Sample records for a2 pla2 enzymes

  1. PLA2 mediated arachidonate free radicals: PLA2 inhibition and neutralization of free radicals by anti-oxidants--a new role as anti-inflammatory molecule.

    PubMed

    Nanda, B L; Nataraju, A; Rajesh, R; Rangappa, K S; Shekar, M A; Vishwanath, B S

    2007-01-01

    PLA2 enzyme catalyses the hydrolysis of cellular phospholipids at the sn-2 position to liberate arachidonic acid and lysophospholipid to generate a family of pro-inflammatory eicosanoids and platelet activating factor. The generation of pro-inflammatory eicosanoids involves a series of free radical intermediates with simultaneous release of reactive oxygen species (superoxide and hydroxyl radicals). Reactive oxygen species formed during arachidonic acid metabolism generates lipid peroxides and the cytotoxic products such as 4-hydroxy nonenal and acrolein, which induces cellular damage. Thus PLA2 catalyzes the rate-limiting step in the production of pro-inflammatory eicosanoids and free radicals. These peroxides and reactive oxygen species in turn activates PLA2 enzyme and further attenuates the inflammatory process. Therefore scavenging these free radicals and inhibition of PLA2 enzyme simultaneously by a single molecule such as antioxidants is of great therapeutic relevance for the development of anti-inflammatory molecules. PLA2 enzymes have been classified into calcium dependent cPLA2 and sPLA2 and calcium independent iPLA2 forms. In several inflammatory diseases sPLA2 group IIA is the most abundant isoform identified. This isoform is therefore targeted for the development of anti-inflammatory molecules. Many secondary metabolites from plants and marine sponges exhibit both anti-inflammatory and antioxidant properties. Some of them include flavonoids, terpenes and alkaloids. But in terms of PLA2 inhibition and antioxidant activity, the structural aspects of flavonoids are well studied rather than terpenes and alkaloids. In this line, molecules having both anti-oxidant and PLA2 inhibitions are reviewed. A single molecule with dual activities may prove to be a powerful anti-inflammatory drug.

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed

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

    2018-03-01

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

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

    PubMed

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

    2014-04-01

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

  5. 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

  6. A two-photon view of an enzyme at work: Crotalus atrox venom PLA2 interaction with single-lipid and mixed-lipid giant unilamellar vesicles.

    PubMed Central

    Sanchez, Susana A; Bagatolli, Luis A; Gratton, Enrico; Hazlett, Theodore L

    2002-01-01

    We describe the interaction of Crotalus atrox-secreted phospholipase A2 (sPLA2) with giant unilamellar vesicles (GUVs) composed of single and binary phospholipid mixtures visualized through two-photon excitation fluorescent microscopy. The GUV lipid compositions that we examined included 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (above their gel-liquid crystal transition temperatures) and two well characterized lipid mixtures, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):DMPC (7:3) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC) (1:1) equilibrated at their phase-coexistence temperature regime. The membrane fluorescence probes, 6-lauroyl-2-(dimethylamino) napthalene, 6-propionyl-2-(dimethylamino) naphthalene, and rhodamine-phosphatidylethanolamine, were used to assess the state of the membrane and specifically mark the phospholipid domains. Independent of their lipid composition, all GUVs were reduced in size as sPLA2-dependent lipid hydrolysis proceeded. The binding of sPLA2 was monitored using a fluorescein-sPLA2 conjugate. The sPLA2 was observed to associate with the entire surface of the liquid phase in the single phospholipid GUVs. In the mixed-lipid GUV's, at temperatures promoting domain coexistence, a preferential binding of the enzyme to the liquid regions was also found. The lipid phase of the GUV protein binding region was verified by the introduction of 6-propionyl-2-(dimethylamino) naphthalene, which partitions quickly into the lipid fluid phase. Preferential hydrolysis of the liquid domains supported the conclusions based on the binding studies. sPLA2 hydrolyzes the liquid domains in the binary lipid mixtures DLPC:DAPC and DMPC:DMPE, indicating that the solid-phase packing of DAPC and DMPE interferes with sPLA2 binding, irrespective of the phospholipid headgroup. These studies

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

    PubMed

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

    2016-05-01

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

  8. 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

  9. Pla a 2 and Pla a 3 reactivities identify plane tree-allergic patients with respiratory symptoms or food allergy.

    PubMed

    Scala, E; Cecchi, L; Abeni, D; Guerra, E C; Pirrotta, L; Locanto, M; Giani, M; Asero, R

    2017-04-01

    Nine hundred and thirty-nine rPla a 1, nPla a 2, and rPla a 3 ImmunoCAP ISAC reactors were studied. nPla a 2 pos MUXF3 pos but Pla a 1/2 neg subjects were excluded from the study because they were cross-reactive carbohydrate determinant reactors. Among the 764 remaining participants, 71.9% were Pla a 3 pos , 54.1% Pla a 2 pos , and 10.9% Pla a 1 pos . Among Pla a 3 reactors, 89.6% were Pru p 3 pos and 86.8% Jug 3 pos , but the strongest IgE recognition relationship was observed between Pla a 3 and Jug r 3. Distinctive clinical subsets could be documented among plane tree-allergic patients. Pla a 3 reactors had both local and systemic food-induced reactions, but lower past respiratory symptoms occurrence. Pla a 2 reactivity was associated with respiratory symptoms but inversely related to systemic reactions to food. Cosensitization to Pla a 2 and Pla a 3 was associated with a lower past incidence of severe food-induced reactions. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Analysis of Several PLA2 mRNA in Human Meningiomas

    PubMed Central

    Denizot, Yves; De Armas, Rafael; Durand, Karine; Robert, Sandrine; Moreau, Jean-Jacques; Caire, François; Weinbreck, Nicolas; Labrousse, François

    2009-01-01

    In view of the important oncogenic action of phospholipase A2(PLA2) we investigated PLA2 transcripts in human meningiomas. Real-time PCR was used to investigate PLA2 transcripts in 26 human meningioma tumors. Results indicated that three Ca2+-dependent high molecular weight PLA2 (PLA2-IVA, PLA2-IVB, PLA2-IVC), one Ca2+-independent high molecular weight PLA2 (PLA2-VI) and five low molecular weight secreted forms of PLA2 (PLA2-IB, PLA2-IIA, PLA2-III, PLA2-V, and PLA2-XII) are expressed with PLA2-IVA, PLA2-IVB, PLA2-VI, and PLA2-XIIA as the major expressed forms. PLA2-IIE, PLA2-IIF, PLA2-IVD, and PLA2-XIIB are not detected. Plasma (PLA2-VIIA) and intracellular (PLA2-VIIB) platelet-activating factor acetylhydrolase transcripts are expressed in human meningiomas. However no difference was found for PLA2 transcript amounts in relation to the tumor grade, the subtype of meningiomas, the presence of inflammatory infiltrated cells, of an associated edema, mitosis, brain invasion, vascularisation or necrosis. In conclusion numerous genes encoding multiples forms of PLA2 are expressed in meningiomas where they might act on the phospholipid remodeling and on the local eicosanoid and/or cytokine networks. PMID:20339511

  11. Identification of a novel antisense long non-coding RNA PLA2G16-AS that regulates the expression of PLA2G16 in pigs.

    PubMed

    Liu, Pengliang; Jin, Long; Zhao, Lirui; Long, Keren; Song, Yang; Tang, Qianzi; Ma, Jideng; Wang, Xun; Tang, Guoqing; Jiang, Yanzhi; Zhu, Li; Li, Xuewei; Li, Mingzhou

    2018-05-31

    Natural antisense transcripts (NATs) are widely present in mammalian genomes and act as pivotal regulator molecules to control gene expression. However, studies on the NATs of pigs are relatively rare. Here, we identified a novel antisense transcript, designated PLA2G16-AS, transcribed from the phospholipase A2 group XVI locus (PLA2G16) in the porcine genome, which is a well-known regulatory molecule of fat deposition. PLA2G16-AS and PLA2G16 were dominantly expressed in porcine adipose tissue, and were differentially expressed between Tibetan pigs and Rongchang pigs. In addition, PLA2G16-AS has a weak sequence conservation among different vertebrates. PLA2G16-AS was also shown to form an RNA-RNA duplex with PLA2G16, and to regulate PLA2G16 expression at the mRNA level. Moreover, the overexpression of PLA2G16-AS increased the stability of PLA2G16 mRNA in porcine cells. We envision that our findings of a NAT for a regulatory gene associated with lipolysis might further our understanding of the molecular regulation of fat deposition. Copyright © 2017. Published by Elsevier B.V.

  12. 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.

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

    PubMed Central

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

    2013-01-01

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

  14. 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.

  15. PLA2G7 genotype, Lp-PLA2 activity and coronary heart disease risk in 10,494 cases and 15,624 controls of European ancestry

    PubMed Central

    Casas, Juan P.; Ninio, Ewa; Panayiotou, Andrie; Palmen, Jutta; Cooper, Jackie A; Ricketts, Sally L; Sofat, Reecha; Nicolaides, Andrew N; Corsetti, James P; Fowkes, F Gerry R; Tzoulaki, Ioanna; Kumari, Meena; Brunner, Eric J; Kivimaki, Mika; Marmot, Michael G; Hoffmann, Michael M; Winkler, Karl; März, Winfred; Ye, Shu; Stirnadel, Heide A; MBBChir, Kay-Tee Khaw; Humphries, Steve E; Sandhu, Manjinder S; Hingorani, Aroon D; Talmud, Philippa J

    2012-01-01

    Background Higher Lp-PLA2 activity is associated with increased risk of coronary heart disease (CHD), making Lp-PLA2 a potential therapeutic target. PLA2G7 variants associated with Lp-PLA2 activity could evaluate whether this relationship is causal. Methods and Results A meta-analysis including a total of 12 studies (5 prospective, 4 case-control, 1 case-only and 2 cross-sectional, n=26,118) was undertaken to examine the association of: (i) LpPLA2 activity vs. cardiovascular biomarkers and risk factors and CHD events (two prospective studies; n=4884); ii) PLA2G7 SNPs and Lp-PLA2 activity (3 prospective, 2 case-control, 2 cross-sectional studies; up to n=6094); and iii) PLA2G7 SNPs and angiographic coronary artery disease (2 case-control, 1 case-only study; n=4971 cases) and CHD events (5 prospective, 2 case-control studies; n=5523). Lp-PLA2 activity correlated with several CHD risk markers. Hazard ratio for CHD events top vs. bottom quartile of Lp-PLA2 activity was 1.61 (95%CI: 1.31, 1.99) and 1.17 (95%CI: 0.91, 1.51) after adjustment for baseline traits. Of seven SNPs, rs1051931 (A379V) showed the strongest association with Lp-PLA2 activity, VV subjects having 7.2% higher activity than AAs. Genotype was not associated with risk markers, angiographic coronary disease (OR 1.03 (95%CI 0.80, 1.32), or CHD events (OR 0.98 (95%CI 0.82, 1.17). Conclusions Unlike Lp-PLA2 activity, PLA2G7 variants associated with modest effects on Lp-PLA2 activity were not associated with cardiovascular risk markers, coronary atheroma or CHD. Larger association studies, identification of SNPs with larger effects, or randomised trials of specific Lp-PLA2 inhibitors are needed to confirm/refute a contributory role for Lp-PLA2 in CHD. PMID:20479152

  16. Stretch-induced ERK2 phosphorylation requires PLA2 activity in skeletal myotubes.

    PubMed

    Burkholder, Thomas J

    2009-08-14

    Mechanical stretch rapidly activates multiple signaling cascades, including phospholipases and kinases, to stimulate protein synthesis and growth. The purpose of this study was to determine whether PLA2 activation contributes to stretch-induced phosphorylation of ERK2 in skeletal muscle myotubes. Myotubes derived from neonatal C57 mice were cultured on silicone membranes and subjected to brief cyclic stretch. Inhibition of PLA2 prevented ERK2 phosphorylation, while inhibition of prostaglandin or leukotriene synthesis did not. ERK2 phosphorylation was also blocked by genistein and PD98059, implicating the canonical raf-MEK-ERK cassette. It appears that PLA2, but not further metabolism of arachidonic acid, is required for stretch-induced activation of ERK2. Exposure to exogenous arachidonic acid had no effect on ERK2 phosphorylation, but exposure to lysophosphatidylcholine, the other metabolite of PLA2, caused a dose-dependent increase in ERK2 phosphorylation. These results suggest that stretch-induced activation of ERK2 may result from an interaction between PLA2 derived lysophosphatidylcholine and membrane receptors.

  17. Stretch-induced ERK2 phosphorylation requires PLA2 activity in skeletal myotubes

    PubMed Central

    Burkholder, Thomas J.

    2009-01-01

    Mechanical stretch rapidly activates multiple signaling cascades, including phospholipases and kinases, to stimulate protein synthesis and growth. The purpose of this study was to determine whether PLA2 activation contributes to stretch-induced phosphorylation of ERK2 in skeletal muscle myotubes. Myotubes derived from neonatal C57 mice were cultured on silicone membranes and subjected to brief cyclic stretch. Inhibition of PLA2 prevented ERK2 phosphorylation, while inhibition of prostaglandin or leukotriene synthesis did not. ERK2 phosphorylation was also blocked by genistein and PD98059, implicating the canonical raf-MEK-ERK cassette. It appears that PLA2, but not further metabolism of arachidonic acid, is required for stretch-induced activation of ERK2. Exposure to exogenous arachidonic acid had no effect on ERK2 phosphorylation, but exposure to lysophosphatidylcholine, the other metabolite of PLA2, caused a dose-dependent increase in ERK2 phosphorylation. These results suggest that stretch-induced activation of ERK2 may result from an interaction between PLA2 derived lysophosphatidylcholine and membrane receptors. PMID:19524551

  18. Involvement of PLA2, COX and LOX in Rhinella arenarum oocyte maturation.

    PubMed

    Ortiz, Maria Eugenia; Bühler, Marta Inés; Zelarayán, Liliana Isabel

    2014-11-01

    In Rhinella arenarum, progesterone is the physiological nuclear maturation inducer that interacts with the oocyte surface and starts a cascade of events that leads to germinal vesicle breakdown (GVBD). Polyunsaturated fatty acids and their metabolites produced through cyclooxygenase (COX) and lipoxygenase (LOX) pathways play an important role in reproductive processes. In amphibians, to date, the role of arachidonic acid (AA) metabolites in progesterone (P4)-induced oocyte maturation has not been clarified. In this work we studied the participation of three enzymes involved in AA metabolism - phospholipase A2 (PLA2), COX and LOX in Rhinella arenarum oocyte maturation. PLA2 activation induced maturation in Rhinella arenarum oocytes in a dose-dependent manner. Oocytes when treated with 0.08 μM melittin showed the highest response (78 ± 6% GVBD). In follicles, PLA2 activation did not significantly induce maturation at the assayed doses (12 ± 3% GVBD). PLA2 inhibition with quinacrine prevented melittin-induced GVBD in a dose-dependent manner, however PLA2 inactivation did not affect P4-induced maturation. This finding suggests that PLA2 is not the only phospholipase involved in P4-induced maturation in this species. P4-induced oocyte maturation was inhibited by the COX inhibitors indomethacin and rofecoxib (65 ± 3% and 63 ± 3% GVBD, respectively), although COX activity was never blocked by their addition. Follicles showed a similar response following the addition of these inhibitors. Participation of LOX metabolites in maturation seems to be correlated with seasonal variation in ovarian response to P4. During the February to June period (low P4 response), LOX inhibition by nordihydroguaiaretic acid or lysine clonixinate increased maturation by up to 70%. In contrast, during the July to January period (high P4 response), LOX inhibition had no effect on hormone-induced maturation.

  19. Acanthamoeba-Cytopathic Protein Induces Apoptosis and Proinflammatory Cytokines in Human Corneal Epithelial Cells by cPLA2α Activation

    PubMed Central

    Tripathi, Trivendra; Smith, Ashley Dawn; Abdi, Mahshid; Alizadeh, Hassan

    2012-01-01

    Purpose. We have shown that Acanthamoeba interacts with a mannosylated protein on corneal epithelial cells and stimulates trophozoites to secrete a mannose-induced 133 kDa protease (MIP-133), which facilitates corneal invasion and induces apoptosis. The mechanism of MIP-133–induced apoptosis is unknown. The aim of this study was to determine if MIP-133 induces apoptosis and proinflammatory cytokines/chemokines in human corneal epithelial (HCE) cells via the cytosolic phospholipase A2α (cPLA2α) pathway. Methods. HCE cells were incubated with or without MIP-133 at doses of 7.5, 15, and 50 μg/mL for 6, 12, and 24 hours. The effects of cPLA2α inhibitors on cPLA2α, arachidonic acid (AA) release, and apoptosis were tested in vitro. Inhibition of cPLA2α involved preincubating HCE cells for 1 hour with cPLA2α inhibitors (10 μM methyl-arachidonyl fluorophosphonate [MAFP] or 20 μM arachidonyl trifluoromethyl ketone [AACOCF3]) with or without MIP-133 for 24 hours. Expression of cPLA2α mRNA and enzyme was examined by RT-PCR and cPLA2 activity assays, respectively. Apoptosis of corneal epithelial cells was determined by caspase-3 and DNA fragmentation assays. Expression of IL-8, IL-6, IL-1β, and IFN-γ was examined by RT-PCR and ELISA. Results. MIP-133 induced significant cPLA2α (approximately two to four times) and AA release (approximately six times) from corneal cells while cPLA2α inhibitors significantly reduced cPLA2α (approximately two to four times) and AA release (approximately three times) (P < 0.05). cPLA2α inhibitors significantly inhibited MIP-133–induced DNA fragmentation approximately 7 to 12 times in HCE cells (P < 0.05). MIP-133 specifically activates cPLA2α enzyme activity in HCE cells, which is blocked by preincubation with anti–MIP-133 antibody. In addition, MIP-133 induced significant IL-8, IL-6, IL-1β, and IFN-γ production, approximately two to three times (P < 0.05). Conclusions. MIP-133 interacts with phospholipids on plasma

  20. PKCalpha regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes.

    PubMed

    Li, Qing; Subbulakshmi, Venkita; Oldfield, Claudine M; Aamir, Rozina; Weyman, Crystal M; Wolfman, Alan; Cathcart, Martha K

    2007-02-01

    Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O(2)(-)) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKCalpha as a kinase pathway required for monocyte O(2)(-) production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKCalpha and cPLA(2) by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA(2) enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA(2) activity. To distinguish between PKCalpha and PKCbeta isoenzymes in regulating cPLA(2) protein phosphorylation and enzymatic activity, we employed our previously characterized PKCalpha or PKCbeta isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764-3771]. Suppression of PKCalpha expression, but not PKCbeta expression, inhibited cPLA(2) protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA(2) phosphorylation and activation. We also found that cPLA(2) co-immunoprecipitated with PKCalpha and vice versa. In vitro studies demonstrated that PKCalpha could directly phosphorylate cPLA(2).and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O(2)(-) in monocytes defective in either PKCalpha or cPLA(2) expression. Taken together, our data suggest that PKCalpha, but not PKCbeta, is the predominant cPKC isoenzyme required for cPLA(2) protein phosphorylation and maximal induction of cPLA(2) enzymatic activity upon activation of human monocytes. Our data also support the

  1. 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

  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. Novel enzymatic method for assaying Lp-PLA2 in serum.

    PubMed

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

    2018-06-01

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

  4. 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.

  5. 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

  6. RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA2 Activation.

    PubMed

    Wu, Xiangbing; Walker, Chandler L; Lu, Qingbo; Wu, Wei; Eddelman, Daniel B; Parish, Jonathan M; Xu, Xiao-Ming

    2017-11-01

    Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A 2 (cPLA 2 ), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA 2 . Inhibition of RhoA, Rho kinase and cPLA 2 significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA 2 activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA 2 . The immunofluorescence staining showed that ROCK 1 or ROCK 2 , two isoforms of Rho kinase, was co-localized with cPLA 2 in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK 1 or ROCK 2 bonded directly with cPLA 2 and phospho-cPLA 2 . When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA 2 activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA 2 activation.

  7. 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

  8. Replacing with whole grains and legumes reduces Lp-PLA2 activities in plasma and PBMCs in patients with prediabetes or T2D1

    PubMed Central

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

    2014-01-01

    To determine dietary effects on circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and enzyme activity in peripheral blood mononuclear cells (PBMCs), 99 patients with impaired fasting glucose, impaired glucose tolerance, or newly-diagnosed T2D were randomly assigned to either a control group (usual diet with refined rice) or the whole grain and legume group. Substitution of whole grains and legumes for refined rice was associated with the replacement of 7% of energy from carbohydrates with energy from protein (about 4%) and fat. After 12 weeks, the whole grain and legume group showed a significant decrease in fasting glucose, insulin, homeostasis model assessment-insulin resistance, hemoglobin A1c, malondialdehyde, plasma Lp-PLA2 activity, and oxidized LDL (ox-LDL), and an increase in LDL particle size. The changes (Δs) in these variables in the whole grain and legume group were significantly different from those in controls after adjustment for the baseline levels. When all subjects were considered, Δ plasma Lp-PLA2 positively correlated with Δ glucose, Δ PBMC Lp-PLA2, Δ ox-LDL, and Δ urinary 8-epi-prostaglandin F2α after being adjusted for confounding factors. The Δ PBMC Lp-PLA2 correlated positively with Δ glucose and Δ ox-LDL, and negatively with Δ LDL particle size and baseline PBMC Lp-PLA2. The substitution of whole grains and legumes for refined rice resulted in a reduction in Lp-PLA2 activities in plasma and PBMCs partly through improved glycemic control, increased consumption of protein relative to carbohydrate, and reduced lipid peroxides. PMID:24904022

  9. Effect of PlA1/A2 glycoprotein IIIa gene polymorphism on the long-term outcome after successful coronary stenting

    PubMed Central

    Le Hello, Claire; Morello, Rémy; Lequerrec, Agnès; Duarte, Christine; Riddell, John; Hamon, Martial

    2007-01-01

    Aim To prospectively determine the role of platelet glycoprotein IIIa (GP IIIa) gene PlA1/PlA2 polymorphism on the long-term clinical outcome in patients with coronary artery disease undergoing coronary stenting. Design and setting Prospective observational study in the University Hospital of Caen (France). Patients and methods 1 111 symptomatic consecutive Caucasian patients treated with percutaneous coronary intervention including stent implantation underwent genotyping for GP IIIa PlA1/A2. Main outcome measures Long-term clinical outcome in terms of the rate of major adverse cardiac events (MACE, ie death from any cause, non-fatal Q wave or non Q wave myocardial infarction, and need for coronary revascularisation) was obtained and subsequently stratified according to the GP IIIa PlA1/A2 polymorphism. Results Three groups of patients were determined according to the GP IIIa PlA1/A2 polymorphism (71.6% had the A1/A1, 25.8% had the A1/A2 and 2.6% had the A2/A2 genotype). These three groups were comparable for all clinical characteristics including sex ratio, mean age, vascular risk factors, previous coronary events, baseline angiographic exam, indication for the percutaneous coronary intervention and drug therapy). The incidence of MACE was similar in these 3 groups of patients during a mean follow-up period of 654+/-152 days. Independent risk factors for MACE were a left ventricular ejection fraction < 40%, absence of treatment with a beta-blocker and absence of treatment with an angiotensin converting enzyme inhibitor during follow-up. Conclusion The GP IIIa PlA1/A2 polymorphism does not influence the clinical long-term outcome in patients with symptomatic coronary disease undergoing percutaneous coronary intervention with stent implantation. PMID:18021403

  10. Differential expression of Lp-PLA2 in obesity and type 2 diabetes and the influence of lipids.

    PubMed

    Jackisch, Laura; Kumsaiyai, Warunee; Moore, Jonathan D; Al-Daghri, Nasser; Kyrou, Ioannis; Barber, Thomas M; Randeva, Harpal; Kumar, Sudhesh; Tripathi, Gyanendra; McTernan, Philip G

    2018-05-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a circulatory macrophage-derived factor that increases with obesity and leads to a higher risk of cardiovascular disease (CVD). Despite this, its role in adipose tissue and the adipocyte is unknown. Therefore, the aims of this study were to clarify the expression of Lp-PLA2 in relation to different adipose tissue depots and type 2 diabetes, and ascertain whether markers of obesity and type 2 diabetes correlate with circulating Lp-PLA2. A final aim was to evaluate the effect of cholesterol on cellular Lp-PLA2 in an in vitro adipocyte model. Analysis of anthropometric and biochemical variables from a cohort of lean (age 44.4 ± 6.2 years; BMI 22.15 ± 1.8 kg/m 2 , n = 23), overweight (age 45.4 ± 12.3 years; BMI 26.99 ± 1.5 kg/m 2 , n = 24), obese (age 49.0 ± 9.1 years; BMI 33.74 ± 3.3 kg/m 2 , n = 32) and type 2 diabetic women (age 53.0 ± 6.13 years; BMI 35.08 ± 8.6 kg/m 2 , n = 35), as part of an ethically approved study. Gene and protein expression of PLA2 and its isoforms were assessed in adipose tissue samples, with serum analysis undertaken to assess circulating Lp-PLA2 and its association with cardiometabolic risk markers. A human adipocyte cell model, Chub-S7, was used to address the intracellular change in Lp-PLA2 in adipocytes. Lp-PLA2 and calcium-independent PLA2 (iPLA2) isoforms were altered by adiposity, as shown by microarray analysis (p < 0.05). Type 2 diabetes status was also observed to significantly alter gene and protein levels of Lp-PLA2 in abdominal subcutaneous (AbdSc) (p < 0.01), but not omental, adipose tissue. Furthermore, multivariate stepwise regression analysis of circulating Lp-PLA2 and metabolic markers revealed that the greatest predictor of Lp-PLA2 in non-diabetic individuals was LDL-cholesterol (p = 0.004). Additionally, in people with type 2 diabetes, oxidised LDL (oxLDL), triacylglycerols and HDL

  11. 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

  12. Cross-reactivity of anti-PLA2R1 autoantibodies to rabbit and mouse PLA2R1 antigens and development of two novel ELISAs with different diagnostic performances in idiopathic membranous nephropathy.

    PubMed

    Seitz-Polski, Barbara; Dolla, Guillaume; Payré, Christine; Tomas, Nicola M; Lochouarn, Marine; Jeammet, Louise; Mariat, Christophe; Krummel, Thierry; Burtey, Stéphane; Courivaud, Cécile; Schlumberger, Wolfgang; Zorzi, Kévin; Benzaken, Sylvia; Bernard, Ghislaine; Esnault, Vincent L M; Lambeau, Gérard

    2015-11-01

    About 70% of patients with idiopathic membranous nephropathy (iMN) have autoantibodies to the phospholipase A2 receptor PLA2R1. We screened sera from iMN patients for their cross-reactivity to human (h), rabbit (rb) and mouse (m) PLA2R1 by western blot (WB) and antigen-specific ELISAs. All iMN patients recognized hPLA2R1 and rbPLA2R1 by WB, and a rbPLA2R1 ELISA was as sensitive as the standardized hPLA2R1 ELISA to monitor anti-PLA2R1 in patients with active disease or in drug-induced remission. In contrast, only 51% of patients were reactive to mPLA2R1 by WB, and a maximum of 78% were weakly to highly positive in the mPLA2R1 ELISA, suggesting that iMN patients exhibit different subsets of anti-PLA2R1 autoantibodies against epitopes that are shared or not among PLA2R1 orthologs. In a cohort of 41 patients with a mean follow-up of 42 months from anti-PLA2R1 assay, the detection of anti-mPLA2R1 autoantibodies was an independent predictor of clinical outcome in multivariate analysis (p = 0.009), and a ROC curve analysis identified a threshold of 605 RU/mL above which 100% of patients (12 patients) had a poor renal outcome (p < 0.001). A similar threshold could not be defined in hPLA2R1 and rbPLA2R1 ELISAs. We conclude that rbPLA2R1 is an alternative antigen to hPLA2R1 to measure anti-PLA2R1 in active disease while mPLA2R1 is a unique antigen that can detect a subset of anti-PLA2R1 autoantibodies present at high levels (>605 RU/mL) only in iMN patients at risk of poor prognosis, and is thus useful to predict iMN outcome. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  13. 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

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

    PubMed Central

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

    2017-01-01

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

  15. Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA2 neurotoxin from the venom of Daboia russelli siamensis.

    PubMed

    Maung-Maung-Thwin; Gopalakrishnakone, P; Yuen, R; Tan, C H

    1996-02-01

    Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations

  16. 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.

  17. Serum anti-PLA2R antibody as a diagnostic biomarker of idiopathic membranous nephropathy: The optimal cut-off value for Chinese patients.

    PubMed

    Liu, Yipeng; Li, Xuan; Ma, Chaoqun; Wang, Ping; Liu, Ju; Su, Hong; Zhuo, Hao; Kong, Xianglei; Xu, Dayu; Xu, Dongmei

    2018-01-01

    The M-type phospholipase A2 receptor (PLA2R) is a specific target autoantigen identified in idiopathic membranous nephropathy (IMN). The autoantibody against PLA2R (anti-PLA2R) may be used to diagnose IMN. However, the appropriate diagnosis cut-off value for Chinese patients with IMN has not been established. In total, 119 patients who underwent renal biopsy (57 patients with IMN and 62 patients with non-IMN glomerulonephritis) and 22 healthy individuals were recruited for our observation study from Qianfoshan Hospital between September 2011 and March 2016. The serum concentration of anti-PLA2R was measured using a quantitative enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and receiver operating characteristic (ROC) curve of anti-PLA2R in diagnosing IMN were analysed based on the ELISA detection. The sensitivity, specificity, PPV, and NPV of anti-PLA2R in the diagnosis of IMN in the Chinese patients were 82.5, 75, 69.1, and 86.3% for the 2RU/ml cut-off value; 78.9, 91.7, 86.5, and 86.5% for the 2.6RU/ml cut-off value; 59.6, 95.2, 89.5, and 77.7% for the 14RU/ml cut-off value; 50.9, 96.4, 90.6, and 74.3% for the 20RU/ml cut-off value; and 47.4, 97.6, 93.1, and 73.2% for the 40RU/ml cut-off value, respectively. The area under the ROC curve was 0.879. The cut-off value of 2.6RU/ml is recommended for the use of anti-PLA2R for the diagnosis of IMN in Chinese patients based on the ELISA. Copyright © 2017. Published by Elsevier B.V.

  18. 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

  19. 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

  20. 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.

  1. The PLA2R1-JAK2 pathway upregulates ERRα and its mitochondrial program to exert tumor-suppressive action.

    PubMed

    Griveau, A; Devailly, G; Eberst, L; Navaratnam, N; Le Calvé, B; Ferrand, M; Faull, P; Augert, A; Dante, R; Vanacker, J M; Vindrieux, D; Bernard, D

    2016-09-22

    Little is known about the biological role of the phospholipase A2 receptor (PLA2R1) transmembrane protein. In recent years, PLA2R1 has been shown to have an important role in regulating tumor-suppressive responses via JAK2 activation, but the underlying mechanisms are largely undeciphered. In this study, we observed that PLA2R1 increases the mitochondrial content, judged by increased levels of numerous mitochondrial proteins, of the mitochondrial structural component cardiolipin, of the mitochondrial DNA content, and of the mitochondrial DNA replication and transcription factor TFAM. This effect of PLA2R1 relies on a transcriptional program controlled by the estrogen-related receptor alpha1 (ERRα) mitochondrial master regulator. Expression of ERRα and of its nucleus-encoded mitochondrial targets is upregulated upon PLA2R1 ectopic expression, and this effect is mediated by JAK2. Conversely, downregulation of PLA2R1 decreases the level of ERRα and of its nucleus-encoded mitochondrial targets. Finally, blocking the ERRα-controlled mitochondrial program largely inhibits the PLA2R1-induced tumor-suppressive response. Together, our data document ERRα and its mitochondrial program as downstream effectors of the PLA2R1-JAK2 pathway leading to oncosuppression.

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-05-01

    PLA2G6 or GVIA calcium-independent PLA2 (iPLA2β) is identified as one of the NAFLD modifier genes in humans, and thought to be a target for NAFLD therapy. iPLA2β is known to play a house-keeping role in phospholipid metabolism and remodeling. However, its role in NAFLD pathogenesis has not been supported by results obtained from high-fat feeding of iPLA2β-null (PKO) mice. Unlike livers of human NAFLD and genetically obese rodents, fatty liver induced by high-fat diet is not associated with depletion of hepatic phospholipids. We therefore tested whether iPLA2β could regulate obesity and hepatic steatosis in leptin-deficient mice by cross-breeding PKO with ob/ob mice to generate ob/ob-PKO mice. Here we observed an improvement in ob/ob-PKO mice with significant reduction in serum enzymes, lipids, glucose, insulin as well as improved glucose tolerance, and reduction in islet hyperplasia. The improvement in hepatic steatosis measured by liver triglycerides, fatty acids and cholesterol esters was associated with decreased expression of PPARγ and de novo lipogenesis genes, and the reversal of β-oxidation gene expression. Notably, ob/ob livers contained depleted levels of lysophospholipids and phospholipids, and iPLA2β deficiency in ob/ob-PKO livers lowers the former, but replenished the latter particularly phosphatidylethanolamine (PE) and phosphatidylcholine (PC) that contained arachidonic (AA) and docosahexaenoic (DHA) acids. Compared with WT livers, PKO livers also contained increased PE and PC containing AA and DHA. Thus, iPLA2β deficiency protected against obesity and ob/ob fatty liver which was associated with hepatic fatty-acyl phospholipid remodeling. Our results support the deleterious role of iPLA2β in severe obesity associated NAFLD. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. The role of cPLA2 in Methylglyoxal-induced cell apoptosis of HUVECs

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

    Yuan, Jie; Zhu, Chao; Hong, Yali

    2017-05-15

    Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is mainly formed as a byproduct of glycolysis. Elevated MGO level is known to induce apoptosis of vascular endothelial cells, which is implicated with progression of atherosclerosis and diabetic complications. However, the underlying mechanisms have not been exhaustively investigated yet. Here, we further characterized the mechanisms how MGO induced apoptosis in human umbilical vein endothelial cells (HUVECs). Our data revealed that cytosolic phospholipase A2 (cPLA2) played an important role in MGO-induced cell apoptosis. It was found that MGO could increase both the activity and expression of cPLA2. Inhibition of cPLA2 by Pyrrophenone (PYR)more » or siRNA significantly attenuated the MGO-induced apoptosis. Additionally, MGO time-dependently decreased the phosphorylation of nuclear factor κB (NF-κB). Pretreatment of the cells with NF-κB inhibitor, BAY11-7082, further increased MGO-induced apoptosis of HUVECs, indicating that NF-κB played a survival role in this MGO-induced apoptosis. Furthermore, in the presence of si-cPLA2 or PYR, MGO no longer decreased NF-κB phosphorylation. Beyond that, the antioxidant N-acetyl cysteine (NAC) could reverse the changes of both cPLA2 and NF-κB caused by MGO. p38, the upstream of cPLA2, was also significantly phosphorylated by MGO. However, p38 inhibitor failed to reverse the apoptosis induced by MGO. This study gives an important insight into the downstream signaling mechanisms of MGO, cPLA2-NF-κB, in endothelial apoptosis. - Highlights: • cPLA2 participated in MGO-induced HUVECs apoptosis. • Inhibition of NF-κB was involved in MGO-cPLA2-mediated cell apoptosis. • Antioxidant NAC attenuated MGO-induced cPLA2 activation and cell apoptosis.« less

  5. 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.

  6. Serum Lp-PLA2: as a novel viewpoint in periodontal treatment of hyperlipidaemics.

    PubMed

    Fentoğlu, Özlem; Kirzioğlu, Fatma Yeşim; Tözüm Bulut, Memduha; Kurgan, Şivge; Koçak, Havva; Sütcü, Recep; Kale Köroğlu, Banu; Günhan, Meral

    2015-01-01

    To evaluate the effects of periodontal treatment on serum lipoprotein-associated phospholipase A2 (Lp-PLA2) and C-reactive protein (CRP) levels in hyperlipidaemic patients with periodontitis. The study included 52 hyperlipidaemics and 28 systemically healthy controls (C) with periodontitis. Of the 52 hyperlipidaemics, 29 received a suggested diet (HD), and 23 of them were prescribed statin (HS). Clinical periodontal parameters, serum lipids, Lp-PLA2, and CRP levels were assessed at the baseline and 2 months after the completion of the nonsurgical periodontal treatment (2MPT). Serum parameters were also evaluated 1 week following the periodontal treatment (1WPT). At the baseline, patients in the HS group had a higher percentage of bleeding on probing than those in the C and HD groups. Hyperlipidaemics had higher serum triglyceride levels than the control group at 2MPT compared to the baseline. At 2MPT, the levels of Lp-PLA2 in the HS group were significantly higher compared to the baseline and 1WPT. There were no statistically significant differences in CRP levels between study periods for all groups. The periodontal treatment may affect the inflammatory control of hyperlipidaemic patients with periodontitis via increased Lp-PLA2 levels and severity of the impaired lipid metabolism. These findings may be important regarding the therapeutic strategies for hyperlipidaemics with periodontitis.

  7. Purification and immunochemical characterization of Pla l 2, the profilin from Plantago lanceolata.

    PubMed

    Moya, Raquel; Rubio, Virginia; Beitia, Juan Mª; Carnés, Jerónimo; López-Matas, M Angeles

    2017-03-01

    Profilins are small actin-binding proteins found in eukaryotes and involved in cell development, cytokinesis, membrane trafficking, and cell motility. From an allergenic point of view, profilins are panallergens usually involved in allergic polysensitization, although they are generally recognized as minor allergens. The objectives of this study were to identify and characterize the profilin from Plantago lanceolata pollen and to investigate the cross-reactivity between profilins from different pollen allergenic sources. Profilins from P. lancelolata (Pla l 2) and palm tree pollen (Pho d 2) were purified by affinity chromatography, deeply characterized and identified by mass spectrometry. Pla l 2 allergenicity was confirmed by immunoblot with serum samples from a patient population sensitized to profilin. Immunoblot inhibition was performed to study IgG reactivity between different pollen profilins. IgE cross-reactivity was demonstrated by ImmunoCAP inhibition. Pla l 2 is the second P. lanceolata allergen included in the IUIS Allergen Nomenclature database. Four peptides from purified Pla l 2 were identified with percentages of homology with other pollen profilins between 73 and 86%. Eighty-six percent (21/24) of the patient population recognized Pla l 2. The allergenic relatedness between Pla l 2, Pho d 2 and six pollen profilins was confirmed, and IgE cross-reactivity of Pla l 2 with rBet v 2 and rPhl p 12 was demonstrated. Pla l 2 is the profilin from P. lanceolata. The demonstrated allergenicity of this protein and its cross-reactivity with other pollen profilins support its use in profilin diagnostic assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. 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

  9. Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

    PubMed Central

    Oliveira, Simone CB; Fonseca, Fabiana V; Antunes, Edson; Camargo, Enilton A; Morganti, Rafael P; Aparício, Ricardo; Toyama, Daniela O; Beriam, Luís OS; Nunes, Eudismar V; Cavada, Benildo S; Nagano, Celso S; Sampaio, Alexandre H; Nascimento, Kyria S; Toyama, Marcos H

    2008-01-01

    Background An interaction between lectins from marine algae and PLA2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA2 and its complex. Results This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24–26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion The

  10. Expression, purification and epitope analysis of Pla a 2 allergen from Platanus acerifolia pollen.

    PubMed

    Wang, De-Wang; Ni, Wei-Wei; Zhou, Yan-Jun; Huang, Wen; Cao, Meng-Da; Meng, Ling; Wei, Ji-Fu

    2018-01-01

    Platanus acerifolia is one of the major sources of outdoor allergens to humans, and can induce allergic asthma, rhinitis, dermatitis and other allergic diseases. Pla a 2 is a polygalacturonase and represents the major allergen identified in P. acerifolia pollen. The aim of the present study was to express and purify Pla a 2, and to predict B and T cell epitopes of Pla a 2. The gene encoding Pla a 2 was cloned into the pET28a vector and subsequently transfected into ArcticExpress™ (DE3) Escherichia coli cells; purified Pla a 2 was analyzed by western blot analysis. The results of the present study revealed that the Pla a 2 allergen has the ability to bind immunoglobulin E within the sera of patients allergic to P. acerifolia pollen. In addition, the B cell epitopes of Pla a 2 were predicted using the DNAStar Protean system, Bioinformatics Predicted Antigenic Peptides and BepiPred 1.0 software; T cell epitopes were predicted using NetMHCIIpan ‑3.0 and ‑2.2. In total, eight B cell epitopes (15‑24, 60‑66, 78‑86, 109‑124, 232‑240, 260‑269, 298‑306 and 315‑322) and five T cell epitopes (62‑67, 86‑91, 125‑132, 217‑222 and 343‑350) were predicted in the present study. These findings may be used to improve allergen immunotherapies and reduce the frequency of pollen‑associated allergic reactions.

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

    PubMed Central

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

    2011-01-01

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

  12. Association of Lp-PLA2-A and early recurrence of vascular events after TIA and minor stroke.

    PubMed

    Lin, Jinxi; Zheng, Hongwei; Cucchiara, Brett L; Li, Jiejie; Zhao, Xingquan; Liang, Xianhong; Wang, Chunxue; Li, Hao; Mullen, Michael T; Johnston, S Claiborne; Wang, Yilong; Wang, Yongjun

    2015-11-03

    To determine the association of lipoprotein-associated phospholipase A2 (Lp-PLA2) measured in the acute period and the short-term risk of recurrent vascular events in patients with TIA or minor stroke. We measured Lp-PLA2 activity (Lp-PLA2-A) in a subset of 3,201 participants enrolled in the CHANCE (Clopidogrel in High-Risk Patients with Acute Non-disabling Cerebrovascular Events) trial. Participants with TIA or minor stroke were enrolled within 24 hours of symptom onset and randomized to single or dual antiplatelet therapy. In the current analysis, the primary outcome was defined as the composite of ischemic stroke, myocardial infarction, or death within 90 days. The composite endpoint occurred in 299 of 3,021 participants (9.9%). The population average Lp-PLA2-A level was 209 ± 59 nmol/min/mL (95% confidence interval [CI] 207-211). Older age, male sex, and current smoking were associated with higher Lp-PLA2-A levels. Lp-PLA2-A was significantly associated with the primary endpoint (adjusted hazard ratio 1.07, 95% CI 1.01-1.13 for every 30 nmol/min/mL increase). Similar results were seen for ischemic stroke alone. Adjustment for low-density lipoprotein cholesterol attenuated the association between Lp-PLA2-A and the primary endpoint (adjusted hazard ratio 1.04, 95% CI 0.97-1.11 for every 30 nmol/min/mL increase). Higher levels of Lp-PLA2-A in the acute period are associated with increased short-term risk of recurrent vascular events. © 2015 American Academy of Neurology.

  13. 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 .

  14. 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.

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

    PubMed

    Dhananjaya, Bhadrapura Lakkappa; Shivalingaiah, Sudharshan

    2016-03-01

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

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

    PubMed

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

    2018-06-01

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

  17. 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

  18. 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

  19. CroFabtrade mark total anti-venom activity measured by SE-HPLC, and anti-PLA(2) activity assayed in vitro at physiological pH.

    PubMed

    Price, Joseph A; Sanny, Charles G

    2007-05-01

    One problem in the development and refinement of anti-venoms is ascertaining both overall anti-venom reactivity and which key toxins are neutralized. Here we show by SE-HPLC that the in vitro reaction of CroFab anti-venin with Crotalus atrox venom asymptotically nears completion (>95%) by 11 min at 4 degrees C by following the change in area under chromatographic peaks. The peaks for reactants decrease and the formation of high molecular weight complexes increases with time. To assay the large number of samples a new microplate format phospholipase A(2) (PLA(2)) assay at an initial pH of 7.5 was developed using phosphotidyl choline as the substrate. The change in absorbance is due to the pH change caused by release of fatty acids, and is linear with dilution of enzyme. This choice of substrate limits detection to PLA(2) and nonspecific esterase (if any) activities. The neutralization mixtures show a dose dependent (CroFab anti-venin) inactivation of C. atrox PLA(2) activity approaching a maximum of 85% neutralization. This approach of revealing antibody binding to venom components coupled with enzyme activity measurements is effective and may lead to greater in vitro assessment of antivenin activity in product development, and less routine use of mouse lethality assays.

  20. Leptin attenuates lipopolysaccharide-induced apoptosis of thymocytes partially via down-regulation of cPLA2 and p38 MAPK activation.

    PubMed

    Liang, Chen; Liao, Jie; Deng, Zihui; Song, Cuihong; Zhang, Jinying; Zabeau, Lennart; Tavernier, Jan; Zhang, Kai; Xue, Hui; Yan, Guangtao

    2013-03-01

    Leptin, a 16-kDa protein that is mainly secreted by adipocytes, plays a protective role in many cell types. It has been shown that leptin acts in the central and peripheral immune system to protect thymocytes. Cytosolic phospholipase A(2) (cPLA(2)) is an enzyme that can specifically initiate the release of arachidonic acid (AA) to produce eicosanoids, which regulate inflammation and immune responses. Our previous work has shown that leptin is important to prevent apoptosis of thymocytes. However, the role of cPLA(2) is still unclear, and the precise mechanism also remains to be elucidated. In this work, we demonstrated that leptin inhibited the LPS-induced toxicity and apoptosis of thymocytes. Western blot and RT-PCR showed that leptin led to a reduction of cPLA(2) activity and mRNA level, as well as caspase-3 cleavage. Moreover, we found that leptin could decrease the activation of p38 MAPK. Accordingly, we pre-treated apoptotic thymocytes with the p38 MAPK inhibitor, SB203580 and observed an effect similar to the leptin alone treated group. SB203580 also suppressed expression of cPLA(2) and cleavage of caspase-3. Based on these results, we suggest that leptin could attenuate LPS-induced apoptotic injury in mouse thymocyte cells, mainly through the p38/cPLA(2) signalling pathway. The study of the regulatory role of leptin in LPS-induced thymocyte apoptosis can help to explain the role of leptin in the immune system and may provide a novel treatment option in cases of severe trauma, infection, shock, organ failure and autoimmune disease caused by thymic atrophy. Copyright © 2013. Published by Elsevier B.V.

