε that links Epac2 activation to Ca2+-dependent exocytosis of insulin. PMID:21478675

  15. Phospholipase C-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate underlies agmatine-induced suppression of N-type Ca2+ channel in rat celiac ganglion neurons.

    PubMed

    Kim, Young-Hwan; Jeong, Ji-Hyun; Ahn, Duck-Sun; Chung, Seungsoo

    2017-03-04

    Agmatine suppresses peripheral sympathetic tone by modulating Cav2.2 channels in peripheral sympathetic neurons. However, the detailed cellular signaling mechanism underlying the agmatine-induced Cav2.2 inhibition remains unclear. Therefore, in the present study, we investigated the electrophysiological mechanism for the agmatine-induced inhibition of Cav2.2 current (I Cav2.2 ) in rat celiac ganglion (CG) neurons. Consistent with previous reports, agmatine inhibited I Cav2.2 in a VI manner. The agmatine-induced inhibition of the I Cav2.2 current was also almost completely hindered by the blockade of the imidazoline I 2 receptor (IR 2 ), and an IR 2 agonist mimicked the inhibitory effect of agmatine on I Cav2.2 , implying involvement of IR 2 . The agmatine-induced I Cav2.2 inhibition was significantly hampered by the blockade of G protein or phospholipase C (PLC), but not by the pretreatment with pertussis toxin. In addition, diC8-phosphatidylinositol 4,5-bisphosphate (PIP 2 ) dialysis nearly completely hampered agmatine-induced inhibition, which became irreversible when PIP 2 resynthesis was blocked. These results suggest that in rat peripheral sympathetic neurons, agmatine-induced IR 2 activation suppresses Cav2.2 channel voltage-independently, and that the PLC-dependent PIP 2 hydrolysis is responsible for the agmatine-induced suppression of the Cav2.2 channel. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Immunohistochemical localization of hepatopancreatic phospholipase in gastropods mollusc, Littorina littorea and Buccinum undatum digestive cells

    PubMed Central

    2011-01-01

    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. PMID:22114916

  17. The phospholipase PNPLA7 functions as a lysophosphatidylcholine hydrolase and interacts with lipid droplets through its catalytic domain.

    PubMed

    Heier, Christoph; Kien, Benedikt; Huang, Feifei; Eichmann, Thomas O; Xie, Hao; Zechner, Rudolf; Chang, Ping-An

    2017-11-17

    Mammalian patatin-like phospholipase domain-containing proteins (PNPLAs) are lipid-metabolizing enzymes with essential roles in energy metabolism, skin barrier development, and brain function. A detailed annotation of enzymatic activities and structure-function relationships remains an important prerequisite to understand PNPLA functions in (patho-)physiology, for example, in disorders such as neutral lipid storage disease, non-alcoholic fatty liver disease, and neurodegenerative syndromes. In this study, we characterized the structural features controlling the subcellular localization and enzymatic activity of PNPLA7, a poorly annotated phospholipase linked to insulin signaling and energy metabolism. We show that PNPLA7 is an endoplasmic reticulum (ER) transmembrane protein that specifically promotes hydrolysis of lysophosphatidylcholine in mammalian cells. We found that transmembrane and regulatory domains in the PNPLA7 N-terminal region cooperate to regulate ER targeting but are dispensable for substrate hydrolysis. Enzymatic activity is instead mediated by the C-terminal domain, which maintains full catalytic competence even in the absence of N-terminal regions. Upon elevated fatty acid flux, the catalytic domain targets cellular lipid droplets and promotes interactions of PNPLA7 with these organelles in response to increased cAMP levels. We conclude that PNPLA7 acts as an ER-anchored lysophosphatidylcholine hydrolase that is composed of specific functional domains mediating catalytic activity, subcellular positioning, and interactions with cellular organelles. Our study provides critical structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

    PubMed

    Husain, S; Abdel-Latif, A A

    1999-08-15

    We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid

  19. Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

    PubMed Central

    Husain, S; Abdel-Latif, A A

    1999-01-01

    We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid

  20. Purification, characterization, molecular cloning and extracellular production of a phospholipase A1 from Streptomyces albidoflavus NA297.

    PubMed

    Sugimori, Daisuke; Kano, Kota; Matsumoto, Yusaku

    2012-01-01

    A novel metal ion-independent phospholipase A1 of Streptomyces albidoflavus isolated from Japanese soil has been purified and characterized. The enzyme consists of a 33-residue N-terminal signal secretion sequence and a 269-residue mature protein with a deduced molecular weight of 27,199. Efficient and extracellular production of the recombinant enzyme was successfully achieved using Streptomyces lividans cells and an expression vector. A large amount (25 mg protein, 14.7 kU) of recombinant enzyme with high specific activity (588 U/mg protein) was purified by simple purification steps. The maximum activity was found at pH 7.2 and 50 °C. At pH 7.2, the enzyme preferably hydrolyzed phosphatidic acid and phosphatidylserine; however, the substrate specificity was dependent on the reaction pH. The enzyme hydrolyzed lysophosphatidylcholine and not triglyceride and the p-nitrophenyl ester of fatty acids. At the reaction equilibrium, the molar ratio of released free fatty acids (sn-1:sn-2) was 63:37. The hydrolysis of phosphatidic acid at 50 °C and pH 7.2 gave apparent V max and k cat values of 1389 μmol min(-1) mg protein(-1) and 630 s(-1), respectively. The apparent K m and k cat/K m values were 2.38 mM and 265 mM(-1) s(-1), respectively. Mutagenesis analysis showed that Ser11 is essential for the catalytic function of the enzyme and the active site may include residues Ser216 and His218.