  1. Enhanced thermal and mechanical properties of PLA/MoS2 nanocomposites synthesized via the in-situ ring-opening polymerization

    NASA Astrophysics Data System (ADS)

    Chen, Pengpeng; Liang, Xiao; Xu, Ying; Zhou, Yifeng; Nie, Wangyan

    2018-05-01

    In this work, MoS2 nanosheets were employed to reinforce PLA. In order to promote the homogeneous dispersion of MoS2 in PLA and form a strong interface between MoS2 and PLA, the MoS2 nanosheets were firstly modified by mercapto-ethylamine, and then functionalized with PLA chains through ring-opening polymerization of poly(L-lactide). The XRD, XPS, TGA and 1H NMR characterizations confirmed the successful amino and PLA functionalization of MoS2 nanosheets. The obtained MoS2-g-PLA nanosheets were then introduced to reinforce PLA. SEM images displayed that the MoS2-g-PLA nanosheets were dispersed in PLA matrix uniformly. TGA results showed that initial decomposition temperature was improved from 275.6 °C to 334.8 °C with 0.5 wt% of MoS2-g-PLA nanosheets. The storage modulus of PLA/MoS2-g-PLA-0.5 wt% in the glass state and rubber state were both greatly enhanced compared with neat PLA.

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

    PubMed Central

    Bolotina, Victoria M

    2008-01-01

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

  3. 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.

  4. PLA2G2A polymorphisms are associated with metabolic syndrome and type 2 diabetes mellitus. Results from the genetics of atherosclerotic disease Mexican study.

    PubMed

    Monroy-Muñoz, Irma Eloisa; Angeles-Martinez, Javier; Posadas-Sánchez, Rosalinda; Villarreal-Molina, Teresa; Alvarez-León, Edith; Flores-Dominguez, Carmina; Cardoso-Saldaña, Guillermo; Medina-Urrutia, Aida; Juárez-Rojas, Juan Gabriel; Posadas-Romero, Carlos; Alarcon, Gilberto Vargas

    2017-10-01

    The secretory phospholipase A 2 II A (sPLA 2 -IIA) encoded by PLA2G2A gene hydrolyzes phospholipids liberating free fatty acids (FFAs) and lysophospholipids. If lipolysis exceeds lipogenesis, the free fatty acids undergo a continuous release into circulation. A sustained excessive increase in this release contributes to metabolic disease. The aim of the present study was to evaluate the role of PLA2G2A gene polymorphisms as susceptibility markers for metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) in Mexican population. Three PLA2G2A gene polymorphisms (rs876018, rs3753827 and rs11573156) were genotyped by 5' exonuclease TaqMan assays in a group of 338 patients with T2DM, 460 individuals with MetS and 366 healthy controls. Under codominant 1(codom1), dominant (dom) and additive (add) models adjusted by age, gender, body mass index (BMI), smoking habit, and hypertension, the rs876018T allele was associated with increased risk of MetS [Odds Ratio (OR)=1.66, P codom1 =0.005; OR=1.67, P dom =0.003; OR=1.49, P add =0.005] as compared to controls. On the other hand, under several models adjusted by the same variables, the rs3753827A (OR=1.52, P codom1 =0.039 and OR=1.49, P dom =0.039) and rs11573156C alleles (OR=6.46, P codom1 =0.013; OR=6.70, P codom2 =0.009; OR=6.65, P dom =0.009) were associated with increased risk of T2DM when compared with controls. In addition, the rs876018T allele was associated with hypercholesterolemia (P dom =0.017, P add =0.009) and risk of subclinical atherosclerosis (SA) (P dom =0.041) in MetS when compared with controls. Also, this allele was associated with SA in T2DM patients (P dom =0.007). The TAG haplotype was significantly associated with increased risk of MetS (OR=1.54, P=0.006). Results suggest that PLA2G2A polymorphisms are involved in the risk of developing MetS and T2D and are associated with SA in this group of patients. Copyright © 2016 Elsevier GmbH. All rights reserved.

  5. 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.

  6. 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.

  7. Phase composition of lipoprotein SM/cholesterol/PtdCho affects FA specificity of sPLA2s.

    PubMed

    Kuksis, Arnis; Pruzanski, Waldemar

    2008-10-01

    We have previously reported preferential release of polyunsaturated FAs during hydrolysis of lipoprotein phosphatidylcholine (PtdCho) by group X secretory phospholipase A2 (sPLA2) and preferential release of oligounsaturated FAs during hydrolysis of lipoprotein PtdCho by group V sPLA2, but the mechanism of this selectivity has remained unknown. We now show that the rate and specificity of hydrolysis are affected by relative increases in endogenous SM and free cholesterol (FC) during the lipase digestion. The highest preference for arachidonate release from LDL and HDL by group X sPLA2 was observed for residual SM/PtdCho molar ratio of 1.2 and 0.4, compared with the respective starting ratios of 0.4 and 0.2, as measured by liquid chromatography/electrospray ionization-mass spectrometry. Group V sPLA2 showed preferential release of linoleate from LDL and HDL at SM/PtdCho ratio 1.5 and 0.6, respectively. We have attributed the change in FA specificity to segregation of molecular species of PtdCho and of sPLA2s between disordered and ordered SM/FC/PtdCho lipid phases. The increases in SM and FC during digestion with group IIA sPLA2 were more limited, and a preferential hydrolysis of any FAs was not observed. The significance of SM and FC SM and FC accumulation during sPLA2 hydrolysis of lipoprotein PtdCho has been previously overlooked.

  8. 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.

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

    PubMed

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

    2017-01-01

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

  10. Lowered iPLA2γ activity causes increased mitochondrial lipid peroxidation and mitochondrial dysfunction in a rotenone-induced model of Parkinson's disease.

    PubMed

    Chao, Honglu; Liu, Yinlong; Fu, Xian; Xu, Xiupeng; Bao, Zhongyuan; Lin, Chao; Li, Zheng; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

    2018-02-01

    iPLA 2 γ, calcium-independent phospholipase A 2 γ, discerningly hydrolyses glycerophospholipids to liberate free fatty acids. iPLA 2 γ-deficiency has been associated with abnormal mitochondrial function. More importantly, the iPLA 2 family is causative proteins in mitochondrial neurodegenerative disorders such as parkinsonian disorders. However, the mechanisms by which iPLA 2 γ affects Parkinson's disease (PD) remain unknown. Mitochondrion stress has a key part in rotenone-induced dopaminergic neuronal degeneration. The present evaluation revealed that lowered iPLA 2 γ function provokes the parkinsonian phenotype and leads to the reduction of dopamine and its metabolites, lowered survival, locomotor deficiencies, and organismal hypersensitivity to rotenone-induced oxidative stress. In addition, lowered iPLA 2 γ function escalated the amount of mitochondrial irregularities, including mitochondrial reactive oxygen species (ROS) regeneration, reduced ATP synthesis, reduced glutathione levels, and abnormal mitochondrial morphology. Further, lowered iPLA 2 γ function was tightly linked with strengthened lipid peroxidation and mitochondrial membrane flaws following rotenone treatment, which can cause cytochrome c release and eventually apoptosis. These results confirmed the important role of iPLA 2 γ, whereby decreasing iPLA 2 γ activity aggravates mitochondrial degeneration to induce neurodegenerative disorders in a rotenone rat model of Parkinson's disease. These findings may be useful in the design of rational approaches for the prevention and treatment of PD-associated symptoms. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-05-01

    The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy. However, the value of anti-PLA2R1 antibody titers in predicting patient outcomes is unknown. Here, we screened serum samples from 50 patients positive for PLA2R1 for immunoreactivity against a series of PLA2R1 deletion mutants covering the extracellular domains. We identified reactive epitopes in the cysteine-rich (CysR), C-type lectin domain 1 (CTLD1), and C-type lectin domain 7 (CTLD7) domains and confirmed the reactivity with soluble forms of each domain. We then used ELISAs to stratify 69 patients positive for PLA2R1 by serum reactivity to one or more of these domains: CysR (n=23), CysRC1 (n=14), and CysRC1C7 (n=32). Median ELISA titers measured using the full-length PLA2R1 antigens were not statistically different between subgroups. Patients with anti-CysR-restricted activity were younger (P=0.008), had less nephrotic range proteinuria (P=0.02), and exhibited a higher rate of spontaneous remission (P=0.03) and lower rates of renal failure progression (P=0.002) and ESRD (P=0.01) during follow-up. Overall, 31 of 69 patients had poor renal prognosis (urinary protein/creatinine ratio >4 g/g or eGFR<45 ml/min per 1.73 m(2) at end of follow-up). High anti-PLA2R1 activity and epitope spreading beyond the CysR epitope were independent risk factors of poor renal prognosis in multivariable Cox regression analysis. Epitope spreading during follow-up associated with disease worsening (n=3), whereas reverse spreading from a CysRC1C7 profile back to a CysR profile associated with favorable outcome (n=1). We conclude that analysis of the PLA2R1 epitope profile and spreading is a powerful tool for monitoring disease severity and stratifying patients by renal prognosis. Copyright © 2016 by the American Society of Nephrology.

  14. H2O2-Activated Mitochondrial Phospholipase iPLA2γ Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein–Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic β-Cells

    PubMed Central

    Ježek, Jan; Dlasková, Andrea; Zelenka, Jaroslav; Jabůrek, Martin

    2015-01-01

    Abstract Aims: Pancreatic β-cell chronic lipotoxicity evolves from acute free fatty acid (FA)–mediated oxidative stress, unprotected by antioxidant mechanisms. Since mitochondrial uncoupling protein-2 (UCP2) plays antioxidant and insulin-regulating roles in pancreatic β-cells, we tested our hypothesis, that UCP2-mediated uncoupling attenuating mitochondrial superoxide production is initiated by FA release due to a direct H2O2-induced activation of mitochondrial phospholipase iPLA2γ. Results: Pro-oxidant tert-butylhydroperoxide increased respiration, decreased membrane potential and mitochondrial matrix superoxide release rates of control but not UCP2- or iPLA2γ-silenced INS-1E cells. iPLA2γ/UCP2-mediated uncoupling was alternatively activated by an H2O2 burst, resulting from palmitic acid (PA) β-oxidation, and it was prevented by antioxidants or catalase overexpression. Exclusively, nascent FAs that cleaved off phospholipids by iPLA2γ were capable of activating UCP2, indicating that the previously reported direct redox UCP2 activation is actually indirect. Glucose-stimulated insulin release was not affected by UCP2 or iPLA2γ silencing, unless pro-oxidant activation had taken place. PA augmented insulin secretion via G-protein–coupled receptor 40 (GPR40), stimulated by iPLA2γ-cleaved FAs (absent after GPR40 silencing). Innovation and Conclusion: The iPLA2γ/UCP2 synergy provides a feedback antioxidant mechanism preventing oxidative stress by physiological FA intake in pancreatic β-cells, regulating glucose-, FA-, and redox-stimulated insulin secretion. iPLA2γ is regulated by exogenous FA via β-oxidation causing H2O2 signaling, while FAs are cleaved off phospholipids, subsequently acting as amplifying messengers for GPR40. Hence, iPLA2γ acts in eminent physiological redox signaling, the impairment of which results in the lack of antilipotoxic defense and contributes to chronic lipotoxicity. Antioxid. Redox Signal. 23, 958–972. PMID:25925080

  15. 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

  16. 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.

  17. Impact of the Pla protease substrate α2-antiplasmin on the progression of primary pneumonic plague.

    PubMed

    Eddy, Justin L; Schroeder, Jay A; Zimbler, Daniel L; Bellows, Lauren E; Lathem, Wyndham W

    2015-12-01

    Many pathogens usurp the host hemostatic system during infection to promote pathogenesis. Yersinia pestis, the causative agent of plague, expresses the plasminogen activator protease Pla, which has been shown in vitro to target and cleave multiple proteins within the fibrinolytic pathway, including the plasmin inhibitor α2-antiplasmin (A2AP). It is not known, however, if Pla inactivates A2AP in vivo; the role of A2AP during respiratory Y. pestis infection is not known either. Here, we show that Y. pestis does not appreciably cleave A2AP in a Pla-dependent manner in the lungs during experimental pneumonic plague. Furthermore, following intranasal infection with Y. pestis, A2AP-deficient mice exhibit no difference in survival time, bacterial burden in the lungs, or dissemination from wild-type mice. Instead, we found that in the absence of Pla, A2AP contributes to the control of the pulmonary inflammatory response during infection by reducing neutrophil recruitment and cytokine production, resulting in altered immunopathology of the lungs compared to A2AP-deficient mice. Thus, our data demonstrate that A2AP is not significantly affected by the Pla protease during pneumonic plague, and although A2AP participates in immune modulation in the lungs, it has limited impact on the course or ultimate outcome of the infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Impact of the Pla Protease Substrate α2-Antiplasmin on the Progression of Primary Pneumonic Plague

    PubMed Central

    Eddy, Justin L.; Schroeder, Jay A.; Zimbler, Daniel L.; Bellows, Lauren E.

    2015-01-01

    Many pathogens usurp the host hemostatic system during infection to promote pathogenesis. Yersinia pestis, the causative agent of plague, expresses the plasminogen activator protease Pla, which has been shown in vitro to target and cleave multiple proteins within the fibrinolytic pathway, including the plasmin inhibitor α2-antiplasmin (A2AP). It is not known, however, if Pla inactivates A2AP in vivo; the role of A2AP during respiratory Y. pestis infection is not known either. Here, we show that Y. pestis does not appreciably cleave A2AP in a Pla-dependent manner in the lungs during experimental pneumonic plague. Furthermore, following intranasal infection with Y. pestis, A2AP-deficient mice exhibit no difference in survival time, bacterial burden in the lungs, or dissemination from wild-type mice. Instead, we found that in the absence of Pla, A2AP contributes to the control of the pulmonary inflammatory response during infection by reducing neutrophil recruitment and cytokine production, resulting in altered immunopathology of the lungs compared to A2AP-deficient mice. Thus, our data demonstrate that A2AP is not significantly affected by the Pla protease during pneumonic plague, and although A2AP participates in immune modulation in the lungs, it has limited impact on the course or ultimate outcome of the infection. PMID:26438794

  19. 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.

  20. 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.

  1. Sequential VEGF and BMP-2 releasing PLA-PEG-PLA scaffolds for bone tissue engineering: I. Design and in vitro tests.

    PubMed

    Eğri, Sinan; Eczacıoğlu, Numan

    2017-03-01

    Biodegradable PLA-PEG-PLA block copolymers were synthesized with desired backbone structures and molecular weights using PEG20000. Rectangular scaffolds were prepared by freeze drying with or without using NaCl particles. Bone morphogenetic protein (BMP)-2 was loaded to the matrix after the scaffold formation for sustained release while vascular endothelial growth factor (VEGF) was loaded within the pores with gelatin solution. VEGF release was quite fast and almost 60% of it was released in 2 d. However, sequential - sustained released was observed for BMP-2 in the following few months. Corporation of VEGF/BMP-2 couple into the scaffolds increased the cell adhesion and proliferation. Neither significant cytotoxicity nor apoptosis/necrosis were observed.

  2. 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.

  3. HLA-DQA1 and PLA2R1 Polymorphisms and Risk of Idiopathic Membranous Nephropathy

    PubMed Central

    Bullich, Gemma; Ballarín, José; Oliver, Artur; Ayasreh, Nadia; Silva, Irene; Santín, Sheila; Díaz-Encarnación, Montserrat M.; Torra, Roser

    2014-01-01

    Summary Background and objectives Single nucleotide polymorphisms (SNPs) within HLA complex class II HLA-DQ α-chain 1 (HLA-DQA1) and M-type phospholipase A2 receptor (PLA2R1) genes were identified as strong risk factors for idiopathic membranous nephropathy (IMN) development in a recent genome-wide association study. Copy number variants (CNVs) within the Fc gamma receptor III (FCGR3) locus have been associated with several autoimmune diseases, but their role in IMN has not been studied. This study aimed to validate the association of HLA-DQA1 and PLA2R1 risk alleles with IMN in a Spanish cohort, test the putative association of FCGR3A and FCGR3B CNVs with IMN, and assess the use of these genetic factors to predict the clinical outcome of the disease. Design, settings, participants, & measurements A Spanish cohort of 89 IMN patients and 286 matched controls without nephropathy was recruited between October of 2009 and July of 2012. Case-control studies for SNPs within HLA-DQA1 (rs2187668) and PLA2R1 (rs4664308) genes and CNVs for FCGR3A and FCGR3B genes were performed. The contribution of these polymorphisms to predict clinical outcome and renal function decline was analyzed. Results This study validated the association of these HLA-DQA1 and PLA2R1 SNPs with IMN in a Spanish cohort and its increased risk when combining both risk genotypes. No significant association was found between FCGR3 CNVs and IMN. These results revealed that HLA-DQA1 and PLA2R1 genotype combination adjusted for baseline proteinuria strongly predicted response to immunosuppressive therapy. HLA-DQA1 genotype adjusted for proteinuria was also linked with renal function decline. Conclusion This study confirms that HLA-DQA1 and PLA2R1 genotypes are risk factors for IMN, whereas no association was identified for FCGR3 CNVs. This study provides, for the first time, evidence of the contribution of these HLA-DQA1 and PLA2R1 polymorphisms in predicting IMN response to immunosuppressors and disease

  4. HLA-DQA1 and PLA2R1 polymorphisms and risk of idiopathic membranous nephropathy.

    PubMed

    Bullich, Gemma; Ballarín, José; Oliver, Artur; Ayasreh, Nadia; Silva, Irene; Santín, Sheila; Díaz-Encarnación, Montserrat M; Torra, Roser; Ars, Elisabet

    2014-02-01

    Single nucleotide polymorphisms (SNPs) within HLA complex class II HLA-DQ α-chain 1 (HLA-DQA1) and M-type phospholipase A2 receptor (PLA2R1) genes were identified as strong risk factors for idiopathic membranous nephropathy (IMN) development in a recent genome-wide association study. Copy number variants (CNVs) within the Fc gamma receptor III (FCGR3) locus have been associated with several autoimmune diseases, but their role in IMN has not been studied. This study aimed to validate the association of HLA-DQA1 and PLA2R1 risk alleles with IMN in a Spanish cohort, test the putative association of FCGR3A and FCGR3B CNVs with IMN, and assess the use of these genetic factors to predict the clinical outcome of the disease. A Spanish cohort of 89 IMN patients and 286 matched controls without nephropathy was recruited between October of 2009 and July of 2012. Case-control studies for SNPs within HLA-DQA1 (rs2187668) and PLA2R1 (rs4664308) genes and CNVs for FCGR3A and FCGR3B genes were performed. The contribution of these polymorphisms to predict clinical outcome and renal function decline was analyzed. This study validated the association of these HLA-DQA1 and PLA2R1 SNPs with IMN in a Spanish cohort and its increased risk when combining both risk genotypes. No significant association was found between FCGR3 CNVs and IMN. These results revealed that HLA-DQA1 and PLA2R1 genotype combination adjusted for baseline proteinuria strongly predicted response to immunosuppressive therapy. HLA-DQA1 genotype adjusted for proteinuria was also linked with renal function decline. This study confirms that HLA-DQA1 and PLA2R1 genotypes are risk factors for IMN, whereas no association was identified for FCGR3 CNVs. This study provides, for the first time, evidence of the contribution of these HLA-DQA1 and PLA2R1 polymorphisms in predicting IMN response to immunosuppressors and disease progression. Future studies are needed to validate and identify prognostic markers.

  5. 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.

  6. 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

  7. Renaturation and one step purification of the chicken GIIA secreted phospholipase A2 from inclusion bodies.

    PubMed

    Karray, Aida; Amara, Sawsan; Carrière, Frédéric; Gargouri, Youssef; Bezzine, Sofiane

    2014-06-01

    The cDNA coding for a mature protein of 123 amino acids, containing all of the structural features of catalytically active group IIA sPLA2, has been amplified from chicken intestine. The gene has been cloned into the bacterial expression vector pET-21a(+), which allows protein over-expression as inclusion bodies and enables about 3mg/l of pure refolded fully active enzyme to be obtained. Recombinant expression of chicken intestinal sPLA2-IIA (ChPLA2-IIA) in Escherichia coli shows that the enzyme is Ca(2+) dependent, maximally active at pH 8-9, and hydrolyses phosphatidylglycerol versus phosphatidylcholine with a 10-fold preference. Indeed, we report in this work, a comparative kinetic study between the wild type and the recombinant ChPLA2-IIA, on zwitterionic head group phospholipids (DDPC) and negatively charged phospholipids (POPG) using the monomolecular film technique. The ability to express reasonably large amounts of the sPLA2 Group IIA, compared to that obtained with the classical purification will provide a basis for future site directed mutagenesis studies of this important enzyme. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. The effect of polyhexamethylene guanidine hydrochloride (PHMG) derivatives introduced into polylactide (PLA) on the activity of bacterial enzymes.

    PubMed

    Walczak, Maciej; Richert, Agnieszka; Burkowska-But, Aleksandra

    2014-11-01

    The present study was aimed at investigating bactericidal properties of polylactide (PLA) films containing three different polyhexamethylene guanidine hydrochloride (PHMG) derivatives and effect of the derivatives on extracellular hydrolytic enzymes and intracellular dehydrogenases. All PHMG derivatives had a slightly stronger bactericidal effect on Staphylococcus aureus than on E. coli but only PHMG granular polyethylene wax (at the concentration of at least 0.6 %) has a bactericidal effect. PHMG derivatives introduced into PLA affected the activity of microbial hydrolases to a small extent. This means that the introduction of PHMG derivatives into PLA will not reduce its enzymatic biodegradation significantly. On the other hand, PHMG derivatives introduced into PLA strongly affected dehydrogenases activity in S. aureus than in E. coli.

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

    PubMed

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

    2018-03-02

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

  10. 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

  11. 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

  12. 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.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Falchi, L; Scaramuzzi, R J

    2013-01-01

    The ovine cervix relaxes at estrus allowing easier entry of spermatozoa into the uterus. The mechanism responsible for this relaxation is not fully elucidated and we hypothesized that cervical relaxation at estrus is induced by ovarian and pituitary hormones stimulating the local production of prostaglandin E(2) via a biosynthetic pathway involving a number of mediators including oxytocin, phospholipase A(2) (cPLA(2)), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor gamma (PPARγ). The aim of this study was to investigate the cervical expression of estradiol receptor alpha (ERα), oxytocin receptor (OTR), cPLA(2), COX-2, and PPARγ at three stages of the estrous cycle (the luteal phase and two times during the follicular phase, just before and just after the LH surge). An experiment was conducted during the breeding season, in 25 ewes to test this hypothesis. Samples of cervical tissue were collected from groups of ewes at three stages of the estrous cycle: the luteal (N = 8), "pre-LH surge" (N = 8), and "post-LH surge" (N = 9) stages. Cervical tissue from uterine, mid, and vaginal regions of the cervix were analyzed by Western immunoblot analysis for ERα, OTR, cPLA(2,) COX-2, and PPARγ. The results showed that the levels of all five proteins were lowest during the luteal phase of the estrous cycle in all regions of the cervix. The levels of all except cPLA(2), increased significantly during the "pre-LH surge" stage. The levels of cPLA(2) and ERα increased in the "post-LH surge" stage and those for OTR and PPARγ were unchanged and those for COX-2 were lower. These data show that the cervical levels of all five of the intermediates in the synthesis of prostaglandin E(2) that were examined in this study were higher in the "pre-" and "post-LH surge" stages compared with the luteal phase of the estrous cycle and these findings are consistent with our hypothesis. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2015-01-01

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

  16. 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.

  17. Evaluation of Phototoxic and Skin Sensitization Potentials of PLA2-Free Bee Venom

    PubMed Central

    Heo, Yunwi; Pyo, Min-Jung; Bae, Seong Kyeong; Lee, Hyunkyoung; Kwon, Young Chul; Kim, Je Hein; Kim, Bokyung; Kim, Choul Goo; Kang, Changkeun; Kim, Euikyung

    2015-01-01

    Bee venom (BV) from honey bee (Apis mellifera L.) has been used in oriental medicine and cosmetic ingredients because of its diverse pharmacological activities. In many studies, among BV components, phospholipase A2 (PLA2) is known as a major player in BV-induced allergic reaction. Therefore, we removed PLA2 from BV using ultrafiltration and then investigated in vitro phototoxicity and in vivo skin sensitization of PLA2-free BV (PBV) in comparison with regular BV. The 3T3 neutral red uptake phototoxicity assay can be appropriated to identify the phototoxic effect of a test substance upon the exposure of ultraviolet A. Chlorpromazine, a positive control, showed high levels of photoirritation factor and mean photo effect values, while BV and PBV had less of these values. Local lymph node assay is an alternative method to evaluate skin sensitization potential of chemicals. BALB/c mice were treated with p-phenylenediamine (PPD, positive control), BV, or PBV. In all of PPD concentrations, stimulation indexes (SI) as sensitizing potential of chemicals were ≥1.6, determined to be sensitizer, while SI levels of BV and PBV were below 1.6. Thus, based on these findings, we propose that both BV and PBV are nonphototoxic compounds and nonsensitizers. PMID:26347784

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  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. Crystallization and preliminary X-ray diffraction analysis of a myotoxic Lys49-PLA{sub 2} from Bothrops jararacussu venom complexed with p-bromophenacyl bromide

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

    Marchi-Salvador, D. P.; Fernandes, C. A. H.; Amui, S. F.

    2006-06-01

    A non-catalytic and myotoxic Lys49-PLA{sub 2} from B. jararacussu venom was crystallized with BPB inhibitor and X-ray diffraction data were collected. Preliminary analysis indicates that the ligand is bound to the His48 residue. Structure determination may provide insights into the myotoxic and cytotoxic mechanisms of Lys49-PLA{sub 2}s. For the first time, a non-catalytic and myotoxic Lys49-PLA{sub 2} (BthTX-I from Bothrops jararacussu venom) has been crystallized with BPB inhibitor. X-ray diffraction data were collected and electron-density calculations showed that the ligand is bound to the His48 residue. BthTX-I with His48 chemically modified by BPB shows strongly reduced myotoxic and cytotoxic activities.more » This suggests a biological correlation between the modification of His48, which is associated with catalytic activity of PLA{sub 2}s, and other toxicological activities of Lys49-PLA{sub 2}s.« less

  5. 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.

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

    USDA-ARS?s Scientific Manuscript database

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

  7. 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.

  8. Novel mutations in PANK2 and PLA2G6 genes in patients with neurodegenerative disorders: two case reports.

    PubMed

    Dastsooz, Hassan; Nemati, Hamid; Fard, Mohammad Ali Farazi; Fardaei, Majid; Faghihi, Mohammad Ali

    2017-08-18

    Neurodegeneration with brain iron accumulation (NBIA) is a genetically heterogeneous group of disorders associated with progressive impairment of movement, vision, and cognition. The disease is initially diagnosed on the basis of changes in brain magnetic resonance imaging which indicate an abnormal brain iron accumulation in the basal ganglia. However, the diagnosis of specific types should be based on both clinical findings and molecular genetic testing for genes associated with different types of NBIA, including PANK2, PLA2G6, C19orf12, FA2H, ATP13A2, WDR45, COASY, FTL, CP, and DCAF17. The purpose of this study was to investigate disease-causing mutations in two patients with distinct NBIA disorders. Whole Exome sequencing using Next Generation Illumina Sequencing was used to enrich all exons of protein-coding genes as well as some other important genomic regions in these two affected patients. A deleterious homozygous four-nucleotide deletion causing frameshift deletion in PANK2 gene (c.1426_1429delATGA, p.M476 fs) was identified in an 8 years old girl with dystonia, bone fracture, muscle rigidity, abnormal movement, lack of coordination and chorea. In addition, our study revealed a novel missense mutation in PLA2G6 gene (c.3G > T:p.M1I) in one and half-year-old boy with muscle weakness and neurodevelopmental regression (speech, motor and cognition). The novel mutations were also confirmed by Sanger sequencing in the proband and their parents. Current study uncovered two rare novel mutations in PANK2 and PLA2G6 genes in patients with NBIA disorder and such studies may help to conduct genetic counseling and prenatal diagnosis more accurately for individuals at the high risk of these types of disorders.

  9. Primate-specific evolution of noncoding element insertion into PLA2G4C and human preterm birth

    PubMed Central

    2010-01-01

    Background The onset of birth in humans, like other apes, differs from non-primate mammals in its endocrine physiology. We hypothesize that higher primate-specific gene evolution may lead to these differences and target genes involved in human preterm birth, an area of global health significance. Methods We performed a comparative genomics screen of highly conserved noncoding elements and identified PLA2G4C, a phospholipase A isoform involved in prostaglandin biosynthesis as human accelerated. To examine whether this gene demonstrating primate-specific evolution was associated with birth timing, we genotyped and analyzed 8 common single nucleotide polymorphisms (SNPs) in PLA2G4C in US Hispanic (n = 73 preterm, 292 control), US White (n = 147 preterm, 157 control) and US Black (n = 79 preterm, 166 control) mothers. Results Detailed structural and phylogenic analysis of PLA2G4C suggested a short genomic element within the gene duplicated from a paralogous highly conserved element on chromosome 1 specifically in primates. SNPs rs8110925 and rs2307276 in US Hispanics and rs11564620 in US Whites were significant after correcting for multiple tests (p < 0.006). Additionally, rs11564620 (Thr360Pro) was associated with increased metabolite levels of the prostaglandin thromboxane in healthy individuals (p = 0.02), suggesting this variant may affect PLA2G4C activity. Conclusions Our findings suggest that variation in PLA2G4C may influence preterm birth risk by increasing levels of prostaglandins, which are known to regulate labor. PMID:21184677

  10. 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

  11. 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.

  12. 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.

  13. [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.

  14. 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.

  15. SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR.

    PubMed

    Thompson, Christopher M; Bloom, Lee R; Ogiue-Ikeda, Mari; Machida, Kazuya

    2015-06-26

    There is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required. We have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1-2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment. We showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein

  16. The association between the levels of CRP, IL-10, PLA2, Fbg and prognosis in traumatic fracture of lower limb.

    PubMed

    Jiao, Jing; Wang, Jun-Wen; Xiao, Fei; Huang, Yu-Cheng

    2016-11-01

    The aim of the present study was to examine changes of sera levels of C-reactive protein (CRP), interleukin-10 (IL-10), phospholipase A2 (PLA2) and fibrinogen β polypeptide chain gene (Fbg) in patients with traumatic fracture of lower limb, and to evaluate their association with prognosis. The changes in sera levels of CRP, IL-10, PLA2 and Fbg were observed at the time of injury, 24 h, and 5 and 7 days after surgery in 90 patients with traumatic fracture of lower limb. In addition, 50 cases, who presented for health examination, were included as the normal controls. The expression of sera levels of CRP, IL-10, PLA2 and Fbg in patients with traumatic fracture of lower limb, was markedly higher than that in the normal controls prior to surgery (P<0.05). The concentration of CRP significantly increased within 24 h after emergency, but decreased gradually as the wound healed, compared to the controls. Pre- and postoperative IL-10 levels increased within 24 h and then decreased gradually. The level of PLA2 in patients before and after surgery was increased, and then decreased gradually. The level of Fbg in patients with trauma was increased after 24 h and then decreased, and increased gradually. The correlation of serum CRP and IL-10 levels (r=0.634, P<0.05), and that of PLA2 and IL-10 levels (r=0.617, P<0.05) were positive. In conclusion, the expression of CRP, IL-10, PLA2 and Fbg levels in traumatic fracture of lower limb markedly increased and was closely associated with prognosis. CRP, IL-10, PLA2 and Fbg levels may therefore serve as useful indexes in determining the progression and prognosis of patients with traumatic fracture of lower limb.

  17. 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

  18. 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.

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

    PubMed

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

    2010-09-15

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

  20. Early-Onset L-dopa-Responsive Parkinsonism with Pyramidal Signs Due to ATP13A2, PLA2G6, FBXO7 and Spatacsin Mutations

    PubMed Central

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

    2018-01-01

    Seven autosomal recessive genes associated with juvenile and young-onset Levodopa-responsive parkinsonism have been identified. Mutations in PRKN, DJ-1, and PINK1 are associated with a rather pure parkinsonian phenotype, and have a more benign course with sustained treatment response and absence of dementia. On the other hand, Kufor-Rakeb syndrome has additional signs, which distinguish it clearly from Parkinson’s disease including supranu-clear vertical gaze palsy, myoclonic jerks, pyramidal signs, and cognitive impairment. Neurodegeneration with brain iron accumulation type I (Hallervorden-Spatz syndrome) due to mutations in PANK2 gene may share similar features with Kufor-Rakeb syndrome. Mutations in three other genes, PLA2G6 (PARK14), FBXO7 (PARK15), and Spatacsin (SPG11) also produce clinical similar phenotypes in that they presented with rapidly progressive parkinsonism, initially responsive to Levodopa treatment but later, developed additional features including cognitive decline and loss of Levodopa responsiveness. Here, using homozygosity mapping and sequence analysis in families with complex parkinsonisms, we identified genetic defects in the ATP13A2 (1 family), PLA2G6 (1 family) FBXO7 (2 families), and SPG11 (1 family). The genetic heterogeneity was surprising given their initially common clinical features. On careful review, we found the FBXO7 cases to have a phenotype more similar to PRKN gene associated parkinsonism. The ATP13A2 and PLA2G6 cases were more seriously disabled with additional swallowing problems, dystonic features, severe in some, and usually pyramidal involvement including pyramidal weakness. These data suggest that these four genes account for many cases of Levodopa responsive parkinsonism with pyramidal signs cases formerly categorized clinically as pallido-pyramidal syndrome. 3 2010 Movement Disorder Society PMID:20669327

  1. Application of phage display for the development of a novel inhibitor of PLA2 activity in Western cottonmouth venom

    PubMed Central

    Titus, James K; Kay, Matthew K; Glaser, CDR Jacob J

    2017-01-01

    Snakebite envenomation is an important global health concern. The current standard treatment approach for snakebite envenomation relies on antibody-based antisera, which are expensive, not universally available, and can lead to adverse physiological effects. Phage display techniques offer a powerful tool for the selection of phage-expressed peptides, which can bind with high specificity and affinity towards venom components. In this research, the amino acid sequences of Phospholipase A2 (PLA2) from multiple cottonmouth species were analyzed, and a consensus peptide synthesized. Three phage display libraries were panned against this consensus peptide, crosslinked to capillary tubes, followed by a modified surface panning procedure. This high throughput selection method identified four phage clones with anti-PLA2 activity against Western cottonmouth venom, and the amino acid sequences of the displayed peptides were identified. This is the first report identifying short peptide sequences capable of inhibiting PLA2 activity of Western cottonmouth venom in vitro, using a phage display technique. Additionally, this report utilizes synthetic panning targets, designed using venom proteomic data, to mimic epitope regions. M13 phages displaying circular 7-mer or linear 12-mer peptides with antivenom activity may offer a novel alternative to traditional antibody-based therapy. PMID:29285351

  2. Application of phage display for the development of a novel inhibitor of PLA2 activity in Western cottonmouth venom.

    PubMed

    Titus, James K; Kay, Matthew K; Glaser, Cdr Jacob J

    2017-01-01

    Snakebite envenomation is an important global health concern. The current standard treatment approach for snakebite envenomation relies on antibody-based antisera, which are expensive, not universally available, and can lead to adverse physiological effects. Phage display techniques offer a powerful tool for the selection of phage-expressed peptides, which can bind with high specificity and affinity towards venom components. In this research, the amino acid sequences of Phospholipase A 2 (PLA 2 ) from multiple cottonmouth species were analyzed, and a consensus peptide synthesized. Three phage display libraries were panned against this consensus peptide, crosslinked to capillary tubes, followed by a modified surface panning procedure. This high throughput selection method identified four phage clones with anti-PLA 2 activity against Western cottonmouth venom, and the amino acid sequences of the displayed peptides were identified. This is the first report identifying short peptide sequences capable of inhibiting PLA 2 activity of Western cottonmouth venom in vitro , using a phage display technique. Additionally, this report utilizes synthetic panning targets, designed using venom proteomic data, to mimic epitope regions. M13 phages displaying circular 7-mer or linear 12-mer peptides with antivenom activity may offer a novel alternative to traditional antibody-based therapy.

  3. Strong synergistic effects in PLA/PCL blends: Impact of PLA matrix viscosity.

    PubMed

    Ostafinska, Aleksandra; Fortelný, Ivan; Hodan, Jiří; Krejčíková, Sabina; Nevoralová, Martina; Kredatusová, Jana; Kruliš, Zdeněk; Kotek, Jiří; Šlouf, Miroslav

    2017-05-01

    Blends of two biodegradable polymers, poly(lactic acid) (PLA) and poly(ϵ-caprolactone) (PCL), with strong synergistic improvement in mechanical performance were prepared by melt-mixing using the optimized composition (80/20) and the optimized preparation procedure (a melt-mixing followed by a compression molding) according to our previous study. Three different PLA polymers were employed, whose viscosity decreased in the following order: PLC ≈ PLA1 > PLA2 > PLA3. The blends with the highest viscosity matrix (PLA1/PCL) exhibited the smallest PCL particles (d∼0.6μm), an elastic-plastic stable fracture (as determined from instrumented impact testing) and the strongest synergistic improvement in toughness (>16× with respect to pure PLA, exceeding even the toughness of pure PCL). According to the available literature, this was the highest toughness improvement in non-compatiblized PLA/PCL blends ever achieved. The decrease in the matrix viscosity resulted in an increase in the average PCL particle size and a dramatic decrease in the overall toughness: the completely stable fracture (for PLA1/PCL) changed to the stable fracture followed by unstable crack propagation (for PLA2/PCL) and finally to the completely brittle fracture (for PLA3/PCL). The stiffness of all blends remained at well acceptable level, slightly above the theoretical predictions based on the equivalent box model. Despite several previous studies, the results confirmed that PLA and PCL could behave as compatible polymers, but the final PLA/PCL toughness is extremely sensitive to the PCL particle size distribution, which is influenced by both processing conditions and PLA viscosity. PLA/PCL blends with high stiffness (due to PLA) and toughness (due to PCL) are very promising materials for medical applications, namely for the bone tissue engineering. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. 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

  5. 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.

  6. 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

  7. 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

    expression of PLA2G10 by eosinophils. These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  8. 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 .

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

    PubMed

    Peng, Ling-Ling; Zhao, Yuan-Qi; Zhou, Zi-Yi; Jin, Jing; Zhao, Min; Chen, Xin-Meng; Chen, Ling-Yan; Cai, Ye-Feng; Li, Jia-Li; Huang, Min

    2016-11-01

    Aspirin resistance has an incidence of 5%-65% in patients with ischemic stroke, who receive the standard dose of aspirin, but the platelet function is inadequately inhibited, thereby leading to thrombotic events. Numerous evidence shows that thromboxane A 2 receptor (TXA 2 receptor, encoded by TBXA2R), lipoprotein-associated phospholipase A 2 (Lp-PLA 2 , encoded by PLA2G7) and platelet endothelial aggregation receptor-1 (PEAR1, encoded by PEAR1) are crucial in regulating platelet activation, and P-glycoprotein (P-gp, encoded by MDR1) influences the absorption of aspirin in the intestine. In this study we examined the correlation between MDR1, TBXA2R, PLA2G7, PEAR1 genetic polymorphisms and platelet activity in Chinese ischemic stroke patients receiving aspirin therapy. A total of 283 ischemic stroke patients receiving 100 mg aspirin for 7 d were genotyped for polymorphisms in MDR1 C3435T, TBXA2R (rs1131882), PLA2G7 (rs1051931, rs7756935), and PEAR1 (rs12566888, rs12041331). The platelet aggregation response was measured using an automatic platelet aggregation analyzer and a commercially available TXB 2 ELISA kit. Thirty-three patients (11.66%) were insensitive to aspirin treatment. MDR1 3435TT genotype carriers, whose arachidonic acid (AA) or adenosine diphosphate (ADP)-induced platelet aggregation was lower than that of CC+CT genotype carriers, were less likely to suffer from aspirin resistance (odds ratio=0.421, 95% CI: 0.233-0.759). The TBXA2R rs1131882 CC genotype, which was found more frequently in the aspirin-insensitive group (81.8% vs 62.4%) than in the sensitive group, was identified as a risk factor for aspirin resistance (odds ratio=2.712, 95% CI: 1.080-6.810) with a higher level of AA-induced platelet aggregation. Due to the combined effects of PLA2G7 rs1051931 and rs7756935, carriers of the AA-CC haplotype had a higher level of ADP-induced platelet aggregation, and were at considerably higher risk of aspirin resistance than noncarriers (odds ratio=8

  10. 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.

  11. 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...

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

    PubMed

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

    2010-01-01

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

  13. Lp-PLA2 silencing protects against ox-LDL-induced oxidative stress and cell apoptosis via Akt/mTOR signaling pathway in human THP1 macrophages.

    PubMed

    Zheng, HuaDong; Cui, DaJiang; Quan, XiaoJuan; Yang, WeiLin; Li, YingNa; Zhang, Lin; Liu, EnQi

    2016-09-02

    Atherosclerosis is a disease of the large- and medium-size arteries that is characterized by the formation of atherosclerotic plaques, in which foam cells are the characteristic pathological cells. However, the key underlying pathomechanisms are still not fully elucidated. In this study, we investigated the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in ox-LDL-induced oxidative stress and cell apoptosis, and further, elucidated the potential machanisms in human THP1 macrophages. Flow cytometry and western blot analyses showed that both cell apoptosis and Lp-PLA2 expression were dose-dependently elevated after ox-LDL treatment for 24 h and also time-dependently increased after 50 mg/L ox-LDL incubation in THP1 macrophages. In addition, Lp-PLA2 silencing decreased ox-LDL-induced Lp-PLA2 and CD36 expression in THP1 macrophages. We also found that the levels of oil red O-staining, triglyceride (TG) and total cholesterol (TC) were significantly upregulated in ox-LDL-treated THP1 cells, but inhibited by Lp-PLA2 silencing. Furthermore, ox-LDL treatment resulted in significant increases of ROS and MDA but a marked decrease of SOD, effects that were reversed by Lp-PLA2 silencing in THP1 cells. Lp-PLA2 silencing reduced ox-LDL-induced cell apoptosis and caspase-3 expression in THP1 cells. Moreover, Lp-PLA2 siRNA transfection dramatically lowered the elevated levels of p-Akt and p-mTOR proteins in ox-LDL-treated THP1 cells. Both PI3K inhibitor LY294002 and mTOR inhibitor rapamycin decreased the augmented caspase-3 expression and TC content induced by ox-LDL, respectively. Taken together, these results revealed that Lp-PLA2 silencing protected against ox-LDL-induced oxidative stress and cell apoptosis via Akt/mTOR signaling pathway in human THP1 macrophages. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2

    PubMed Central

    Tyurin, V A; Balasubramanian, K; Winnica, D; Tyurina, Y Y; Vikulina, A S; He, R R; Kapralov, A A; Macphee, C H; Kagan, V E

    2014-01-01

    Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H2O2 was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A2 (Lp-PLA2), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA2 was blocked by a selective inhibitor of Lp-PLA2, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic ‘eat-me' signal that participates in the engulfment of cells undergoing intrinsic apoptosis. PMID:24464221

  15. AtsPLA2-alpha nuclear relocalization by the Arabidopsis transcription factor AtMYB30 leads to repression of the plant defense response.

    PubMed

    Froidure, Solène; Canonne, Joanne; Daniel, Xavier; Jauneau, Alain; Brière, Christian; Roby, Dominique; Rivas, Susana

    2010-08-24

    The hypersensitive response (HR), characterized by a rapid and localized cell death at the inoculation site, is one of the most efficient resistance reactions to pathogen attack in plants. The transcription factor AtMYB30 was identified as a positive regulator of the HR and resistance responses during interactions between Arabidopsis and bacteria. Here, we show that AtMYB30 and the secreted phospholipase AtsPLA(2)-alpha physically interact in vivo, following the AtMYB30-mediated specific relocalization of AtsPLA(2)-alpha from cytoplasmic vesicles to the plant cell nucleus. This protein interaction leads to repression of AtMYB30 transcriptional activity and negative regulation of plant HR. Moreover, Atspla(2)-alpha mutant plants are more resistant to bacterial inoculation, whereas AtsPLA(2)-alpha overexpression leads to decreased resistance, confirming that AtsPLA(2)-alpha is a negative regulator of AtMYB30-mediated defense. These data underline the importance of cellular dynamics and, particularly, protein translocation to the nucleus, for defense-associated gene regulation in plants.

  16. 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

  17. [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.

  18. ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications.

    PubMed

    Tong, Qing-He; Tao, Tao; Xie, Li-Qi; Lu, Hao-Jie

    2016-06-15

    Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. 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.

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

    PubMed

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

    2017-08-01

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

  1. 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 sPLA

  2. 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.

  3. NFATC3-PLA2G15 Fusion Transcript Identified by RNA Sequencing Promotes Tumor Invasion and Proliferation in Colorectal Cancer Cell Lines.

    PubMed

    Jang, Jee-Eun; Kim, Hwang-Phill; Han, Sae-Won; Jang, Hoon; Lee, Si-Hyun; Song, Sang-Hyun; Bang, Duhee; Kim, Tae-You

    2018-06-14

    This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer lines. We performed paired-end RNA sequencing of 28 colorectal cancer (CRC) cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed. 1,380 FT candidates were detected through bioinformatics filtering. We selected 6 candidate FTs, including 4 inter-chromosomal and 2 intra-chromosomal FTs and each FT was found in at least 1 of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3-PLA2G15 FT was found in 2. Knockdown of NFATC3-PLA2G15 using siRNA reduced mRNA expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal-epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3-PLA2G15 FT. The NFATC3-PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation. These results suggest that that NFATC3-PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation.

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

    PubMed Central

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

    2013-01-01

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

  5. Case report of a novel homozygous splice site mutation in PLA2G6 gene causing infantile neuroaxonal dystrophy in a Sudanese family.

    PubMed

    Elsayed, Liena E O; Mohammed, Inaam N; Hamed, Ahlam A A; Elseed, Maha A; Salih, Mustafa A M; Yahia, Ashraf; Siddig, Rayan A; Amin, Mutaz; Koko, Mahmoud; Elbashir, Mustafa I; Ibrahim, Muntaser E; Brice, Alexis; Ahmed, Ammar E; Stevanin, Giovanni

    2018-05-08

    Infantile neuroaxonal dystrophy (INAD) is a rare hereditary neurological disorder caused by mutations in PLA2G6. The disease commonly affects children below 3 years of age and presents with delay in motor skills, optic atrophy and progressive spastic tetraparesis. Studies of INAD in Africa are extremely rare, and genetic studies from Sub Saharan Africa are almost non-existent. Two Sudanese siblings presented, at ages 18 and 24 months, with regression in both motor milestones and speech development and hyper-reflexia. Brain MRI showed bilateral and symmetrical T2/FLAIR hyperintense signal changes in periventricular areas and basal ganglia and mild cerebellar atrophy. Whole exome sequencing with confirmatory Sanger sequencing were performed for the two patients and healthy family members. A novel variant (NM_003560.2 c.1427 + 2 T > C) acting on a splice donor site and predicted to lead to skipping of exon 10 was found in PLA2G6. It was found in a homozygous state in the two patients and homozygous reference or heterozygous in five healthy family members. This variant has one very strong (loss of function mutation) and three supporting evidences for its pathogenicity (segregation with the disease, multiple computational evidence and specific patients' phenotype). Therefore this variant can be currently annotated as "pathogenic". This is the first study to report mutations in PLA2G6 gene in patients from Sudan.

  6. Influence of Mussel-Derived Bioactive BMP-2-Decorated PLA on MSC Behavior in Vitro and Verification with Osteogenicity at Ectopic Sites in Vivo.

    PubMed

    Chen, Zhuoyue; Zhang, Zhen; Feng, Juantao; Guo, Yayuan; Yu, Yuan; Cui, Jihong; Li, Hongmin; Shang, Lijun

    2018-04-11

    Osteoinductive activity of the implant in bone healing and regeneration is still a challenging research topic. Therapeutic application of recombinant human bone morphogenetic protein-2 (BMP-2) is a promising approach to enhance osteogenesis. However, high dose and uncontrolled burst release of BMP-2 may introduce edema, bone overgrowth, cystlike bone formation, and inflammation. In this study, low-dose BMP-2 of 1 μg was used to design PLA-PD-BMP for functionalization of polylactic acid (PLA) implants via mussel-inspired polydopamine (PD) assist. For the first time, the binding property and efficiency of the PD coating with BMP-2 were directly demonstrated and analyzed using an antigen-antibody reaction. The obtained PLA-PD-BMP surface immobilized with this low BMP-2 dose can endow the implants with abilities of introducing strong stem cell adhesion and enhanced osteogenicity. Furthermore, in vivo osteoinduction of the PLA-PD-BMP-2 scaffolds was confirmed by a rat ectopic bone model, which is marked as the "gold standard" for the evidence of osteoinductive activity. The microcomputed tomography, Young's modulus, and histology analyses were also employed to demonstrate that PLA-PD-BMP grafted with 1 μg of BMP-2 can induce bone formation. Therefore, the method in this study can be used as a model system to immobilize other growth factors onto various different types of polymer substrates. The highly biomimetic mussel-derived strategy can therefore improve the clinical outcome of polymer-based medical implants in a facile, safe, and effective way.

  7. Genetic ontogeny of pancreatic enzymes in Labrus bergylta larvae and the effect of feed type on enzyme activities and gene expression.

    PubMed

    Hansen, Truls Wergeland; Folkvord, Arild; Grøtan, Espen; Sæle, Øystein

    2013-03-01

    A newly cultivated wrasse species, Labrus bergylta, have shown great potential for use in Atlantic salmon (Salmo salar) farms in the battle against sea lice (Lepeoptheirus salmonis) infections. Hatchery reared L. bergylta were studied from 2 to 55 DPH to examine the molecular basis of digestive ontogeny related to the pancreas. An isolated feeding trial was performed on 27-34 DPH larvae to compare the effect of diet on enzyme activity and the possible exogenous contribution by live feed. The following genes coding for key pancreatic enzymes were analyzed by qPCR: trypsin, Cyp7 A1, BAL, sPLA(2) 1B, amylase and pancreatic chitinase. Enzyme activity was measured on trypsin, neutral lipase, sPLA(2), amylase and chitinase in fed and unfed larvae. We did not observe any effects of the formulated diet v.s. rotifers on enzyme activities of neutral lipase, chitinase and sPLA(2). However, a probable feed-dependency was observed at a transcriptional level, where rotifers seem to stimulate upregulation. The regulation of BAL was the only exception, where an upregulation was observed after weaning both in the ontogeny series and the experimental part. Our data on pancreatic chitinase and amylase mRNA levels suggest the importance of carbohydrates in the diet of early larval and juvenile L. bergylta. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Ageing sensitized by iPLA2β deficiency induces liver fibrosis and intestinal atrophy involving suppression of homeostatic genes and alteration of intestinal lipids and bile acids.

    PubMed

    Jiao, Li; Gan-Schreier, Hongying; Zhu, Xingya; Wei, Wang; Tuma-Kellner, Sabine; Liebisch, Gerhard; Stremmel, Wolfgang; Chamulitrat, Walee

    2017-12-01

    Ageing is a major risk factor for various forms of liver and gastrointestinal (GI) disease and genetic background may contribute to the pathogenesis of these diseases. Group VIA phospholipase A2 or iPLA 2 β is a homeostatic PLA 2 by playing a role in phospholipid metabolism and remodeling. Global iPLA 2 β -/- mice exhibit aged-dependent phenotypes with body weight loss and abnormalities in the bone and brain. We have previously reported the abnormalities in these mutant mice showing susceptibility for chemical-induced liver injury and colitis. We hypothesize that iPLA 2 β deficiency may sensitize with ageing for an induction of GI injury. Male wild-type and iPLA 2 β -/- mice at 4 and 20-22months of age were studied. Aged, but not young, iPLA 2 β -/- mice showed increased hepatic fibrosis and biliary ductular expansion as well as severe intestinal atrophy associated with increased apoptosis, pro-inflammation, disrupted tight junction, and reduced number of mucin-containing globlet cells. This damage was associated with decreased expression of intestinal endoplasmic stress XBP1 and its regulator HNF1α, FATP4, ACSL5, bile-acid transport genes as well as nuclear receptors LXRα and FXR. By LC/MS-MS profiling, iPLA 2 β deficiency in aged mice caused an increase of intestinal arachidonate-containing phospholipids concomitant with a decrease in ceramides. By the suppression of intestinal FXR/FGF-15 signaling, hepatic bile-acid synthesis gene expression was increased leading to an elevation of secondary and hydrophobic bile acids in liver, bile, and intestine. In conclusions, ageing sensitized by iPLA 2 β deficiency caused a decline of key intestinal homeostatic genes resulting in the development of GI disease in a gut-to-liver manner. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. 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

  10. 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.

  11. Expression of Clonorchis sinensis GIIIsPLA2 protein in baculovirus-infected insect cells and its overexpression facilitating epithelial-mesenchymal transition in Huh7 cells via AKT pathway.

    PubMed

    Shang, Mei; Xie, Zhizhi; Tang, Zeli; He, Lei; Wang, Xiaoyun; Wang, Caiqin; Wu, Yinjuan; Li, Ye; Zhao, Lu; Lv, Zhiyue; Wu, Zhongdao; Huang, Yan; Yu, Xinbing; Li, Xuerong

    2017-04-01

    Although prior studies confirmed that group III secretory phospholipase A 2 of Clonorchis sinensis (CsGIIIsPLA 2 ) had stimulating effect on liver fibrosis by binding to LX-2 cells, large-scale expression of recombinant protein and its function in the progression of hepatoma are worth exploring. Because of high productivity and low lipopolysaccharides (LPS) in the Sf9-baculovirus expression system, we firstly used this system to express the coding region of CsGIIIsPLA 2 . The molecular weight of recombinant CsGIIIsPLA 2 protein was about 34 kDa. Further investigation showed that most of the recombinant protein presented intracellular expression in Sf9 insect cell nucleus and could be detected only into cell debris, which made the protein purification and further functional study difficult. Therefore, to study the role of CsGIIIsPLA 2 in hepatocellular carcinoma (HCC) progression, CsGIIIsPLA 2 overexpression Huh7 cell model was applied. Cell proliferation, migration, and the expression level of epithelial-mesenchymal transition (EMT)-related molecules (E-cadherin, N-cadherin, α-catenin, Vimentin, p300, Snail, and Slug) along with possible mechanism were measured. The results indicated that CsGIIIsPLA 2 overexpression not only inhibited cell proliferation and promoted migration and EMT but also enhanced the phosphorylation of AKT in HCC cells. In conclusion, this study supported that CsGIIIsPLA 2 overexpression suppressed cell proliferation and induced EMT through the AKT pathway.

  12. Proteomic Characterization of Plasmid pLA1 for Biodegradation of Polycyclic Aromatic Hydrocarbons in the Marine Bacterium, Novosphingobium pentaromativorans US6-1

    PubMed Central

    Yun, Sung Ho; Choi, Chi-Won; Lee, Sang-Yeop; Lee, Yeol Gyun; Kwon, Joseph; Leem, Sun Hee; Chung, Young Ho; Kahng, Hyung-Yeel; Kim, Sang Jin; Kwon, Kae Kyoung; Kim, Seung Il

    2014-01-01

    Novosphingobium pentaromativorans US6-1 is a halophilic marine bacterium able to degrade polycyclic aromatic hydrocarbons (PAHs). Genome sequence analysis revealed that the large plasmid pLA1 present in N. pentaromativorans US6-1 consists of 199 ORFs and possess putative biodegradation genes that may be involved in PAH degradation. 1-DE/LC-MS/MS analysis of N. pentaromativorans US6-1 cultured in the presence of different PAHs and monocyclic aromatic hydrocarbons (MAHs) identified approximately 1,000 and 1,400 proteins, respectively. Up-regulated biodegradation enzymes, including those belonging to pLA1, were quantitatively compared. Among the PAHs, phenanthrene induced the strongest up-regulation of extradiol cleavage pathway enzymes such as ring-hydroxylating dioxygenase, putative biphenyl-2,3-diol 1,2-dioxygenase, and catechol 2,3-dioxygenase in pLA1. These enzymes lead the initial step of the lower catabolic pathway of aromatic hydrocarbons through the extradiol cleavage pathway and participate in the attack of PAH ring cleavage, respectively. However, N. pentaromativorans US6-1 cultured with p-hydroxybenzoate induced activation of another extradiol cleavage pathway, the protocatechuate 4,5-dioxygenase pathway, that originated from chromosomal genes. These results suggest that N. pentaromativorans US6-1 utilizes two different extradiol pathways and plasmid pLA1 might play a key role in the biodegradation of PAH in N. pentaromativorans US6-1. PMID:24608660

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

    PubMed Central

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

    2013-01-01

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

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. Influence of dynamic compressive loading on the in vitro degradation behavior of pure PLA and Mg/PLA composite.

    PubMed

    Li, Xuan; Qi, Chenxi; Han, Linyuan; Chu, Chenglin; Bai, Jing; Guo, Chao; Xue, Feng; Shen, Baolong; Chu, Paul K

    2017-12-01

    The effects of dynamic compressive loading on the in vitro degradation behavior of pure poly-lactic acid (PLA) and PLA-based composite unidirectionally reinforced with micro-arc oxidized magnesium alloy wires (Mg/PLA) are investigated. Dynamic compressive loading is shown to accelerate degradation of pure PLA and Mg/PLA. As the applied stress is increased from 0.1MPa to 0.9MPa or frequency from 0.5Hz to 2.5Hz, the overall degradation rate goes up. After immersion for 21days at 0.9MPa and 2.5Hz, the bending strength retention of the composite and pure PLA is 60.1% and 50%, respectively. Dynamic loading enhances diffusion of small acidic molecules resulting in significant pH decrease in the immersion solution. The synergistic reaction between magnesium alloy wires and PLA in the composite is further clarified by electrochemical tests. The degradation behavior of the pure PLA and PLA matrix in the composite under dynamic conditions obey the first order degradation kinetics and a numerical model is postulated to elucidate the relationship of the bending strength, stress, frequency, and immersion time under dynamic conditions. We systematically study the influence of dynamic loading on the degradation behavior of pure PLA and Mg/PLA. Dynamic compressive loading is shown to accelerate degradation of pure PLA and Mg/PLA. The synergistic reaction between magnesium alloy wires and PLA in the composite is firstly clarified by electrochemical tests. The degradation behavior of the pure PLA and PLA matrix in the composite under dynamic conditions obey the first order degradation kinetics. Then, a numerical model is postulated to elucidate the relationship of the bending strength, stress, frequency, and immersion time under dynamic conditions. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  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. Creating a Positive PLA Experience: A Step-by-Step Look at University PLA

    ERIC Educational Resources Information Center

    Leiste, Sara M.; Jensen, Kathryn

    2011-01-01

    A prior learning assessment (PLA) can be an intimidating process for adult learners. Capella University's PLA team has developed best practices, resources, and tools to foster a positive experience and to remove barriers in PLA and uses three criteria to determine how to best administer the assessment. First, a PLA must be motivating, as described…

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

    PubMed Central

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

    2016-01-01

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

  2. Characteristics and cytotoxicity of folate-modified curcumin-loaded PLA-PEG micellar nano systems with various PLA:PEG ratios.

    PubMed

    Phan, Quoc Thong; Le, Mai Huong; Le, Thi Thu Huong; Tran, Thi Hong Ha; Xuan, Phuc Nguyen; Ha, Phuong Thu

    2016-06-30

    Targeting delivery system use natural drugs for tumor cells is an appealing platform help to reduce the side effects and enhance the therapeutic effects of the drug. In this study, we synthesized curcumin (Cur) loaded (D, L Poly lactic - Poly ethylenglycol) micelle (Cur/PLA-PEG) with the ratio of PLA/PEG of 3:1 2:1 1:1 1:2 and 1:3 (w/w) and another micelle modified by folate (Cur/PLA-PEG-Fol) for targeting cancer therapy. The PLA-PEG copolymer was synthesized by ring opening polymerization method. After loading onto the micelle, solubility of Cur increased from 0.38 to 0.73mgml(-1). The average size of prepared Cur/PLA-PEG micelles was from 60 to 69nm (corresponding to the ratio difference of PLA/PEG) and the drug encapsulating efficiency was from 48.8 to 91.3%. Compared with the Cur/PLA-PEG micelles, the size of Cur/PLA-PEG-Fol micelles were from 80 to 86nm and showed better in vitro cellular uptake and cytotoxicity towards HepG2 cells. The cytotoxicity of the NPs however depends much on the PEG component. The results demonstrated that Folate-modified micelles could serve as a potential nano carrier to improve solubility, anti-cancer activity of Cur and targeting ability of the system. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Evaluation of self-assembled HCPT-loaded PEG-b-PLA nanoparticles by comparing with HCPT-loaded PLA nanoparticles.

    PubMed

    Yang, Xiangrui; Wu, Shichao; Wang, Yange; Li, Yang; Chang, Di; Luo, Yin; Ye, Shefang; Hou, Zhenqing

    2014-12-01

    We present a dialysis technique to prepare the 10-hydroxycamptothecin (HCPT)-loaded nanoparticles (NPs) using methoxypolyethylene glycol-poly(D,L-lactide) (PEG-b-PLA) and PLA, respectively. Both HCPT-loaded PEG-b-PLA NPs and HCPT-loaded PLA NPs were characterized by differential scanning calorimetry (DSC), dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The results showed that the HCPT-loaded PEG-b-PLA NPs and HCPT-loaded PLA NPs presented a hydrodynamic particle size of 120.1 and 226.8 nm, with a polydispersity index of 0.057 and 0.207, a zeta potential of -31.2 and -45.7 mV, drug encapsulation efficiency of 44.52% and 44.94%, and drug-loaded content of 7.42% and 7.49%, respectively. The HCPT-loaded PEG-b-PLA NPs presented faster drug release rate compared to the HCPT-loaded PLA NPs. The HCPT-loaded PEG-b-PLA NPs presented higher cytotoxicity than the HCPT-loaded PLA NPs. These results suggested that the HCPT-loaded PEG-b-PLA NPs presented better characteristics for drug delivery compared to HCPT-loaded PLA NPs.

  4. Single phase dynamic CMOS PLA using charge sharing technique

    NASA Technical Reports Server (NTRS)

    Dhong, Y. B.; Tsang, C. P.

    1991-01-01

    A single phase dynamic CMOS NOR-NOR programmable logic array (PLA) using triggered decoders and charge sharing techniques for high speed and low power is presented. By using the triggered decoder technique, the ground switches are eliminated, thereby, making this new design much faster and lower power dissipation than conventional PLA's. By using the charge-sharing technique in a dynamic CMOS NOR structure, a cascading AND gate can be implemented. The proposed PLA's are presented with a delay-time of 15.95 and 18.05 nsec, respectively, which compare with a conventional single phase PLA with 35.5 nsec delay-time. For a typical example of PLA like the Signetics 82S100 with 16 inputs, 48 input minterms (m) and 8 output minterms (n), the 2-SOP PLA using the triggered 2-bit decoder is 2.23 times faster and has 2.1 times less power dissipation than the conventional PLA. These results are simulated using maximum drain current of 600 micro-A, gate length of 2.0 micron, V sub DD of 5 V, the capacitance of an input miniterm of 1600 fF, and the capacitance of an output minterm of 1500 fF.

  5. 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.

  6. Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays.

    PubMed

    Fortes-Dias, Consuelo Latorre; Santos, Roberta Márcia Marques dos; Magro, Angelo José; Fontes, Marcos Roberto de Mattos; Chávez-Olórtegui, Carlos; Granier, Claude

    2009-01-01

    Crotoxin (CA.CB) is a beta-neurotoxin from Crotalus durissus terrificus snake venom that is responsible for main envenomation effects upon biting by this snake. It is a heterodimer of an acidic protein (CA) devoid of any biological activity per se and a basic, enzymatically active, PLA(2) counterpart (CB). Both lethal and enzymatic activities of crotoxin have been shown to be inhibited by CNF, a protein from the blood of C. d. terrificus snakes. CNF replaces CA in the CA.CB complex, forming a stable, non-toxic complex CNF.CB. The molecular sites involved in the tight interfacial protein-protein interactions in these PLA(2)-based complexes have not been clearly determined. To help address this question, we used the peptide arrays approach to map possible interfacial interaction sites in CA.CB and CNF.CB. Amino acid stretches putatively involved in these interactions were firstly identified in the primary structure of CB. Further analysis of the interfacial availability of these stretches in the presumed biologically active structure of CB, suggested two interaction main sites, located at the amino-terminus and beta-wing regions. Peptide segments at the carboxyl-terminus of CB were also suggested to play a secondary role in the binding of both CA and CNF.

  7. 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

  8. 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

  9. 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.

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

    PubMed

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

    2018-03-01

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

  11. 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.

  12. Inhibition mechanism of P-glycoprotein mediated efflux by mPEG-PLA and influence of PLA chain length on P-glycoprotein inhibition activity.

    PubMed

    Li, Wenjing; Li, Xinru; Gao, Yajie; Zhou, Yanxia; Ma, Shujin; Zhao, Yong; Li, Jinwen; Liu, Yan; Wang, Xinglin; Yin, Dongdong

    2014-01-06

    The present study aimed to investigate the effect of monomethoxy poly(ethylene glycol)-block-poly(D,L-lactic acid) (mPEG-PLA) on the activity of P-glycoprotein (P-gp) in Caco-2 cells and further unravel the relationship between PLA chain length in mPEG-PLA and influence on P-gp efflux and the action mechanism. The transport results of rhodamine 123 (R123) across Caco-2 cell monolayers suggested that mPEG-PLA unimers were responsible for its P-gp inhibitory effect. Furthermore, transport studies of R123 revealed that the inhibitory potential of P-gp efflux by mPEG-PLA analogues was strongly correlated with their structural features and showed that the hydrophilic mPEG-PLA copolymers with an intermediate PLA chain length and 10.20 of hydrophilic-lipophilic balance were more effective at inhibiting P-gp efflux in Caco-2 cells. The fluorescence polarization measurement results ruled out the plasma membrane fluidization as a contributor for inhibition of P-gp by mPEG-PLA. Concurrently, mPEG-PLA inhibited neither basal P-gp ATPase (ATP is adenosine triphosphate) activity nor substrate stimulated P-gp ATPase activity, suggesting that mPEG-PLA seemed not to be a substrate of P-gp and a competitive inhibitor. No evident alteration in P-gp surface level was detected by flow cytometry upon exposure of the cells to mPEG-PLA. The depletion of intracellular ATP, which was likely to be a result of partial inhibition of cellular metabolism, was directly correlated with inhibitory potential for P-gp mediated efflux by mPEG-PLA analogues. Hence, intracellular ATP-depletion appeared to be possible explanation to the inhibition mechanism of P-gp by mPEG-PLA. Taken together, the establishment of a relationship between PLA chain length and impact on P-gp efflux activity and interpretation of action mechanism of mPEG-PLA on P-gp are of fundamental importance and will facilitate future development of mPEG-PLA in the drug delivery area.

  13. Leptin rapidly activates PPARs in C2C12 muscle cells

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

    Bendinelli, Paola; Piccoletti, Roberta; Maroni, Paola

    2005-07-08

    Experimental evidence suggests that leptin operates on the tissues, including skeletal muscle, also by modulating gene expression. Using electrophoretic mobility shift assays, we have shown that physiological doses of leptin promptly increase the binding of C2C12 cell nuclear extracts to peroxisome proliferator-activated receptor (PPAR) response elements in oligonucleotide probes and that all three PPAR isoforms participate in DNA-binding complexes. We pre-treated C2C12 cells with AACOCF{sub 3}, a specific inhibitor of cytosolic phospholipase A{sub 2} (cPLA{sub 2}), an enzyme that supplies ligands to PPARs, and found that it abrogates leptin-induced PPAR DNA-binding activity. Leptin treatment significantly increased cPLA{sub 2} activity, evaluatedmore » as the release of [{sup 3}H]arachidonic acid from pre-labelled C2C12 cells, as well as phosphorylation. Further, using MEK1 inhibitor PD-98059 we showed that leptin activates cPLA{sub 2} through ERK induction. These results support a direct effect of leptin on skeletal muscle cells, and suggest that the hormone may modulate muscle transcription also by precocious activation of PPARs through ERK-cPLA{sub 2} pathway.« less

  14. 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

  15. Mutants of phospholipase A (pPLA-I) have a red light and auxin phenotype.

    PubMed

    Effendi, Yunus; Radatz, Katrin; Labusch, Corinna; Rietz, Steffen; Wimalasekera, Rinukshi; Helizon, Hanna; Zeidler, Mathias; Scherer, Günther F E

    2014-07-01

    pPLA-I is the evolutionarily oldest patatin-related phospholipase A (pPLA) in plants, which have previously been implicated to function in auxin and defence signalling. Molecular and physiological analysis of two allelic null mutants for pPLA-I [ppla-I-1 in Wassilewskija (Ws) and ppla-I-3 in Columbia (Col) ] revealed pPLA-I functions in auxin and light signalling. The enzyme is localized in the cytosol and to membranes. After auxin application expression of early auxin-induced genes is significantly slower compared with wild type and both alleles show a slower gravitropic response of hypocotyls, indicating compromised auxin signalling. Additionally, phytochrome-modulated responses like abrogation of gravitropism, enhancement of phototropism and growth in far red-enriched light are decreased in both alleles. While early flowering, root coils and delayed phototropism are only observed in the Ws mutant devoid of phyD, the light-related phenotypes observed in both alleles point to an involvement of pPLA-I in phytochrome signalling. © 2014 John Wiley & Sons Ltd.

  16. Indole cytosolic phospholipase A2 alpha inhibitors: discovery and in vitro and in vivo characterization of 4-{3-[5-chloro-2-(2-{[(3,4-dichlorobenzyl)sulfonyl]amino}ethyl)-1-(diphenylmethyl)-1H-indol-3-yl]propyl}benzoic acid, efipladib.

    PubMed

    McKew, John C; Lee, Katherine L; Shen, Marina W H; Thakker, Paresh; Foley, Megan A; Behnke, Mark L; Hu, Baihua; Sum, Fuk-Wah; Tam, Steve; Hu, Yonghan; Chen, Lihren; Kirincich, Steven J; Michalak, Ronald; Thomason, Jennifer; Ipek, Manus; Wu, Kun; Wooder, Lane; Ramarao, Manjunath K; Murphy, Elizabeth A; Goodwin, Debra G; Albert, Leo; Xu, Xin; Donahue, Frances; Ku, M Sherry; Keith, James; Nickerson-Nutter, Cheryl L; Abraham, William M; Williams, Cara; Hegen, Martin; Clark, James D

    2008-06-26

    The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.

  17. 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.

  18. Coordination of gene expression of arachidonic and docosahexaenoic acid cascade enzymes during human brain development and aging.

    PubMed

    Ryan, Veronica H; Primiani, Christopher T; Rao, Jagadeesh S; Ahn, Kwangmi; Rapoport, Stanley I; Blanchard, Helene

    2014-01-01

    The polyunsaturated arachidonic and docosahexaenoic acids (AA and DHA) participate in cell membrane synthesis during neurodevelopment, neuroplasticity, and neurotransmission throughout life. Each is metabolized via coupled enzymatic reactions within separate but interacting metabolic cascades. AA and DHA pathway genes are coordinately expressed and underlie cascade interactions during human brain development and aging. The BrainCloud database for human non-pathological prefrontal cortex gene expression was used to quantify postnatal age changes in mRNA expression of 34 genes involved in AA and DHA metabolism. Expression patterns were split into Development (0 to 20 years) and Aging (21 to 78 years) intervals. Expression of genes for cytosolic phospholipases A2 (cPLA2), cyclooxygenases (COX)-1 and -2, and other AA cascade enzymes, correlated closely with age during Development, less so during Aging. Expression of DHA cascade enzymes was less inter-correlated in each period, but often changed in the opposite direction to expression of AA cascade genes. Except for the PLA2G4A (cPLA2 IVA) and PTGS2 (COX-2) genes at 1q25, highly inter-correlated genes were at distant chromosomal loci. Coordinated age-related gene expression during the brain Development and Aging intervals likely underlies coupled changes in enzymes of the AA and DHA cascades and largely occur through distant transcriptional regulation. Healthy brain aging does not show upregulation of PLA2G4 or PTGS2 expression, which was found in Alzheimer's disease.

  19. Potential of a newly developed high-speed near-infrared (NIR) camera (Compovision) in polymer industrial analyses: monitoring crystallinity and crystal evolution of polylactic acid (PLA) and concentration of PLA in PLA/Poly-(R)-3-hydroxybutyrate (PHB) blends.

    PubMed

    Ishikawa, Daitaro; Nishii, Takashi; Mizuno, Fumiaki; Sato, Harumi; Kazarian, Sergei G; Ozaki, Yukihiro

    2013-12-01

    This study was carried out to evaluate a new high-speed hyperspectral near-infrared (NIR) camera named Compovision. Quantitative analyses of the crystallinity and crystal evolution of biodegradable polymer, polylactic acid (PLA), and its concentration in PLA/poly-(R)-3-hydroxybutyrate (PHB) blends were investigated using near-infrared (NIR) imaging. This NIR camera can measure two-dimensional NIR spectral data in the 1000-2350 nm region obtaining images with wide field of view of 150 × 250 mm(2) (approximately 100  000 pixels) at high speeds (in less than 5 s). PLA with differing crystallinities between 0 and 50% blended samples with PHB in ratios of 80/20, 60/40, 40/60, 20/80, and pure films of 100% PLA and PHB were prepared. Compovision was used to collect respective NIR spectra in the 1000-2350 nm region and investigate the crystallinity of PLA and its concentration in the blends. The partial least squares (PLS) regression models for the crystallinity of PLA were developed using absorbance, second derivative, and standard normal variate (SNV) spectra from the most informative region of the spectra, between 1600 and 2000 nm. The predicted results of PLS models achieved using the absorbance and second derivative spectra were fairly good with a root mean square error (RMSE) of less than 6.1% and a determination of coefficient (R(2)) of more than 0.88 for PLS factor 1. The results obtained using the SNV spectra yielded the best prediction with the smallest RMSE of 2.93% and the highest R(2) of 0.976. Moreover, PLS models developed for estimating the concentration of PLA in the blend polymers using SNV spectra gave good predicted results where the RMSE was 4.94% and R(2) was 0.98. The SNV-based models provided the best-predicted results, since it can reduce the effects of the spectral changes induced by the inhomogeneity and the thickness of the samples. Wide area crystal evolution of PLA on a plate where a temperature slope of 70-105 °C had occurred was also

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. Mitigation of N2O Emission from Aquaponics by Optimizing the Nitrogen Transformation Process: Aeration Management and Exogenous Carbon (PLA) Addition.

    PubMed

    Zou, Yina; Hu, Zhen; Zhang, Jian; Fang, Yingke; Li, Minying; Zhang, Jianda

    2017-10-11

    N 2 O production in aquaponics is an inevitable concern when aquaponics is developed as a future production system. In the present study, two attempts were applied to mitigate N 2 O emission from aquaponics, i.e., aeration in hydroponic bed (HA) and addition of polylactic acid (PLA) into fillers (PA). Results showed that N 2 O emission from HA and PA was decreased by 47.1-58.1% and 43.2-74.9% respectively compared with that in control. Denitrification was proved to be the main emission pathway in all treatments, representing 62.4%, 86.4%, and 75.8% of the total N 2 O emission in HA, PA, and control, respectively. However, production of plants in HA was severely impaired, which was only 3.04 ± 0.39 kg/m 2 , while in PA and control, plants yields were 4.87 ± 0.56 kg/m 2 and 4.33 ± 0.58 kg/m 2 . Combining the environmental and economic benefits, adding PLA in aquaponics may have a better future when developing and applying aquaponics systems.

  7. 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.

  8. Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA{sub 2} activity

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

    Takatani-Nakase, Tomoka, E-mail: nakase@mukogawa-u.ac.jp; Takahashi, Koichi, E-mail: koichi@mukogawa-u.ac.jp

    Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA{sub 2}, which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death.more » - Highlights: • Myocardial ischemia-model induces in caspase-independent, non-apoptotic cell death. • Leptin strongly inhibits ischemic-augmented non-apoptotic cell death. • Leptin reduces iPLA{sub 2} activity, leading to avoidance of non-apoptotic cell death.« less

  9. Common rs5918 (PlA1/A2) polymorphism in the ITGB3 gene and risk of coronary artery disease

    PubMed Central

    Heidari, Mohammad Mehdi; Soheilyfar, Sorour

    2016-01-01

    Introduction The T to C transition at nucleotide 1565 of the human glycoprotein IIIa (ITGB3) gene represents a genetic polymorphism (PlA1/A2) that can influence both platelet activation and aggregation and that has been associated with many types of disease. Here, we present a newly designed multiplex tetra-primer amplification refractory mutation system – polymerase chain reaction (T-ARMS-PCR) for genotyping a single nucleotide polymorphism (SNP) (dbSNP ID: rs5918) in the human ITGB3 gene. Material and methods We set up T-ARMS-PCR for the rs5918 SNP in a single-step PCR and the results were validated by the PCR-RFLP method in 132 coronary artery disease (CAD) patients and 122 unrelated healthy individuals. Results Full accordance was found for genotype determination by the PCR-RFLP method. The multiple logistic regression analysis showed a significant association of the rs5918 polymorphism and CAD according to dominant and recessive models (dominant model OR: 2.40, 95% CI: 1.33–4.35; p = 0.003, recessive model OR: 4.71, 95% CI: 1.32–16.80; p = 0.0067). Conclusions Our T-ARMS-PCR in comparison with RFLP and allele-specific PCR is more advantageous because this PCR method allows the evaluation of both the wild type and the mutant allele in the same tube. Our results suggest that the rs5918 (PlA1/A2) polymorphism in the ITGB3 gene may contribute to the susceptibility of sporadic Iranian coronary artery disease (CAD) patients. PMID:28905013

  10. Coordination of Gene Expression of Arachidonic and Docosahexaenoic Acid Cascade Enzymes during Human Brain Development and Aging

    PubMed Central

    Ryan, Veronica H.; Primiani, Christopher T.; Rao, Jagadeesh S.; Ahn, Kwangmi; Rapoport, Stanley I.; Blanchard, Helene

    2014-01-01

    Background The polyunsaturated arachidonic and docosahexaenoic acids (AA and DHA) participate in cell membrane synthesis during neurodevelopment, neuroplasticity, and neurotransmission throughout life. Each is metabolized via coupled enzymatic reactions within separate but interacting metabolic cascades. Hypothesis AA and DHA pathway genes are coordinately expressed and underlie cascade interactions during human brain development and aging. Methods The BrainCloud database for human non-pathological prefrontal cortex gene expression was used to quantify postnatal age changes in mRNA expression of 34 genes involved in AA and DHA metabolism. Results Expression patterns were split into Development (0 to 20 years) and Aging (21 to 78 years) intervals. Expression of genes for cytosolic phospholipases A2 (cPLA2), cyclooxygenases (COX)-1 and -2, and other AA cascade enzymes, correlated closely with age during Development, less so during Aging. Expression of DHA cascade enzymes was less inter-correlated in each period, but often changed in the opposite direction to expression of AA cascade genes. Except for the PLA2G4A (cPLA2 IVA) and PTGS2 (COX-2) genes at 1q25, highly inter-correlated genes were at distant chromosomal loci. Conclusions Coordinated age-related gene expression during the brain Development and Aging intervals likely underlies coupled changes in enzymes of the AA and DHA cascades and largely occur through distant transcriptional regulation. Healthy brain aging does not show upregulation of PLA2G4 or PTGS2 expression, which was found in Alzheimer's disease. PMID:24963629

  11. Antibody production of wild-type and enzyme V279F variants of PAF-AH as a risk factor for Cardiovascular disease

    NASA Astrophysics Data System (ADS)

    Ramadhani, Anggia N.; Puspitarini, Sapti; Sari, Anissa N.; Widodo

    2017-11-01

    Coronary artery disease (CAD) has emerged as a leading cause of death in Indonesia nowadays. WHO data in 2012 revealed that 37% of the Indonesian population died from this disease. CAD occurs because of endothelial dysfunction in the arteries. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a phospholipase A2 enzyme, encoded by the PLA2G7 gene. This protein is predicted to be involved in inflammatory phospholipid metabolism so it can be used as a biomarker of CAD in the early phase. Thus, the purpose of this research is to discover the difference in antibody production between wild-type and mutant V279F. The PAF-AH enzyme was isolated from mice lymphocyte cells in order to develop this enzyme as a biomarker of cardiovascular disease. PAF-AH migrates at 55kDa according to SDS-PAGE analysis. Flow cytometry analysis showed that mutant PAF-AH (V279F) is more antigenic than wild-type PAF-AH. The missense mutation of V279F PAF-AH means this enzyme cannot catabolize the acetyl group at the sn-2 position of PAF.

  12. 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

  13. Exploring the in vitro and in vivo compatibility of PLA, PLA/GNP and PLA/CNT-COOH biodegradable nanocomposites: Prospects for tendon and ligament applications.

    PubMed

    Correia Pinto, Viviana; Costa-Almeida, Raquel; Rodrigues, Ilda; Guardão, Luísa; Soares, Raquel; Miranda Guedes, Rui

    2017-08-01

    Anterior cruciate ligament (ACL) reconstructive surgeries are the most frequent orthopedic procedures in the knee. Currently, existing strategies fail in completely restoring tissue functionality and have a high failure rate associated, presenting a compelling argument towards the development of novel materials envisioning ACL reinforcement. Tendons and ligaments, in general, have a strong demand in terms of biomechanical features of developed constructs. We have previously developed polylactic acid (PLA)-based biodegradable films reinforced either with graphene nanoplatelets (PLA/GNP) or with carboxyl-functionalized carbon nanotubes (PLA/CNT-COOH). In the present study, we comparatively assessed the biological performance of PLA, PLA/GNP, and PLA/CNT-COOH by seeding human dermal fibroblasts (HFF-1) and studying cell viability and proliferation. In vivo tests were also performed by subcutaneous implantation in 6-week-old C57Bl/6 mice. Results showed that all formulations studied herein did not elicit cytotoxic responses in seeded HFF-1, supporting cell proliferation up to 3 days in culture. Moreover, animal studies indicated no physiological signs of severe inflammatory response after 1 and 2 weeks after implantation. Taken together, our results present a preliminary assessment on the compatibility of PLA reinforced with GNP and CNT-COOH nanofillers, highlighting the potential use of these carbon-based nanofillers for the fabrication of reinforced synthetic polymer-based structures for ACL reinforcement. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2182-2190, 2017. © 2017 Wiley Periodicals, Inc.

  14. 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

  15. 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.

  16. 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.

  17. A DNA enzyme with Mg(2+)-Dependent RNA Phosphoesterase Activity

    NASA Technical Reports Server (NTRS)

    Breaker, Ronald R.; Joyce, Gerald F.

    1995-01-01

    Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approx. 10(exp -4)/ min in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 C. The Mg(2+) - dependent reaction is more difficult, with an uncatalyzed rate of approx. 10(exp -7)/ min under comparable conditions. Mg(2+) - dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+) - dependent reaction. Results: We generated a population of greater than 10(exp 13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg(2+), Mn(2+), Zn(2+) or Pb(2+). Examination of individual clones from the Mg(2+) lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction.This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01/ min. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. Conclusions: We have generated a Mg(2+) - dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approx. 10(exp 5) - fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.

  18. Roles of Human CYP2A6 and Monkey CYP2A24 and 2A26 Cytochrome P450 Enzymes in the Oxidation of 2,5,2',5'-Tetrachlorobiphenyl.

    PubMed

    Shimada, Tsutomu; Kakimoto, Kensaku; Takenaka, Shigeo; Koga, Nobuyuki; Uehara, Shotaro; Murayama, Norie; Yamazaki, Hiroshi; Kim, Donghak; Guengerich, F Peter; Komori, Masayuki

    2016-12-01

    2,5,2',5'-Tetrachlorobiphenyl (TCB) induced type I binding spectra with cytochrome P450 (P450) 2A6 and 2A13, with K s values of 9.4 and 0.51 µM, respectively. However, CYP2A6 oxidized 2,5,2',5'-TCB to form 4-hydroxylated products at a much higher rate (∼1.0 minute -1 ) than CYP2A13 (∼0.02 minute -1 ) based on analysis by liquid chromatography-tandem mass spectrometry. Formation of 4-hydroxy-2,5,2',5'-TCB by CYP2A6 was greater than that of 3-hydroxy-2,5,2',5'-TCB and three other hydroxylated products. Several human P450 enzymes, including CYP1A1, 1A2, 1B1, 2B6, 2D6, 2E1, 2C9, and 3A4, did not show any detectable activities in oxidizing 2,5,2',5'-TCB. Cynomolgus monkey CYP2A24, which shows 95% amino acid identity to human CYP2A6, catalyzed 4-hydroxylation of 2,5,2',5'-TCB at a higher rate (∼0.3 minute -1 ) than CYP2A26 (93% identity to CYP2A6, ∼0.13 minute -1 ) and CYP2A23 (94% identity to CYP2A13, ∼0.008 minute -1 ). None of these human and monkey CYP2A enzymes were catalytically active in oxidizing other TCB congeners, such as 2,4,3',4'-, 3,4,3',4'-, and 3,5,3',5'-TCB. Molecular docking analysis suggested that there are different orientations of interaction of 2,5,2',5'-TCB with the active sites (over the heme) of human and monkey CYP2A enzymes, and that ligand interaction energies (U values) of bound protein-ligand complexes show structural relationships of interaction of TCBs and other ligands with active sites of CYP2A enzymes. Catalytic differences in human and monkey CYP2A enzymes in the oxidation of 2,5,2',5'-TCB are suggested to be due to amino acid changes at substrate recognition sites, i.e., V110L, I209S, I300F, V365M, S369G, and R372H, based on the comparison of primary sequences. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  19. In vitro degradation kinetics of pure PLA and Mg/PLA composite: Effects of immersion temperature and compression stress.

    PubMed

    Li, Xuan; Chu, Chenglin; Wei, Yalin; Qi, Chenxi; Bai, Jing; Guo, Chao; Xue, Feng; Lin, Pinghua; Chu, Paul K

    2017-01-15

    The effects of the immersion temperature and compression stress on the in vitro degradation behavior of pure poly-lactic acid (pure-PLA) and PLA-based composite unidirectionally reinforced with micro-arc oxidized magnesium alloy wires (Mg/PLA or MAO-MAWs/PLA) are investigated. The degradation kinetics of pure-PLA and the PLA matrix in MAO-MAWs/PLA exhibit an Arrhenius-type behavior. For the composite, the synergic degradation of MAO-MAWs maintains a steady pH and mitigates the degradation of PLA matrix during immersion. However, the external compression stress decreases the activation energy (E a ) and pre-exponential factor (k 0 ) consequently increasing the degradation rate of PLA. Under a compression stress of 1MPa, E a and k 0 of pure PLA are 57.54kJ/mol and 9.74×10 7 day -1 , respectively, but 65.5kJ/mol and 9.81×10 8 day -1 for the PLA matrix in the composite. Accelerated tests are conducted in rising immersion temperature in order to shorten the experimental time. Our analysis indicates there are well-defined relationships between the bending strength of the specimens and the PLA molecular weight during immersion, which are independent of the degradation temperature and external compression stress. Finally, a numerical model is established to elucidate the relationship of bending strength, the PLA molecular weight, activation energy, immersion time and temperature. We systematically evaluate the effects of compression stress and temperature on the degradation properties of two materials: (pure-PLA) and MAO-MAWs/PLA (or Mg/PLA). The initial in vitro degradation kinetics of the unstressed or stressed pure-PLA and MAO-MAWs/PLA composite is confirmed to be Arrhenius-like. MAO-MAWs and external compression stress would influence the degradation activation energy (E a ) and pre-exponential factor (k 0 ) of PLA, and we noticed there is a linear relationship between E a and ln k 0 . Thereafter, we noticed that Mg 2+ , not H + , plays a significant role on the

  20. 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.

  1. Biochemical differences in the mass and activity tests of lipoprotein-associated phospholipase A2 explain the discordance in results between the two assay methods.

    PubMed

    Zhuo, Shaoqiu; Wolfert, Robert L; Yuan, Chong

    2017-12-01

    There are two platforms for the detection of Lp-PLA 2 in sera or plasmas: by its enzymatic activity (PLAC® activity test) and by its mass concentration (PLAC® mass test). It has been long recognized that these two platforms are not correlated well. The underlying cause for this is therefore investigated by the biochemical characterization of the two PLAC tests. Human sera with and without the treatment by detergent were fractionated by using a Superose-6 column in phosphate buffered saline and the phospholipid associated Lp-PLA 2 was assessed by both PLAC mass and activity tests. The Lp-PLA 2 values of the two PLAC tests were compared under such conditions. Fractionation of sera and plasmas indicates that the association of Lp-PLA 2 with phospholipids, especially LDL and other large size phospholipid vesicles, may block the detection of the enzyme by antibodies in the immunoassay format under the conditions of the PLAC mass test. Inclusion of high concentration (>CMC, critical micelle concentration) of detergents in the assay buffer of PLAC mass test dissociates Lp-PLA 2 from phospholipid vesicles and results in the full detection of all Lp-PLA 2 in sera or plasmas for concentration. Such assay modification significantly improves the correlation between the PLAC mass and PLAC activity tests. PLAC mass test only detects a small portion of the total Lp-PLA 2 , mainly the Lp-PLA 2 associated with HDL. This is the main cause of the discordance and poor correlation between the PLAC mass and activity tests. Our results demonstrate the PLAC activity test is more accurate in assessing the total level of circulating Lp-PLA 2 . Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  2. Phospholipids and their degrading enzyme in the tears of soft contact lens wearers.

    PubMed

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

    2006-12-01

    Low tear phospholipids levels are associated with tear film instability in soft contact lens wearers. We assayed levels of phospholipids and their degrading enzyme secretory phospholipase A2 (sPLA2) both in tears and deposited on contact lenses composed of 2 hydrophilic materials after 1 day of routine use. Polymacon (Medalist; FDA group 1, low water/nonionic) and Etafilcon A (One Day Acuvue; group 4, high water/ionic) contact lenses were worn for 12 hours by 16 experienced contact lens wearers. Phospholipids in tear fluids and deposited on contact lenses were estimated by phosphorus determination with ammonium molybdate through enzymatic digestion. Double-antibody sandwich ELISA was used to determine group IIa sPLA2 concentrations, and sPLA2 activity was assayed using 1,2-diheptanoyl thio-phosphatidylcholine as substrate. Phospholipids concentrations in tears with Polymacon and Etafilcon A were 186 +/- 39 and 162 +/- 33 microg/mL, respectively. The latter concentration was significantly lower than that observed in the same subjects when not wearing contact lenses (P = 0.0023). In tears, both group IIa sPLA2 concentrations and enzymatic activity remained unchanged, regardless of lens wearing. However, Etafilcon A (0.57 +/- 0.09 microg/lens) showed more group IIa sPLA2 deposition than Polymacon (0.01 +/- 0.01 microg/lens; P < 0.001). Furthermore, group IIa sPLA2 deposited on Etafilcon A but not on Polymacon lenses retained its enzymatic activity. Significant differences of group IIa sPLA2 deposition were found in the 2 lenses tested. Such deposition might induce phospholipid hydrolysis in tears and thereby promote tear film instability in hydrophilic contact lens wearers.

  3. Effect of TiO2-Crystal Forms on the Photo-Degradation of EVA/PLA Blend Under Accelerated Weather Testing

    NASA Astrophysics Data System (ADS)

    Van Cong, Do; Trang, Nguyen Thi Thu; Giang, Nguyen Vu; Lam, Tran Dai; Hoang, Thai

    2016-05-01

    Photo-degradation of poly (ethylene-co-vinyl acetate) (EVA)/poly (lactic acid) (PLA) blend and EVA/PLA/TiO2 nanocomposites was carried out under accelerated weather testing conditions by alternating cycles of ultraviolet (UV) light and moisture at controlled and elevated temperatures. The characters, properties, and morphology of these materials before and after accelerated weather testing were determined by Fourier transform infrared spectroscopy, colour changes, viscosity, tensile test, thermogravimetric analysis, and field emission scanning electron microscopy. The increases in the content of oxygen-containing groups, colour changes; the decreases in viscosity, tensile properties, and thermal stability of these materials after accelerated weather testing are the evidence for the photo-degradation of the blend and nanocomposites. After accelerated weather testing, the appearance of many micro-holes and micro-pores on the surface of the collected samples was observed. The photo-degradation degree of the nanocomposites depended on the TiO2-crystal form. Rutile TiO2 do not enhance the degradation, but anatase and mixed crystals TiO2 nanoparticles promoted the degradation of the nanocomposites. Particularly, the mixed crystals TiO2 nanoparticles showed the highest photo-catalytic activity of the nanocomposites.

  4. Preparation of Chitin-PLA laminated composite for implantable application.

    PubMed

    Nasrin, Romana; Biswas, Shanta; Rashid, Taslim Ur; Afrin, Sanjida; Jahan, Rumana Akhter; Haque, Papia; Rahman, Mohammed Mizanur

    2017-12-01

    The present study explores the possibilities of using locally available inexpensive waste prawn shell derived chitin reinforced and bioabsorbable polylactic acid (PLA) laminated composites to develop new materials with excellent mechanical and thermal properties for implantable application such as in bone or dental implant. Chitin at different concentration (1-20% of PLA) reinforced PLA films (CTP) were fabricated by solvent casting process and laminated chitin-PLA composites (LCTP) were prepared by laminating PLA film (obtained by hot press method) with CTP also by hot press method at 160 °C. The effect of variation of chitin concentration on the resulting laminated composite's behavior was investigated. The detailed physico-mechanical, surface morphology and thermal were assessed with different characterization technique such as FT-IR, XRD, SEM and TGA. The FTIR spectra showed the characteristic peaks for chitin and PLA in the composites. SEM images showed an excellent dispersion of chitin in the films and composites. Thermogravimetric analysis (TGA) showed that the complete degradation of chitin, PLA film, 5% chitin reinforced PLA film (CTP2) and LCTP are 98%, 95%, 87% and 98% respectively at temperature of 500 °C. The tensile strength of the LCTP was found 25.09 MPa which is significantly higher than pure PLA film (18.55 MPa) and CTP2 film (8.83 MPa). After lamination of pure PLA and CTP2 film, the composite (LCTP) yielded 0.265-1.061% water absorption from 30 min to 24 h immerse in water that is much lower than PLA and CTP. The increased mechanical properties of the laminated films with the increase of chitin content indicated good dispersion of chitin into PLA and strong interfacial actions between the polymer and chitin. The improvement of mechanical properties and the results of antimicrobial and cytotoxicity of the composites also evaluated and revealed the composite would be a suitable candidate for implant application in biomedical sector.

  5. 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

  6. 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

  7. Platelet indirect radioactive Coombs test. Its utilization for PLA1 grouping.

    PubMed

    Soulier, J P; Patereau, C; Drouet, J

    1975-01-01

    A platelet indirect radioactive Coombs test (PIRC) has been described. The technique for purification and labelling the antiglobulin has been precised. This test allowed the typing of platelets in the PLA system by using an absorbed serum from a mother of a thrombocytopenic child. Six other families of neonatal thrombocytopenias were tested. In three of them, the mothers were found PLA1 negative (PLA2, PLA2). Among a panel of 93 platelets, two (0.022) were found PLA1, negative. This PIRC test has many advantages compared to other tests such as platelet complement fixation, assay for blocking antibodies or antiglobulin consumption: it gives objective and quantitative results and is highly reproducible; anticomplementary serum may be tested.

  8. 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.

  9. Development of polylactide (PLA) and PLA nanocomposite foams in injection molding for automotive applications

    NASA Astrophysics Data System (ADS)

    Najafi Chaloupli, Naqi

    foaming behavior of PLA. To this end, LCB-PLAs were prepared in the presence of a multifunctional chain extender (CE) using two different processing strategies. In the first strategy, the dried PLA was directly mixed in the molten state with various quantities of CE (the formation of LCB structure). To further examine the impact of CE and molecular topology, a LCB-PLA was also prepared using a second approach, strategy S2. In this approach, a highly branched PLA was first prepared and then mixed with the neat PLA at a weight ratio of 50:50 (the introduction of LCB structure). The steady and transient rheological properties of the linear and LCB-PLAs revealed that the LCB-PLAs exhibited an increased viscosity, shear sensitivity and longer relaxation time in comparison with the linear PLA. The presence of the LCB structure, moreover, led to a strong strain-hardening behavior in uniaxial elongational flow whereas no strain hardening was observed for the linear PLA. The batch foaming of the samples was conducted using CO2 at different foaming temperatures ranging from 130 to 155 °C. The impact of molecular structure and foaming temperature on the void fraction, cell density, and cell size were examined. It was found that the increased melt strength and elasticity, resulting from branching, strongly affected the cell uniformity, cell density and void fraction. Among the investigated compositions, LCB-PLA prepared by strategy S2 provided smaller cell size and higher cell density than the other compositions. In most polymer processing operations such as extrusion and injection molding the polymeric chains are subjected to complex flow fields (elongation, shear, and mixed flows). Shearing the molten polymer during processing plays an essential role on crystallization and, thus, on the final properties of the product. The impact of the LCB structure and shear on the isothermal shear-induced crystallization kinetics, and the crystal morphology of PLA were studied in the second

  10. 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

  11. 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

  12. 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

  13. 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

  14. Influence of the Formulation Parameters on the Particle Size and Encapsulation Efficiency of Resveratrol in PLA and PLA-PEG Blend Nanoparticles: A Factorial Design.

    PubMed

    Lindner, Gabriela da Rocha; Dalmolin, Luciana Facco; Khalil, Najeh Maissar; Mainardes, Rubiana Mara

    2015-12-01

    Polymeric nanoparticles are colloidal systems that promote protection and modification of physicochemical characteristics of a drug and that also ensure controlled and extended drug release. This paper reports a 2(3) factorial design study to optimize poly(lactide) (PLA) and poly(lactide)-polyethylene glycol (PLA-PEG) blend nanoparticles containing resveratrol (RVT) for prolonged release. The independent variables analyzed were solvent composition, surfactant concentration and ratio of aqueous to organic phase (two levels each factor). Mean particle size and RVT encapsulation efficiency were set as the dependent variables. The selected optimized parameters were set as organic phase comprised of a mixture of dichloromethane and ethyl acetate, 1% of surfactant polyvinyl alcohol and a 3:1 ratio of aqueous to organic phase, for both PLA and PLA-PEG blend nanoparticles. This formulation originated nanoparticles with size of 228 ± 10 nm and 185 ± 70 nm and RVT encapsulation efficiency of 82 ± 10% and 76 ± 7% for PLA and PLA-PEG blend nanoparticles, respectively. The in vitro release study showed a biphasic pattern with prolonged RVT release and PEG did not influence the RVT release. The in vitro release data were in favor of Higuchi-diffusion kinetics for both nanoformulations and the Kossmeyer-Peppas coefficient indicated that anomalous transport was the main release mechanism of RVT. PLA and PLA-PEG blend nanoparticles produced with single emulsion-solvent evaporation technology were found to be a promising approach for the incorporation of RVT and promoted its controlled release. The factorial design is a tool of great value in choosing formulations with optimized parameters.

  15. O 2 Activation by Non-Heme Iron Enzymes

    DOE PAGES

    Solomon, Edward I.; Goudarzi, Serra; Sutherlin, Kyle D.

    2016-10-28

    The non-heme Fe enzymes are ubiquitous in nature and perform a wide range of functions involving O 2 activation. These had been difficult to study relative to heme enzymes; however, spectroscopic methods have now been developed that provide significant insight into the correlation of structure with function. This Current Topics article summarizes both the molecular mechanism these enzymes use to control O 2 activation in the presence of cosubstrates and the oxygen intermediates these reactions generate. Three types of O 2 activation are observed. First, non-heme reactivity is shown to be different from heme chemistry where a low-spin Fe III-OOHmore » non-heme intermediate directly reacts with substrate. Also, two subclasses of non-heme Fe enzymes generate high-spin Fe IV=O intermediates that provide both σ and π frontier molecular orbitals that can control selectivity. Lastly, for several subclasses of non-heme Fe enzymes, substrate binding to the Fe II site leads to the one electron reductive activation of O 2 to an Fe III-superoxide capable of H-atom abstraction and electrophilic attack.« less

  16. O 2 Activation by Non-Heme Iron Enzymes

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

    Solomon, Edward I.; Goudarzi, Serra; Sutherlin, Kyle D.

    The non-heme Fe enzymes are ubiquitous in nature and perform a wide range of functions involving O 2 activation. These had been difficult to study relative to heme enzymes; however, spectroscopic methods have now been developed that provide significant insight into the correlation of structure with function. This Current Topics article summarizes both the molecular mechanism these enzymes use to control O 2 activation in the presence of cosubstrates and the oxygen intermediates these reactions generate. Three types of O 2 activation are observed. First, non-heme reactivity is shown to be different from heme chemistry where a low-spin Fe III-OOHmore » non-heme intermediate directly reacts with substrate. Also, two subclasses of non-heme Fe enzymes generate high-spin Fe IV=O intermediates that provide both σ and π frontier molecular orbitals that can control selectivity. Lastly, for several subclasses of non-heme Fe enzymes, substrate binding to the Fe II site leads to the one electron reductive activation of O 2 to an Fe III-superoxide capable of H-atom abstraction and electrophilic attack.« less

  17. 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

  18. 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.

  19. 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

  20. Dual Enzyme-Responsive Capsules of Hyaluronic Acid-block-Poly(Lactic Acid) for Sensing Bacterial Enzymes.

    PubMed

    Tücking, Katrin-Stephanie; Grützner, Verena; Unger, Ronald E; Schönherr, Holger

    2015-07-01

    The synthesis of novel amphiphilic hyaluronic acid (HYA) and poly(lactic acid) (PLA) block copolymers is reported as the key element of a strategy to detect the presence of pathogenic bacterial enzymes. In addition to the formation of defined HYA-block-PLA assemblies, the encapsulation of fluorescent reporter dyes and the selective enzymatic degradation of the capsules by hyaluronidase and proteinase K are studied. The synthesis of the dual enzyme-responsive HYA-b-PLA is carried out by copper-catalyzed Huisgen 1,3-dipolar cycloaddition. The resulting copolymers are assembled in water to form vesicular structures, which are characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering (DLS), and fluorescence lifetime imaging microscopy (FLIM). DLS measurements show that both enzymes cause a rapid decrease in the hydrodynamic diameter of the nanocapsules. Fluorescence spectroscopy data confirm the liberation of encapsulated dye, which indicates the disintegration of the capsules and validates the concept of enzymatically triggered payload release. Finally, cytotoxicity assays confirm that the HYA-b-PLA nanocapsules are biocompatible with primary human dermal microvascular endothelial cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

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

    2015-08-01

    Chronic bowel disease can co-exist with severe autoimmune hepatitis (AIH) in an absence of primary sclerosing cholangitis. Genetic background may contribute to this overlap syndrome. We previously have shown that the deficiency of iPLA2β causes an accumulation of hepatocyte apoptosis, and renders susceptibility for acute liver injury. We here tested whether AIH induction in iPLA2β-null mice could result in intestinal injury, and whether bile acid metabolism was altered. Control wild-type (WT) and female iPLA2β-null (iPLA2β(-/-)) mice were intravenously injected with 10mg/kg concanavalinA (ConA) or saline for 24h. ConA treatment of iPLA2β(-/-) mice caused massive liver injury with increased liver enzymes, fibrosis, and necrosis. While not affecting WT mice, ConA treatment of iPLA2β(-/-) mice caused severe duodenal villous atrophy concomitant with increased apoptosis, cell proliferation, globlet cell hyperplasia, and endotoxin leakage into portal vein indicating a disruption of intestinal barrier. With the greater extent than in WT mice, ConA treatment of iPLA2β(-/-) mice increased jejunal expression of innate response cytokines CD14, TNF-α, IL-6, and SOCS3 as well as chemokines CCL2 and the CCL3 receptor CCR5. iPLA2β deficiency in response to ConA-induced AIH caused a significant decrease in hepatic and biliary bile acids, and this was associated with suppression of hepatic Cyp7A1, Ntcp and ABCB11/Bsep and upregulation of intestinal FXR/FGF15 mRNA expression. The suppression of hepatic Ntcp expression together with the loss of intestinal barrier could account for the observed bile acid leakage into peripheral blood. Thus, enteropathy may result from acute AIH in a susceptible host such as iPLA2β deficiency. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Relationship between compatibilizer and yield strength of PLA/PP Blend

    NASA Astrophysics Data System (ADS)

    Jariyakulsith, Pattanun; Puajindanetr, Somchai

    2018-01-01

    The aim of this research is to study the relationship between compatibilizer and yield strength of polylactic acid (PLA) and polypropylene (PP) blend. The PLA is blended with PP (PLA/PP) at the ratios of 70/30, 50/50 and 30/70. In addition, (1) polypropylene grafted maleic anhydride (PP-g-MAH) as a compatibilizer at 0.3 and 0.7 part per hundred of PLA/PP resin (phr) and (2) dicumyl peroxide (DCP) being an initiator at 0.03 and 0.07 phr are added in each composition. Yield strength is characterized to study the interaction between compatibilizer, initiator and yield strength by using experimental design of multilevel full factorial. The results show that (1) the yield strength of PLA/PP blend are increased after addition of compatibilizer. Because the adding of PP-g-MAH and DCP resulted in improving compatibility between PLA and PP. (2) there are interaction between PP-g-MAH and DCP that have affected the final properties of PLA/PP blend. The highest yield strength of 27.68 MPa is provided at the ratio of 70/30 blend by using the 0.3 phr of PP-g-MAH and 0.03 phr of DCP. Linear regression model is fitted and follow the assumptions of normal distribution.

  3. 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.

  4. EFICAz2.5: application of a high-precision enzyme function predictor to 396 proteomes.

    PubMed

    Kumar, Narendra; Skolnick, Jeffrey

    2012-10-15

    High-quality enzyme function annotation is essential for understanding the biochemistry, metabolism and disease processes of organisms. Previously, we developed a multi-component high-precision enzyme function predictor, EFICAz(2) (enzyme function inference by a combined approach). Here, we present an updated improved version, EFICAz(2.5), that is trained on a significantly larger data set of enzyme sequences and PROSITE patterns. We also present the results of the application of EFICAz(2.5) to the enzyme reannotation of 396 genomes cataloged in the ENSEMBL database. The EFICAz(2.5) server and database is freely available with a use-friendly interface at http://cssb.biology.gatech.edu/EFICAz2.5.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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).

  11. The role of cyclooxygenase-2 and inflammatory cytokines in pain induction of herniated lumbar intervertebral disc.

    PubMed

    Miyamoto, H; Saura, R; Harada, T; Doita, M; Mizuno, K

    2000-04-01

    Lumbar disc herniation (LDH) is the disease which is the major cause of radiculopathy. In terms of the pathogenesis of disease, it is reported that prostaglandinE2 (PGE2) plays an important role to induce radiculopathy. Arachidonate cascade, which is the process of PGE2 synthesis, is mainly regulated by two kinds of enzymes, phospholipaseA2 (PLA2) and cyclooxy genase (COX). Previously, PLA2 was recognized as the rate-limiting enzyme of this cascade, and some authors reported the clinical significance of PLA2 at the site of LDH concerning the radicular pain. Recently, COX was elucidated to consist of 2 types of isoform, a constitutive form of COX-1 and an inducible form of COX-2. COX-2 has been focused as a key enzyme to regulate PGE2 synthesis and plays an important role in inflammation, because COX-2 was induced in many types of cells by the stimulation of inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha). However, it is not fully discussed whether or not, COX-2 is induced in lumbar disc tissue and if it plays a significant role in the pathogenesis of LDH. To clarify the role of COX-2 in the pathomechanism of radiculopathy of LDH, we have investigated the expression of COX-2, IL-1 beta and TNF alpha in herniated lumbar disc tissue. Immunohistologically, they were detected in the cytosol of chondrocytes constituting the disc tissue. RT-PCR showed that herniated lumbar disc-derived cells expressed mRNA of COX-2, IL-1 beta and TNF alpha in the presence of inflammatory cytokines in vitro. The disc-derived cells also produced much PGE2 by stimulating of inflammatory cytokines at the same time and this PGE2 production was distinctly suppressed by a selective inhibitor of COX-2, 6-methoxy-2-naphtyl acetic acids (6MNA). These results suggest that COX-2 and inflammatory cytokines might play a causative role in the radiculopathy of LDH through upregulating PGE2 synthesis.

  12. Involvement of Lipid Metabolism in the Action of Phospholipase A2 Neurotoxins

    DTIC Science & Technology

    1992-04-15

    activity by 50% toward an egg yolk substrate, but had no effect on hydrolysis of a lecithin substrate (Rosenberg et al., 1989). Lethality was not...etc.) hydrolyzed , not molecular species as evidenced in a recent review (Harris, 1985). The existence of reacylating enzymes in tissues that "restore...34 the hydrolyzed PL by attaching another FFA on to it would mean PLs will appear not to have been hydrolyzed at levels of PLA 2 activity less than the

  13. Electro-spun PLA-PEG-yarns for tissue engineering applications.

    PubMed

    Kruse, Magnus; Greuel, Marc; Kreimendahl, Franziska; Schneiders, Thomas; Bauer, Benedict; Gries, Thomas; Jockenhoevel, Stefan

    2018-06-27

    Electro-spinning is widely used in tissue-engineered applications mostly in form of non-woven structures. The development of e-spun yarn opens the door for textile fabrics which combine the micro to nanoscale dimension of electro-spun filaments with three-dimensional (3D) drapable textile fabrics. Therefore, the aim of the study was the implementation of a process for electro-spun yarns. Polylactic acid (PLA) and polyethylene glycol (PEG) were spun from chloroform solutions with varying PLA/PEG ratios (100:0, 90:10, 75:25 and 50:50). The yarn samples produced were analyzed regarding their morphology, tensile strength, water uptake and cytocompatibility. It was found that the yarn diameter decreased when the funnel collector rotation was increasd, however, the fiber diameter was not influenced. The tensile strength was also found to be dependent on the PEG content. While samples composed of 100% PLA showed a tensile strength of 2.5±0.7 cN/tex, the tensile strength increased with a decreasing PLA content (PLA 75%/PEG 25%) to 6.2±0.5 cN/tex. The variation of the PEG content also influenced the viscosity of the spinning solutions. The investigation of the cytocompatibility with endothelial cells was conducted for PLA/PEG 90:10 and 75:25 and indicated that the samples are cytocompatible.

  14. 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

  15. The Saccharomyces cerevisiae Lipin Homolog is a Mg2+-dependent Phosphatidate Phosphatase Enzyme*

    PubMed Central

    Han, Gil-Soo; Wu, Wen-I; Carman, George M.

    2006-01-01

    Mg2+-dependent phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of PA to yield diacylglycerol and Pi. In this work, we identified the Saccharomyces cerevisiae PAH1 (previously known as SMP2) gene that encodes Mg2+-dependent PA phosphatase using amino acid sequence information derived from a purified preparation of the enzyme (Lin, Y.-P., and Carman, G.M. (1989) J. Biol. Chem. 264, 8641–8645). Overexpression of PAH1 in S. cerevisiae directed elevated levels of Mg2+-dependent PA phosphatase activity, whereas the pah1Δ mutation caused reduced levels of enzyme activity. Heterologous expression of PAH1 in Escherichia coli confirmed that Pah1p is a Mg2+-dependent PA phosphatase enzyme, and showed that its enzymological properties were very similar to those of the enzyme purified from S. cerevisiae. The PAH1-encoded enzyme activity was associated with both the membrane and cytosolic fractions of the cell, and the membrane-bound form of the enzyme was salt-extractable. Lipid analysis showed that mutants lacking PAH1 accumulated PA, and had reduced amounts of diacylglycerol and its derivative triacylglycerol. The PAH1-encoded Mg2+-dependent PA phosphatase shows homology to mammalian lipin, a fat-regulating protein whose molecular function is unknown. Heterologous expression of human LPIN1 in E. coli showed that lipin 1 is also a Mg2+-dependent PA phosphatase enzyme. PMID:16467296

  16. 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.

  17. Inducing PLA/starch compatibility through butyl-etherification of waxy and high amylose starch.

    PubMed

    Wokadala, Obiro Cuthbert; Emmambux, Naushad Mohammad; Ray, Suprakas Sinha

    2014-11-04

    In this study, waxy and high amylose starches were modified through butyl-etherification to facilitate compatibility with polylactide (PLA). Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy and wettability tests showed that hydrophobic butyl-etherified waxy and high amylose starches were obtained with degree of substitution values of 2.0 and 2.1, respectively. Differential scanning calorimetry, tensile testing, and scanning electron microscopy (SEM) demonstrated improved PLA/starch compatibility for both waxy and high amylose starch after butyl-etherification. The PLA/butyl-etherified waxy and high amylose starch composite films had higher tensile strength and elongation at break compared to PLA/non-butyl-etherified composite films. The morphological study using SEM showed that PLA/butyl-etherified waxy starch composites had a more homogenous microstructure compared to PLA/butyl-etherified high amylose starch composites. Thermogravimetric analysis showed that PLA/starch composite thermal stability decreased with starch butyl-etherification for both waxy and high amylose starches. This study mainly demonstrates that PLA/starch compatibility can be improved through starch butyl-etherification. Copyright © 2014 Elsevier Ltd. All rights reserved.

  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. 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

  20. 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.

  1. Mechanical behaviour׳s evolution of a PLA-b-PEG-b-PLA triblock copolymer during hydrolytic degradation.

    PubMed

    Breche, Q; Chagnon, G; Machado, G; Girard, E; Nottelet, B; Garric, X; Favier, D

    2016-07-01

    PLA-b-PEG-b-PLA is a biodegradable triblock copolymer that presents both the mechanical properties of PLA and the hydrophilicity of PEG. In this paper, physical and mechanical properties of PLA-b-PEG-b-PLA are studied during in vitro degradation. The degradation process leads to a mass loss, a decrease of number average molecular weight and an increase of dispersity index. Mechanical experiments are made in a specific experimental set-up designed to create an environment close to in vivo conditions. The viscoelastic behaviour of the material is studied during the degradation. Finally, the mechanical behaviour is modelled with a linear viscoelastic model. A degradation variable is defined and included in the model to describe the hydrolytic degradation. This variable is linked to physical parameters of the macromolecular polymer network. The model allows us to describe weak deformations but become less accurate for larger deformations. The abilities and limits of the model are discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Nanoarmored Enzymes for Organic Enzymology: Synthesis and Characterization of Poly(2-Alkyloxazoline)-Enzyme Conjugates.

    PubMed

    Leurs, Melanie; Tiller, Joerg C

    2017-01-01

    The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH 2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein. The resulting PECs are capable of serving as surfactants for unmodified proteins, rendering the whole mixture organosoluble. Depending on the nature of the POx and the molecular weight and the nature of the enzyme, the PECs are soluble in chloroform or even toluene. Another advantage of this method is that the poly(2-alkyloxazoline) can be activated with the coupling agent and used for the enzyme conjugation without further purification. The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. © 2017 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  5. 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.

  6. Multiple E2 ubiquitin-conjugating enzymes regulate human cytomegalovirus US2-mediated immunoreceptor downregulation.

    PubMed

    van de Weijer, Michael L; Schuren, Anouk B C; van den Boomen, Dick J H; Mulder, Arend; Claas, Frans H J; Lehner, Paul J; Lebbink, Robert Jan; Wiertz, Emmanuel J H J

    2017-09-01

    Misfolded endoplasmic reticulum (ER) proteins are dislocated towards the cytosol and degraded by the ubiquitin-proteasome system in a process called ER-associated protein degradation (ERAD). During infection with human cytomegalovirus (HCMV), the viral US2 protein targets HLA class I molecules (HLA-I) for degradation via ERAD to avoid elimination by the immune system. US2-mediated degradation of HLA-I serves as a paradigm of ERAD and has facilitated the identification of TRC8 (also known as RNF139) as an E3 ubiquitin ligase. No specific E2 enzymes had previously been described for cooperation with TRC8. In this study, we used a lentiviral CRISPR/Cas9 library targeting all known human E2 enzymes to assess their involvement in US2-mediated HLA-I downregulation. We identified multiple E2 enzymes involved in this process, of which UBE2G2 was crucial for the degradation of various immunoreceptors. UBE2J2, on the other hand, counteracted US2-induced ERAD by downregulating TRC8 expression. These findings indicate the complexity of cellular quality control mechanisms, which are elegantly exploited by HCMV to elude the immune system. © 2017. Published by The Company of Biologists Ltd.

  7. Synthetic CO2-fixation enzyme cascades immobilized on self-assembled nanostructures that enhance CO2/O2 selectivity of RubisCO.

    PubMed

    Satagopan, Sriram; Sun, Yuan; Parquette, Jon R; Tabita, F Robert

    2017-01-01

    With increasing concerns over global warming and depletion of fossil-fuel reserves, it is attractive to develop innovative strategies to assimilate CO 2 , a greenhouse gas, into usable organic carbon. Cell-free systems can be designed to operate as catalytic platforms with enzymes that offer exceptional selectivity and efficiency, without the need to support ancillary reactions of metabolic pathways operating in intact cells. Such systems are yet to be exploited for applications involving CO 2 utilization and subsequent conversion to valuable products, including biofuels. The Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) play a pivotal role in global CO 2 fixation. We hereby demonstrate the co-assembly of two RubisCO-associated multienzyme cascades with self-assembled synthetic amphiphilic peptide nanostructures. The immobilized enzyme cascades sequentially convert either ribose-5-phosphate (R-5-P) or glucose, a simpler substrate, to ribulose 1,5-bisphosphate (RuBP), the acceptor for incoming CO 2 in the carboxylation reaction catalyzed by RubisCO. Protection from proteolytic degradation was observed in nanostructures associated with the small dimeric form of RubisCO and ancillary enzymes. Furthermore, nanostructures associated with a larger variant of RubisCO resulted in a significant enhancement of the enzyme's selectivity towards CO 2 , without adversely affecting the catalytic activity. The ability to assemble a cascade of enzymes for CO 2 capture using self-assembling nanostructure scaffolds with functional enhancements show promise for potentially engineering entire pathways (with RubisCO or other CO 2 -fixing enzymes) to redirect carbon from industrial effluents into useful bioproducts.

  8. 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.

  9. 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.

  10. 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

  11. Supercritical CO2 impregnation of PLA/PCL films with natural substances for bacterial growth control in food packaging.

    PubMed

    Milovanovic, Stoja; Hollermann, Gesa; Errenst, Cornelia; Pajnik, Jelena; Frerich, Sulamith; Kroll, Stephen; Rezwan, Kurosch; Ivanovic, Jasna

    2018-05-01

    Biodegradable polymers with antibacterial properties are highly desirable materials for active food packaging applications. Thymol, a dietary monoterpene phenol with a strong antibacterial activity is abundant in plants belonging to the genus Thymus. This study presents two approaches for supercritical CO 2 impregnation of poly(lactic acid)(PLA)/poly(ε-caprolactone)(PCL) blended films to induce antibacterial properties of the material: (i) a batch impregnation process for loading pure thymol, and (ii) an integrated supercritical extraction-impregnation process for isolation of thyme extract and its incorporation into the films, operated in both batch or semi-continuous modes with supercritical solution circulation. The PCL content in films, impregnation time and CO 2 flow regime were varied to maximize loading of the films with thymol or thyme extract with preserving films' structure and thermal stability. Representative film samples impregnated with thymol and thyme extract were tested against Gram (-) (Escherichia coli) and Gram(+) (Bacillus subtilis) model strains, by measuring their metabolic activity and re-cultivation after exposure to the films. The film containing thymol (35.8 wt%) showed a strong antibacterial activity leading to a total reduction of bacterial cell viability. Proposed processes enable fast, controlled and organic solvent-free fabrication of the PLA/PCL films containing natural antibacterial substances at moderately low temperature, with a compact structure and a good thermal stability, for potential use as active food packaging materials. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Drug-conjugated PLA-PEG-PLA copolymers: a novel approach for controlled delivery of hydrophilic drugs by micelle formation.

    PubMed

    Danafar, H; Rostamizadeh, K; Davaran, S; Hamidi, M

    2017-12-01

    A conjugate of the antihypertensive drug, lisinopril, with triblock poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA) copolymer was synthesized by the reaction of PLA-PEG-PLA copolymer with lisinopril in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. The conjugated copolymer was characterized in vitro by hydrogen nuclear magnetic resonance (HNMR), Fourier transform infrared (FTIR), differential scanning calorimetry (DSC) and gel permeation chromatography (GPC) techniques. Then, the lisinopril conjugated PLA-PEG-PLA were self-assembled into micelles in aqueous solution. The resulting micelles were characterized further by various techniques such as dynamic light scattering (DLS) and atomic force microscopy (AFM). The results revealed that the micelles formed by the lisinopril-conjugated PLA-PEG-PLA have spherical structure with the average size of 162 nm. The release behavior of conjugated copolymer, micelles and micelles physically loaded by lisinopril were compared in different media. In vitro release study showed that in contrast to physically loaded micelles, the release rate of micelles consisted of the conjugated copolymer was dependent on pH of media where it was higher at lower pH compared to the neutral medium. Another feature of the conjugated micelles was their more sustained release profile compared to the lisinopril-conjugated copolymer and physically loaded micelles.

  13. 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

  14. 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

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

    PubMed

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

    2015-01-01

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

  16. 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.

  17. Synthesis of protein-coated biocompatible methotrexate-loaded PLA-PEG-PLA nanoparticles for breast cancer treatment

    PubMed Central

    Massadeh, Salam; Alaamery, Manal; Al-Qatanani, Shatha; Alarifi, Saqer; Bawazeer, Shahad; Alyafee, Yusra

    2016-01-01

    Background PLA-PEG-PLA triblock polymer nanoparticles are promising tools for targeted dug delivery. The main aim in designing polymeric nanoparticles for drug delivery is achieving a controlled and targeted release of a specific drug at the therapeutically optimal rate and choosing a suitable preparation method to encapsulate the drug efficiently, which depends mainly on the nature of the drug (hydrophilic or hydrophobic). In this study, methotrexate (MTX)-loaded nanoparticles were prepared by the double emulsion method. Method Biodegradable polymer polyethylene glycol-polylactide acid tri-block was used with poly(vinyl alcohol) as emulsifier. The resulting methotrexate polymer nanoparticles were coated with bovine serum albumin in order to improve their biocompatibility. This study focused on particle size distribution, zeta potential, encapsulation efficiency, loading capacity, and in vitro drug release at various concentrations of PVA (0.5%, 1%, 2%, and 3%). Results Reduced particle size of methotrexate-loaded nanoparticles was obtained using lower PVA concentrations. Enhanced encapsulation efficiency and loading capacity was obtained using 1% PVA. FT-IR characterization was conducted for the void polymer nanoparticles and for drug-loaded nanoparticles with methotrexate, and the protein-coated nanoparticles in solid state showed the structure of the plain PEG-PLA and the drug-loaded nanoparticles with methotrexate. The methotrexate-loaded PLA-PEG-PLA nanoparticles have been studied in vitro; the drug release, drug loading, and yield are reported. Conclusion The drug release profile was monitored over a period of 168 hours, and was free of burst effect before the protein coating. The results obtained from this work are promising; this work can be taken further to develop MTX based therapies.

  18. Peroxiredoxin 6 is the primary antioxidant enzyme for the maintenance of viability and DNA integrity in human spermatozoa.

    PubMed

    Fernandez, Maria C; O'Flaherty, Cristian

    2018-06-15

    Are all components of the peroxiredoxins (PRDXs) system important to control the levels of reactive oxygen species (ROS) to maintain viability and DNA integrity in spermatozoa? PRDX6 is the primary player of the PRDXs system for maintaining viability and DNA integrity in human spermatozoa. Mammalian spermatozoa are sensitive to high levels of ROS and PRDXs are antioxidant enzymes proven to control the levels of ROS generated during sperm capacitation to avoid oxidative damage in the spermatozoon. Low amounts of PRDXs are associated with male infertility. The absence of PRDX6 promotes sperm oxidative damage and infertility in mice. Semen samples were obtained over a period of one year from a cohort of 20 healthy non-smoking volunteers aged 22-30 years old. Sperm from healthy donors was incubated for 2 h in the absence or presence of inhibitors for the 2-Cys PRDXs system (peroxidase, reactivation system and NADPH-enzymes suppliers) or the 1-Cys PRDX system (peroxidase and calcium independent-phospholipase A2 (Ca2+-iPLA2) activity). Sperm viability, DNA oxidation, ROS levels, mitochondrial membrane potential and 4-hydroxynonenal production were determined by flow cytometry. We observed a significant decrease in viable cells due to inhibitors of the 2-Cys PRDXs, PRDX6 Ca2+-iPLA2 activity or the PRDX reactivation system compared to controls (P ≤ 0.05). PRDX6 Ca2+-iPLA2 activity inhibition had the strongest detrimental effect on sperm viability and DNA oxidation compared to controls (P ≤ 0.05). The 2-Cys PRDXs did not compensate for the inhibition of PRDX6 peroxidase and Ca2+-iPLA2 activities. Not applicable. Players of the reactivation systems may differ among mammalian species. The Ca2+-iPLA2 activity of PRDX6 is the most important and first line of defense against oxidative stress in human spermatozoa. Peroxynitrite is scavenged mainly by the PRDX6 peroxidase activity. These findings can help to design new diagnostic tools and therapies for male infertility. This

  19. Complete regression of xenograft tumors using biodegradable mPEG-PLA-SN38 block copolymer micelles.

    PubMed

    Lu, Lu; Zheng, Yan; Weng, Shuqiang; Zhu, Wenwei; Chen, Jinhong; Zhang, Xiaomin; Lee, Robert J; Yu, Bo; Jia, Huliang; Qin, Lunxiu

    2016-06-01

    7-Ethyl-10-hydroxy-comptothecin (SN38) is an active metabolite of irinotecan (CPT-11) and the clinical application of SN38 is limited by its hydrophobicity and instability. To address these issues, a series of novel amphiphilic mPEG-PLA-SN38-conjugates were synthesized by linking SN38 to mPEG-PLA-SA, and they could form micelles by self-assembly. The effects of mPEG-PLA composition were studied in vitro and in vivo. The mean diameters of mPEG2K-PLA-SN38 micelles and mPEG4K-PLA-SN38 micelles were 10-20nm and 120nm, respectively, and mPEG2K-PLA-SN38 micelles showed greater antitumor efficacy than mPEG4K-PLA-SN38 micelles both in vitro and in vivo. These data suggest that the lengths of mPEG and PLA chains had a major impact on the physicochemical characteristics and antitumor activity of SN38-conjugate micelles. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Plasma modified PLA electrospun membranes for actinorhodin production intensification in Streptomyces coelicolor immobilized-cell cultivations.

    PubMed

    Scaffaro, Roberto; Lopresti, Francesco; Sutera, Alberto; Botta, Luigi; Fontana, Rosa Maria; Gallo, Giuseppe

    2017-09-01

    Most of industrially relevant bioproducts are produced by submerged cultivations of actinomycetes. The immobilization of these Gram-positive filamentous bacteria on suitable porous supports may prevent mycelial cell-cell aggregation and pellet formation which usually negatively affect actinomycete submerged cultivations, thus, resulting in an improved biosynthetic capability. In this work, electrospun polylactic acid (PLA) membranes, subjected or not to O 2 -plasma treatment (PLA-plasma), were used as support for immobilized-cell submerged cultivations of Streptomyces coelicolor M145. This strain produces different bioactive compounds, including the blue-pigmented actinorhodin (ACT) and red-pigmented undecylprodigiosin (RED), and constitutes a model for the study of antibiotic-producing actinomycetes. Wet contact angles and X-ray photoelectron spectroscopy analysis confirmed the increased wettability of PLA-plasma due to the formation of polar functional groups such as carboxyl and hydroxyl moieties. Scanning electron microscope observations, carried out at different incubation times, revealed that S. coelicolor immobilized-cells created a dense "biofilm-like" mycelial network on both kinds of PLA membranes. Cultures of S. coelicolor immobilized-cells on PLA or PLA-plasma membranes produced higher biomass (between 1.5 and 2 fold) as well as higher levels of RED and ACT than planktonic cultures. In particular, cultures of immobilized-cells on PLA and PLA-plasma produced comparable levels of RED that were approximatively 4 and 5 fold higher than those produced by planktonic cultures, respectively. In contrast, levels of ACT produced by immobilized-cell cultures on PLA and PLA-plasma were different, being 5 and 10 fold higher than those of planktonic cultures, respectively. Therefore, this is study demonstrated the positive influence of PLA membrane on growth and secondary metabolite production in S. coelicolor and also revealed that O 2 -plasma treated PLA membranes

  1. Preparation and characterization of collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering.

    PubMed

    Haaparanta, Anne-Marie; Järvinen, Elina; Cengiz, Ibrahim Fatih; Ellä, Ville; Kokkonen, Harri T; Kiviranta, Ilkka; Kellomäki, Minna

    2014-04-01

    In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.

  2. Effects of prostaglandin F(2alpha)and carbachol on MAP kinases, cytosolic phospholipase A(2)and arachidonic acid release in cat iris sphincter smooth muscle cells.

    PubMed

    Husain, S; Abdel-Latif, A A

    2001-05-01

    The signal transduction pathways initiated by Ca(2+)-mobilizing agonists, such as prostaglandin F(2alpha)(PGF(2alpha)) and carbachol (CCh), leading to activation of cytosolic phospholipase A(2)(cPLA(2)) and arachidonic acid (AA) release in a wide variety of tissues remain obscure. To further define the role of protein kinases in receptor mediated stimulation of cPLA(2)and consequently AA release we have investigated the role of mitogen-activated protein (MAP) kinases and protein kinase C (PKC) in PGF(2alpha)- and CCh-induced cPLA(2)phosphorylation and AA release in cat iris sphincter smooth muscle (CISM) cells. The cells were prelabeled with [(3)H]AA for 24 hr and incubated in the absence or presence of the agonist for 5-10 min as indicated. MAP kinases activities and cPLA(2)phosphorylation were determined in immunoprecipitates obtained by using anti-p38 MAP kinase and anti-cPLA(2)antibodies. We found that: (a) PGF(2alpha)and CCh increased p38 MAP kinase activity by 197 and 215%, respectively, and increased p42/p44 MAP kinase activity by 200 and 125%, respectively. (b) SB202190, a p38 MAP kinase specific inhibitor, inhibited PGF(2alpha)- and CCh-induced cPLA(2)phosphorylation by 92 and 85%, respectively, and AA release by 62 and 78%, respectively. (c) PD98059, a p42/p44 MAP kinase inhibitor, inhibited CCh-induced cPLA(2)phosphorylation by 70% and AA release by 71%, but had no effect on that of PGF(2alpha). (d) Inhibition of PKC activity by RO 31-8220 inhibited both PGF(2alpha)- and CCh-stimulation of p38 MAP kinase, p42/p44 MAP kinases and cPLA(2)phosphorylation. We conclude from these results that in CISM cells PGF(2alpha)-induced cPLA(2)phosphorylation and AA release is mediated through p38 MAP kinase, but not through p42/p44 MAP kinases, whereas that of CCh is mediated through both p38 MAP kinase and p42/p44 MAP kinases. These effects of PGF(2alpha)and CCh are regulated by the MAP kinases in a PKC-dependent manner. Studies aimed at elucidating the role of

  3. PLA/CS/Nifedipine Nanocomposite Films: Properties and the In Vitro Release of Nifedipine

    NASA Astrophysics Data System (ADS)

    Trang, Nguyen Thi Thu; Chinh, Nguyen Thuy; Giang, Nguyen Vu; Thanh, Dinh Thi Mai; Lam, Tran Dai; Hoang, Thai

    2016-07-01

    The polylactic acid (PLA)/chitosan (CS) films containing a drug, nifedipine (NIF), in the presence of polyethylene oxide (PEO) as a compatibilizer were prepared by the solution method. This method has not been used to form films containing four components (PLA, CS, NIF, PEO) up to now. The CS, PEO, and NIF contents are 25 wt.%, 6-8 wt.%, and 10-50 wt.% in comparison with PLA weight, respectively. Fourier transform infrared spectroscopy (FTIR), thermo-gravimetric analysis (TGA), differential scanning calorimetry (DSC), and field emission scanning electron microscopy (FESEM) were used to characterize the interactions, properties, and morphology of the PLA/CS/PEO/NIF films. The FTIR, TGA, and DSC results show that NIF carried by PLA/CS/PEO films and PLA, CS, NIF had better interaction and were more compatible when using PEO. The surface morphology of PLA/CS/PEO/NIF films was similar to that of PLA/CS/PEO films. Moreover, this was the first time drug loading and NIF release content from PLA/CS/PEO films were determined by the ultraviolet-visible (UV-Vis) spectroscopy method. The drug loading of PLA/CS/PEO/NIF films was from 80.99% to 93.61%. The in vitro NIF release studies were carried out in pH 2, 6.8, and 7.4 solutions corresponding to the pH of stomach, colon, and duodenum regions in the human body, respectively. The NIF release content in different pH solutions is in the order: pH 2 > pH 6.8 > pH 7.4 and increases when there is increasing NIF loading. The PLA/CS/PEO films are potential materials to apply for long-circulating systems for NIF delivery.

  4. Polymer Film-Based Screening and Isolation of Polylactic Acid (PLA)-Degrading Microorganisms.

    PubMed

    Kim, Mi Yeon; Kim, Changman; Moon, Jungheun; Heo, Jinhee; Jung, Sokhee P; Kim, Jung Rae

    2017-02-28

    Polylactic acid (PLA) has been highlighted as an alternative renewable polymer for the replacement of petroleum-based plastic materials, and is considered to be biodegradable. On the other hand, the biodegradation of PLA by terminal degraders, such as microorganisms, requires a lengthy period in the natural environment, and its mechanism is not completely understood. PLA biodegradation studies have been conducted using mainly undefined mixed cultures, but only a few bacterial strains have been isolated and examined. For further characterization of PLA biodegradation, in this study, the PLA-degrading bacteria from digester sludge were isolated and identified using a polymer film-based screening method. The enrichment of sludge on PLA granules was conducted with the serial transference of a subculture into fresh media for 40 days, and the attached biofilm was inoculated on a PLA film on an agar plate. 3D optical microscopy showed that the isolates physically degraded the PLA film due to bacterial degradation. 16S rRNA gene sequencing identified the microbial colonies to be Pseudomonas sp. MYK1 and Bacillus sp. MYK2. The two isolates exhibited significantly higher specific gas production rates from PLA biodegradation compared with that of the initial sludge inoculum.

  5. 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

  6. 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.

  7. Additive Manufacturing of PLA and CF/PLA Binding Layer Specimens via Fused Deposition Modeling

    NASA Astrophysics Data System (ADS)

    Li, Yuhang; Gao, Shiyou; Dong, Rongmei; Ding, Xuebing; Duan, Xiaoxi

    2018-02-01

    As one of the most popular additive manufacturing techniques, fused deposition modeling (FDM) is successfully applied in aerospace, automotive, architecture, and other fields to fabricate thermoplastic parts. Unfortunately, as a result of the limited nature of the mechanical properties and mass in raw materials, there is a pressing need to improve mechanical properties and reduce weight for FDM parts. Therefore, this paper presents an experiment of a special polylactic acid (PLA) and carbon fiber (CF)/PLA-laminated experimental specimen fabricated using the FDM process. The mechanical properties and mass analysis of the new composites for the PLA and CF/PLA binding layer specimen are investigated experimentally. Through the experimental analysis, one can conclude that the mass of laminated specimen is lighter than the CF/PLA specimen, and the tensile and flexural mechanical properties are higher than the pure PLA specimen.

  8. 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.

  9. Docetaxel (DTX)-loaded polydopamine-modified TPGS-PLA nanoparticles as a targeted drug delivery system for the treatment of liver cancer.

    PubMed

    Zhu, Dunwan; Tao, Wei; Zhang, Hongling; Liu, Gan; Wang, Teng; Zhang, Linhua; Zeng, Xiaowei; Mei, Lin

    2016-01-01

    Polydopamine-based surface modification is a simple way to functionalize polymeric nanoparticle (NP) surfaces with ligands and/or additional polymeric layers. In this work, we developed DTX-loaded formulations using polydopamine-modified NPs synthesized using D-α-tocopherol polyethylene glycol 1000 succinate-poly(lactide) (pD-TPGS-PLA/NPs). To target liver cancer cells, galactosamine was conjugated on the prepared NPs (Gal-pD-TPGS-PLA/NPs) to enhance the delivery of DTX via ligand-mediated endocytosis. The size and morphology of pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs changed obviously compared with TPGS-PLA/NPs. In vitro studies showed that TPGS-PLA/NPs, pD-TPGS-PLA/NPs and Gal-pD-TPGS-PLA/NPs had similar release profiles of DTX. Both confocal laser scanning microscopy and flow cytometric results showed that coumarin 6-loaded Gal-pD-TPGS-PLA/NPs had the highest cellular uptake efficiency in liver cancer cell line HepG2. Moreover, DTX-loaded Gal-pD-TPGS-PLA/NPs inhibited the growth of HepG2 cells more potently than TPGS-PLA/NPs, pD-TPGS-PLA/NPs, and a clinically available DTX formulation (Taxotere®). The in vivo biodistribution experiments show that the Gal-pD-TPGS-PLA/NPs are specifically targeted to the tumor. Furthermore, the in vivo anti-tumor effects study showed that injecting DTX-loaded Gal-pD-TPGS-PLA/NPs reduced the tumor size most significantly on hepatoma-bearing nude mice. These results suggest that Gal-pD-TPGS-PLA/NPs prepared in the study specifically interacted with the hepatocellular carcinoma cells through ligand-receptor recognition and they may be used as a potentially eligible drug delivery system targeting liver cancers. Polydopamine-based surface modification is a simple way to functionalize polymeric nanoparticle surfaces with ligands and/or additional polymeric layers. In this work, we developed docetaxel (DTX)-loaded formulations using polydopamine-modified NPs synthesized from D-α-tocopherol polyethylene glycol 1000 succinate

  10. ER trapping reveals Golgi enzymes continually revisit the ER through a recycling pathway that controls Golgi organization

    PubMed Central

    Sengupta, Prabuddha; Satpute-Krishnan, Prasanna; Seo, Arnold Y.; Burnette, Dylan T.; Patterson, George H.; Lippincott-Schwartz, Jennifer

    2015-01-01

    Whether Golgi enzymes remain localized within the Golgi or constitutively cycle through the endoplasmic reticulum (ER) is unclear, yet is important for understanding Golgi dependence on the ER. Here, we demonstrate that the previously reported inefficient ER trapping of Golgi enzymes in a rapamycin-based assay results from an artifact involving an endogenous ER-localized 13-kD FK506 binding protein (FKBP13) competing with the FKBP12-tagged Golgi enzyme for binding to an FKBP-rapamycin binding domain (FRB)-tagged ER trap. When we express an FKBP12-tagged ER trap and FRB-tagged Golgi enzymes, conditions precluding such competition, the Golgi enzymes completely redistribute to the ER upon rapamycin treatment. A photoactivatable FRB-Golgi enzyme, highlighted only in the Golgi, likewise redistributes to the ER. These data establish Golgi enzymes constitutively cycle through the ER. Using our trapping scheme, we identify roles of rab6a and calcium-independent phospholipase A2 (iPLA2) in Golgi enzyme recycling, and show that retrograde transport of Golgi membrane underlies Golgi dispersal during microtubule depolymerization and mitosis. PMID:26598700

  11. Effect of Commercial SiO2 and SiO2 from rice husk ash loading on biodegradation of Poly (lactic acid) and crosslinked Poly (lactic acid)

    NASA Astrophysics Data System (ADS)

    Prapruddivongs, C.; Apichartsitporn, M.; Wongpreedee, T.

    2017-09-01

    In this work, biodegradation behavior of poly (lactic acid) (PLA) and crosslinked PLA filled with two types of SiO2, precipitated SiO2 (commercial SiO2) and SiO2 from rice husk ash, were studied. Rice husks were first treated with 2 molar hydrochloric acid (HCl) to produce high purity SiO2, before burnt in a furnace at 800°C for 6 hours. All components were melted bending by an internal mixer then hot pressed using compression molder to form tested specimens. FTIR spectra of SiO2 and PLA samples were investigated. The results showed the lack of silanol group (Si-OH) of rice husk ash after steric acid surface modification, while the addition of particles can affect the crosslinking of the PLA. For biodegradation test by evaluating total amount of carbon dioxide (CO2) evolved during 60 days incubation at a controlled temperature of 58±2°C, the results showed that the biodegradation of crosslinked PLA occurred slower than the neat PLA. However, SiO2 incorporation enhanced the degree of biodegradation In particular, introducing commercial SiO2 in PLA and crosslinked PLA tended to clearly increase the degree of biodegradation as a consequence of the more accelerated hydrolysis degradation.

  12. 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.

  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. CO2-H2O based pretreatment and enzyme hydrolysis of soybean hulls.

    PubMed

    Islam, S M Mahfuzul; Li, Qian; Loman, Abdullah Al; Ju, Lu-Kwang

    2017-11-01

    The high carbohydrate content of soybean hull makes it an attractive biorefinery resource. But hydrolyzing its complex structure requires concerted enzyme activities, at least cellulase, xylanase, pectinase and α-galactosidase. Effective pretreatment that generates minimal inhibitory products is important to facilitate enzymatic hydrolysis. Combined CO 2 -H 2 O pretreatment and enzymatic hydrolysis by Aspergillus niger and Trichoderma reesei enzyme broths was studied here. The pretreatment was evaluated at 80°C-180°C temperature and 750psi-1800psi pressure, with fixed moisture content (66.7%) and pretreatment time (30min). Ground hulls without and with different pretreatments were hydrolyzed by enzyme at 50°C and pH 4.8 and compared for glucose, xylose, galactose, arabinose, mannose and total reducing sugar release. CO 2 -H 2 O pretreatment at 1250psi and 130°C was found to be optimal. Compared to the unpretreated hulls hydrolyzed with 2.5-fold more enzyme, this pretreatment improved glucose, xylose, galactose, arabinose and mannose releases by 55%, 35%, 105%, 683% and 52%, respectively. Conversions of 97% for glucose, 98% for xylose, 41% for galactose, 59% for arabinose, 87% for mannose and 89% for total reducing sugar were achieved with Spezyme CP at 18FPU/g hull. Monomerization of all carbohydrate types was demonstrated. At the optimum pretreatment condition, generation of inhibitors acetic acid, furfural and hydroxymethylfurfural (HMF) was negligible, 1.5mg/g hull in total. The results confirmed the effective CO 2 -H 2 O pretreatment of soybean hulls at much lower pressure and temperature than those reported for biomass of higher lignin contents. The lower pressure requirement reduces the reactor cost and makes this new pretreatment method more practical and economical. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. All Work and No PLA Makes Jack a Dull Boy

    ERIC Educational Resources Information Center

    Popova-Gonci, Viktoria

    2009-01-01

    In this article, the author presents an assortment of approaches--building blocks--commonly used in Prior Learning Assessment (PLA) practices (this list is certainly not comprehensive): (1) course matching (comparing course requirements to a student's prior learning experiences); (2) student's ability to articulate learning gained from…

  17. 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.

  18. 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.

  19. Polymer blends of polylactic acid (PLA) and polybutylene succinate-adipate

    NASA Astrophysics Data System (ADS)

    Ma, Wenguang

    results show that BionolleRTM #3000 has a very strong ability to form the continuous phase in the blends and in films made from the blends. A partial continuous net structure with very thin wall thickness (0.1˜0.2 mum) can form in blends with 20 part of BionolleRTM #3000. The reason why PLA/BionolleRTM #6000 blends do not have good mechanical properties is that the size of the phase domain is too big (five times that of PLA/BionolleRTM #3000 blends).

  20. In-vitro evaluation of Polylactic acid (PLA) manufactured by fused deposition modeling.

    PubMed

    Wurm, Matthias C; Möst, Tobias; Bergauer, Bastian; Rietzel, Dominik; Neukam, Friedrich Wilhelm; Cifuentes, Sandra C; Wilmowsky, Cornelius von

    2017-01-01

    With additive manufacturing (AM) individual and biocompatible implants can be generated by using suitable materials. The aim of this study was to investigate the biological effects of polylactic acid (PLA) manufactured by Fused Deposition Modeling (FDM) on osteoblasts in vitro according to European Norm / International Organization for Standardization 10,993-5. Human osteoblasts (hFOB 1.19) were seeded onto PLA samples produced by FDM and investigated for cell viability by fluorescence staining after 24 h. Cell proliferation was measured after 1, 3, 7 and 10 days by cell-counting and cell morphology was evaluated by scanning electron microscopy. For control, we used titanium samples and polystyrene (PS). Cell viability showed higher viability on PLA (95,3% ± 2.1%) than in control (91,7% ±2,7%). Cell proliferation was highest in the control group (polystyrene) and higher on PLA samples compared to the titanium samples. Scanning electron microscopy revealed homogenous covering of sample surface with regularly spread cells on PLA as well as on titanium. The manufacturing of PLA discs from polylactic acid using FDM was successful. The in vitro investigation with human fetal osteoblasts showed no cytotoxic effects. Furthermore, FDM does not seem to alter biocompatibility of PLA. Nonetheless osteoblasts showed reduced growth on PLA compared to the polystyrene control within the cell experiments. This could be attributed to surface roughness and possible release of residual monomers. Those influences could be investigated in further studies and thus lead to improvement in the additive manufacturing process. In addition, further research focused on the effect of PLA on bone growth should follow. In summary, PLA processed in Fused Deposition Modelling seems to be an attractive material and method for reconstructive surgery because of their biocompatibility and the possibility to produce individually shaped scaffolds.

  1. Computational Insights into an Enzyme-Catalyzed [4+2] Cycloaddition

    PubMed Central

    2017-01-01

    The enzyme SpnF, involved in the biosynthesis of spinosyn A, catalyzes a formal [4+2] cycloaddition of a 22-membered macrolactone, which may proceed as a concerted [4+2] Diels–Alder reaction or a stepwise [6+4] cycloaddition followed by a Cope rearrangement. Quantum mechanics/molecular mechanics (QM/MM) calculations combined with free energy simulations show that the Diels–Alder pathway is favored in the enzyme environment. OM2/CHARMM free energy simulations for the SpnF-catalyzed reaction predict a free energy barrier of 22 kcal/mol for the concerted Diels–Alder process and provide no evidence of a competitive stepwise pathway. Compared with the gas phase, the enzyme lowers the Diels–Alder barrier significantly, consistent with experimental observations. Inspection of the optimized geometries indicates that the enzyme may prearrange the substrate within the active site to accelerate the [4+2] cycloaddition and impede the [6+4] cycloaddition through interactions with active-site residues. Judging from partial charge analysis, we find that the hydrogen bond between the Thr196 residue of SpnF and the substrate C15 carbonyl group contributes to the enhancement of the rate of the Diels–Alder reaction. QM/MM simulations show that the substrate can easily adopt a reactive conformation in the active site of SpnF because interconversion between the C5–C6 s-trans and s-cis conformers is facile. Our QM/MM study suggests that the enzyme SpnF does behave as a Diels-Alderase. PMID:29131960

  2. Improved thermal stability of polylactic acid (PLA) composite film via PLA-β-cyclodextrin-inclusion complex systems.

    PubMed

    Byun, Youngjae; Rodriguez, Katia; Han, Jung H; Kim, Young Teck

    2015-11-01

    The effects of the incorporation of PLA-β-cyclodextrin-inclusion complex (IC) and β-cyclodextrin (β-CD) on biopolyester PLA films were investigated. Thermal stability, surface morphology, barrier, and mechanical properties of the films were measured at varying IC (1, 3, 5, and 7%) and β-CD (1 and 5%) concentrations. The PLA-IC-composite films (IC-PLA-CFs) showed uniform morphological structure, while samples containing β-CD (β-CD-PLA-CFs) showed high agglomeration of β-CD due to poor interfacial interaction between β-CD and PLA moieties. According to the thermal property analysis, the 5% IC-PLA-CFs showed 6.6 times lower dimensional changes (6.5%) at the temperature range of 20-80°C than that of pure PLA film (43.0%). The increase of IC or β-CD content in the PLA-composite films shifted the glass transition and crystallization temperature to higher temperature regions. The crystallinity of both composite films improved by increasing IC or β-CD content. Both composite films had higher oxygen and water vapor permeability as IC or β-CD content increased in comparison to pure PLA film. All the composite films had less flexibility and lower tensile strength than the pure PLA film. In conclusion, this study shows that the IC technique is valuable to improve the thermal expansion stability of PLA-based films. Published by Elsevier B.V.

  3. 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.

  4. Characterization of prostaglandin E2 generation through the cyclooxygenase (COX)-2 pathway in human neutrophils

    PubMed Central

    St-Onge, Mireille; Flamand, Nicolas; Biarc, Jordane; Picard, Serge; Bouchard, Line; Dussault, Andrée-Anne; Laflamme, Cynthia; James, Michael J.; Caughey, Gillian E.; Cleland, Leslie G.; Borgeat, Pierre; Pouliot, Marc

    2010-01-01

    In the present study, we characterized the generation of prostaglandin (PG)E2 in human neutrophils. We found that the Ca2+-dependent type IV cytosolic phospholipase A2 (cPLA2) was pivotally involved in the COX-2-mediated generation of PGE2 in response to a calcium ionophore, as determined by the use of selected PLA2 inhibitors. PGE2 biosynthesis elicited by bacterial-derived peptides or by phagocytic stimuli acting on cell surface receptors also showed to be dependent on cPLA2 activity. We then assessed metabolism of unesterified arachidonic acid (AA), and observed that PGE2 production becomes favored over that of LTB4 with higher AA concentrations. Withdrawal of calcium prevented the generation of PGE2 in response to a calcium ionophore but did not affect the up-regulation of COX-2 or its capacity to convert AA, thus limiting its implication at the level of cPLA2 activation. Of the main eicosanoids produced by neutrophils, only LTB4 was able to up-regulate COX-2 expression. Finally, the only PGE synthase isoform found in neutrophils is microsomal PGE synthase-1; it co-localized with COX-2 and its expression appeared mainly constitutive. These results highlight key differences in regulatory processes of the 5-LO and COX pathways, and enhance our knowledge at several levels in the PGE2 biosynthesis in neutrophils. PMID:17643350

  5. Angiotensin-converting enzyme 2 activation improves endothelial function.

    PubMed

    Fraga-Silva, Rodrigo A; Costa-Fraga, Fabiana P; Murça, Tatiane M; Moraes, Patrícia L; Martins Lima, Augusto; Lautner, Roberto Q; Castro, Carlos H; Soares, Célia Maria A; Borges, Clayton L; Nadu, Ana Paula; Oliveira, Marilene L; Shenoy, Vinayak; Katovich, Michael J; Santos, Robson A S; Raizada, Mohan K; Ferreira, Anderson J

    2013-06-01

    Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

  6. One small step for Mot1; one giant leap for other Swi2/Snf2 enzymes?

    PubMed Central

    Viswanathan, Ramya; Auble, David T.

    2011-01-01

    The TATA-binding protein (TBP) is a major target for transcriptional regulation. Mot1, a Swi2/Snf2-related ATPase, dissociates TBP from DNA in an ATP dependent process. The experimental advantages of this relatively simple reaction have been exploited to learn more about how Swi2/Snf2 ATPases function biochemically. However, many unanswered questions remain and fundamental aspects of the Mot1 mechanism are still under debate. Here, we review the available data and integrate the results with structural and biochemical studies of related enzymes to derive a model for Mot1’s catalytic action consistent with the broad literature on enzymes in this family. We propose that the Mot1 ATPase domain is tethered to TBP by a flexible, spring-like linker of alpha helical hairpins. The linker juxtaposes the ATPase domain such that it can engage duplex DNA on one side of the TBP-DNA complex. This allows the ATPase to employ short-range, nonprocessive ATP-driven DNA tracking to pull or push TBP off its DNA site. DNA translocation is a conserved property of ATPases in the broader enzyme family. As such, the model explains how a structurally and functionally conserved ATPase domain has been put to use in a very different context than other enzymes in the Swi2/Snf2 family. PMID:21658482

  7. 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.

  8. Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis.

    PubMed

    Tsuji, Daisuke; Akeboshi, Hiromi; Matsuoka, Kazuhiko; Yasuoka, Hiroko; Miyasaki, Eri; Kasahara, Yoshiko; Kawashima, Ikuo; Chiba, Yasunori; Jigami, Yoshifumi; Taki, Takao; Sakuraba, Hitoshi; Itoh, Kohji

    2011-04-01

    Novel recombinant human lysosomal β-hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay-Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency. A recombinant human HexA (Om4HexA) with a high mannose 6-phosphate (M6P)-type-N-glycan content, which was produced by a methylotrophic yeast strain, Ogataea minuta, overexpressing the OmMNN4 gene, was intracerebroventricularly (ICV) administered to Sandhoff disease model mice (Hexb⁻/⁻ mice) at different doses (0.5-2.5 mg/kg), and then the replacement and therapeutic effects were examined. The Om4HexA was widely distributed across the ependymal cell layer, dose-dependently restored the enzyme activity due to uptake via cell surface cation-independent M6P receptor (CI-M6PR) on neural cells, and reduced substrates, including GM2 ganglioside (GM2), asialo GM2 (GA2), and oligosaccharides with terminal N-acetylglucosamine residues (GlcNAc-oligosaccharides), accumulated in brain parenchyma. A significant inhibition of chemokine macrophage inflammatory protein-1 α (MIP-1α) induction was also revealed, especially in the hindbrain (< 63%). The decrease in central neural storage correlated with an improvement of motor dysfunction as well as prolongation of the lifespan. This lysosome-directed recombinant human enzyme drug derived from methylotrophic yeast has the high therapeutic potential to improve the motor dysfunction and quality of life of the lysosomal storage diseases (LSDs) patients with neurological manifestations. We emphasize the importance of neural cell surface M6P receptor as a delivery target of neural cell-directed enzyme replacement therapy (NCDERT) for neurodegenerative metabolic diseases. Copyright © 2010 American Neurological Association.

  9. Synthesis and study of sericin-g-PLA

    NASA Astrophysics Data System (ADS)

    Saetae, S.; Magaraphan, R.

    2015-05-01

    In this paper we present an experiment for bulk synthesis of the sericin-g-PLA by using Sn(Oct)2 as catalyst and study the effect of Thai silk cocoon species (Dok Bua, Luang Pirote, Nang Noi and Nang Lai) on properties of the sericin-g-PLA. We investigated the chemical structure of the grafted copolymers by using FTIR and GPC. Moreover, the grafting percentage was determined by soxhlet extaction. The IR spectra of extracted sample showed peaks at 1188 and 1215 cm-1 that assigned to the symmetric C-O-C stretching modes of the ester group. The methyl rocking stretching and C-CH3 vibration of polylactide appeared at 1130 and 1045 cm-1, respectively. The peak positioned 3440 cm-1 belonged to the hydroxyl group and the amino group of sericin which became less after polymerized with lactide. These evidences suggested that the lactide was reacted with sericin. Also, the molecular weight of the grafted copolymers were in range from 5.2 to 6.1 kg/mole. And Nang Lai-g-PLA showed the highest grafting percentage of the grafted copolymers.

  10. 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.

  11. Structurally characterized 1,1,3,3-tetramethylguanidine solvated magnesium aryloxide complexes: [Mg(mu-OEt)(DBP)(H-TMG)]2, [Mg(mu-OBc)(DBP)(H-TMG)]2, [Mg(mu-TMBA)(DBP)(H-TMG)]2, [Mg(mu-DPP)(DBP)(H-TMG)]2, [Mg(BMP)2(H-TMG)2], [Mg(O-2,6-Ph2C6H3)2 (H-TMG)2].

    PubMed

    Monegan, Jessie D; Bunge, Scott D

    2009-04-06

    The synthesis and structural characterization of several 1,1,3,3-tetramethylguanidine (H-TMG) solvated magnesium aryloxide complexes are reported. Bu(2)Mg was successfully reacted with H-TMG, HOC(6)H(3)(CMe(3))(2)-2,6 (H-DBP), and either ethanol, a carboxylic acid, or diphenyl phosphate in a 1:1 ratio to yield the corresponding [Mg(mu-L)(DBP)(H-TMG)](2) where L = OCH(2)CH(3) (OEt, 1), O(2)CC(CH(3))(3) (OBc, 2), O(2)C(C(6)H(2)-2,4,6-(CH(3))(3)) (TMBA, 3), or O(2)P(OC(6)H(5))(2) (DPP, 4). Bu(2)Mg was also reacted with two equivalents of H-TMG and HOC(6)H(3)(CMe(3))-2-(CH(3))-6 (BMP) or HO-2,6-Ph(2)C(6)H(3) to yield [Mg(BMP)(2)(H-TMG)(2)] (5) and [Mg(O-2,6-Ph(2)C(6)H(3))(2)(H-TMG)(2)] (6). Compounds 1-6 were characterized by single-crystal X-ray diffraction. Polymerization of l- and rac-lactide with 1 was found to generate polylactide (PLA). A discussion concerning the relevance of compounds 2 - 4 to the structure of Mg-activated phosphatase enzymes is also provided. The bulk powders for all complexes were found to be in agreement with the crystal structures based on elemental analyses, FT-IR spectroscopy, and (1)H, (13)C and (31)P NMR studies.

  12. Morphology control of PLA microfibers and spheres via melt electrospinning

    NASA Astrophysics Data System (ADS)

    Yu, Shu-Xin; Zheng, Jie; Yan, Xu; Wang, Xiao-Xiong; Nie, Guang-Di; Tan, Ye-Qiang; Zhang, Jun; Sui, Kun-Yan; Long, Yun-Ze

    2018-04-01

    In conventional solution electrospinning, the morphologies (e.g., spheres, beaded fibers, and fibers) of electrospun products can be controlled by solution concentration. Here, we report that the morphologies and structures of polylactic acid (PLA) via melt electrospinning also can be adjusted from microfibers to microspheres by simply increasing the spinning temperature. It was found that with temperature increasing from 200 °C to 240 °C, the average diameter of melt-electrospun PLA fibers decreased from 58.46 to 2.96 μm. Then, beaded fibers and microspheres about 14.5 μm in diameter were collected when the spinning temperature was increased to 250 °C and 260 °C. In addition, we also found that the average PLA fiber diameter decreased with increasing the applied spinning voltage, and increased with the increase of spinning distance. To explain the formation mechanism of different PLA microstructures, rheological property and infrared spectra of PLA under different spinning temperatures were also tested.

  13. Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis

    PubMed Central

    Zimbler, Daniel L.; Eddy, Justin L.; Schroeder, Jay A.

    2015-01-01

    Pneumonic plague represents the most severe form of disease caused by Yersinia pestis due to its ease of transmission, rapid progression, and high mortality rate. The Y. pestis outer membrane Pla protease is essential for the development of pneumonic plague; however, the complete repertoire of substrates cleaved by Pla in the lungs is not known. In this study, we describe a proteomic screen to identify host proteins contained within the bronchoalveolar lavage fluid of mice that are cleaved and/or processed by Y. pestis in a Pla-dependent manner. We identified peroxiredoxin 6 (Prdx6), a host factor that contributes to pulmonary surfactant metabolism and lung defense against oxidative stress, as a previously unknown substrate of Pla. Pla cleaves Prdx6 at three distinct sites, and these cleavages disrupt both the peroxidase and phospholipase A2 activities of Prdx6. In addition, we found that infection with wild-type Y. pestis reduces the abundance of extracellular Prdx6 in the lungs compared to that after infection with Δpla Y. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or Δpla Y. pestis, Prdx6-deficient mice exhibit no differences in bacterial burden, host immune response, or lung damage from wild-type mice. Thus, while Pla is able to disrupt Prdx6 function in vitro and reduce Prdx6 levels in vivo, the cleavage of Prdx6 has little detectable impact on the progression or outcome of pneumonic plague. PMID:26553463

  14. Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis.

    PubMed

    Zimbler, Daniel L; Eddy, Justin L; Schroeder, Jay A; Lathem, Wyndham W

    2016-01-01

    Pneumonic plague represents the most severe form of disease caused by Yersinia pestis due to its ease of transmission, rapid progression, and high mortality rate. The Y. pestis outer membrane Pla protease is essential for the development of pneumonic plague; however, the complete repertoire of substrates cleaved by Pla in the lungs is not known. In this study, we describe a proteomic screen to identify host proteins contained within the bronchoalveolar lavage fluid of mice that are cleaved and/or processed by Y. pestis in a Pla-dependent manner. We identified peroxiredoxin 6 (Prdx6), a host factor that contributes to pulmonary surfactant metabolism and lung defense against oxidative stress, as a previously unknown substrate of Pla. Pla cleaves Prdx6 at three distinct sites, and these cleavages disrupt both the peroxidase and phospholipase A2 activities of Prdx6. In addition, we found that infection with wild-type Y. pestis reduces the abundance of extracellular Prdx6 in the lungs compared to that after infection with Δpla Y. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or Δpla Y. pestis, Prdx6-deficient mice exhibit no differences in bacterial burden, host immune response, or lung damage from wild-type mice. Thus, while Pla is able to disrupt Prdx6 function in vitro and reduce Prdx6 levels in vivo, the cleavage of Prdx6 has little detectable impact on the progression or outcome of pneumonic plague. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. 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.

  16. The Role of Human Cytochrome P450 Enzymes in the Formation of 2-Hydroxymetronidazole: CYP2A6 is the High Affinity (Low Km) Catalyst

    PubMed Central

    Cohen-Wolkowiez, Michael; Sampson, Mario R.; Kearns, Gregory L.

    2013-01-01

    Despite metronidazole’s widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a “therapeutic concentration” of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo. PMID:23813797

  17. Plant Extract Synthesized PLA Nanoparticles for Controlled and Sustained Release of Quercetin: A Green Approach

    PubMed Central

    Yadav, Sudesh Kumar

    2012-01-01

    Background Green synthesis of metallic nanoparticles (NPs) has been extensively carried out by using plant extracts (PEs) which have property of stabilizers/ emulsifiers. To our knowledge, there is no comprehensive study on applying a green approach using PEs for fabrication of biodegradable PLA NPs. Conventional methods rely on molecules like polyvinyl alcohol, polyethylene glycol, D-alpha-tocopheryl poly(ethylene glycol 1000) succinate as stabilizers/emulsifiers for the synthesis of such biodegradable NPs which are known to be toxic. So, there is urgent need to look for stabilizers which are biogenic and non-toxic. The present study investigated use of PEs as stabilizers/emulsifiers for the fabrication of stable PLA NPs. Synthesized PLA NPs through this green process were explored for controlled release of the well known antioxidant molecule quercetin. Methodology/Principal Findings Stable PLA NPs were synthesized using leaf extracts of medicinally important plants like Syzygium cumini (1), Bauhinia variegata (2), Cedrus deodara (3), Lonicera japonica (4) and Eleaocarpus sphaericus (5). Small and uniformly distributed NPs in the size range 70±30 nm to 143±36 nm were formed with these PEs. To explore such NPs for drugs/ small molecules delivery, we have successfully encapsulated quercetin a lipophilic molecule on a most uniformly distributed PLA-4 NPs synthesized using Lonicera japonica leaf extract. Quercetin loaded PLA-4 NPs were observed for slow and sustained release of quercetin molecule. Conclusions This green approach based on PEs mediated synthesis of stable PLA NPs pave the way for encapsulating drug/small molecules, nutraceuticals and other bioactive ingredients for safer cellular uptake, biodistribution and targeted delivery. Hence, such PEs synthesized PLA NPs would be useful to enhance the therapeutic efficacy of encapsulated small molecules/drugs. Furthermore, different types of plants can be explored for the synthesis of PLA as well as other

  18. 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

  19. 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.

  20. [Effect of glyceryl triacetate on properties of PLA/PBAT blends].

    PubMed

    Yang, Nan; Wang, Xiyuan; Weng, Yunxuan; Jin, Yujuan; Zhang, Min

    2016-06-25

    Poly lactic acid (PLA)/Poly (butyleneadipate-co-terephthalate)(PBAT) and glyceryl triacetate (GTA) blend were prepared by torque rheometer, and the effect of GTA on thermodynamical performance, mechanical properties and microstructure of PLA/PBAT composites were studied using differential scanning calorimeter(DSC), dynamic mechanical analysis(DMA), universal testing machine, impact testing machine and scanning electron microscope(SEM). After adding GTA, Tg values of the two phases gradually became closer, blends cold crystallization temperature and melting temperature decreased. When with 3 phr GTA, the dispersed phase particle size of PLA/PBAT blend decreased. Mechanics performance test showed that the elongation at break and impact strength of the PLA/PBAT blend was greatly increased with 3 phr GTA, and the elongation at break increased 2.6 times, improved from 17.7% to 64.1%.

  1. Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour.

    PubMed

    Mathy, Nathalie; Hébert, Agnès; Mervelet, Peggy; Bénard, Lionel; Dorléans, Audrey; Li de la Sierra-Gallay, Inés; Noirot, Philippe; Putzer, Harald; Condon, Ciarán

    2010-01-01

    Ribonucleases J1 and J2 are recently discovered enzymes with dual 5'-to-3' exoribonucleolytic/endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild-type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5'-to-3' exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.

  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. PLA composites: From production to properties.

    PubMed

    Murariu, Marius; Dubois, Philippe

    2016-12-15

    Poly(lactic acid) or polylactide (PLA), a biodegradable polyester produced from renewable resources, is used for various applications (biomedical, packaging, textile fibers and technical items). Due to its inherent properties, PLA has a key-position in the market of biopolymers, being one of the most promising candidates for further developments. Unfortunately, PLA suffers from some shortcomings, whereas for the different applications specific end-use properties are required. Therefore, the addition of reinforcing fibers, micro- and/or nanofillers, and selected additives within PLA matrix is considered as a powerful method for obtaining specific end-use characteristics and major improvements of properties. This review highlights recent developments, current results and trends in the field of composites based on PLA. It presents the main advances in PLA properties and reports selected results in relation to the preparation and characterization of the most representative PLA composites. To illustrate the possibility to design the properties of composites, a section is devoted to the production and characterization of innovative PLA-based products filled with thermally-treated calcium sulfate, a by-product from the lactic acid production process. Moreover, are emphasized the last tendencies strongly evidenced in the case of PLA, i.e., the high interest to diversify its uses by moving from biomedical and packaging (biodegradation properties, "disposables") to technical applications ("durables"). Copyright © 2016 Elsevier B.V. All rights reserved.

  4. A study on properties of PLA/PBAT from blown film process

    NASA Astrophysics Data System (ADS)

    Hongdilokkul, P.; Keeratipinit, K.; Chawthai, S.; Hararak, B.; Seadan, M.; Suttiruengwong, S.

    2015-07-01

    The aim of this work was to study the properties of films based on PLA/PBAT blend prepared by the reactive compounding. PLA/PBAT blends were prepared at the weight ratio of 80:20 together with peroxide as a reactive agent in a twin screw extruder with temperature profile of 160/170/180/210/220/190/175/150°C from feed to die zone. All blended samples, neat PLA, and neat PBAT were characterized for morphology, mechanical and rheological properties. SEM micrographs showed finely dispersed phases of PBAT in PLA in all cases. The particle sizes of PBAT were around 1 μm. The results indicated that the drawability and toughness properties of PLA were greatly improved when blended with 20%wt PBAT. The interface adhesion, and mechanical properties of PLA/PBAT blends were also improved when adding a very small quantity of peroxide. PLA/PBAT blends were then used to produce films. The film characteristics and mechanical properties were examined. Tensile strength of films was significantly improved in the machine direction in PLA/PBAT/peroxide blends whereas the good optical transparent property were remained the same compared with neat PLA.

  5. EFICAz2: enzyme function inference by a combined approach enhanced by machine learning.

    PubMed

    Arakaki, Adrian K; Huang, Ying; Skolnick, Jeffrey

    2009-04-13

    We previously developed EFICAz, an enzyme function inference approach that combines predictions from non-completely overlapping component methods. Two of the four components in the original EFICAz are based on the detection of functionally discriminating residues (FDRs). FDRs distinguish between member of an enzyme family that are homofunctional (classified under the EC number of interest) or heterofunctional (annotated with another EC number or lacking enzymatic activity). Each of the two FDR-based components is associated to one of two specific kinds of enzyme families. EFICAz exhibits high precision performance, except when the maximal test to training sequence identity (MTTSI) is lower than 30%. To improve EFICAz's performance in this regime, we: i) increased the number of predictive components and ii) took advantage of consensual information from the different components to make the final EC number assignment. We have developed two new EFICAz components, analogs to the two FDR-based components, where the discrimination between homo and heterofunctional members is based on the evaluation, via Support Vector Machine models, of all the aligned positions between the query sequence and the multiple sequence alignments associated to the enzyme families. Benchmark results indicate that: i) the new SVM-based components outperform their FDR-based counterparts, and ii) both SVM-based and FDR-based components generate unique predictions. We developed classification tree models to optimally combine the results from the six EFICAz components into a final EC number prediction. The new implementation of our approach, EFICAz2, exhibits a highly improved prediction precision at MTTSI < 30% compared to the original EFICAz, with only a slight decrease in prediction recall. A comparative analysis of enzyme function annotation of the human proteome by EFICAz2 and KEGG shows that: i) when both sources make EC number assignments for the same protein sequence, the assignments tend to

  6. 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

  7. 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.

  8. 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

  9. 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

  10. E2 enzyme inhibition by stabilization of a low affinity interface with ubiquitin

    PubMed Central

    St-Cyr, Daniel J.; Ziemba, Amy; Garg, Pankaj; Plamondon, Serge; Auer, Manfred; Sidhu, Sachdev; Marinier, Anne; Kleiger, Gary; Tyers, Mike; Sicheri, Frank

    2014-01-01

    Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small molecule inhibitor of the E2 ubiquitin conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester, without overtly affecting the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities. PMID:24316736

  11. Using every trick in the book: the Pla surface protease of Yersinia pestis.

    PubMed

    Suomalainen, Marjo; Haiko, Johanna; Ramu, Päivi; Lobo, Leandro; Kukkonen, Maini; Westerlund-Wikström, Benita; Virkola, Ritva; Lähteenmäki, Kaarina; Korhonen, Timo K

    2007-01-01

    The Pla surface protease of Yersinia pestis, encoded by the Y. pestis-specific plasmid pPCP1, is a versatile virulence factor. In vivo studies have shown that Pla is essential in the establishment of bubonic plague, and in vitro studies have demonstrated various putative virulence functions for the Pla molecule. Pla is a surface protease of the omptin family, and its proteolytic targets include the abundant, circulating human zymogen plasminogen, which is activated by Pla to the serine protease plasmin. Plasmin is important in cell migration, and Pla also proteolytically inactivates the main circulating inhibitor of plasmin, alpha2-antiplasmin. Pla also is an adhesin with affinity for laminin, a major glycoprotein of mammalian basement membranes, which is degraded by plasmin but not by Pla. Together, these functions create uncontrolled plasmin proteolysis targeted at tissue barriers. Other proteolytic targets for Pla include complement proteins. Pla also mediates bacterial invasion into human endothelial cell lines; the adhesive and invasive charateristics of Pla can be genetically dissected from its proteolytic activity. Pla is a 10-stranded antiparallel beta-barrel with five surface-exposed short loops, where the catalytic residues are oriented inwards at the top of the beta-barrel. The sequence of Pla contains a three-dimensional motif for protein binding to lipid A of the lipopolysaccharide. Indeed, the proteolytic activity of Pla requires rough lipopolysaccharide but is sterically inhibited by the O antigen in smooth LPS, which may be the selective advantage of the loss of O antigen in Y. pestis. Members of the omptin family are highly similar in structure but differ in functions and virulence association. The catalytic residues of omptins are conserved, but the variable substrate specificities in proteolysis by Pla and other omptins are dictated by the amino acid sequences near or at the surface loops, and hence reflect differences in substrate binding. The

  12. 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.

  13. Electrospun PBLG/PLA nanofiber membrane for constructing in vitro 3D model of melanoma.

    PubMed

    Wang, Yaping; Qian, Junmin; Liu, Ting; Xu, Weijun; Zhao, Na; Suo, Aili

    2017-07-01

    Though much progress in utilizing tissue engineering technology to investigate tumor development in vitro has been made, the effective management of human melanoma is still a challenge in clinic due to lack of suitable 3D culture systems. In this study, we prepared a poly(γ-benzyl-l-glutamate)/poly(lactic acid) (PBLG/PLA) nanofiber membrane by electrospinning and demonstrated its suitability as a matrix for 3D culture of melanoma cells in vitro. The electrospun PBLG/PLA nanofiber membrane displayed a smooth and uniform fibrous morphology and had a desirable water contact angle of 79.3±0.6°. The average diameter of PBLG/PLA nanofibers was 320.3±95.1nm that was less than that (516.2±163.3nm) of pure PLA nanofibers. The addition of PBLG into PLA decreased the cold crystallization peak of PLA fibers from 93 to 75°C. The in vitro biocompatibility of PBLG/PLA nanofiber membrane was evaluated with B16F10 cells using PLA nanofiber membrane as control. It was found that, compared to PLA nanofiber membrane, PBLG/PLA nanofiber membrane could better support cell viability and proliferation, as indicated by MTT assay and live-dead staining. SEM results revealed that PBLG/PLA rather than PLA nanofiber membrane promoted the generation of tumoroid-like structures. These findings clearly demonstrated that the electrospun PBLG/PLA nanofiber membrane could mimick the extracellular matrix of melanoma microenvironment and be a promising platform for 3D cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Blends of low molecular weight of poly lactic acid (PLA) with gondorukem (gum rosin)

    NASA Astrophysics Data System (ADS)

    Kaavessina, Mujtahid; Distantina, Sperisa; Chafidz, Achmad; Utama, Aditya; Anggraeni, Venisa Mega Puteri

    2018-02-01

    The utilization of plastic was increasing as well as the increasing its demand in wide range application. Consequently, the number of plastic litter will increase and make more serious environmental problems. This research concerns to minimize waste problems by designing biodegradable plastic. In this research, biodegradable plastic was made of poly lactic acid (PLA) and gondorukem (Gum rosin, Resina colophonium) as the plasticizer. The effect of gondorukem towards PLA properties such as rheology and degradability was investigated. The research divided into two steps: (i) the polycondensation of lactic acid (LA) and (ii) modification of obtained poly lactic acid. In the first step, polycondensation was done in N2 atmosphere (138°C) for 30 hours and added 0.1 %w of SnCl2 as catalyst. Bulk modification was conducted by blending of gondurukem in varied weight (0.5, 1, and 2 g in 10 g of PLA). Furthermore, the modified PLA was analyzed its molecular structure, biodegradability and rheological property. The presence of gondorukem enhanced the biodegradability of poly lactic acid. Gondorukem could act as the plasticizer. It is confirmed that the complex viscosity of PLA melt decreased upon the addition of gondorukem

  15. 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

  16. Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody.

    PubMed

    Ni, Wei-Wei; Huang, Wen; Wu, De-Qin; Zhou, Yan-Jun; Ji, Chun-Mei; Cao, Meng-Da; Guo, Miao; Sun, Jin-Lu; Wei, Ji-Fu

    2017-09-01

    Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET‑28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1. Hybridoma cells were screened by indirect ELISA and limiting dilution. Positive cells were used to induce the formation of antibody‑containing ascites fluid, and the antibodies were purified using protein A‑agarose. The results of the present study demonstrated that recombinant Pla a 1 was successfully expressed and purified, and exhibited positive immunoglobulin E‑binding to serum from patients allergic to P. acerifolia. A total of 11 hybridomas that steadily secreted anti‑Pla a 1 antibody were obtained and an immunoblotting analysis indicated that all of these monoclonal antibodies specifically recognized the Pla a 1 protein. These results suggested that specific anti‑Pla a 1 antibodies may be obtained, which can be used for the rapid detection of Pla a 1 allergens and in the preparation of vaccines against P. acerifolia pollen.

  17. 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

  18. Antibacterial poly(lactic acid) (PLA) films grafting electrospun PLA/Ally isothioscyanate (AITC) fibers for food packaging

    USDA-ARS?s Scientific Manuscript database

    Poly(lactic acid) (PLA) fibers of submicron sizes encapsulating allyl isothiocyanate (AITC) (PfA) were made and electrospun onto the surfaces of PLA films (PfA-g-film). SEM examination confirmed that the fibers were grafted to the PLA film after the (PfA-g-film) underwent air blowing and water washi...

  19. Combination analysis in genetic polymorphisms of drug-metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population.

    PubMed

    Ota, Tomoko; Kamada, Yuka; Hayashida, Mariko; Iwao-Koizumi, Kyoko; Murata, Shigenori; Kinoshita, Kenji

    2015-01-01

    The Cytochrome P450 is the major enzyme involved in drug metabolism. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is one important factor that contributes to drug therapy failure. We have developed a new straightforward TaqMan PCR genotyping assay to investigate the prevalence of the most common allelic variants of polymorphic CYP enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population. Moreover, we focused on the combination of each genotype for clinical treatment. The genotype analysis identified a total of 139 out of 483 genotype combinations of five genes in the 1,003 Japanese subjects. According to our results, most of subjects seemed to require dose modification during clinical treatment. In the near future, modifications should be considered based on the individual patient genotype of each treatment.

  20. 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

  1. Polylactic acid (PLA)/Silver-NP/VitaminE bionanocomposite electrospun nanofibers with antibacterial and antioxidant activity

    NASA Astrophysics Data System (ADS)

    Munteanu, Bogdanel Silvestru; Aytac, Zeynep; Pricope, Gina M.; Uyar, Tamer; Vasile, Cornelia

    2014-10-01

    The antibacterial property of silver nanoparticles (Ag-NPs) and the antioxidant activity of Vitamin E have been combined by incorporation of these two active components within polylactic acid (PLA) nanofibers via electrospinning (PLA/Ag-NP/VitaminE nanofibers). The morphological and structural characterizations of PLA/Ag-NP/VitaminE nanofibers were performed by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy and X-ray diffraction. The average fiber diameter was 140 ± 60 nm, and the size of the Ag-NP was 2.7 ± 1.5 nm. PLA/Ag-NP/VitaminE nanofibers inhibited growth of Escherichia coli, Listeria monocytogenes and Salmonella typhymurium up to 100 %. The amount of released Ag ions from the nanofibers immersed in aqueous solution was determined by Inductively Coupled Plasma Mass Spectrometry, and it has been observed that the release of Ag ions was kept approximately constant after 10 days of immersion. The antioxidant activity of PLA/Ag-NP/VitaminE nanofibers was evaluated according to DPPH (2,2-diphenyl-1-picrylhydrazyl) method and determined as 94 %. The results of the tests on fresh apple and apple juice indicated that the PLA/Ag/VitaminE nanofiber membrane actively reduced the polyphenol oxidase activity. The multifunctional electrospun PLA nanofibers incorporating Ag-NP and Vitamin E may be quite applicable in food packaging due to the extremely large surface area of nanofibers along with antibacterial and antioxidant activities. These materials could find application in food industry as a potential preservative packaging for fruits and juices.

  2. Hydrophobic-modified nano-cellulose fiber/PLA biodegradable composites for lowering water vapor transmission rate (WVTR) of paper.

    PubMed

    Song, Zhaoping; Xiao, Huining; Zhao, Yi

    2014-10-13

    New biodegradable nanocomposites have been successfully prepared by incorporating modified nano-cellulose fibers (NCF) in a biodegradable polylactic acid (PLA) matrix in this work. The hydrophobic-modified NCF was obtained by grafting hydrophobic monomers on NCF to improve the compatibility between NCF and PLA during blending. The resulting NCF/PLA composites were then applied on paper surface via a cast-coating process in an attempt to reduce the water vapor transmission rate (WVTR) of paper. The WVTR tests, conducted under various testing conditions and with different coating weights, demonstrated that the modified NCF/PLA composites coating played a critical role in lowering WVTR of paper. The lowest WVTR value was 34 g/m(2)/d, which was obtained with an addition of 1% of modified NCF to PLA and the composites coating weight at 40 g/m(2) and substantially lower than the control value at 1315 g/m(2)/d. The paper coated with the modified biodegradable composite is promising as green-based packaging materials. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. 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

  4. Production, characterisation, and in vitro nebulisation performance of budesonide-loaded PLA nanoparticles.

    PubMed

    Amini, Mohammad Ali; Faramarzi, Mohammad Ali; Gilani, Kambiz; Moazeni, Esmaeil; Esmaeilzadeh-Gharehdaghi, Elina; Amani, Amir

    2014-01-01

    The aim of this study is to prepare a nanosuspension of budesonide for respiratory delivery using nebuliser by optimising its particle size and characterising its in vitro deposition behaviour. PLA (poly lactic acid)-budesonide nanosuspension (BNS) was prepared using high-pressure emulsification/solvent evaporation method. To optimise particle size, different parameters such as PLA concentration, sonication time, and amplitude were investigated. Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and scanning electron microscope (SEM) analyses were performed to characterise the prepared PLA-budesonide nanoparticles. The in vitro aerodynamic characteristics of the PLA-BNS using a jet nebuliser were estimated and compared with that of commercially available suspension formulation of budesonide. Budesonide-loaded PLA nanoparticles with fine particle size (an average size of 224-360 nm), narrow size distribution, and spherical and smooth surface were prepared. The optimum condition for preparation of fine particle size for aerosolisation was found to be at PLA concentration of 1.2 mg/ml and amplitude of 70 for 75 s sonication time. The in vitro aerosolisation performance of PLA-BNS compared to that of commercial budesonide indicated that it has significantly (p < 0.05) smaller mass median aerodynamic diameter (MMAD) value with an enhancement in fine particle fraction (FPF) value. Improving the in vitro deposition of budesonide, PLA-BNS could be considered as a promising alternative suspension formulation for deep lung delivery of the drug using nebuliser.

  5. 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.

  6. 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

  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. Computational Study of a Model System of Enzyme-Mediated [4+2] Cycloaddition Reaction

    PubMed Central

    2015-01-01

    A possible mechanistic pathway related to an enzyme-catalyzed [4+2] cycloaddition reac-tion was studied by theoretical calculations at density functional (B3LYP, O3LYP, M062X) and semiempirical levels (PM6-DH2, PM6) performed on a model system. The calculations were carried out for the key [4+2] cycloaddition step considering enzyme-catalyzed biosynthesis of Spinosyn A in a model reaction, where a reliable example of a biological Diels-Alder reaction was reported experimentally. In the present study it was demonstrated that the [4+2] cycloaddition reaction may benefit from moving along the energetically balanced reaction coordinate, which enabled the catalytic rate enhancement of the [4+2] cycloaddition pathway involving a single transition state. Modeling of such a system with coordination of three amino acids indicated a reliable decrease of activation energy by ~18.0 kcal/mol as compared to a non-catalytic transformation. PMID:25853669

  9. 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.

  10. Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2.

    PubMed Central

    Hernández-Ruiz, J; Arnao, M B; Hiner, A N; García-Cánovas, F; Acosta, M

    2001-01-01

    H2O2 is the usual oxidizing substrate of horseradish peroxidase C (HRP-C). In the absence in the reaction medium of a one-electron donor substrate, H2O2 is able to act as both oxidizing and reducing substrate. However, under these conditions the enzyme also undergoes a progressive loss of activity. There are several pathways that maintain the activity of the enzyme by recovering the ferric form, one of which is the decomposition of H2O2 to molecular oxygen in a similar way to the action of catalase. This production of oxygen has been kinetically characterized with a Clark-type electrode coupled to an oxygraph. HRP-C exhibits a weak catalase-like activity, the initial reaction rate of which is hyperbolically dependent on the H2O2 concentration, with values for K(2) (affinity of the first intermediate, compound I, for H2O2) and k(3) (apparent rate constant controlling catalase activity) of 4.0 +/- 0.6 mM and 1.78 +/- 0.12 s(-1) respectively. Oxygen production by HRP-C is favoured at pH values greater than approx. 6.5; under similar conditions HRP-C is also much less sensitive to inactivation during incubations with H2O2. We therefore suggest that this pathway is a major protective mechanism of HRP-C against such inactivation. PMID:11171085

  11. 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.

  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. Effect of orientation on the morphology and mechanical properties of PLA/starch composite filaments

    USDA-ARS?s Scientific Manuscript database

    PLA/starch fibers were produced by twin screw extrusion of PLA with granular or gelatinized starch/glycerol followed by drawing through a set of winders with an intermediate oven. At 30% starch, fibers drawn 2-5x were highly flexible (elongation 20-100%) while undrawn filaments were brittle (elonga...

  14. 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.

  15. Constitutive expression of CaPLA1 conferred enhanced growth and grain yield in transgenic rice plants.

    PubMed

    Park, Ki Youl; Kim, Eun Yu; Seo, Young Sam; Kim, Woo Taek

    2016-03-01

    Phospholipids are not only important components of cell membranes, but participate in diverse processes in higher plants. In this study, we generated Capsicum annuum phospholipiase A1 (CaPLA1) overexpressing transgenic rice (Oryza sativa L.) plants under the control of the maize ubiquitin promoter. The T4 CaPLA1-overexpressing rice plants (Ubi:CaPLA1) had a higher root:shoot mass ratio than the wild-type plants in the vegetative stage. Leaf epidermal cells from transgenic plants had more cells than wild-type plants. Genes that code for cyclin and lipid metabolic enzymes were up-regulated in the transgenic lines. When grown under typical paddy field conditions, the transgenic plants produced more tillers, longer panicles and more branches per panicle than the wild-type plants, all of which resulted in greater grain yield. Microarray analysis suggests that gene expressions that are related with cell proliferation, lipid metabolism, and redox state were widely altered in CaPLA1-overexpressing transgenic rice plants. Ubi:CaPLA1 plants had a reduced membrane peroxidation state, as determined by malondialdehyde and conjugated diene levels and higher peroxidase activity than wild-type rice plants. Furthermore, three isoprenoid synthetic genes encoding terpenoid synthase, hydroxysteroid dehydrogenase and 3-hydroxy-3-methyl-glutaryl-CoA reductase were up-regulated in CaPLA1-overexpressing plants. We suggest that constitutive expression of CaPLA1 conferred increased grain yield with enhanced growth in transgenic rice plants by alteration of gene activities related with cell proliferation, lipid metabolism, membrane peroxidation state and isoprenoid biosynthesis.

  16. 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

  17. Visualizing Active Enzyme Complexes Using a Photoreactive Inhibitor for Proximity Ligation – Application on γ-Secretase

    PubMed Central

    Schedin-Weiss, Sophia; Inoue, Mitsuhiro; Teranishi, Yasuhiro; Yamamoto, Natsuko Goto; Karlström, Helena; Winblad, Bengt; Tjernberg, Lars O.

    2013-01-01

    Here, we present a highly sensitive method to study protein-protein interactions and subcellular location selectively for active multicomponent enzymes. We apply the method on γ-secretase, the enzyme complex that catalyzes the cleavage of the amyloid precursor protein (APP) to generate amyloid β-peptide (Aβ), the major causative agent in Alzheimer disease (AD). The novel assay is based on proximity ligation, which can be used to study protein interactions in situ with very high sensitivity. In traditional proximity ligation assay (PLA), primary antibody recognition is typically accompanied by oligonucleotide-conjugated secondary antibodies as detection probes. Here, we first performed PLA experiments using antibodies against the γ-secretase components presenilin 1 (PS1), containing the catalytic site residues, and nicastrin, suggested to be involved in substrate recognition. To selectively study the interactions of active γ-secretase, we replaced one of the primary antibodies with a photoreactive γ-secretase inhibitor containing a PEG linker and a biotin group (GTB), and used oligonucleotide-conjugated streptavidin as a probe. Interestingly, significantly fewer interactions were detected with the latter, novel, assay, which is a reasonable finding considering that a substantial portion of PS1 is inactive. In addition, the PLA signals were located more peripherally when GTB was used instead of a PS1 antibody, suggesting that γ-secretase matures distal from the perinuclear ER region. This novel technique thus enables highly sensitive protein interaction studies, determines the subcellular location of the interactions, and differentiates between active and inactive γ-secretase in intact cells. We suggest that similar PLA assays using enzyme inhibitors could be useful also for other enzyme interaction studies. PMID:23717518

  18. Two enzyme immunoassays to screen for 2,4-dichlorophenoxyacetic acid in water.

    PubMed

    Fleeker, J

    1987-01-01

    Two solid-phase enzyme immunoassays were developed to measure 2,4-dichlorophenoxyacetic acid (2,4-D), using 2 sets of structurally distinct immunogens and enzyme ligands. The 2,4-D analog, 2-methyl-4-chlorophenoxyacetic acid (MCPA), gave a similar response with both methods, whereas other phenoxy herbicides cross-reacted differently. In method A, the aromatic moiety of 2,4-D was distal from the carrier protein and labeled enzyme, whereas in method B, the acetic acid portion of the herbicide was distal. The use of both methods to screen for this herbicide in ground water and municipal and river water reduced the number of false-positive responses. Water sources having a low background response could be monitored with either method alone. When a concentration step, with disposable C18 extraction columns, was used, the limit of sensitivity was 5 micrograms/L. Method A was the more sensitive of the 2 methods with a limit of detection of 10 micrograms/L without the concentration step.

  19. 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.

  20. 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.

  1. Portable Enzyme-Paper Biosensors Based on Redox-Active CeO2 Nanoparticles.

    PubMed

    Karimi, A; Othman, A; Andreescu, S

    2016-01-01

    Portable, nanoparticle (NP)-enhanced enzyme sensors have emerged as powerful devices for qualitative and quantitative analysis of a variety of analytes for biomedicine, environmental applications, and pharmaceutical fields. This chapter describes a method for the fabrication of a portable, paper-based, inexpensive, robust enzyme biosensor for the detection of substrates of oxidase enzymes. The method utilizes redox-active NPs of cerium oxide (CeO2) as a sensing platform which produces color in response to H2O2 generated by the action of oxidase enzymes on their corresponding substrates. This avoids the use of peroxidases which are routinely used in conjunction with glucose oxidase. The CeO2 particles serve dual roles, as high surface area supports to anchor high loadings of the enzyme as well as a color generation reagent, and the particles are recycled multiple times for the reuse of the biosensor. These sensors are small, light, disposable, inexpensive, and they can be mass produced by standard, low-cost printing methods. All reagents needed for the analysis are embedded within the paper matrix, and sensors stored over extended periods of time without performance loss. This novel sensor is a general platform for the in-field detection of analytes that are substrates for oxidase enzymes in clinical, food, and environmental samples. © 2016 Elsevier Inc. All rights reserved.

  2. 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.

  3. Coalescence in PLA-PBAT blends under shear flow: Effects of blend preparation and PLA molecular weight

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

    Nofar, M.; Heuzey, M. C.; Carreau, P. J., E-mail: pierre.carreau@polymtl.ca

    Blends containing 75 wt. % of an amorphous polylactide (PLA) with two different molecular weights and 25 wt. % of a poly[(butylene adipate)-co-terephthalate] (PBAT) were prepared using either a Brabender batch mixer or a twin-screw extruder. These compounds were selected because blending PLA with PBAT can overcome various drawbacks of PLA such as its brittleness and processability limitations. In this study, we investigated the effects of varying the molecular weight of the PLA matrix and of two different mixing processes on the blend morphology and, further, on droplet coalescence during shearing. The rheological properties of these blends were investigated and the interfacialmore » properties were analyzed using the Palierne emulsion model. Droplet coalescence was investigated by applying shear flows of 0.05 and 0.20 s{sup −1} at a fixed strain of 60. Subsequently, small amplitude oscillatory shear tests were conducted to investigate changes in the viscoelastic properties. The morphology of the blends was also examined using scanning electron microscope (SEM) micrographs. It was observed that the PBAT droplets were much smaller when twin-screw extrusion was used for the blend preparation. Shearing at 0.05 s{sup −1} induced significant droplet coalescence in all blends, but coalescence and changes in the viscoelastic properties were much more pronounced for the PLA-PBAT blend based on a lower molecular weight PLA. The viscoelastic responses were also somehow affected by the thermal degradation of the PLA matrix during the experiments.« less

  4. Enzyme-based CO2 capture for advanced life support

    NASA Technical Reports Server (NTRS)

    Ge, Jijun; Cowan, Robert M.; Tu, Chingkuang; McGregor, Martin L.; Trachtenberg, Michael C.

    2002-01-01

    Elevated CO2 levels in air can lead to impaired functioning and even death to humans. Control of CO2 is critical in confined spaces that have little physical or biological buffering capacity (e.g., spacecraft, submarines, or aircraft). A novel enzyme-based contained liquid membrane bioreactor was designed for CO2 capture and certain application cases are reported in this article. The results show that the liquid layer accounts for the major transport resistance. With addition of carbonic anhydrase, the transport resistance decreased by 71%. Volatile organic compounds of the type and concentration expected to be present in either the crew cabin or a plant growth chamber did not influence carbonic anhydrase activity or reactor operation during 1-day operation. Alternative sweep method studies, examined as a means of eliminating consumables, showed that the feed gas could be used successfully in a bypass mode when combined with medium vacuum pressure (-85 kPa) to achieve CO2 separation comparable to that with an inert sweep gas. The reactor exhibited a selectivity for CO2 versus N2 of 1400:1 and CO2 versus O2 is 866:1. The CO2 permeance was 1.44 x 10(-7) mol m-2 Pa-1 s-1 (4.3 x 10(-4) cm3 cm-2 s-1 cmHg-1) at a feed concentration of 0.1% CO2. These data show that the enzyme-based contained liquid membrane is a promising candidate technology that may be suitable for NASA applications to control CO2 in the crew or plant chambers.

  5. 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.

  6. 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.

  7. 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

  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. 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

  10. 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.

  11. 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

  12. Cloning and molecular ontogeny of digestive enzymes in fed and food-deprived developing gilthead seabream (Sparus aurata) larvae.

    PubMed

    Mata-Sotres, José Antonio; Martos-Sitcha, Juan Antonio; Astola, Antonio; Yúfera, Manuel; Martínez-Rodríguez, Gonzalo

    2016-01-01

    We have determined the expression pattern of key pancreatic enzymes precursors (trypsinogen, try; chymotrypsinogen, ctrb; phospholipase A2, pla2; bile salt-activated lipase, cel; and α-amylase, amy2a) during the larval stage of gilthead seabream (Sparus aurata) up to 60days after hatching (dph). Previously, complete sequences of try, cel, and amy2a were cloned and phylogenetically analyzed. One new isoform was found for cel transcript (cel1b). Expression of all enzyme precursors was detected before the mouth opening. Expression of try and ctrb increased during the first days of development and then maintained high values with some fluctuations during the whole larval stage. The prolipases pla2 and cel1b increased from first-feeding with irregular fluctuation until the end of the experiment. Contrarily, cel1a maintained low expression values during most of the larval stage increasing at the end of the period. Nevertheless, cel1a expression was negligible as compared with cel1b. The expression of amy2a sharply increased during the first week followed by a gradual decrease. In addition, a food-deprivation experiment was performed to find the differences in relation to presence/absence of gut content after the opening of the mouth. The food-deprived larvae died at 10dph. The expression levels of all digestive enzymes increased up to 7dph, declining sharply afterwards. This expression pattern up to 7dph was the same observed in fed larvae, confirming the genetic programming during the early development. Main digestive enzymes in gilthead seabream larvae exhibited the same expression profiles than other marine fish with carnivorous preferences in their juvenile stages. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Preparation of poly(lactic acid)/sintered hydroxyapatite composite biomaterial by supercritical CO2.

    PubMed

    Zhang, Yumin; Wang, Jianru; Ma, Yanmiao; Han, Bo; Niu, Xiaojun; Liu, Jianchun; Gao, Lan; Wang, Jue; Zhai, Xiaoyan; Chu, Kaibo; Yang, Liwang

    2018-01-01

    Based on a kind of sintered hydroxyapatite (HA) with a good cytocompatibility, a series of polylactic acid (PLA) and PLA/HA with the various PLA:HA weight ratio (5:5, 4:6, 3:7, 2:8, 1:9) were fabricated by supercritical CO2. The physical and chemical properties were evaluated by pH, degradation, water absorption, porosity, density, mechanical property, and cytotoxicity respectively. With the increase of HA content, the pH value and porosity increased gradually, while weight loss rate and the density showed a gradual downward trend. Existence of HA can drastically improve the hydroscopicity of PLA scaffolds. The compression strength values slightly increased (p>0.05) from 39.96 MPa of PLA to 45.00 MPa of PLA/HA with the ratio of 7:3, subsequently, the values decreased (p<0.05) from 43.29 MPa (8:2) to 19.00 MPa (9:1). While the modulus of elasticity decreased (p<0.05) from 5.89 to 1.84 GPa with increasing HA content. The PLA/HA (8:2) promoted cell proliferation more significantly than any of other groups (p<0.05). Based on the results, the overall properties of porous scaffolds are the optimal when the weight ratio of PLA/HA is 8:2. Its pH, porosity, density, compression strength, and elasticity modulus are 7.39, 83.0%, 0.60g/cm-3, 34.1 MPa and 2.63 GPa, respectively. SEM observation presented a homogeneous distribution of HA in PLA matrix and a foam-like structure comprising interconnected pores.

  14. Prevalence of abnormal plasma liver enzymes in older people with Type 2 diabetes.

    PubMed

    Morling, J R; Strachan, M W J; Hayes, P C; Butcher, I; Frier, B M; Reynolds, R M; Price, J F

    2012-04-01

    To determine the prevalence and distribution of abnormal plasma liver enzymes in a representative sample of older adults with Type 2 diabetes. Plasma concentrations of alanine aminotransferase, aspartate aminotransferase and γ-glutamyltransferase were measured in a randomly selected, population-based cohort of 1066 men and women aged 60-75 years with Type 2 diabetes (the Edinburgh Type 2 Diabetes Study). Overall, 29.1% (95% CI 26.1-31.8) of patients had one or more plasma liver enzymes above the upper limit of the normal reference range. Only 10.1% of these patients had a prior history of liver disease and a further 12.4% reported alcohol intake above recommended limits. Alanine aminotransferase was the most commonly raised liver enzyme (23.1% of patients). The prevalence of abnormal liver enzymes was significantly higher in men (odds ratio 1.40, 95% CI 1.07-1.83), in the youngest 5-year age band (odds ratio 2.02, 95% CI 1.44-2.84), in patients with diabetes duration < 5 years (odds ratio 1.38, 95% CI 1.01-1.90), plasma HbA(1c) ≥ 58 mmol/mol (7.5%) (odds ratio 1.43, 95% CI 1.09-1.88), obese BMI (odds ratio 2.84, 95% CI 1.59-3.06) and secondary care management for their diabetes (odds ratio 1.40, 95% CI 1.05-1.87). However, all these factors combined accounted for only 7.6% of the variation in liver enzyme abnormality. The prevalence of elevated liver enzymes in people with Type 2 diabetes is high, with only modest variation between clinically defined patient groups. Further research is required to determine the prognostic value of raised, routinely measured liver enzymes to inform decisions on appropriate follow-up investigations. © 2011 The Authors. Diabetic Medicine © 2011 Diabetes UK.

  15. DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

    PubMed Central

    Bernard, Mark A.; Wang, Leyu; Tachado, Souvenir D.

    2015-01-01

    Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K m=74 nM) with substrate inhibition kinetics (K i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong

  16. Studies on non-steroidal inhibitors of aromatase enzyme; 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives.

    PubMed

    Sahin, Zafer; Ertas, Merve; Berk, Barkın; Biltekin, Sevde Nur; Yurttas, Leyla; Demirayak, Seref

    2018-05-01

    Steroidal and non-steroidal aromatase inhibitors target the suppression of estrogen biosynthesis in the treatment of breast cancer. Researchers have increasingly focused on developing non-steroidal derivatives for their potential clinical use avoiding steroidal side-effects. Non-steroidal derivatives generally have planar aromatic structures attached to the azole ring system. One part of this ring system comprises functional groups that inhibit aromatization through the coordination of the haem group of the aromatase enzyme. Replacement of the triazole ring system and development of aromatic/cyclic structures of the side chain can increase selectivity over aromatase enzyme inhibition. In this study, 4-(aryl/heteroaryl)-2-(pyrimidin-2-yl)thiazole derivatives were synthesized and physical analyses and structural determination studies were performed. The IC 50 values were determined by a fluorescence-based aromatase inhibition assay and compound 1 (4-(2-hydroxyphenyl)-2-(pyrimidine-2-yl)thiazole) were found potent inhibitor of enzyme (IC 50 :0.42 nM). Then, their antiproliferative activity over MCF-7 and HEK-293 cell lines was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds 1, 7, 8, 13, 15, 18, 21 were active against MCF-7 breast cancer cells. Lastly, a series of docking experiments were undertaken to analyze the crystal structure of human placental aromatase and identify the possible interactions between the most active structure and the active site. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. 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.

  18. Improved design and characterization of PLGA/PLA-coated Chitosan based micro-implants for controlled release of hydrophilic drugs.

    PubMed

    Manna, Soumyarwit; Donnell, Anna M; Kaval, Necati; Al-Rjoub, Marwan F; Augsburger, James J; Banerjee, Rupak K

    2018-05-29

    Repetitive intravitreal injections of Methotrexate (MTX), a hydrophilic chemotherapeutic drug, are currently used to treat selected vitreoretinal (VR) diseases, such as intraocular lymphoma. To avoid complications associated with the rapid release of MTX from the injections, a Polylactic acid (PLA) and Chitosan (CS)-based MTX micro-implant prototype was fabricated in an earlier study, which showed a sustained therapeutic release rate of 0.2-2.0 µg/day of MTX for a period ∼1 month in vitro and in vivo. In the current study, different combinations of Poly(lactic-co-glycolic) acid (PLGA)/PLA coatings were used for lipophilic surface modification of the CS-MTX micro-implant, such as PLGA 5050, PLGA 6535 and PLGA 7525 (PLA: PGA - 50:50, 65:35, 75:25, respectively; M.W: 54,400 - 103,000) and different PLA, such as PLA 100 and PLA 250 (MW: 102,000 and 257,000, respectively). This improved the duration of total MTX release from the coated CS-MTX micro-implants to ∼3-5 months. With an increase in PLA content in PLGA and molecular weight of PLA, a) the initial burst of MTX and the mean release rate of MTX can be reduced; and b) the swelling and biodegradation of the micro-implants can be delayed. The controlled drug release mechanism is caused by a combination of diffusion process and hydrolysis of the polymer coating, which can be modulated by a) PLA content in PLGA and b) molecular weight of PLA, as inferred from Korsmeyer Peppas model, Zero order, First order and Higuchi model fits. This improved micro-implant formulation has the potential to serve as a platform for controlled release of hydrophilic drugs to treat selected VR diseases. Copyright © 2018. Published by Elsevier B.V.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. Interplay between the chalcone cardamonin and selenium in the biosynthesis of Nrf2-regulated antioxidant enzymes in intestinal Caco-2 cells.

    PubMed

    De Spirt, Silke; Eckers, Anna; Wehrend, Carina; Micoogullari, Mustafa; Sies, Helmut; Stahl, Wilhelm; Steinbrenner, Holger

    2016-02-01

    Selenoenzymes and nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated phase II enzymes comprise key components of the cellular redox and antioxidant systems, which show multiple interrelations. Deficiency of the micronutrient selenium (Se) and impaired biosynthesis of selenoproteins have been reported to result in induction of Nrf2 target genes. Conversely, transcription of the selenoenzymes glutathione peroxidase 2 (GPx2) and thioredoxin reductase 1 (TrxR1) is up-regulated upon Nrf2 activation. Here, we have studied the interplay between Se and the secondary plant metabolite cardamonin, an Nrf2-activating chalcone, in the regulation of Nrf2-controlled antioxidant enzymes. Se-deficient and Se-repleted (sodium selenite-supplemented) human intestinal Caco-2 cells were exposed to cardamonin. Uptake of cardamonin by the Caco-2 cells was independent of their Se status. Cardamonin strongly induced gene expression of GPx2 and TrxR1. However, cardamonin treatment did not result in elevated GPx or TrxR activity and protein levels, possibly relating to a concomitant down-regulation of O-phosphoseryl-tRNA(Sec) kinase (PSTK), an enzyme involved in translation of selenoprotein mRNAs. On the other hand, induction of the Nrf2-regulated enzyme heme oxygenase 1 (HO-1) by cardamonin was diminished in Se-replete compared to Se-deficient cells. Our findings suggest that cardamonin interferes with the biosynthesis of Nrf2-regulated selenoenzymes, in contrast to the Nrf2-activating isothiocyanate compound sulforaphane, which has been shown earlier to synergize with Se-mediated cytoprotection. Conversely, the cellular Se status apparently affects the cardamonin-mediated induction of non-selenoprotein antioxidant enzymes such as HO-1. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

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

    Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen

    Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or asmore » enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. The E1/E2-independent ubiquitination catalyzed by these enzymes requires NAD but not ATP and Mg2+. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. These results establish that ubiquitination can be catalyzed by a single enzyme.« less

  5. 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.

  6. Binding of Diverse Environmental Chemicals with Human Cytochromes P450 2A13, 2A6, and 1B1 and Enzyme Inhibition

    PubMed Central

    Shimada, Tsutomu; Kim, Donghak; Murayama, Norie; Tanaka, Katsuhiro; Takenaka, Shigeo; Nagy, Leslie D.; Folkman, Lindsay M.; Foroozesh, Maryam K.; Komori, Masayuki; Yamazaki, Hiroshi; Guengerich, F. Peter

    2014-01-01

    A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low- to high-spin state shift) with P450 2A13 than those seen with P450 2A6, i.e. the spectral binding intensities (ΔAmax/Ks ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2’,5’-tetrachlorobiphenyl, 7-hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2’-methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2’-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low Ks values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals interacted with both P450s 2A13 and 1B1, indicating that the two enzymes have some similarty of structural features regarding these chemicals. Molecular docking analyses suggest similarities at the active sites of these P450 enzymes. These results indicate that P450 2A13, as well as Family

  7. 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

  8. Kinetics and mechanism of the biodegradation of PLA/clay nanocomposites during thermophilic phase of composting process.

    PubMed

    Stloukal, Petr; Pekařová, Silvie; Kalendova, Alena; Mattausch, Hannelore; Laske, Stephan; Holzer, Clemens; Chitu, Livia; Bodner, Sabine; Maier, Guenther; Slouf, Miroslav; Koutny, Marek

    2015-08-01

    The degradation mechanism and kinetics of polylactic acid (PLA) nanocomposite films, containing various commercially available native or organo-modified montmorillonites (MMT) prepared by melt blending, were studied under composting conditions in thermophilic phase of process and during abiotic hydrolysis and compared to the pure polymer. Described first order kinetic models were applied on the data from individual experiments by using non-linear regression procedures to calculate parameters characterizing aerobic composting and abiotic hydrolysis, such as carbon mineralization, hydrolysis rate constants and the length of lag phase. The study showed that the addition of nanoclay enhanced the biodegradation of PLA nanocomposites under composting conditions, when compared with pure PLA, particularly by shortening the lag phase at the beginning of the process. Whereas the lag phase of pure PLA was observed within 27days, the onset of CO2 evolution for PLA with native MMT was detected after just 20days, and from 13 to 16days for PLA with organo-modified MMT. Similarly, the hydrolysis rate constants determined tended to be higher for PLA with organo-modified MMT, particularly for the sample PLA-10A with fastest degradation, in comparison with pure PLA. The acceleration of chain scission in PLA with nanoclays was confirmed by determining the resultant rate constants for the hydrolytical chain scission. The critical molecular weight for the hydrolysis of PLA was observed to be higher than the critical molecular weight for onset of PLA mineralization, suggesting that PLA chains must be further shortened so as to be assimilated by microorganisms. In conclusion, MMT fillers do not represent an obstacle to acceptance of the investigated materials in composting facilities. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. H2A-DUBbing the mammalian epigenome: expanding frontiers for histone H2A deubiquitinating enzymes in cell biology and physiology.

    PubMed

    Belle, Jad I; Nijnik, Anastasia

    2014-05-01

    Posttranslational modifications of histone H2A through the attachment of ubiquitin or poly-ubiquitin conjugates are common in mammalian genomes and play an important role in the regulation of chromatin structure, gene expression, and DNA repair. Histone H2A deubiquitinases (H2A-DUBs) are a group of structurally diverse enzymes that catalyze the removal ubiquitin from histone H2A. In this review we provide a concise summary of the mechanisms that mediate histone H2A ubiquitination in mammalian cells, and review our current knowledge of mammalian H2A-DUBs, their biochemical activities, and recent developments in our understanding of their functions in mammalian physiology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. The association among lipoprotein-associated phospholipase A2 levels, total antioxidant capacity and arousal in male patients with OSA.

    PubMed

    Bekci, Taha T; Kayrak, Mehmet; Kiyici, Aysel; Maden, Emin; Ari, Hatem; Kaya, Zeynettin; Teke, Turgut; Akilli, Hakan

    2011-01-01

    The mechanisms of the increased cardiac and vascular events in patients with OSA are not well understood. Arousal which is an important component of OSA was associated with increased sympathetic activation and electrocardiographic changes which prone to arrhythmias. We planned to examine the association among arousal, circulating Lp-PLA2 and total antioxidant capacity in male patients with OSA. Fifty male patients with newly diagnosed OSA were enrolled the study. A full-night polysomnography was performed and arousal index was obtained. Lp-PLA2 concentrations were measured in serum samples with the PLAC Test. Total antioxidant capacity in patients was determined with Antioxidant Assay Kit. Arousal was positively correlated with LP-PLA2 levels (r=0.43, p=0.002) and was negatively correlated with total antioxidant capacity (r= -0.29, p=0.04). Elevated LP-PLA2 levels and decreased total antioxidant activities were found in the highest arousal quartile compared with the lowest and 2nd quartiles (p=0.02, p=0.05, respectively). LP-PLA2 was an independently predictor of arousal index in regression model (β=0.357, p=0.002) This study demonstrated a moderate linear relationship between arousal and LP-PLA2 levels. Also, total antioxidant capacities were decreased in the higher arousal index. Based on the study result, the patients with higher arousal index may be prone to vascular events.

  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. 2-Keto-3-fluoroglutarate: a useful mechanistic probe of 2-keto-glutarate-dependent enzyme systems.

    PubMed

    Grissom, C B; Cleland, W W

    1987-12-18

    2-Keto-3-fluoroglutaric acid prepared by acid hydrolysis of its diethyl ester is stable, as the free acid in aqueous solution at pH 2, and can be stored at -20 degrees C for several years. Both enantiomers are reduced by NADH in the presence of glutamate dehydrogenase (EC 1.4.1.2) to the two diastereomers of 3-fluoro-L-glutamate, which are stable at neutral pH and at high pH unless heated. 2-Keto-3-fluoroglutarate exists in solution almost entirely as a hydrate both at low and neutral pH. Both enantiomers of ketofluoroglutarate react with the pyridoxamine forms of aspartate, alanine and 4-aminobutyrate transaminases to give fluoride release. 2 mol of cosubstrate amino acid react for each mol of ketofluoroglutarate (KFG) when starting from the pyridoxamine form of the enzyme: 2 RCHNH2COOH + KFG + H2O----F- + NH4+ + glutamate + 2 RCOCOOH. Both diastereomers of fluoroglutamate are decarboxylated by glutamate decarboxylase (EC 4.1.1.15) with fluoride release: KFG + H2O----CO2 + F- + HCOCH2CH2COOH. By contrast, only one isomer of fluoroglutamate will react with the pyridoxal form of glutamate-oxalacetate transaminase to give fluoride release: HOOCCHNH2CHFCH2COOH + H2O----4F- + NH4+ + HOOCCOCH2CH2COOH. The enzymatic decarboxylation of 3-fluoroisocitrate produces only one enantiomer of ketofluoroglutarate, which is reduced to threo (2R,3R)-3-fluoroglutamate by NADH and glutamate dehydrogenase: [2R,3S]-HOOCCH(OH)CF(COOH)CH2COOH + NADP+----[3R]-KFG + CO2 + NADPH + H+. The proton, 13C, and 19F-NMR parameters of ketofluoroglutarate and the two fluoroglutamate diastereomers are presented. These molecules are useful probes of enzymatic mechanisms thought to involve carbanion intermediates.

  14. Preparation and mechanical properties of modified nanocellulose/PLA composites from cassava residue

    NASA Astrophysics Data System (ADS)

    Huang, Lijie; Zhang, Xiaoxiao; Xu, Mingzi; Chen, Jie; Shi, Yinghan; Huang, Chongxing; Wang, Shuangfei; An, Shuxiang; Li, Chunying

    2018-02-01

    Nanocellulose was prepared by a mechanochemical method using cassava residue as a raw material and phosphoric acid as the auxiliary agent. The prepared nanocellulose was hydrophobically modified with stearic acid to improve its dispersibility. This modified nanocellulose was added to polylactic acid (PLA) film-forming liquids at concentrations of 0%, 0.5%, 1.0%, 1.5% and 2.0%, and the effect of modified nanocellulose on the mechanical properties of polylactic acid (PLA) films were investigated. When at least 0.5% modified nanocellulose is added, more active groups of modified nanocellulose are adsorbed onto the PLA molecular chain. Although the tensile strength of the film is only improved by 13.59%, the flexibility of the film decreases, and the elastic modulus decreases by 28.91%. When 1% modified nanocellulose is added, the modified nanocellulose and PLA are tangled together through molecular chains and they co-crystallize to form a stable network structure. The tensile strength of the nanocomposite films is enhanced by 40.03%, the elastic modulus is enhanced by 55.65%, and the flexibility of the film decreases.

  15. Dioxygen Binding, Activation, and Reduction to H2O by Cu Enzymes.

    PubMed

    Solomon, Edward I

    2016-07-05

    Oxygen intermediates in copper enzymes exhibit unique spectroscopic features that reflect novel geometric and electronic structures that are key to reactivity. This perspective will describe: (1) the bonding origin of the unique spectroscopic features of the coupled binuclear copper enzymes and how this overcomes the spin forbiddenness of O2 binding and activates monooxygenase activity, (2) how the difference in exchange coupling in the non-coupled binuclear Cu enzymes controls the reaction mechanism, and (3) how the trinuclear Cu cluster present in the multicopper oxidases leads to a major structure/function difference in enabling the irreversible reductive cleavage of the O-O bond with little overpotential and generating a fully oxidized intermediate, different from the resting enzyme studied by crystallography, that is key in enabling fast PCET in the reductive half of the catalytic cycle.

  16. 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

  17. 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.

  18. 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.

  19. Student Perceptions of and Experiences with a PLA Course and Portfolio Review

    ERIC Educational Resources Information Center

    Rust, Dianna Z.; Brinthaupt, Thomas M.

    2017-01-01

    Prior learning assessment (PLA) is the process whereby a student's outside-of-the-classroom learning is evaluated for college-level credit. PLA has been gaining momentum across the United States as part of the efforts to improve institutional completion rates for adult learners. In the present article, we provide an overview of the PLA portfolio…

  20. Endothelin-converting enzyme 2 differentially regulates opioid receptor activity

    PubMed Central

    Gupta, A; Fujita, W; Gomes, I; Bobeck, E; Devi, L A

    2015-01-01

    BACKGROUND AND PURPOSE Opioid receptor function is modulated by post-activation events such as receptor endocytosis, recycling and/or degradation. While it is generally understood that the peptide ligand gets co-endocytosed with the receptor, relatively few studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor function. In this study, we focused on endothelin-converting enzyme 2 (ECE2), a member of the neprilysin family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively processes neuropeptides including opioid peptides in vitro, and examined its role in modulating μ receptor recycling and resensitization. EXPERIMENTAL APPROACH The effect of ECE2 inhibition on hydrolysis of the endocytosed peptide was examined using thin-layer chromatography and on μ opioid receptor trafficking using either elisa or microscopy. The effect of ECE2 inhibition on receptor signalling was measured using a cAMP assay and, in vivo, on antinociception induced by intrathecally administered opioids by the tail-flick assay. KEY RESULTS The highly selective ECE2 inhibitor, S136492, significantly impaired μ receptor recycling and signalling by only those ligands that are ECE2 substrates and this was seen both in heterologous cells and in cells endogenously co-expressing μ receptors with ECE2. We also found that ECE2 inhibition attenuated antinociception mediated only by opioid peptides that are ECE2 substrates. CONCLUSIONS AND IMPLICATIONS These results suggest that ECE2, by selectively processing endogenous opioid peptides in the endocytic compartment, plays a role in modulating opioid receptor activity. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24990314

  1. [Pharmacokinetics of α-asarone after intranasal and intravenous administration with PLA-α-asarone nanoparticles].

    PubMed

    Lu, Jin; Guo, Li-Wei; Fu, Ting-Ming; Zhu, Guo-Long; Dai, Zhen-Nan; Zhan, Guan-Jun; Chen, Li-Li

    2017-06-01

    PLA-α-asarone nanoparticles were prepared by using organic solvent evaporation method, and their in vivo distribution and brain targeting after intranasal administration were studied as compared with intravenous administration. The results showed that brain targeting coefficient of PLA-α-asarone nanoparticles after intranasal and intravenous administration was 1.65 and 1.16 respectively. The absolute bioavailability, brain-targeting efficiency and the percentage of nasal-brain delivery of PLA-α-asarone nanoparticles were 74.2%, 142.24 and 29.83%, respectively after intranasal administration. The results of fluorescence labeling showed that the fluorescent intensity of coumarin-6 in the brain tissue was the highest after intranasal administration of PLA-α-asarone fluorescent nanoparticles, achieving the purpose of brain-targeted drug delivery. The fluorescent intensity of coumarin-6 in liver tissue after intravenous administration of PLA-α-asarone nanoparticles was much higher than that after intranasal administration, indicating that intranasal administration of PLA-α-asarone nanoparticles could decrease drug-induced hepatotoxicity. In addition, the fluorescent intensity of coumarin-6 in lung tissue was weaker after intranasal administration, which solved the shortcomings of intranasal administration of α-asarone dry powder prepared by airflow pulverization method. In vivo studies indicated that PLA-α-asarone nanoparticles after intranasal administration had a stronger brain targeting as compared with intravenous administration. Copyright© by the Chinese Pharmaceutical Association.

  2. Patterns Cancer Prevention Through Induction of Phase 2 Enzymes

    DTIC Science & Technology

    2003-04-01

    2) enzymes. During our Phase I Award, we identified sulforaphane as the most potent inducer of carcinogen defenses in the prostate cell. We have...characterized global effects of sulforaphane in prostate cancer cell lines using cDNA microarray technology that allows large-scale determination of...changes in gene expression. These findings argue strongly for a preventive intervention trial involving with sulforaphane . During our Phase 2 Award, we used

  3. 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.

  4. Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

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

    Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai

    Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement bothmore » defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.« less

  5. 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.

  6. ChaC2, an Enzyme for Slow Turnover of Cytosolic Glutathione*

    PubMed Central

    Kaur, Amandeep; Gautam, Ruchi; Srivastava, Ritika; Chandel, Avinash; Kumar, Akhilesh; Karthikeyan, Subramanian; Bachhawat, Anand Kumar

    2017-01-01

    Glutathione degradation plays an important role in glutathione and redox homeostasis, and thus it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of γ-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammalian cells. ChaC2 is distinct from the previously described ChaC1, to which ChaC2 shows ∼50% sequence identity. Human and mouse ChaC2 proteins purified in vitro show 10–20-fold lower catalytic efficiency than ChaC1, although they showed comparable Km values (Km of 3.7 ± 0.4 mm and kcat of 15.9 ± 1.0 min−1 toward glutathione for human ChaC2; Km of 2.2 ± 0.4 mm and kcat of 225.2 ± 15 min−1 toward glutathione for human ChaC1). The ChaC1 and ChaC2 proteins also shared the same specificity for reduced glutathione, with no activity against either γ-glutamyl amino acids or oxidized glutathione. The ChaC2 proteins were found to be expressed constitutively in cells, unlike the tightly regulated ChaC1. Moreover, lower eukaryotes have a single member of the ChaC family that appears to be orthologous to ChaC2. In addition, we determined the crystal structure of yeast ChaC2 homologue, GCG1, at 1.34 Å resolution, which represents the first structure of the ChaC family of proteins. The catalytic site is defined by a fortuitous benzoic acid molecule bound to the crystal structure. The mechanism for binding and catalytic activity of this new enzyme of glutathione degradation, which is involved in continuous but basal turnover of cytosolic glutathione, is proposed. PMID:27913623

  7. Effect of solvents on the enzyme mediated degradation of copolymers

    NASA Astrophysics Data System (ADS)

    Banerjee, Aditi; Chatterjee, Kaushik; Madras, Giridhar

    2015-09-01

    The biodegradation of polycaprolactone (PCL), polylactic acid (PLA), polyglycolide (PGA) and their copolymers, poly (lactide-co-glycolide) and poly (D, L-lactide-co-caprolactone) (PLCL) was investigated. The influence of different solvents on the degradation of these polymers at 37 °C in the presence of two different lipases namely Novozym 435 and the free lipase of porcine pancreas was investigated. The rate coefficients for the polymer degradation and enzyme deactivation were determined using continuous distribution kinetics. Among the homopolymers, the degradation of PGA was nearly an order of magnitude lower than that for PCL and PLA. The overall rate coefficients of the copolymers were higher than their respective homopolymers. Thus, PLCL degraded faster than either PCL or PLA. The degradation was highly dependent on the viscosity of the solvent used with the highest degradation observed in acetone. The degradation of the polymers in acetone was nearly twice that observed in dimethyl sulfoxide indicating that the degradation decreases with increase in the solvent viscosity. The degradation of the polymers in water-solvent mixtures indicated an optimal water content of 2.5 wt% of water.

  8. Dynamic regulation of a metabolic multi-enzyme complex by protein kinase CK2.

    PubMed

    An, Songon; Kyoung, Minjoung; Allen, Jasmina J; Shokat, Kevan M; Benkovic, Stephen J

    2010-04-09

    The reversible association and dissociation of a metabolic multi-enzyme complex participating in de novo purine biosynthesis, the purinosome, was demonstrated in live cells to respond to the levels of purine nucleotides in the culture media. We also took advantage of in vitro proteomic scale studies of cellular substrates of human protein kinases (e.g. casein kinase II (CK2) and Akt), that implicated several de novo purine biosynthetic enzymes as kinase substrates. Here, we successfully identified that purinosome formation in vivo was significantly promoted in HeLa cells by the addition of small-molecule CK2-specific inhibitors (i.e. 4,5,6,7-tetrabromo-1H-benzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, tetrabromocinammic acid, 4,4',5,5',6,6'-hexahydroxydiphenic acid 2,2',6,6'-dilactone (ellagic acid) as well as by silencing the endogenous human CK2alpha catalytic subunit with small interfering RNA. However, 4,5,6,7-tetrabromobenzotriazole, another CK2-specific inhibitor, triggered the dissociation of purinosome clusters in HeLa cells. Although the mechanism by which 4,5,6,7-tetrabromobenzotriazole affects purinosome clustering is not clear, we were capable of chemically reversing purinosome formation in cells by the sequential addition of two CK2 inhibitors. Collectively, we provide compelling cellular evidence that CK2-mediated pathways reversibly regulate purinosome assembly, and thus the purinosome may be one of the ultimate targets of kinase inhibitors.

  9. Gadolinium-conjugated PLA-PEG nanoparticles as liver targeted molecular MRI contrast agent.

    PubMed

    Chen, Zhijin; Yu, Dexin; Liu, Chunxi; Yang, Xiaoyan; Zhang, Na; Ma, Chunhong; Song, Jibin; Lu, Zaijun

    2011-09-01

    A nanoparticle magnetic resonance imaging (MRI) contrast agent targeted to liver was developed by conjugation of gadolinium (Gd) chelate groups onto the biocompatible poly(l-lactide)-block-poly (ethylene glycol) (PLA-PEG) nanoparticles. PLA-PEG conjugated with diethylenetriaminopentaacetic acid (DTPA) was used to formulate PLA-PEG-DTPA nanoparticles by solvent diffusion method, and then Gd was loaded onto the nanoparticles by chelated with the unfolding DTPA on the surface of the PLA-PEG-DTPA nanoparticles. The mean size of the nanoparticles was 265.9 ± 6.7 nm. The relaxivity of the Gd-labeled nanoparticles was measured, and the distribution in vivo was evaluated in rats. Compared with conventional contrast agent (Magnevist), the Gd-labeled PLA-PEG nanoparticles showed significant enhancement both on liver targeting ability and imaging signal intensity. The T(1) and T(2) relaxivities per [Gd] of the Gd-labeled nanoparticles was 18.865 mM(-1) s(-1) and 24.863 mM(-1) s(-1) at 3 T, respectively. In addition, the signal intensity in vivo was stronger comparing with the Gd-DTPA and the T(1) weight time was lasting for 4.5 h. The liver targeting efficiency of the Gd-labeled PLA-PEG nanoparticles in rats was 14.57 comparing with Magnevist injection. Therefore, the Gd-labeled nanoparticles showed the potential as targeting molecular MRI contrast agent for further clinical utilization.

  10. mPEG-PLA Micelle for Delivery of Effective Parts of Andrographis Paniculata.

    PubMed

    Yao, Hailu; Song, Shiyong; Miao, Xiaolu; Liu, Xiao; Zhao, Junli; Wang, Zhen; Shao, Xiaoting; Zhang, Yu; Han, Guang

    2018-01-01

    Many studies have shown that Andrographis paniculata (Burm. f.) Nees has a good anti-tumor effect, but poor solubility in water and poor bioavailability hinder the modernization of it. To formulate the effective parts (mainly diterpene lactones) of Andrographis paniculata (AEP) into targeting drug delivery system, a series of poly(ethylene glycol)-poly(D.L-lactic acid)(mPEG-PLA) with different ratio of hydrophilic and hydrophobic segment was synthetized to encapsulate AEP. AEP micelles were prepared by a simple solvent-evaporation method. According to the loading capacity, the best polymer was chosen. mPEG-PLA micelles were characterized in terms of drug entrapping efficiency, loading capacity, size, the crystalline state of AEP, stability and release profile. Meanwhile, the cytotoxicity of micelles on mouse breast cancer 4T-1 was investigated. These micelle (mPEG-PLA-AEP) particles had a size of (92.84±5.63) nm and a high entrapping efficiency and loading capacity of (91.00±11.53)% and (32.14±3.02)%(w/w), respectively. The powder DSC showed that drugs were well encapsulated in the core of micelles. mPEG-PLA-AEP had a good stability against salt dissociation, protein adsorption and anion substitution and the solubility of andrographolide (AG) and 14-deoxy-11,12-didehydroandrographolide(DDAG) in AEP increased 4.51 times and 2.12 times in water, and the solubility of DAG showed no difference. mPEG-PLA-AEP had the same release profile in different dissolution medium. Cytotoxicity testing in vitro demonstrated that mPEG-PLA-AEP exhibited higher cell viability inhibition in mouse breast cancer 4T-1 than free AEP. mPEG-PLA micelles offer a promising alternative for TCM therapy with higher solubility and improved antitumor effect. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. In Vitro Characterization and Evaluation of the Cytotoxicity Effects of Nisin and Nisin-Loaded PLA-PEG-PLA Nanoparticles on Gastrointestinal (AGS and KYSE-30), Hepatic (HepG2) and Blood (K562) Cancer Cell Lines.

    PubMed

    Goudarzi, Fariba; Asadi, Asadollah; Afsharpour, Maryam; Jamadi, Robab Hassanvand

    2018-05-01

    The aim of this study was an in vitro evaluation and comparison of the cytotoxic effects of free nisin and nisin-loaded PLA-PEG-PLA nanoparticles on gastrointestinal (AGS and KYSE-30), hepatic (HepG2), and blood (K562) cancer cell lines. To create this novel anti-cancer drug delivery system, the nanoparticles were synthesized and then loaded with nisin. Subsequently, their biocompatibility, ability to enter cells, and physicochemical properties, including formation, size, and shape, were studied using hemolysis, fluorescein isothiocyanate (FITC), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM), respectively. Then, its loading efficiency and release kinetics were examined to assess the potential impact of this formulation for the nanoparticle carrier candidacy. The cytotoxicities of nisin and nisin-loaded nanoparticles were evaluated by using the MTT and Neutral Red (NR) uptake assays. Detections of the apoptotic cells were done via Ethidium Bromide (EB)/Acridine Orange (AO) staining. The FTIR spectra, SEM images, and DLS graph confirmed the formations of the nanoparticles and nisin-loaded nanoparticles with spherical, distinct, and smooth surfaces and average sizes of 100 and 200 nm, respectively. The loading efficiency of the latter nanoparticles was about 85-90%. The hemolysis test represented their non-cytotoxicities and the FITC images indicated their entrance inside the cells. An increase in the percentage of apoptotic cells was observed through EB/AO staining. These results demonstrated that nisin had a cytotoxic effect on AGS, KYSE-30, HepG2, and K562 cancer cell lines, while the cytotoxicity of nisin-loaded nanoparticles was more than that of the free nisin.

  12. 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.

  13. New Method to Prepare Mitomycin C Loaded PLA-Nanoparticles with High Drug Entrapment Efficiency

    NASA Astrophysics Data System (ADS)

    Hou, Zhenqing; Wei, Heng; Wang, Qian; Sun, Qian; Zhou, Chunxiao; Zhan, Chuanming; Tang, Xiaolong; Zhang, Qiqing

    2009-07-01

    The classical utilized double emulsion solvent diffusion technique for encapsulating water soluble Mitomycin C (MMC) in PLA nanoparticles suffers from low encapsulation efficiency because of the drug rapid partitioning to the external aqueous phase. In this paper, MMC loaded PLA nanoparticles were prepared by a new single emulsion solvent evaporation method, in which soybean phosphatidylcholine (SPC) was employed to improve the liposolubility of MMC by formation of MMC-SPC complex. Four main influential factors based on the results of a single-factor test, namely, PLA molecular weight, ratio of PLA to SPC (wt/wt) and MMC to SPC (wt/wt), volume ratio of oil phase to water phase, were evaluated using an orthogonal design with respect to drug entrapment efficiency. The drug release study was performed in pH 7.2 PBS at 37 °C with drug analysis using UV/vis spectrometer at 365 nm. MMC-PLA particles prepared by classical method were used as comparison. The formulated MMC-SPC-PLA nanoparticles under optimized condition are found to be relatively uniform in size (594 nm) with up to 94.8% of drug entrapment efficiency compared to 6.44 μm of PLA-MMC microparticles with 34.5% of drug entrapment efficiency. The release of MMC shows biphasic with an initial burst effect, followed by a cumulated drug release over 30 days is 50.17% for PLA-MMC-SPC nanoparticles, and 74.1% for PLA-MMC particles. The IR analysis of MMC-SPC complex shows that their high liposolubility may be attributed to some weak physical interaction between MMC and SPC during the formation of the complex. It is concluded that the new method is advantageous in terms of smaller size, lower size distribution, higher encapsulation yield, and longer sustained drug release in comparison to classical method.

  14. 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

  15. Characterization and properties of acetylated nanocrystalline cellulose (aNC) reinforced polylactic acid (PLA) polymer

    NASA Astrophysics Data System (ADS)

    Kasa, Siti Norbaya; Omar, Mohd Firdaus; Ismail, Ismarul Nizam

    2017-12-01

    Nanocrystalline cellulose (NCC) was synthesized from banana stem through strong acid hydrolysis with measured length of approximately 287.0 ± 56.4 nm and diameter of 26.6 ± 4.8 nm. Modification of NCC was carried by acetylation reaction in order to increase the compatibility during reinforcement with polylactic acid (PLA) polymer. The reinforcing effect towards morphology, crystallinity, mechanical and thermal properties of bio-nanocomposites was investigated. Scanning Electron Microscope (SEM) micrograph reveals the uniform dispersion achieved at 1 %, 3 % and 5% aNC loading while agglomeration was found at 7 % aNC loading. Disappearance of crystallinity peak at 2θ = 22.7⁰ for low aNC loading during elemental analysis using X-Ray Diffraction (XRD) indicates the proper dispersion of aNC in PLA polymer. From the tensile test, 1 % aNC loading gives the highest mechanical properties of bio-nanocomposite film with 82.71 %, 118.7 % and 24.18 % increment in tensile strength, tensile modulus and elongation at break. However, 7 % aNC loading gives the highest increment in TGA of aNC-PLA nanocomposites which is from 310 °C to 320 °C.

  16. PS-b-PMMA/PLA blends for nanoporous templates with hierarchical and tunable pore size

    NASA Astrophysics Data System (ADS)

    Nguyen, Thi-Hoa; Vayer, Marylène; Sinturel, Christophe

    2018-01-01

    Blends of poly(styrene)-block-poly(methyl methacrylate) (PS-b-PMMA) and poly(lactide) (PLA) were deposited in the form of thin films on the surface of modified silicon wafers and exposed to tetrahydrofuran (THF) vapor annealing. It was shown that in specific experimental conditions, a core-shell morphology consisting in cylinders with a PMMA shell and a PLA core, within a continuous matrix of PS, was formed. In this case, PLA naturally segregated in the core of the PMMA cylinders, minimizing the PS/PLA interaction, which constitutes the most incompatible pair (the interaction strength between the various components was confirmed in thin films of the corresponding polymer blends). Compared to other block copolymer/homopolymer blends described in the literature, this system exhibits unexpected high increase of the characteristic lengths of the system (center-to-center distance and diameter). This was attributed to a partial solubilization of the PLA in the PMMA corona (the two polymers are highly compatible), inducing an enhanced level of PS and PLA stretching caused by the strong repulsion between these two polymers. The selective extraction of the PLA yielded to porous domains with small dimensions (6 ± 2.5 nm), reaching the performances that are currently attained in highly incompatible block polymers with low molecular weight. Further PMMA removal revealed a second porosity level, with higher pores diameter and center-to-center distance compared to the neat PS-b-PMMA system. This work highlights how PS-b-PMMA, that currently represents one of the industrial standards nanoporous template precursors, can be modified in an easy and costless approach using PLA homopolymer addition.

  17. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes.

    PubMed

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-09-01

    Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.

  18. 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.

  19. PEG-PLA-PEG block copolymeric nanoparticles for oral immunization against hepatitis B.

    PubMed

    Jain, Arvind K; Goyal, Amit K; Mishra, Neeraj; Vaidya, Bhuvaneshwar; Mangal, Sharad; Vyas, Suresh P

    2010-03-15

    PLA/PLGA nanoparticles are well known as efficient vaccine delivery systems, but they have got limitation in oral vaccine delivery because of their sensitivity to harsh gastric environment. The aim of present study was to improve the stability of PLA nanoparticles in such environment by copolymerizing PLA with PEG. Nanoparticles were formulated using different block copolymers AB, ABA and BAB (where 'A' is PLA and 'B' is PEG) encapsulating hepatitis B surface antigen (HBsAg) to evaluate their efficacy as oral vaccine delivery system. The results of in vitro studies engrave the efficiency of copolymeric nanoparticles to retain encapsulated antigen and average particle size even after 2 h incubation in simulated gastric fluid and simulated intestinal fluid. Fluorescence microscopic studies indicated efficient uptake of copolymeric nanoparticles by gut mucosa of immunized mice model as compared to control. Finally copolymeric and PLA nanoparticles, encapsulating HBsAg, were evaluated for their adjuvancity in generating immune response after oral administration. PLA nanoparticles could not generate an effective immune response due to stability issues. On the other hand, oral administration of copolymeric nanoparticles exhibited effective levels of humoral immunity along with the mucosal (sIgA) and cellular immune response (T(H)1). The results of in vitro and in vivo studies demonstrate that BAB nanoparticles depict enhanced mucosal uptake leading to effective immune response as compared to other copolymeric nanoparticles. Present study indicates the efficacy of BAB nanoparticles as a promising carrier for oral immunization. 2009 Elsevier B.V. All rights reserved.

  20. Increased Activity of Rhizosphere and Hyphosphere Enzymes under Elevated CO2 in a Loblolly Pine Stand

    NASA Astrophysics Data System (ADS)

    Meier, I.; Phillips, R.

    2012-12-01

    The stimulatory effect of elevated atmospheric CO2 under global climate change on forest productivity has been predicted to decrease over time as pools of available N in soil become depleted, but empirical support for such progressive N limitation has been lacking. Increased N acquisition from soil depleted in inorganic nitrogen requires stimulation of the microbial processing of organic N, possibly through increasing C supply to soil by plant roots or mycorrhizal hyphae. Increases in (mycorr)rhizosphere C fluxes could stimulate microbes to produce extra-cellular enzymes that release N from SOM, feeding back from soil microsites to ecosystem-scale processes. We investigated the influence of elevated CO2 on root exudation and soil enzyme activity at the Duke Forest FACE site, USA, where loblolly pine (Pinus taeda L.) stands have been exposed to elevated CO2 for 14 years and N fertilization for five years. In each plot, root boxes containing acetate windows were installed in 2008. Two years after installation, we collected soils adjacent to root tips (the rhizosphere), hyphal tips (the hyphosphere) and bulk soil. We measured in situ root exudation rates from intact pine roots. Study objectives were to analyze (i) the influence of atmospheric CO2 on root exudation and extra-cellular enzyme activities, (ii) the influence of soil N availability in regulating these activities, and (iii) the relationship between the activities of enzymes involved in N cycling in soils and gross N transformations at soil microsites. Elevated atmospheric CO2 significantly increased the activity of β-1-4-N-acetylglucosaminidase (NAG) in the rhizosphere by almost 2.5 times (39 to 95 nmol h-1 g-1), and 1.6fold in the hyphosphere relative to ambient plots. NAG is an enzyme involved in the degradation of chitin from the cell walls of soil organisms, releasing absorbable forms of nitrogen. The activity of peroxidase, which degrades aromatic C compounds of SOM, increased significantly in the

  1. Design and evaluation of mPEG-PLA micelles functionalized with drug-interactive domains as improved drug carriers for docetaxel delivery.

    PubMed

    Qi, Dingqing; Gong, Feirong; Teng, Xin; Ma, Mingming; Wen, Huijing; Yuan, Weihao; Cheng, Yi; Lu, Chong

    2017-10-01

    Polymeric micelles are very attractive drug delivery systems for hydrophobic agents, owing to their readily tailorable chemical structure and ease for scale-up preparation. However, the intrinsic poor stability of drug-loaded micelles presents one of the major challenges for most micellar systems in the translation to clinical applications. In this study, a simple, well-defined, and easy-to-scale up 9-Fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc) containing lysine dendronized mPEG-PLA (mPEG-PLA-Lys(FB) 2 ) micellar formulation was designed and prepared for docetaxel (DTX) delivery, in an effort to improve the stability of the micelles, and its physicochemical properties, pharmacokinetics, and anti-tumor efficacy against SKOV-3 ovarian cancer were evaluated. MPEG-PLA-Lys(FB) 2 was synthesized via a three-step synthetic route, and it actively interacted with DTX in aqueous media to form stable micelles with small particle sizes (~17-19 nm) and narrow size distribution (PI < 0.1), which can be lyophilized and easily reconstituted in saline without significant change in particle size distribution. In vitro drug-release study demonstrated that mPEG-PLA-Lys(FB) 2 micelles achieved delayed and sustained release manner of DTX in comparison with mPEG-PLA micelles. Further in vivo xenograft tumor model in nude mice DTX/mPEG-PLA-Lys(FB) 2 micelles demonstrated significantly higher inhibitory effect on tumor growth than the marketed formulation Taxotere. Thus, our system may hold promise as a simple and effective delivery system for DTX with a potential for translation into clinical study.

  2. Compostability and biodegradation study of PLA-wheat straw and PLA-soy straw based green composites in simulated composting bioreactor.

    PubMed

    Pradhan, Ranjan; Misra, Manjusri; Erickson, Larry; Mohanty, Amar

    2010-11-01

    A laboratory scale simulated composting facility (as per ASTM D 5338) was designed and utilized to determine and evaluate the extent of degradation of polylactic acid (PLA), untreated wheat and soy straw and injection moulded composites of PLA-wheat straw (70:30) and PLA-soy straw (70:30). The outcomes of the study revealed the suitability of the test protocol, validity of the test system and defined the compostability of the composites of PLA with unmodified natural substrate. The study would help to design composites using modified soy straw and wheat straw as reinforcement/filler to satisfy ASTM D 6400 specifications. Copyright 2010 Elsevier Ltd. All rights reserved.

  3. 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.

  4. PlaMoM: a comprehensive database compiles plant mobile macromolecules

    PubMed Central

    Guan, Daogang; Yan, Bin; Thieme, Christoph; Hua, Jingmin; Zhu, Hailong; Boheler, Kenneth R.; Zhao, Zhongying; Kragler, Friedrich; Xia, Yiji; Zhang, Shoudong

    2017-01-01

    In plants, various phloem-mobile macromolecules including noncoding RNAs, mRNAs and proteins are suggested to act as important long-distance signals in regulating crucial physiological and morphological transition processes such as flowering, plant growth and stress responses. Given recent advances in high-throughput sequencing technologies, numerous mobile macromolecules have been identified in diverse plant species from different plant families. However, most of the identified mobile macromolecules are not annotated in current versions of species-specific databases and are only available as non-searchable datasheets. To facilitate study of the mobile signaling macromolecules, we compiled the PlaMoM (Plant Mobile Macromolecules) database, a resource that provides convenient and interactive search tools allowing users to retrieve, to analyze and also to predict mobile RNAs/proteins. Each entry in the PlaMoM contains detailed information such as nucleotide/amino acid sequences, ortholog partners, related experiments, gene functions and literature. For the model plant Arabidopsis thaliana, protein–protein interactions of mobile transcripts are presented as interactive molecular networks. Furthermore, PlaMoM provides a built-in tool to identify potential RNA mobility signals such as tRNA-like structures. The current version of PlaMoM compiles a total of 17 991 mobile macromolecules from 14 plant species/ecotypes from published data and literature. PlaMoM is available at http://www.systembioinfo.org/plamom/. PMID:27924044

  5. Benign thyroid nodules treatment using percutaneous laser ablation (PLA) and radiofrequency ablation (RFA).

    PubMed

    Mauri, Giovanni; Cova, Luca; Monaco, Cristian Giuseppe; Sconfienza, Luca Maria; Corbetta, Sabrina; Benedini, Stefano; Ambrogi, Federico; Milani, Valentina; Baroli, Alberto; Ierace, Tiziana; Solbiati, Luigi

    2016-11-15

    To evaluate the reduction over time of benign thyroid nodules treated using percutaneous laser ablation (PLA) and radiofrequency ablation (RFA) by the same equipe. Ninety patients (age 55.6 ± 14.1 years) underwent ablation for benign thyroid nodule causing compression/aesthetic dissatisfaction from 2011. Fifty-nine (age 55.8 ± 14.1 years) underwent RFA and 31 (age 55.2 ± 14.2 years) PLA, ultrasound guided. Technical success, complications, duration of ablation and treatment, energy deployed, volumetric percentage reduction at 1, 6 and 12 months were derived. A regression model for longitudinal measurements was used with random intercept and random slope. Values are expressed as mean ± standard deviation or N (%). Technical success was always obtained. No major complications occurred. Mean ablation time was 30.1 ± 13.8 vs. 13.9 ± 5.9 min (p < .0001) and mean energy deployment was 5422.3 ± 2484.5 J vs. 34 662.7 ± 15 812.3 J in PLA vs. RFA group. Mean volume reduced from 20.3 ± 16.4 ml to 13.17 ± 10.74 ml (42% ± 17% reduction) at 1st month, 8.7 ± 7.4 ml (60% ± 15% reduction) at 6th month and 7.1 ± 7.7 ml (70%% ± 16% reduction) at 12th month, in PLA group, and from 32.7 ± 19.5 ml to 17.2 ± 12.9 ml (51%±15% reduction) at 1st month, 12.8 ± 9.6 ml (64 ± 14% reduction) at 6th month and 9.9 ± 9.2 ml (74% ± 14% reduction) at 12th month in RFA group. No difference in time course of the relative volume reduction between the two techniques was found. RFA and PLA are similarly feasible, safe and effective in treating benign thyroid nodules when performed by the same equipe. RFA is faster than PLA but require significantly higher energy.

  6. Mode of Action of Membrane Perturbing Agents: Snake Venom Cardiotoxins and Phospholipases A

    DTIC Science & Technology

    1990-06-30

    with the PLA2 neurotoxins. CTXs are potent membrane perturbing agents and PLA2s hydrolyze diacylphosphoglycerides at the two position, generating two...The bee and cobra (Naja naJa) venom PLA2 enzymes readily hydrolyze biological phospholipid substrates, but are unable to penetrate membrane bilayers...Zwaal et al., 1975; Sundler et al., 1978; Fletcher et al., 1987). The inability to hydrolyze the inner phospholipids of the bilayer does not relate to

  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. Blending Novatein¯ thermoplastic protein with PLA for carbon dioxide assisted batch foaming

    NASA Astrophysics Data System (ADS)

    Walallavita, Anuradha; Verbeek, Casparus J. R.; Lay, Mark

    2016-03-01

    The convenience of polymeric foams has led to their widespread utilisation in everyday life. However, disposal of synthetic petroleum-derived foams has had a detrimental effect on the environment which needs to be addressed. This study uses a clean and sustainable approach to investigate the foaming capability of a blend of two biodegradable polymers, polylactic acid (PLA) and Novatein® Thermoplastic Protein (NTP). PLA, derived from corn starch, can successfully be foamed using a batch technique developed by the Biopolymer Network Ltd. NTP is a patented formulation of bloodmeal and chemical additives which can be extruded and injection moulded similar to other thermoplastics. However, foaming NTP is a new area of study and its interaction with blowing agents in the batch process is entirely unknown. Subcritical and supercritical carbon dioxide have been examined individually in two uniquely designed pressure vessels to foam various compositions of NTP-PLA blends. Foamed material were characterised in terms of expansion ratio, cell size, and cellular morphology in order to study how the composition of NTP-PLA affects foaming with carbon dioxide. It was found that blends with 5 wt. % NTP foamed using subcritical CO2 expanded up to 11 times due to heterogeneous nucleation. Morphology analysis using scanning electron microscopy showed that foams blown with supercritical CO2 had a finer cell structure with consistent cell size, whereas, foams blown with subcritical CO2 ranged in cell size and showed cell wall rupture. Ultimately, this research would contribute to the production of a biodegradable foam material to be used in packaging applications, thereby adding to the application potential of NTP.

  9. PDZ Binding Domains, Structural Disorder and Phosphorylation: A Menage-a-trois Tailing Dcp2 mRNA Decapping Enzymes.

    PubMed

    Gunawardana, Dilantha

    2016-01-01

    Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein-protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer's disease, Parkinson's disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes.

  10. Preparation of porous PLA/DBM composite biomaterials and experimental research of repair rabbit radius segmental bone defect.

    PubMed

    Zhang, Yumin; Wang, Jianru; Wang, Jue; Niu, Xiaojun; Liu, Jianchun; Gao, Lan; Zhai, Xiaoyan; Chu, Kaibo

    2015-12-01

    Bone substitutes are used in wide range of orthopaedic application. An ideal bone substitute should exhibit superior osteoinductive and osteoconductive properties. Neither bio-derived materials nor synthetic materials can meet the needs of an ideal bone substitute. Preparation of composite materials is a promising way to improve properties of biomaterial. In this study, the porous poly lactic acid (PLA)/demineralized bone matrix (DBM) composite biomaterials prepared by supercritical CO2 technique were implanted to repair rabbit radius segmental bone defect. By comparing with PLA and bone autograft, the X-ray result and histological analysis showed the repair effect of PLA/DBM porous composite materials is significantly better than that of the PLA group and the blank control group, and is similar to autologous bone. The PLA/DBM can promote the healing of bone defects and can be used as a kind of ideal alternative materials to repair bone defects.

  11. 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.

  12. ChaC2, an Enzyme for Slow Turnover of Cytosolic Glutathione.

    PubMed

    Kaur, Amandeep; Gautam, Ruchi; Srivastava, Ritika; Chandel, Avinash; Kumar, Akhilesh; Karthikeyan, Subramanian; Bachhawat, Anand Kumar

    2017-01-13

    Glutathione degradation plays an important role in glutathione and redox homeostasis, and thus it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of γ-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammalian cells. ChaC2 is distinct from the previously described ChaC1, to which ChaC2 shows ∼50% sequence identity. Human and mouse ChaC2 proteins purified in vitro show 10-20-fold lower catalytic efficiency than ChaC1, although they showed comparable K m values (K m of 3.7 ± 0.4 mm and k cat of 15.9 ± 1.0 min -1 toward glutathione for human ChaC2; K m of 2.2 ± 0.4 mm and k cat of 225.2 ± 15 min -1 toward glutathione for human ChaC1). The ChaC1 and ChaC2 proteins also shared the same specificity for reduced glutathione, with no activity against either γ-glutamyl amino acids or oxidized glutathione. The ChaC2 proteins were found to be expressed constitutively in cells, unlike the tightly regulated ChaC1. Moreover, lower eukaryotes have a single member of the ChaC family that appears to be orthologous to ChaC2. In addition, we determined the crystal structure of yeast ChaC2 homologue, GCG1, at 1.34 Å resolution, which represents the first structure of the ChaC family of proteins. The catalytic site is defined by a fortuitous benzoic acid molecule bound to the crystal structure. The mechanism for binding and catalytic activity of this new enzyme of glutathione degradation, which is involved in continuous but basal turnover of cytosolic glutathione, is proposed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer

    NASA Astrophysics Data System (ADS)

    Nilsson, Roland; Jain, Mohit; Madhusudhan, Nikhil; Sheppard, Nina Gustafsson; Strittmatter, Laura; Kampf, Caroline; Huang, Jenny; Asplund, Anna; Mootha, Vamsi K.

    2014-01-01

    Metabolic remodeling is now widely regarded as a hallmark of cancer, but it is not clear whether individual metabolic strategies are frequently exploited by many tumours. Here we compare messenger RNA profiles of 1,454 metabolic enzymes across 1,981 tumours spanning 19 cancer types to identify enzymes that are consistently differentially expressed. Our meta-analysis recovers established targets of some of the most widely used chemotherapeutics, including dihydrofolate reductase, thymidylate synthase and ribonucleotide reductase, while also spotlighting new enzymes, such as the mitochondrial proline biosynthetic enzyme PYCR1. The highest scoring pathway is mitochondrial one-carbon metabolism and is centred on MTHFD2. MTHFD2 RNA and protein are markedly elevated in many cancers and correlated with poor survival in breast cancer. MTHFD2 is expressed in the developing embryo, but is absent in most healthy adult tissues, even those that are proliferating. Our study highlights the importance of mitochondrial compartmentalization of one-carbon metabolism in cancer and raises important therapeutic hypotheses.

  14. Electroactive Shape Memory Property of a Cu-decorated CNT Dispersed PLA/ESO Nanocomposite

    PubMed Central

    Alam, Javed; Khan, Aslam; Alam, Manawwer; Mohan, Raja

    2015-01-01

    Shape memory polymer (SMP) nanocomposites with a fast electro-actuation speed were prepared by dispersing Cu-decorated carbon nanotubes (CNTs) (Cu-CNTs, 1 wt %, 2 wt %, and 3 wt %) in a polylactic acid (PLA)/epoxidized soybean oil (ESO) blend matrix. The shape memory effect (SME) induced by an electrical current was investigated by a fold-deploy “U”-shape bending test. In addition, the Cu-CNT dispersed PLA/ESO nanocomposite was characterized by atomic force microscopy (AFM), dynamic mechanical analysis (DMA) and tensile and electrical measurements. The results demonstrated that the SME was dependent on the Cu-CNT content in the nanocomposites. When comparing the SMEs of the nanocomposite specimens with different Cu-CNT contents, the 2 wt % Cu-CNT dispersed system exhibited a shape recovery as high as 98% within 35 s due to its higher electrical conductivity that results from uniform Cu-CNT dispersion. However, the nanocomposites that contained 1 wt % and 3 wt % Cu-CNTs required 75 s and 63 s, respectively, to reach a maximum recovery level. In addition, the specimens exhibited better mechanical properties after the addition of Cu-CNTs. PMID:28793570

  15. PHEA-PLA biocompatible nanoparticles by technique of solvent evaporation from multiple emulsions.

    PubMed

    Cavallaro, Gennara; Craparo, Emanuela Fabiola; Sardo, Carla; Lamberti, Gaetano; Barba, Anna Angela; Dalmoro, Annalisa

    2015-11-30

    Nanocarriers of amphiphilic polymeric materials represent versatile delivery systems for poorly water soluble drugs. In this work the technique of solvent evaporation from multiple emulsions was applied to produce nanovectors based on new amphiphilic copolymer, the α,β-poly(N-2-hydroxyethyl)-DL-aspartamide-polylactic acid (PHEA-PLA), purposely synthesized to be used in the controlled release of active molecules poorly soluble in water. To this aim an amphiphilic derivative of PHEA, a hydrophilic polymer, was synthesized by derivatization of the polymeric backbone with hydrophobic grafts of polylactic acid (PLA). The achieved copolymer was thus used to produce nanoparticles loaded with α tocopherol (vitamin E) adopted as lipophilic model molecule. Applying a protocol based on solvent evaporation from multiple emulsions assisted by ultrasonic energy and optimizing the emulsification process (solvent selection/separation stages), PHEA-PLA nanostructured particles with total α tocopherol entrapment efficiency (100%), were obtained. The drug release is expected to take place in lower times with respect to PLA due to the presence of the hydrophilic PHEA, therefore the produced nanoparticles can be used for semi-long term release drug delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. PLA recycling by hydrolysis at high temperature

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

    Cristina, Annesini Maria; Rosaria, Augelletti; Sara, Frattari, E-mail: sara.frattari@uniroma1.it

    In this work the process of PLA hydrolysis at high temperature was studied, in order to evaluate the possibility of chemical recycling of this polymer bio-based. In particular, the possibility to obtain the monomer of lactic acid from PLA degradation was investigated. The results of some preliminary tests, performed in a laboratory batch reactor at high temperature, are presented: the experimental results show that the complete degradation of PLA can be obtained in relatively low reaction times.

  17. 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.

  18. PlaMoM: a comprehensive database compiles plant mobile macromolecules.

    PubMed

    Guan, Daogang; Yan, Bin; Thieme, Christoph; Hua, Jingmin; Zhu, Hailong; Boheler, Kenneth R; Zhao, Zhongying; Kragler, Friedrich; Xia, Yiji; Zhang, Shoudong

    2017-01-04

    In plants, various phloem-mobile macromolecules including noncoding RNAs, mRNAs and proteins are suggested to act as important long-distance signals in regulating crucial physiological and morphological transition processes such as flowering, plant growth and stress responses. Given recent advances in high-throughput sequencing technologies, numerous mobile macromolecules have been identified in diverse plant species from different plant families. However, most of the identified mobile macromolecules are not annotated in current versions of species-specific databases and are only available as non-searchable datasheets. To facilitate study of the mobile signaling macromolecules, we compiled the PlaMoM (Plant Mobile Macromolecules) database, a resource that provides convenient and interactive search tools allowing users to retrieve, to analyze and also to predict mobile RNAs/proteins. Each entry in the PlaMoM contains detailed information such as nucleotide/amino acid sequences, ortholog partners, related experiments, gene functions and literature. For the model plant Arabidopsis thaliana, protein-protein interactions of mobile transcripts are presented as interactive molecular networks. Furthermore, PlaMoM provides a built-in tool to identify potential RNA mobility signals such as tRNA-like structures. The current version of PlaMoM compiles a total of 17 991 mobile macromolecules from 14 plant species/ecotypes from published data and literature. PlaMoM is available at http://www.systembioinfo.org/plamom/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. 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.

  20. Enhanced effect of folated pluronic F87-PLA/TPGS mixed micelles on targeted delivery of paclitaxel.

    PubMed

    Xiong, Xiang Yuan; Pan, Xiaoqian; Tao, Long; Cheng, Feng; Li, Zi Ling; Gong, Yan Chun; Li, Yu Ping

    2017-10-01

    Targeted drug delivery systems have great potential to overcome the side effect and improve the bioavailability of conventional anticancer drugs. In order to further improve the antitumor efficacy of paclitaxel (PTX) loaded in folated Pluronic F87/poly(lactic acid) (FA-F87-PLA) micelles, D-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS or Vitamin E TPGS) were added into FA-F87-PLA to form FA-F87-PLA/TPGS mixed micelles. The LE of PTX-loaded mixed micelles (13.5%) was highest in the mass ratio 5 to 3 of FA-F87-PLA to TPGS. The in vitro cytotoxicity assays indicated that the IC50 values for free PTX injections, PTX-loaded FA-F87-PLA micelles and PTX-loaded FA-F87-PLA/TPGS mixed micelles after 72h of incubation were 1.52, 0.42 and 0.037mg/L, respectively. The quantitative cellular uptake of coumarin 6-loaded FA-F87-PLA/TPGS and FA-F87-PLA micelles showed that the cellular uptake efficiency of mixed micelles was higher for 2 and 4h incubation, respectively. In vivo pharmacokinetic studies found that the AUC of PTX-loaded FA-F87-PLA/TPGS mixed micelles is almost 1.4 times of that of PTX-loaded FA-F87-PLA micelles. The decreased particle size and inhibition of P-glycoprotein effect induced by the addition of TPGS could result in enhancing the cellular uptake and improving the antitumor efficiency of PTX-loaded FA-F87-PLA/TPGS mixed micelles. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Bilateral PLA/alginate membranes for the prevention of postsurgical adhesions.

    PubMed

    Kessler, Martina; Esser, Eva; Groll, Jürgen; Tessmar, Jörg

    2016-11-01

    A bilateral barrier membrane for the prevention of postsurgical adhesions was developed. Thereby, a smooth PLA side was supposed to keep the affected tissues glidingly separated, while a mucoadhesive side made of alginate was meant to keep the barrier resident on the site of injury so that suturing becomes redundant or at least the membrane stays long enough to facilitate surgical handling. Because hydrophilic alginate and lipophilic PLA films show only low cohesion, solution electrospun meshes of PLA and PLA-PEG-PLA triblock copolymers with varying poly(ethylene glycol) [PEG] content were investigated as cohesion promoter to avoid an easy separation of the functionally different layers. Using direct electrospinning onto the PLA film, a modified contact surface of the mesh was created, which allowed the tested alginate solutions (3%, 5%) to infiltrate to different extents. Thereby, an increasing content of hydrophilic PEG within the mesh copolymer and a lower alginate concentration facilitated the infiltration. As a result, the PLA film with a PLA35k-PEG10k-PLA35k (racemic PLA chains) mesh and an alginate layer cast from a 3% alginate solution appeared to be the most effective combination as examined by means of a t peel test, a mucoadhesion test, a tensile test and optical evaluations. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1563-1570, 2016. © 2015 Wiley Periodicals, Inc.

  2. Polylactic acid (PLA) controlled delivery carriers for biomedical applications.

    PubMed

    Tyler, Betty; Gullotti, David; Mangraviti, Antonella; Utsuki, Tadanobu; Brem, Henry

    2016-12-15

    Polylactic acid (PLA) and its copolymers have a long history of safety in humans and an extensive range of applications. PLA is biocompatible, biodegradable by hydrolysis and enzymatic activity, has a large range of mechanical and physical properties that can be engineered appropriately to suit multiple applications, and has low immunogenicity. Formulations containing PLA have also been Food and Drug Administration (FDA)-approved for multiple applications making PLA suitable for expedited clinical translatability. These biomaterials can be fashioned into sutures, scaffolds, cell carriers, drug delivery systems, and a myriad of fabrications. PLA has been the focus of a multitude of preclinical and clinical testing. Three-dimensional printing has expanded the possibilities of biomedical engineering and has enabled the fabrication of a myriad of platforms for an extensive variety of applications. PLA has been widely used as temporary extracellular matrices in tissue engineering. At the other end of the spectrum, PLA's application as drug-loaded nanoparticle drug carriers, such as liposomes, polymeric nanoparticles, dendrimers, and micelles, can encapsulate otherwise toxic hydrophobic anti-tumor drugs and evade systemic toxicities. The clinical translation of these technologies from preclinical experimental settings is an ever-evolving field with incremental advancements. In this review, some of the biomedical applications of PLA and its copolymers are highlighted and briefly summarized. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. 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

  4. A performance comparison of choline biosensors: anodic or cathodic detections of H2O2 generated by enzyme immobilized on a conducting polymer.

    PubMed

    Rahman, Md Aminur; Park, Deog-Soo; Shim, Yoon-Bo

    2004-07-15

    Amperometric choline biosensors were fabricated by the covalent immobilization of an enzyme of choline oxidase (ChO) and a bi-enzyme of ChO/horseradish peroxidase (ChO/HRP) onto poly-5,2':5',2"-terthiophene-3'-carboxylic acid (poly-TTCA) modified electrodes (CPMEs). A sensor modified with ChO utilized the oxidation process of enzymatically generated H(2)O(2) in a choline solution at +0.6V. The other one modified with ChO/HRP utilized the reduction process of H(2)O(2) in a choline solution at -0.2V. Experimental parameters affecting the sensitivity of sensors, such as pH, applied potential, and temperature were optimized. A performance comparison of two sensors showed that one based on ChO/HRP/CPME had a linear range from 1.0 x 10(-6) to 8.0 x 10(-5) M and the other based on ChO/CPME from 1.0 x 10(-6) to 5.0 x 10(-5) M. The detection limits for choline employing ChO/HRP/CPME and ChO/CPME were determined to be about 1.0 x 10(-7) and 4.0 x 10(-7) M, respectively. The response time of sensors was less than 5s. Sensors showed good selectivity to interfering species. The long-term storage stability of the sensor based on ChO/HRP/CPME was longer than that based on ChO/CPME.

  5. The internalization of fluorescence-labeled PLA nanoparticles by macrophages.

    PubMed

    Li, Fengjuan; Zhu, Aiping; Song, Xiaoli; Ji, Lijun; Wang, Juan

    2013-09-10

    Rhodamine B (RhB)-labeled PLA nanoparticles were prepared through surface grafting copolymerization of glycidyl methacrylate (GMA) onto PLA nanoparticles during the emulsion/evaporation process. RhB firstly interacts with sodium dodecyl sulfate (SDS) through electrostatic interaction to form hydrophobic complex (SDS-RhB). Due to the high-affinity of SDS-RhB with GMA, hydrophilic RhB can be successfully combined into PLA nanoparticles. The internalization of RhB-labeled PLA nanoparticles by macrophages was investigated with fluorescence microscope technology. The effects of the PLA nanoparticle surface nature and size on the internalization were investigated. The results indicate that the PLA particles smaller than 200 nm can avoid the uptake of phagocytosis. The bigger PLA particles (300 nm) with polyethylene glycol (PEG) surface showed less internalization by macrophage compared with those with poly(ethylene oxide-propylene oxide) copolymer (F127) or poly(vinyl alcohol) (PVA) surface. The "stealth" function of PEG on the PLA nanoparticles from internalization of macrophages due to the low protein adsorption is revealed by electrochemical impedance technology. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Structural comparison of cytochromes P450 2A6, 2A13, and 2E1 with pilocarpine

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

    DeVore, Natasha M.; Meneely, Kathleen M.; Bart, Aaron G.

    2013-11-20

    Human xenobiotic-metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drugmore » pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 {angstrom}), CYP2A13 (3.0 {angstrom}), CYP2E1 (2.35 {angstrom}), and the CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 {angstrom}) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine.« less

  7. Is the Chinese Army the Real Winner in PLA Reforms

    DTIC Science & Technology

    2016-10-01

    44 Commentary / The Chinese Army and PLA Reforms JFQ 83, 4th Quarter 2016 Is the Chinese Army the Real Winner in PLA Reforms? By Phillip C. Saunders...and John Chen G round force officers run China’s military, the People’s Liberation Army ( PLA ). About 70 percent of PLA soldiers serve in the PLA ...Saunders and Chen 45 services and arms of the PLA ” has meant reductions in “technologically backward” PLAA units and personnel increases for the other

  8. Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn 2+/Mn 2+ heterobinucleation

    DOE PAGES

    Ransey, Elizabeth; Paredes, Eduardo; Dey, Sourav K.; ...

    2017-05-17

    Here, the RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2'-5' phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni 2+, Mn 2+, Mg 2+, Fe 2+, and Zn 2+. While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn 2+ ion), recent structures determined a Zn 2+/Fe 2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn 2+/Mn 2+ occupancy.more » ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.« less

  9. Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn 2+/Mn 2+ heterobinucleation

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

    Ransey, Elizabeth; Paredes, Eduardo; Dey, Sourav K.

    Here, the RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2'-5' phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni 2+, Mn 2+, Mg 2+, Fe 2+, and Zn 2+. While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn 2+ ion), recent structures determined a Zn 2+/Fe 2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn 2+/Mn 2+ occupancy.more » ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.« less

  10. Albumin Stimulates the Activity of the Human UDP-Glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the Effects Are Enzyme and Substrate Dependent

    PubMed Central

    Svaluto-Moreolo, Paolo; Dziedzic, Klaudyna; Yli-Kauhaluoma, Jari; Finel, Moshe

    2013-01-01

    Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro–in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2–4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction’s K m, increasing its V max, or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions’ K m are concerned. In the cases of V max values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V max increases. Additionally, the BSA effects may be UGT subfamily dependent since K m decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V max increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs. PMID:23372764

  11. Effect of inhibition of microsomal Ca(2+)-ATPase on cytoplasmic calcium and enzyme secretion in pancreatic acini.

    PubMed

    Metz, D C; Pradhan, T K; Mrozinski, J E; Jensen, R T; Turner, R J; Patto, R J; Gardner, J D

    1994-01-13

    We used thapsigargin (TG), 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA), each of which inhibits microsomal Ca(2+)-ATPase, to evaluate the effects of this inhibition on cytoplasmic free calcium ([Ca2+]i) and secretagogue-stimulated enzyme secretion in rat pancreatic acini. Using single-cell microspectrofluorimetry of fura-2-loaded acini we found that all three agents caused a sustained increase in [Ca2+]i by mobilizing calcium from inositol-(1,4,5)-trisphosphate-sensitive intracellular calcium stores and by promoting influx of extracellular calcium. Concentrations of all three agents that increased [Ca2+]i potentiated the stimulation of enzyme secretion caused by secretagogues that activate adenylate cyclase but inhibited the stimulation of enzyme secretion caused by secretagogues that activate phospholipase C. With BHQ, potentiation of adenylate cyclase-mediated enzyme secretion occurred immediately whereas inhibition of phospholipase C-mediated enzyme secretion occurred only after several min of incubation. In addition, the effects of BHQ and CPA on both [Ca2+]i and secretagogue-stimulated enzyme secretion were reversed completely by washing whereas the actions of TG could not be reversed by washing. Concentrations of BHQ in excess of those that caused maximal changes in [Ca2+]i inhibited all modes of stimulated enzyme secretion by a mechanism that was apparently unrelated to changes in [Ca2+]i. Finally, in contrast to the findings with TG and BHQ, CPA inhibited bombesin-stimulated enzyme secretion over a range of concentrations that was at least 10-fold lower than the range of concentrations over which CPA potentiated VIP-stimulated enzyme secretion.

  12. In vitro degradation of a 3D porous Pennisetum purpureum/PLA biocomposite scaffold.

    PubMed

    Revati, R; Majid, M S Abdul; Ridzuan, M J M; Basaruddin, K S; Rahman Y, M N; Cheng, E M; Gibson, A G

    2017-10-01

    The in vitro degradation and mechanical properties of a 3D porous Pennisetum purpureum (PP)/polylactic acid (PLA)-based scaffold were investigated. In this study, composite scaffolds with PP to PLA ratios of 0%, 10%, 20%, and 30% were immersed in a PBS solution at 37°C for 40 days. Compression tests were conducted to evaluate the compressive strength and modulus of the scaffolds, according to ASTM F451-95. The compression strength of the scaffolds was found to increase from 1.94 to 9.32MPa, while the compressive modulus increased from 1.73 to 5.25MPa as the fillers' content increased from 0wt% to 30wt%. Moreover, field emission scanning electron microscopy (FESEM) and X-ray diffraction were employed to observe and analyse the microstructure and fibre-matrix interface. Interestingly, the degradation rate was reduced for the PLA/PP 20 scaffold, though insignificantly, this could be attributed to the improved mechanical properties and stronger fibre-matrix interface. Microstructure changes after degradation were observed using FESEM. The FESEM results indicated that a strong fibre-matrix interface was formed in the PLA/PP 20 scaffold, which reflected the addition of P. purpureum into PLA decreasing the degradation rate compared to in pure PLA scaffolds. The results suggest that the P. purpureum/PLA scaffold degradation rate can be altered and controlled to meet requirements imposed by a given tissue engineering application. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Blending Novatein{sup ®} thermoplastic protein with PLA for carbon dioxide assisted batch foaming

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

    Walallavita, Anuradha, E-mail: asw15@students.waikato.ac.nz; Verbeek, Casparus J. R., E-mail: jverbeek@waikato.ac.nz; Lay, Mark, E-mail: mclay@waikato.ac.nz

    2016-03-09

    The convenience of polymeric foams has led to their widespread utilisation in everyday life. However, disposal of synthetic petroleum-derived foams has had a detrimental effect on the environment which needs to be addressed. This study uses a clean and sustainable approach to investigate the foaming capability of a blend of two biodegradable polymers, polylactic acid (PLA) and Novatein® Thermoplastic Protein (NTP). PLA, derived from corn starch, can successfully be foamed using a batch technique developed by the Biopolymer Network Ltd. NTP is a patented formulation of bloodmeal and chemical additives which can be extruded and injection moulded similar to othermore » thermoplastics. However, foaming NTP is a new area of study and its interaction with blowing agents in the batch process is entirely unknown. Subcritical and supercritical carbon dioxide have been examined individually in two uniquely designed pressure vessels to foam various compositions of NTP-PLA blends. Foamed material were characterised in terms of expansion ratio, cell size, and cellular morphology in order to study how the composition of NTP-PLA affects foaming with carbon dioxide. It was found that blends with 5 wt. % NTP foamed using subcritical CO{sub 2} expanded up to 11 times due to heterogeneous nucleation. Morphology analysis using scanning electron microscopy showed that foams blown with supercritical CO{sub 2} had a finer cell structure with consistent cell size, whereas, foams blown with subcritical CO{sub 2} ranged in cell size and showed cell wall rupture. Ultimately, this research would contribute to the production of a biodegradable foam material to be used in packaging applications, thereby adding to the application potential of NTP.« less

  14. Investigation of Ultrasonics as a tool for energy efficient recycling of Lactic acid from postconsumer PLA products

    NASA Astrophysics Data System (ADS)

    Srinivasan, Gowrishankar

    crystallinity also was an important factor that affected the reaction kinetics of depolymerization. It was found that amorphous PLA de-polymerized faster compared to semi-crystalline PLA under the same conditions. While the depolymerization of PLA was anticipated to require 15 to 30 minutes, or extreme conditions [40], it was determined that with K2CO3 or NaOH catalysts and methanol media as the conditions, PLA could be fully de-polymerized within a few minutes. This information provided insight for effective pathways for the depolymerization of PLA, reducing the environmental impact of material use on the environment. The effects of the ultrasonics were modeled with finite element analysis based on fundamental concepts. The predictions from the modeling were confirmed by studying real-time streaming and fluid flow inside the treatment cell utilizing particle image Velocimetry (PIV). The FEA (finite element analysis) models of ultrasonic streaming were verified and were in reasonable agreement with the experimental values, validating simple assumptions for future researchers.

  15. Large-scale aerosol-assisted synthesis of biofriendly Fe2O3 yolk-shell particles: a promising support for enzyme immobilization

    NASA Astrophysics Data System (ADS)

    Patel, Sanjay K. S.; Choi, Seung Ho; Kang, Yun Chan; Lee, Jung-Kul

    2016-03-01

    Multiple-shelled Fe2O3 yolk-shell particles were synthesized using the spray drying method and intended as a suitable support for the immobilization of commercial enzymes such as glucose oxidase (GOx), horseradish peroxidase (HRP), and laccase as model enzymes. Yolk-shell particles have an average diameter of 1-3 μm with pore diameters in the range of 16 to 28 nm. The maximum immobilization of GOx, HRP, and laccase resulted in the enzyme loading of 292, 307 and 398 mg per g of support, respectively. After cross-linking of immobilized laccase by glutaraldehyde, immobilization efficiency was improved from 83.5% to 90.2%. Km and Vmax values were 41.5 μM and 1722 μmol min-1 per mg protein for cross-linked laccase and those for free laccase were 29.3 μM and 1890 μmol min-1 per mg protein, respectively. The thermal stability of the enzyme was enhanced up to 18-fold upon cross-linking, and the enzyme retained 93.1% of residual activity after ten cycles of reuse. The immobilized enzyme has shown up to 32-fold higher stability than the free enzyme towards different solvents and it showed higher efficiency than free laccase in the decolorization of dyes and degradation of bisphenol A. The synthesized yolk-shell particles have 3-fold higher enzyme loading efficiency and lower acute toxicity than the commercial Fe2O3 spherical particles. Therefore, the use of unique yolk-shell structure Fe2O3 particles with multiple-shells will be promising for the immobilization of various enzymes in biotechnological applications with improved electrochemical properties. To the best of our knowledge, this is the first report on the use of one pot synthesized Fe2O3 yolk-shell structure particles for the immobilization of enzymes.Multiple-shelled Fe2O3 yolk-shell particles were synthesized using the spray drying method and intended as a suitable support for the immobilization of commercial enzymes such as glucose oxidase (GOx), horseradish peroxidase (HRP), and laccase as model enzymes

  16. Structural Characterization of Myotoxic Ecarpholin S From Echis carinatus Venom

    PubMed Central

    Zhou, Xingding; Tan, Tien-Chye; Valiyaveettil, S.; Go, Mei Lin; Kini, R. Manjunatha; Velazquez-Campoy, Adrian; Sivaraman, J.

    2008-01-01

    Phospholipase A2 (PLA2), a common toxic component of snake venom, has been implicated in various pharmacological effects. Ecarpholin S, isolated from the venom of the snake Echis carinatus sochureki, is a phospholipase A2 (PLA2) belonging to the Ser49-PLA2 subgroup. It has been characterized as having low enzymatic but potent myotoxic activities. The crystal structures of native ecarpholin S and its complexes with lauric acid, and its inhibitor suramin, were elucidated. This is the first report of the structure of a member of the Ser49-PLA2 subgroup. We also examined interactions of ecarpholin S with phosphatidylglycerol and lauric acid, using surface plasmon resonance, and of suramin with isothermal titration calorimetry. Most Ca2+-dependent PLA2 enzymes have Asp in position 49, which plays a crucial role in Ca2+ binding. The three-dimensional structure of ecarpholin S reveals a unique conformation of the Ca2+-binding loop that is not favorable for Ca2+ coordination. Furthermore, the endogenously bound fatty acid (lauric acid) in the hydrophobic channel may also interrupt the catalytic cycle. These two observations may account for the low enzymatic activity of ecarpholin S, despite full retention of the catalytic machinery. These observations may also be applicable to other non-Asp49-PLA2 enzymes. The interaction of suramin in its complex with ecarpholin S is quite different from that reported for the Lys49-PLA2/suramin complex, where the interfacial recognition face (i-face), C-terminal region, and N-terminal region of ecarpholin S play important roles. This study provides significant structural and functional insights into the myotoxic activity of ecarpholin S and, in general, of non-Asp49-PLA2 enzymes. PMID:18586854

  17. Structural Characterization of Myotoxic Ecarpholin S From Echis carinatus Venom

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

    Zhou, X.; Tan, T; Valiyaveettil, S

    2008-01-01

    Phospholipase A2 (PLA2), a common toxic component of snake venom, has been implicated in various pharmacological effects. Ecarpholin S, isolated from the venom of the snake Echis carinatus sochureki, is a phospholipase A2 (PLA2) belonging to the Ser49-PLA2 subgroup. It has been characterized as having low enzymatic but potent myotoxic activities. The crystal structures of native ecarpholin S and its complexes with lauric acid, and its inhibitor suramin, were elucidated. This is the first report of the structure of a member of the Ser49-PLA2 subgroup. We also examined interactions of ecarpholin S with phosphatidylglycerol and lauric acid, using surface plasmonmore » resonance, and of suramin with isothermal titration calorimetry. Most Ca2+-dependent PLA2 enzymes have Asp in position 49, which plays a crucial role in Ca2+ binding. The three-dimensional structure of ecarpholin S reveals a unique conformation of the Ca2+-binding loop that is not favorable for Ca2+ coordination. Furthermore, the endogenously bound fatty acid (lauric acid) in the hydrophobic channel may also interrupt the catalytic cycle. These two observations may account for the low enzymatic activity of ecarpholin S, despite full retention of the catalytic machinery. These observations may also be applicable to other non-Asp49-PLA2 enzymes. The interaction of suramin in its complex with ecarpholin S is quite different from that reported for the Lys49-PLA2/suramin complex, where the interfacial recognition face (i-face), C-terminal region, and N-terminal region of ecarpholin S play important roles. This study provides significant structural and functional insights into the myotoxic activity of ecarpholin S and, in general, of non-Asp49-PLA2 enzymes.« less

  18. E2 potentializes benzo(a)pyrene-induced hepatic cytochrome P450 enzyme activities in Nile tilapia at high concentrations.

    PubMed

    Rodrigues, Aline Cristina Ferreira; Moneró, Tatiana de Oliveira; Frighetto, Rosa Toyoko Shiraishi; de Almeida, Eduardo Alves

    2015-11-01

    In the aquatic environment, biotransformation enzymes are established biomarkers for assessing PAH exposure in fish, but little is known about the effect of 17β-estradiol (E2) on these enzymes during exposure to benzo(a)pyrene (BaP). In this study, Nile tilapia (Oreochromis niloticus) were exposed for 3, 5, and 10 days to BaP (300 μg L(-1)) and E2 (5 μg L(-1)). These substances were applied isolated or mixed. In the mixture experiment, fish were analyzed pre- and postexposure in order to better understand whether preexposure to the hormone masks the responses activated by PAH or vice versa. Phase I enzymes ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-depenthylase (PROD), and benzyloxyresorufin-O-debenzylase (BROD) activities as well as the phase II enzyme glutathione S-transferase (GST) were analyzed. Isolated E2 treatment decreased EROD activity after 3 days, but this enzyme activity returned to control values after 5 and 10 days of exposure. Isolated BaP treatment significantly induced EROD activity after 3 and 5 days, and the activity returned to control levels after ten exposure days. Combined treatment (E2 + Bap) significantly increased EROD activity, both in the pre- and postexposure. This increase was even higher than in the isolated BaP treatment, suggesting a synergism between these two compounds. When E2 and BaP were used singly, they did not change BROD and PROD activities. However, combined treatment (E2 + Bap) significantly increased PROD activity. Isolated BaP treatment increased GST activity after 10 days. However, this response was not observed in the mixture treatment, suggesting that E2 suppressed the GST induction modulated by BaP. The results put together indicated that E2 altered the biotransformation pathway regarding enzymes activated by BaP in Nile tilapia.

  19. Conformational Dynamics Modulate Activation of the Ubiquitin Conjugating Enzyme Ube2g2

    PubMed Central

    2017-01-01

    The ubiquitin conjugating enzyme Ube2g2 together with its cognate E3 ligase gp78 catalyzes the synthesis of lysine-48 polyubiquitin chains constituting signals for the proteasomal degradation of misfolded proteins in the endoplasmic reticulum. Here, we employ NMR spectroscopy in combination with single-turnover diubiquitin formation assays to examine the role of the RING domain from gp78 in the catalytic activation of Ube2g2∼Ub conjugates. We find that approximately 60% of the Ube2g2∼Ub conjugates occupy a closed conformation in the absence of gp78-RING, with the population increasing to 82% upon gp78-RING binding. As expected, strong mutations in the hydrophobic patch residues of the ∼Ub moiety result in Ube2g2∼Ub populating only open states with corresponding loss of the ubiquitin conjugation activity. Less disruptive mutations introduced into the hydrophobic patch of the ∼Ub moiety also destabilize the closed conformational state, yet the corresponding effect on the ubiquitin conjugation activity ranges from complete loss to an enhancement of the catalytic activity. These results present a picture in which Ube2g2’s active site is in a state of continual dynamic flux with the organization of the active site into a catalytically viable conformation constituting the rate-limiting step for a single ubiquitin ligation event. Ube2g2’s function as a highly specific K48-polyubiquitin chain elongator leads us to speculate that this may be a strategy by which Ube2g2 reduces the probability of nonproductive catalytic outcomes in the absence of available substrate. PMID:28884161

  20. Composite porous scaffold of PEG/PLA support improved bone matrix deposition in vitro compared to PLA-only scaffolds.

    PubMed

    Bhaskar, Birru; Owen, Robert; Bahmaee, Hossein; Wally, Zena; Sreenivasa Rao, Parcha; Reilly, Gwendolen C

    2018-05-01

    Controllable pore size and architecture are essential properties for tissue-engineering scaffolds to support cell ingrowth colonization. To investigate the effect of polyethylene glycol (PEG) addition on porosity and bone-cell behavior, porous polylactic acid (PLA)-PEG scaffolds were developed with varied weight ratios of PLA-PEG (100/0, 90/10, 75/25) using solvent casting and porogen leaching. Sugar 200-300 µm in size was used as a porogen. To assess scaffold suitability for bone tissue engineering, MLO-A5 murine osteoblast cells were cultured and cell metabolic activity, alkaline phosphatase (ALP) activity and bone-matrix production determined using (alizarin red S staining for calcium and direct red 80 staining for collagen). It was found that metabolic activity was significantly higher over time on scaffolds containing PEG, ALP activity and mineralized matrix production were also significantly higher on scaffolds containing 25% PEG. Porous architecture and cell distribution and penetration into the scaffold were analyzed using SEM and confocal microscopy, revealing that inclusion of PEG increased pore interconnectivity and therefore cell ingrowth in comparison to pure PLA scaffolds. The results of this study confirmed that PLA-PEG porous scaffolds support mineralizing osteoblasts better than pure PLA scaffolds, indicating they have a high potential for use in bone tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1334-1340, 2018. © 2018 Wiley Periodicals, Inc.

  1. Construction and Characterization of a Chitosan-Immobilized-Enzyme and β-Cyclodextrin-Included-Ferrocene-Based Electrochemical Biosensor for H2O2 Detection

    PubMed Central

    Dong, Wenbo; Wang, Kaiyin; Chen, Yu; Li, Weiping; Ye, Yanchun; Jin, Shaohua

    2017-01-01

    An electrochemical detection biosensor was prepared with the chitosan-immobilized-enzyme (CTS-CAT) and β-cyclodextrin-included-ferrocene (β-CD-FE) complex for the determination of H2O2. Ferrocene (FE) was included in β-cyclodextrin (β-CD) to increase its stability. The structure of the β-CD-FE was characterized. The inclusion amount, inclusion rate, and electrochemical properties of inclusion complexes were determined to optimize the reaction conditions for the inclusion. CTS-CAT was prepared by a step-by-step immobilization method, which overcame the disadvantages of the conventional preparation methods. The immobilization conditions were optimized to obtain the desired enzyme activity. CTS-CAT/β-CD-FE composite electrodes were prepared by compositing the CTS-CAT with the β-CD-FE complex on a glassy carbon electrode and used for the electrochemical detection of H2O2. It was found that the CTS-CAT could produce a strong reduction peak current in response to H2O2 and the β-CD-FE could amplify the current signal. The peak current exhibited a linear relationship with the H2O2 concentration in the range of 1.0 × 10−7–6.0 × 10−3 mol/L. Our work provided a novel method for the construction of electrochemical biosensors with a fast response, good stability, high sensitivity, and a wide linear response range based on the composite of chitosan and cyclodextrin. PMID:28773229

  2. A short history of RubisCO: the rise and fall (?) of Nature's predominant CO2 fixing enzyme.

    PubMed

    Erb, Tobias J; Zarzycki, Jan

    2018-02-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is arguably one of the most abundant proteins in the biosphere and a key enzyme in the global carbon cycle. Although RubisCO has been intensively studied, its evolutionary origins and rise as Nature's most dominant carbon dioxide (CO 2 )-fixing enzyme still remain in the dark. In this review we will bring together biochemical, structural, physiological, microbiological, as well as phylogenetic data to speculate on the evolutionary roots of the CO 2 -fixation reaction of RubisCO, the emergence of RubisCO-based autotrophic CO 2 -fixation in the context of the Calvin-Benson-Bassham cycle, and the further evolution of RubisCO into the 'RubisCOsome', a complex of various proteins assembling and interacting with the enzyme to improve its operational capacity (functionality) under different biological and environmental conditions. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Tissue Expressions of Soluble Human Epoxide Hydrolase-2 Enzyme in Patients with Temporal Lobe Epilepsy.

    PubMed

    Ahmedov, Merdin Lyutviev; Kemerdere, Rahsan; Baran, Oguz; Inal, Berrin Bercik; Gumus, Alper; Coskun, Cihan; Yeni, Seher Naz; Eren, Bulent; Uzan, Mustafa; Tanriverdi, Taner

    2017-10-01

    We sought to simply demonstrate how levels of soluble human epoxide hydrolase-2 show changes in both temporal the cortex and hippocampal complex in patients with temporal lobe epilepsy. A total of 20 patients underwent anterior temporal lobe resection due to temporal lobe epilepsy. The control group comprised 15 people who died in traffic accidents or by falling from a height, and their autopsy findings were included. Adequately sized temporal cortex and hippocampal samples were removed from each patient during surgery, and the same anatomic structures were removed from the control subjects during the autopsy procedures. Each sample was stored at -80°C as rapidly as possible until the enzyme assay. The temporal cortex in the epilepsy patients had a significantly higher enzyme level than did the temporal cortex of the control group (P = 0.03). Correlation analysis showed that as the enzyme level increases in the temporal cortex, it also increases in the hippocampal complex (r 2  = 0.06, P = 0.00001). More important, enzyme tissue levels showed positive correlations with seizure frequency in both the temporal cortex and hippocampal complex in patients (r 2  = 0.7, P = 0.00001 and r 2  = 0.4, P = 0.003, respectively). The duration of epilepsy was also positively correlated with the hippocampal enzyme level (r 2  = 0.06, P = 0.00001). Soluble human epoxy hydrolase enzyme-2 is increased in both lateral and medial temporal tissues in temporal lobe epilepsy. Further studies should be conducted as inhibition of this enzyme has resulted in a significant decrease in or stopping of seizures and attenuated neuroinflammation in experimental epilepsy models in the current literature. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. 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.

  5. Characterization and Structural Analysis of a Novel exo-Type Enzyme Acting on β-1,2-Glucooligosaccharides from Parabacteroides distasonis.

    PubMed

    Shimizu, Hisaka; Nakajima, Masahiro; Miyanaga, Akimasa; Takahashi, Yuta; Tanaka, Nobukiyo; Kobayashi, Kaito; Sugimoto, Naohisa; Nakai, Hiroyuki; Taguchi, Hayao

    2018-05-25

    β-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-β-1,2-glucanase from Chitinophaga pinensis ( CpSGL) as an enzyme comprising a new family. Here, we report the characteristics and crystal structure of a CpSGL homologue from Parabacteroides distasonis, an intestinal bacterium (BDI_3064 protein), which exhibits distinctive properties of known β-1,2-glucan-degrading enzymes. BDI_3064 hydrolyzed linear β-1,2-glucan and β-1,2-glucooligosaccharides with degrees of polymerization (DPs) of ≥4 to produce sophorose specifically but did not hydrolyze cyclic β-1,2-glucan. This result indicates that BDI_3064 is a new exo-type enzyme. BDI_3064 also produced sophorose from β-1,2-glucooligosaccharide analogues that have a modified reducing end, indicating that BDI_3064 acts on its substrates from the nonreducing end. The crystal structure showed that BDI_3064 possesses additional N-terminal domains 1 and 2, unlike CpSGL. Superimposition of BDI_3064 and CpSGL complexed with ligands showed that R93 in domain 1 overlapped subsite -3 in CpSGL. Docking analysis involving a β-1,2-glucooligosaccharide with DP4 showed that R93 completely blocks the nonreducing end of the docked β-1,2-glucooligosaccharide. This indicates that BDI_3064 employs a distinct mechanism of recognition at the nonreducing end of substrates to act as an exo-type enzyme. Thus, we propose 2-β-d-glucooligosaccharide sophorohydrolase (nonreducing end) as a systematic name for BDI_3064.

  6. A Study of the Crystallization, Melting, and Foaming Behaviors of Polylactic Acid in Compressed CO2

    PubMed Central

    Zhai, Wentao; Ko, Yoorim; Zhu, Wenli; Wong, Anson; Park, Chul B.

    2009-01-01

    The crystallization and melting behaviors of linear polylactic acid (PLA) treated by compressed CO2 was investigated. The isothermal crystallization test indicated that while PLA exhibited very low crystallization kinetics under atmospheric pressure, CO2 exposure significantly increased PLA’s crystallization rate; a high crystallinity of 16.5% was achieved after CO2 treatment for only 1 min at 100 °C and 6.89 MPa. One melting peak could be found in the DSC curve, and this exhibited a slight dependency on treatment times, temperatures, and pressures. PLA samples tended to foam during the gas release process, and a foaming window as a function of time and temperature was established. Based on the foaming window, crystallinity, and cell morphology, it was found that foaming clearly reduced the needed time for PLA’s crystallization equilibrium. PMID:20054476

  7. Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM.

    PubMed

    Zheng, Zhaojuan; Ma, Cuiqing; Gao, Chao; Li, Fengsong; Qin, Jiayang; Zhang, Haiwei; Wang, Kai; Xu, Ping

    2011-04-20

    Phenyllactic acid (PLA), a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA) into PLA are equivocal. A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l(-1)) and high productivity (2.3 g l(-1) h(-1)) under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA. Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering.

  8. In vitro degradation of MAO/PLA coating on Mg-1.21Li-1.12Ca-1.0Y alloy

    NASA Astrophysics Data System (ADS)

    Zeng, Rong-Chang; Qi, Wei-Chen; Song, Ying-Wei; He, Qin-Kun; Cui, Hong-Zhi; Han, En-Hou

    2014-12-01

    Magnesium and its alloys are promising biomaterials due to their biocompatibility and osteoinduction. The plasticity and corrosion resistance of commercial magnesium alloys cannot meet the requirements for degradable biomaterials completely at present. Particularly, the alkalinity in the microenvironment surrounding the implants, resulting from the degradation, arouses a major concern. Micro-arc oxidation (MAO) and poly(lactic acid) (PLA) composite (MAO/PLA) coating on biomedical Mg-1.21Li-1.12Ca-1.0Y alloy was prepared to manipulate the pH variation in an appropriate range. Surface morphologies were discerned using SEM and EMPA. And corrosion resistance was evaluated via electrochemical polarization and impedance and hydrogen volumetric method. The results demonstrated that the MAO coating predominantly consisted of MgO, Mg2SiO4 and Y2O3. The composite coating markedly improved the corrosion resistance of the alloy. The rise in solution pH for the MAO/PLA coating was tailored to a favorable range of 7.5-7.8. The neutralization caused by the alkalinity of MAO and Mg substrate and acidification of PLA was probed. The result designates that MAO/PLA composite coating on Mg-1.21Li-1.12Ca-1.0Y alloys may be a promising biomedical coating.

  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. Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia.

    PubMed

    Smith, Clyde A; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B

    2014-06-01

    The bifunctional acetyltransferase(6')-Ie-phosphotransferase(2'')-Ia [AAC(6')-Ie-APH(2'')-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2'')-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2'')-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2'')-IIa and APH(2'')-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2'')-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2'')-IIIa enzyme. In APH(2'')-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2'')-Ia into a dual-specificity enzyme.

  11. Beyond the Strait: PLA Missions Other Than Taiwan

    DTIC Science & Technology

    2009-04-01

    modernization efforts through increased operational and cultural experience. In some cases , China also seeks specific diplomatic gains vis-à- vis...strategic ambiguity about the use of the PLA in cases of domestic unrest. The resolution of this dilemma surrounding the minimal use of the PLA in support...much of the PLA was destroyed and how much of China’s infrastructure or economic assets were destroyed). 34 In any case , the outcome would be a “major

  12. 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.

  13. AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) METHOD FOR THE URINARY BIOMONITORING OF 2,4-DICHLOROPHRENOCYACETIC ACID (2,4-D)

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline, 0.05% Tween 20, with 0.02% sodium azide, and analyzed by a 96-microwekk pl...

  14. Physical and mechanical properties of PLA, and their functions in widespread applications - A comprehensive review.

    PubMed

    Farah, Shady; Anderson, Daniel G; Langer, Robert

    2016-12-15

    Poly(lactic acid) (PLA), so far, is the most extensively researched and utilized biodegradable aliphatic polyester in human history. Due to its merits, PLA is a leading biomaterial for numerous applications in medicine as well as in industry replacing conventional petrochemical-based polymers. The main purpose of this review is to elaborate the mechanical and physical properties that affect its stability, processability, degradation, PLA-other polymers immiscibility, aging and recyclability, and therefore its potential suitability to fulfill specific application requirements. This review also summarizes variations in these properties during PLA processing (i.e. thermal degradation and recyclability), biodegradation, packaging and sterilization, and aging (i.e. weathering and hygrothermal). In addition, we discuss up-to-date strategies for PLA properties improvements including components and plasticizer blending, nucleation agent addition, and PLA modifications and nanoformulations. Incorporating better understanding of the role of these properties with available improvement strategies is the key for successful utilization of PLA and its copolymers/composites/blends to maximize their fit with worldwide application needs. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Bone sialoprotein binding to matrix metalloproteinase-2 alters enzyme inhibition kinetics†

    PubMed Central

    Jain, Alka; Fisher, Larry W.; Fedarko, Neal S.

    2008-01-01

    Bone sialoprotein (BSP) is a secreted glycophosphoprotein normally restricted in expression to skeletal tissue that is also induced by multiple neoplasms in vivo. Previous work has shown that BSP can bind to matrix metalloproteinase-2 (MMP-2). Because of MMP-2 activity in promoting tumor progression, potential therapeutic inhibitors were developed, but clinical trials have been disappointing. The effect of BSP on MMP-2 modulation by inhibitors was determined with purified components and in cell culture. Enzyme inhibition kinetics were studied using a low-molecular weight freely diffusable substrate and purified MMP-2, BSP, and natural (tissue inhibitor of matrix metalloproteinase-2) and synthetic (ilomastat and oleoyl-N-hydroxylamide) inhibitors. We determined parameters of enzyme kinetics by varying substrate concentrations at different fixed inhibitor concentrations added to MMP-2 alone, MMP-2 and BSP, or preformed MMP-2-BSP complexes and solving a general linear mixed inhibition rate equation with a global curve fitting program. Two in vitro angiogenesis model systems employing human umbilical vein endothelial cells (HUVECs) were used to follow BSP modulation of MMP-2 inhibition and tubule formation. The presence of BSP increased the competitive KI values between 15- and 47-fold for natural and synthetic inhibitors. The extent of tubule formation by HUVECs cocultured with dermal fibroblasts was reduced in the presence of inhibitors, while the addition of BSP restored vessel formation. A second HUVEC culture system demonstrated that tubule formation by cells expressing BSP could be inhibited by an activity blocking antibody against MMP-2. BSP modulation of MMP-2 activity and inhibition may define its biological role in promoting tumor progression. PMID:18465841

  16. Bone sialoprotein binding to matrix metalloproteinase-2 alters enzyme inhibition kinetics.

    PubMed

    Jain, Alka; Fisher, Larry W; Fedarko, Neal S

    2008-06-03

    Bone sialoprotein (BSP) is a secreted glycophosphoprotein normally restricted in expression to skeletal tissue that is also induced by multiple neoplasms in vivo. Previous work has shown that BSP can bind to matrix metalloproteinase-2 (MMP-2). Because of MMP-2 activity in promoting tumor progression, potential therapeutic inhibitors were developed, but clinical trials have been disappointing. The effect of BSP on MMP-2 modulation by inhibitors was determined with purified components and in cell culture. Enzyme inhibition kinetics were studied using a low-molecular weight freely diffusable substrate and purified MMP-2, BSP, and natural (tissue inhibitor of matrix metalloproteinase-2) and synthetic (ilomastat and oleoyl- N-hydroxylamide) inhibitors. We determined parameters of enzyme kinetics by varying substrate concentrations at different fixed inhibitor concentrations added to MMP-2 alone, MMP-2 and BSP, or preformed MMP-2-BSP complexes and solving a general linear mixed inhibition rate equation with a global curve fitting program. Two in vitro angiogenesis model systems employing human umbilical vein endothelial cells (HUVECs) were used to follow BSP modulation of MMP-2 inhibition and tubule formation. The presence of BSP increased the competitive K I values between 15- and 47-fold for natural and synthetic inhibitors. The extent of tubule formation by HUVECs cocultured with dermal fibroblasts was reduced in the presence of inhibitors, while the addition of BSP restored vessel formation. A second HUVEC culture system demonstrated that tubule formation by cells expressing BSP could be inhibited by an activity blocking antibody against MMP-2. BSP modulation of MMP-2 activity and inhibition may define its biological role in promoting tumor progression.

  17. Prostate Cancer Prevention Through Induction of Phase 2 Enzymes

    DTIC Science & Technology

    1999-10-01

    hypothesis. We have identified sulforaphane , a dietary isothiocyanate found in cucifers, as the most potent phase 2 enzyme inducing agent in human prostate...cancer cell lines compared to over 50 other compounds screened in our laboratory. Sulforaphane readily induced increased expression of quinone...characterizing global changes in mRNA expression for nearly 10,000 genes simultaneously using cDNA microarrays after treatment of prostate cells with sulforaphane

  18. Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity[S

    PubMed Central

    Jaishy, Bharat; Zhang, Quanjiang; Chung, Heaseung S.; Riehle, Christian; Soto, Jamie; Jenkins, Stephen; Abel, Patrick; Cowart, L. Ashley; Van Eyk, Jennifer E.; Abel, E. Dale

    2015-01-01

    Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs. PMID:25529920

  19. Involvement of MAPKs, NF-{kappa}B and p300 co-activator in IL-1{beta}-induced cytosolic phospholipase A{sub 2} expression in canine tracheal smooth muscle cells

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

    Luo, S.-F.; Lin, C.-C.; Chen, H.-C.

    2008-11-01

    Cytosolic phospholipase A{sub 2} (cPLA{sub 2}) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during stimulation with interleukin-1{beta} (IL-1{beta}). However, the mechanisms underlying IL-1{beta}-induced cPLA{sub 2} expression and PGE{sub 2} synthesis by canine tracheal smooth muscle cells (CTSMCs) have not been defined. IL-1{beta} induced cPLA{sub 2} protein and mRNA expression, PGE{sub 2} production, and phosphorylation of p42/p44 MAPK, p38 MAPK (ATF{sub 2}), and JNK (c-Jun) in a time- and concentration-dependent manner, determined by Western blotting, RT-PCR, and ELISA, which was attenuated by the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), ormore » transfection with dominant negative mutants of MEK1/2, p38, and JNK, respectively. Furthermore, IL-1{beta}-induced cPLA{sub 2} expression and PGE{sub 2} synthesis was inhibited by a selective NF-{kappa}B inhibitor (helenalin) or transfection with dominant negative mutants of NF-{kappa}B inducing kinase (NIK), I{kappa}B kinase (IKK)-{alpha}, and IKK-{beta}. Consistently, IL-1{beta} stimulated both I{kappa}B-{alpha} degradation and NF-{kappa}B translocation into nucleus in these cells. NF-{kappa}B translocation was blocked by helenalin, but not by U0126, SB202190, and SP600125. MAPKs together with NF-{kappa}B-activated p300 recruited to cPLA{sub 2} promoter thus facilitating the binding of NF-{kappa}B to cPLA{sub 2} promoter region and expression of cPLA{sub 2} mRNA. IL-1{beta}-induced cPLA{sub 2} expression and PGE{sub 2} production was inhibited by actinomycin D and cycloheximide, indicating the involvement of transcriptional and translational events in these responses. These results suggest that in CTSMCs, IL-1{beta}-induced cPLA{sub 2} expression and PGE{sub 2} synthesis was independently mediated through activation of MAPKs and NF-{kappa}B pathways and was connected to p300 recruitment and activation.« less

  20. CYP2J2 and CYP2C19 Are the Major Enzymes Responsible for Metabolism of Albendazole and Fenbendazole in Human Liver Microsomes and Recombinant P450 Assay Systems

    PubMed Central

    Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk

    2013-01-01

    Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo. PMID:23959307

  1. CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

    PubMed

    Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk; Lee, Taeho; Liu, Kwang-Hyeon

    2013-11-01

    Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

  2. Associated liver enzymes with hyperlipidemic profile in type 2 diabetes patients.

    PubMed

    Al-Jameil, Noura; Khan, Farah A; Arjumand, Sadia; Khan, Mohammad F; Tabassum, Hajera

    2014-01-01

    Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia and is associated with dyslipidemia and disturbed liver function. Aim of the present work is to assess the liver enzymes and to find its association with hyperlipidemic profile in T2DM. Total of 157 subjects were studied and divided into two groups; diabetes (n=81) and non-diabetes (n=76). Various biochemical parameters like fasting glucose, post prandial glucose, HbA1c, total cholesterol (TC), triglycerides (Tg), high density lipoprotein cholesterol (HDL-C), alanine amino transferase (ALT), aspartate amino transferase (AST) and gamma-glutamyl transferase (GGT) were analyzed by ROCHE module Cobas 6000 (C501 & C601) analyzer, kits were procured by ROCHE diagnostics. Low density lipoprotein cholesterol (LDL-C) was estimated by Freidwald's formula. Statistical analysis was performed by applying student t test and Pearson's correlation coefficient, at 0.0001 and 0.05 level of significance, respectively. All the glycemic control parameters, lipid profile parameters except HDL-C and liver enzymes were found increased in diabetes group and significantly differ from non-diabetes group (p>0.0001). ALT showed significant positive correlation with fasting glucose, post prandial glucose, HbA1c, TC, Tg, LDL-C and GGT at p>0.05. AST showed very weak relation with all parameters while GGT was positively associated with fasting glucose, post prandial glucose, HbA1c, TC, Tg, LDL-C and ALT at p>0.05. In conclusion, T2DM incline to elevate liver enzymes, especially ALT and GGT were of significance. Routine screening of ALT and GGT in T2DM patients may assists early detection of liver abnormalities and to arrest the progress of disease.

  3. 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

  4. 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

  5. Experiment K-6-21. Effect of microgravity on 1) metabolic enzymes of type 1 and type 2 muscle fibers and on 2) metabolic enzymes, neutransmitter amino acids, and neurotransmitter associated enzymes in motor and somatosensory cerebral cortex. Part 1: Metabolic enzymes of individual muscle fibers; part 2: metabolic enzymes of hippocampus and spinal cord

    NASA Technical Reports Server (NTRS)

    Lowry, O.; Mcdougal, D., Jr.; Nemeth, Patti M.; Maggie, M.-Y. Chi; Pusateri, M.; Carter, J.; Manchester, J.; Norris, Beverly; Krasnov, I.

    1990-01-01

    The individual fibers of any individual muscle vary greatly in enzyme composition, a fact which is obscured when enzyme levels of a whole muscle are measured. The purpose of this study was therefore to assess the changes due to weightless on the enzyme patterns composed by the individual fibers within the flight muscles. In spite of the limitation in numbers of muscles examined, it is apparent that: (1) that the size of individual fibers (i.e., their dry weight) was reduced about a third, (2) that this loss in dry mass was accompanied by changes in the eight enzymes studied, and (3) that these changes were different for the two muscles, and different for the two enzyme groups. In the soleus muscle the absolute amounts of the three enzymes of oxidative metabolism decreased about in proportion to the dry weight loss, so that their concentration in the atrophic fibers was almost unchanged. In contrast, there was little loss among the four enzymes of glycogenolysis - glycolysis so that their concentrations were substantially increased in the atrophic fibers. In the TA muscle, these seven enzymes were affected in just the opposite direction. There appeared to be no absolute loss among the oxidative enzymes, whereas the glycogenolytic enzymes were reduced by nearly half, so that the concentrations of the first metabolic group were increased within the atrophic fibers and the concentrations of the second group were only marginally decreased. The behavior of hexokinase was exceptional in that it did not decrease in absolute terms in either type of muscle and probably increased as much as 50 percent in soleus. Thus, their was a large increase in concentration of this enzyme in the atrophied fibers of both muscles. Another clear-cut finding was the large increase in the range of activities of the glycolytic enzymes among individual fibers of TA muscles. This was due to the emergence of TA fibers with activities for enzymes of this group extending down to levels as low as

  6. RNA editing enzyme ADAR2 is a mediator of neuropathic pain after peripheral nerve injury.

    PubMed

    Uchida, Hitoshi; Matsumura, Shinji; Okada, Shunpei; Suzuki, Tsutomu; Minami, Toshiaki; Ito, Seiji

    2017-05-01

    Transcriptional and post-translational regulations are important in peripheral nerve injury-induced neuropathic pain, but little is known about the role of post-transcriptional modification. Our objective was to determine the possible effect of adenosine deaminase acting on RNA (ADAR) enzymes, which catalyze post-transcriptional RNA editing, in tactile allodynia, a hallmark of neuropathic pain. Seven days after L5 spinal nerve transection (SNT) in adult mice, we found an increase in ADAR2 expression and a decrease in ADAR3 expression in the injured, but not in the uninjured, dorsal root ganglions (DRGs). These changes were accompanied by elevated levels of editing at the D site of the serotonin (5-hydroxytryptamine) 2C receptor (5-HT 2C R), at the I/V site of coatomer protein complex subunit α (COPA), and at the R/G site of AMPA receptor subunit GluA2 in the injured DRG. Compared to Adar2 +/+ /Gria2 R/R littermate controls, Adar2 -/- /Gria2 R/R mice completely lacked the increased editing of 5-HT 2C R, COPA, and GluA2 transcripts in the injured DRG and showed attenuated tactile allodynia after SNT. Furthermore, the antidepressant fluoxetine inhibited neuropathic allodynia after injury and reduced the COPA I/V site editing in the injured DRG. These findings suggest that ADAR2 is a mediator of injury-induced tactile allodynia and thus a potential therapeutic target for the treatment of neuropathic pain.-Uchida, H., Matsumura, S., Okada, S., Suzuki, T., Minami, T., Ito, S. RNA editing enzyme ADAR2 is a mediator of neuropathic pain after peripheral nerve injury. © FASEB.

  7. Activation of angiotensin-converting enzyme 2 (ACE2) attenuates allergic airway inflammation in rat asthma model

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

    Dhawale, Vaibhav Shrirang; Amara, Venkateswara Rao

    Angiotensin-I converting enzyme (ACE) is positively correlated to asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS) and is highly expressed in lungs. ACE2, the counteracting enzyme of ACE, was proven to be protective in pulmonary, cardiovascular diseases. In the present study we checked the effect of ACE2 activation in animal model of asthma. Asthma was induced in male wistar rats by sensitization and challenge with ovalbumin and then treated with ACE2 activator, diminazene aceturate (DIZE) for 2 weeks. 48 h after last allergen challenge, animals were anesthetized, blood, BALF, femoral bone marrow lavage were collected for leucocytemore » count; trachea for measuring airway responsiveness to carbachol; lungs and heart were isolated for histological studies and western blotting. In our animal model, the characteristic features of asthma such as altered airway responsiveness to carbachol, eosinophilia and neutrophilia were observed. Western blotting revealed the increased pulmonary expression of ACE1, IL-1β, IL-4, NF-κB, BCL2, p-AKT, p-p38 and decreased expression of ACE2 and IκB. DIZE treatment prevented these alterations. Intraalveolar interstitial thickening, inflammatory cell infiltration, interstitial fibrosis, oxidative stress and right ventricular hypertrophy in asthma control animals were also reversed by DIZE treatment. Activation of ACE2 by DIZE conferred protection against asthma as evident from biochemical, functional, histological and molecular parameters. To the best of our knowledge, we report for the first time that activation of ACE2 by DIZE prevents asthma progression by altering AKT, p38, NF-κB and other inflammatory markers. - Highlights: • Diminazene aceturate (DIZE), an ACE2 activator prevents ovalbumin-induced asthma. • DIZE acted by upregulating ACE2, downregulating ACE1, MAPKs, markers of inflammation, apoptosis. • DIZE reduced airway inflammation, fibrosis, right ventricular

  8. Functional Characterization of the Vitamin K2 Biosynthetic Enzyme UBIAD1

    PubMed Central

    Hirota, Yoshihisa; Nakagawa, Kimie; Sawada, Natsumi; Okuda, Naoko; Suhara, Yoshitomo; Uchino, Yuri; Kimoto, Takashi; Funahashi, Nobuaki; Kamao, Maya; Tsugawa, Naoko; Okano, Toshio

    2015-01-01

    UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a significant role in vitamin K2 (MK-4) synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg2+/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1. PMID:25874989

  9. Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization.

    PubMed

    Behera, Rabindra K; Torres, Rodrigo; Tosha, Takehiko; Bradley, Justin M; Goulding, Celia W; Theil, Elizabeth C

    2015-09-01

    Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe(2+) through intracage ion channels to cage-embedded enzyme (2Fe(2+)/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe(2+) movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one -CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650 nm (DFP λ max). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe(3+)-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: (1) narrower interior ion channel openings/pores; (2) increased numbers of ion channel protein-metal binding sites, and (3) a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells.

  10. Fe2+ Substrate Transport through Ferritin Protein Cage Ion Channels Influences Enzyme Activity and Biomineralization

    PubMed Central

    Behera, Rabindra K.; Torres, Rodrigo; Tosha, Takehiko; Bradley, Justin M.; Goulding, Celia W.; Theil, Elizabeth C.

    2015-01-01

    Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3.H2O), by moving cytoplasmic Fe2+ through intracage ion channels to cage-embedded enzyme (2Fe2+/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe2+ movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one – CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650nm (DFP λmax). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe3+-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: 1. narrower interior ion channel openings/pores, 2. increased numbers of ion channel protein-metal binding sites, and 3. a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells. PMID:26202907

  11. Fracture Surface Morphology and Impact Strength of Cellulose/PLA Composites

    PubMed Central

    Gao, Honghong; Qiang, Tao

    2017-01-01

    Polylactide (PLA)-based composite materials reinforced with ball-milled celluloses were manufactured by extrusion blending followed by injection molding. Their surface morphology from impact fracture were imaged with scanning electron microscopy (SEM) and investigated by calculating their fractal dimensions. Then, linear regression was used to explore the relationship between fractal dimension and impact strength of the resultant cellulose/PLA composite materials. The results show that filling the ball-milled celluloses into PLA can improve the impact toughness of PLA by a minimum of 38%. It was demonstrated that the fracture pattern of the cellulose/PLA composite materials is different from that of pristine PLA. For the resultant composite materials, the fractal dimension of the impact fractured surfaces increased with increasing filling content and decreasing particle size of the ball-milled cellulose particles. There were highly positive correlations between fractal dimension of the fractured surfaces and impact strength of the cellulose/PLA composites. However, the linearity between fractal dimension and impact strength were different for the different methods, due to their different R-squared values. The approach presented in this work will help to understand the structure–property relationships of composite materials from a new perspective. PMID:28772983

  12. Fracture Surface Morphology and Impact Strength of Cellulose/PLA Composites.

    PubMed

    Gao, Honghong; Qiang, Tao

    2017-06-07

    Polylactide (PLA)-based composite materials reinforced with ball-milled celluloses were manufactured by extrusion blending followed by injection molding. Their surface morphology from impact fracture were imaged with scanning electron microscopy (SEM) and investigated by calculating their fractal dimensions. Then, linear regression was used to explore the relationship between fractal dimension and impact strength of the resultant cellulose/PLA composite materials. The results show that filling the ball-milled celluloses into PLA can improve the impact toughness of PLA by a minimum of 38%. It was demonstrated that the fracture pattern of the cellulose/PLA composite materials is different from that of pristine PLA. For the resultant composite materials, the fractal dimension of the impact fractured surfaces increased with increasing filling content and decreasing particle size of the ball-milled cellulose particles. There were highly positive correlations between fractal dimension of the fractured surfaces and impact strength of the cellulose/PLA composites. However, the linearity between fractal dimension and impact strength were different for the different methods, due to their different R-squared values. The approach presented in this work will help to understand the structure-property relationships of composite materials from a new perspective.

  13. Photobiodegradation of chlorinated water pollutants by a combined TiO2-polyaniline-enzyme catalytic system

    NASA Astrophysics Data System (ADS)

    Campanella, Luigi; Crescentini, G.; Militerno, S.

    1995-10-01

    The removal of xenobiotic compounds, such as chlorophenols and pesticides, from municipal and industrial wastewaters is an important task because of the toxicity and the tendency to bioaccumulation of these compounds. Among the several methods proposed, photodegradation catalyzed by suspended inorganic semiconductors (i.e. TiO2) has lately received wide attention because this process is fast, leads to non-toxic final products and shows a high degradation efficiency. In this work, the results obtained in the photodegradation of monochlorophenols using a new catalyst, made of TiO2 and polyaniline both immobilized on a polyvinylchloride (PVC) membrane, in presence (and in absence) of an enzyme are presented. Different enzymes have been tested by adding 5, 10 or 15 U/mL to 50 mL of aqueous solution (1 multiplied by 10-4 mol/L) of o-chloro-phenol containing the catalytic membrane. The samples were irradiated using a QUV panel accelerated weathering tester, which simulates very well the solar radiation up to lambda equals 400 nm and HPLC was used to measure the variation of the compound's concentration with the time. While some enzymes (i.e., peroxidase) do not improve the photodegradation process since they do not survive under the irradiation conditions used, some of them show marked effect both in terms of rate degradation and time required to reach the total degradation of the compound examined. For example, the addition of Laccase reduces the 100% degradation time from 35 hrs to about 20 hrs. Attempts to immobilize the enzyme on the catalytic membrane (by adsorption) have been carried out and the performance of the catalyst with non-immobilized and immobilized enzyme has been studied.

  14. 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.

  15. Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase[OPEN

    PubMed Central

    Niehaus, Thomas D.; Hasnain, Ghulam; Gidda, Satinder K.; Nguyen, Thuy N.D.; Anderson, Erin M.; Brown, Greg; Yakunin, Alexander F.; de Crécy-Lagard, Valérie; Gregory, Jesse F.

    2016-01-01

    To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470. Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms. PMID:27677881

  16. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

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

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

  17. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    DOE PAGES

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; ...

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

  18. A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes

    PubMed Central

    Mulder, Monique P.C.; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N.; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C.O.; Schulman, Brenda A.; Komander, David; Neefjes, Jacques; Oualid, Farid El; Ovaa, Huib

    2016-01-01

    Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers, orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a staggering breadth of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Akin to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe ‘hops’ and ‘traps’ catalytically active ubiquitin-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activities in living cells, presents novel and versatile tools to interrogate the Ub/Ubl cascades. PMID:27182664

  19. Quantitative Determination of Fluorine Content in Blends of Polylactide (PLA)–Talc Using Near Infrared Spectroscopy

    PubMed Central

    Tamburini, Elena; Tagliati, Chiara; Bonato, Tiziano; Costa, Stefania; Scapoli, Chiara; Pedrini, Paola

    2016-01-01

    Near-infrared spectroscopy (NIRS) has been widely used for quantitative and/or qualitative determination of a wide range of matrices. The objective of this study was to develop a NIRS method for the quantitative determination of fluorine content in polylactide (PLA)-talc blends. A blending profile was obtained by mixing different amounts of PLA granules and talc powder. The calibration model was built correlating wet chemical data (alkali digestion method) and NIR spectra. Using FT (Fourier Transform)-NIR technique, a Partial Least Squares (PLS) regression model was set-up, in a concentration interval of 0 ppm of pure PLA to 800 ppm of pure talc. Fluorine content prediction (R2cal = 0.9498; standard error of calibration, SEC = 34.77; standard error of cross-validation, SECV = 46.94) was then externally validated by means of a further 15 independent samples (R2EX.V = 0.8955; root mean standard error of prediction, RMSEP = 61.08). A positive relationship between an inorganic component as fluorine and NIR signal has been evidenced, and used to obtain quantitative analytical information from the spectra. PMID:27490548

  20. Development and evaluation of nanoparticles based on mPEG-PLA for controlled delivery of vinpocetine: in vitro and in vivo studies.

    PubMed

    Wang, Run; Xu, Yong

    2017-02-01

    The aim of present study was to develop VIN-loaded mPEG-PLA nanoparticle systems. The VIN mPEG-PLA nanoparticles were prepared using an emulsion solvent evaporation method, and studied their particle size, morphology, encapsulation efficiency and drug-loading coefficient. Moreover, the nanoparticles were evaluated on the drug release behaviors in vitro and bioavailability in vivo. The results show that the spherical nanoparticles obtained were negatively charged with a zeta potential of about -23.4 mV and characterized ∼110 nm with a narrow size distribution. The encapsulation efficiency and drug loading of prepared NPs were 76.4 ± 6.3 and 9.2 ± 2.2% (n=5), respectively. The in vitro release showed that the percent of accumulated dissolution of VIN NPs in phosphate-buffered saline 6.8 over 24 h was <80%, which was almost 100% of VIN in commercial injections. The in vivo study indicated that systemic absorption of VIN was significantly enhanced by incorporating into mPEG-PLA NPs compared with VIN injection (2.87-fold in AUC 0- t ). The results suggested that the form of VIN in mPEG-PLA NPs could enter the body circulation to perform sustained release in vitro and in vivo.

  1. Biodegradability Evaluation of Polymers by ISO 14855-2

    PubMed Central

    Funabashi, Masahiro; Ninomiya, Fumi; Kunioka, Masao

    2009-01-01

    Biodegradabilities of polymers and their composites in a controlled compost were described. Polycaprolactone (PCL) and poly(lactic acid) (PLA) were employed as biodegradable polymers. Biodegradabilities of PCL and PLA samples in a controlled compost were measured using a Microbial Oxidative Degradation Analyzer (MODA) according to ISO 14855-2. Sample preparation method for biodegradation test according to ISO/DIS 10210 was also described. Effects of sizes and shapes of samples on biodegradability were studied. Reproducibility of biodegradation test of ISO 14855-2 by MODA was confirmed. Validity of sample preparation method for polymer pellets, polymer film, and polymer products of ISO/DIS 10210 for ISO 14855-2 was confirmed. PMID:20111676

  2. Hybrid nanocatalysts containing enzymes and metallic nanoparticles for ethanol/O2 biofuel cell

    NASA Astrophysics Data System (ADS)

    Aquino Neto, S.; Almeida, T. S.; Palma, L. M.; Minteer, S. D.; de Andrade, A. R.

    2014-08-01

    We report the preparation of hybrid nanostructured bioanodes containing the enzyme alcohol dehydrogenase (ADH) with either Au, Pt, or Pt0.75Sn0.25 nanoparticles for use in ethanol/O2 hybrid biofuel cells. We describe two different methodologies for the preparation of the bioanodes: in a first case, multi walled carbon nanotubes (MWCNTs) were employed as a support for the metallic nanoparticles and TBAB-modified Nafion® aided enzyme immobilization. In the second case, we immobilized the enzymes using dendrimers-encapsulated nanoparticles as the agent for enzyme anchoring. The biofuel cell tests showed that the addition of metallic nanoparticles to the bioanode structure enhanced the overall biofuel cell performance. The bioelectrode containing Au nanoparticles displaying the best performance, with an open circuit potential of 0.61 ± 0.05 V and a maximum power density of 155 ± 11 μW cm-2. NADH cyclic voltammetric experiments indicated that Au nanoparticles behaved as a catalyst toward NADH oxidation. Comparing the two protocols we used to synthetized nanoparticles, the sample containing the Au nanoparticles supported on MWCNTs furnished fourfold higher values. Therefore, from the satisfactory results obtained, it can be inferred that the combination of small amounts of metallic nanoparticles with enzymes improve bioanode performance.

  3. OTUB1 Co-opts Lys48-Linked Ubiquitin Recognition to Suppress E2 Enzyme Function

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

    Juang, Yu-Chi; Landry, Marie-Claude; Sanches, Mario

    2012-03-26

    Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibitedmore » E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.« less

  4. 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.

  5. Production, purification and characterization of fibrinolytic enzyme from Serratia sp. KG-2-1 using optimized media.

    PubMed

    Taneja, Kapila; Bajaj, Bijender Kumar; Kumar, Sandeep; Dilbaghi, Neeraj

    2017-07-01

    Intravascular thrombosis is one of the major causes of variety of cardiovascular disorders leading to high mortality worldwide. Fibrinolytic enzymes from microbial sources possess ability to dissolve these clots and help to circumvent these problems in more efficient and safer way. In the present study, fibrinolytic protease with higher fibrinolytic activity than plasmin was obtained from Serratia sp. KG-2-1 isolated from garbage dump soil. Response surface methodology was used to study the interactive effect of concentration of maltose, yeast extract + peptone (1:1), incubation time, and pH on enzyme production and biomass. Maximum enzyme production was achieved at 33 °C after 24 h at neutral pH in media containing 1.5% Maltose, 4.0% yeast extract + peptone and other trace elements resulting in 1.82 folds increased production. The enzyme was purified from crude extract using ammonium sulfate precipitation and DEAE-Sephadex chromatography resulting in 12.9 fold purification with 14.9% yield. The purified enzyme belongs to metalloprotease class and had optimal activity in conditions similar to physiological environment with temperature optima of 40 °C and pH optima of 8. The enzyme was found to be stable in various solvents and its activity was enhanced in presence of Na + , K + , Ba 2+ , Cu 2+ , Mn 2+ , Hg 2+ but inhibited by Ca 2+ and Fe 3+ . Hence, the obtained enzyme may be used as potential therapeutic agent in combating various thrombolytic disorders.

  6. Fabrication of PLA Filaments and its Printable Performance

    NASA Astrophysics Data System (ADS)

    Liu, Wenjie; Zhou, Jianping; Ma, Yuming; Wang, Jie; Xu, Jie

    2017-12-01

    Fused deposition modeling (FDM) is a typical 3D printing technology and preparation of qualified filaments is the basis. In order to prepare polylactic acid (PLA) filaments suitable for personalized FDM 3D printing, this article investigated the effect of factors such as extrusion temperature and screw speed on the diameter, surface roughness and ultimate tensile stress of the obtained PLA filaments. The optimal process parameters for fabrication of qualified filaments were determined. Further, the printable performance of the obtained PLA filaments for 3D objects was preliminarily explored.

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

    PubMed

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

    2016-02-01

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

  8. Purification and Characterization of a Fibrinolytic Enzyme from Bacillus pumilus 2.g Isolated from Gembus, an Indonesian Fermented Food

    PubMed Central

    Afifah, Diana Nur; Sulchan, Muhammad; Syah, Dahrul; Yanti; Suhartono, Maggy Thenawidjaja; Kim, Jeong Hwan

    2014-01-01

    Bacillus pumilus 2.g isolated from gembus, an Indonesian fermented soybean cake, secretes several proteases that have strong fibrinolytic activities. A fibrinolytic enzyme with an apparent molecular weight of 20 kDa was purified from the culture supernatant of B. pumilus 2.g by sequential application of ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic chromatography. The partially purified enzyme was stable between pH 5 and pH 9 and temperature of less than 60°C. Fibrinolytic activity was increased by 5 mM MgCl2 and 5 mM CaCl2 but inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic acid (EDTA). The partially purified enzyme quickly degraded the α and β chains of fibrinogen but was unable to degrade the γ chain. PMID:25320719

  9. Electrospinning synthesis and characterization of PLA-PEG-MNPs composite fibrous membranes

    NASA Astrophysics Data System (ADS)

    Kumar, M.; Klimke, S.; Preiss, A.; Unruh, D.; Wengerowsky, D.; Lehmann, R.; Sindelar, R.; Klingelhöfer, G.; Boča, R.; Renz, F.

    2017-11-01

    An electrospinning technique was used to fabricate PLA, PLA-PEG and PLA-PEG-MNPs composite fibrous membranes. The morphology of electrospun composite membranes were characterized by scanning electron microscope. To test the potential availability of MNPs in PLA-PEG composite membranes, TG, Raman, Mössbauer, VSM and ICP-OES analysis were used. The PLA-PEG composite fibrous membranes showed the presence of MNPs, hence offers the possibility for magnetically triggered on-demand drug delivery.

  10. In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology.

    PubMed

    Fang, Huan; Dong, Huina; Cai, Tao; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

    2016-01-01

    In order to maximize the production of biologically-derived chemicals, kinetic analyses are first necessary for predicting the role of enzyme components and coordinating enzymes in the same reaction system. Precorrin-2 is a key precursor of cobalamin and siroheme synthesis. In this study, we sought to optimize the concentrations of several molecules involved in precorrin-2 synthesis in vitro: porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and S-adenosyl-l-methionine-dependent urogen III methyltransferase (SUMT). Response surface methodology was applied to develop a kinetic model designed to maximize precorrin-2 productivity. The optimal molar ratios of PBGS, PBGD, UROS, and SUMT were found to be approximately 1:7:7:34, respectively. Maximum precorrin-2 production was achieved at 0.1966 ± 0.0028 μM/min, agreeing with the kinetic model's predicted value of 0.1950 μM/min. The optimal concentrations of the cofactor S-adenosyl-L-methionine (SAM) and substrate 5-aminolevulinic acid (ALA) were also determined to be 200 μM and 5 mM, respectively, in a tandem-enzyme assay. By optimizing the relative concentrations of these enzymes, we were able to minimize the effects of substrate inhibition and feedback inhibition by S-adenosylhomocysteine on SUMT and thereby increase the production of precorrin-2 by approximately five-fold. These results demonstrate the effectiveness of kinetic modeling via response surface methodology for maximizing the production of biologically-derived chemicals.

  11. In-Solution SH2 Domain Binding Assay Based on Proximity Ligation.

    PubMed

    Machida, Kazuya

    2017-01-01

    Protein-protein interactions mediated by SH2 domains confer specificity in tyrosine kinase pathways. Traditional assays for assessing interactions between an SH2 domain and its interacting protein such as far-Western and pull-down are inherently low throughput. We developed SH2-PLA, an in-solution SH2 domain binding assay, that takes advantage of the speed and sensitivity of proximity ligation and real-time PCR. SH2-PLA allows for rapid assessment of SH2 domain binding to a target protein using only a few microliters of cell lysate, thereby making it an attractive new tool to study tyrosine kinase signaling.

  12. Characterization of PLA parts made with AM process

    NASA Astrophysics Data System (ADS)

    Spina, Roberto; Cavalcante, Bruno; Lavecchia, Fulvio

    2018-05-01

    The main objective of the presented work is to evaluate the thermal behavior of Poly-lactic acid (PLA) parts made with a Fused Deposition Modelling (FDM) process. By using a robust framework for the testing sequence of PLA parts, with the aim of establishing a standard testing cycle for the optimization of the part performance and quality. The research involves study the materials before and after 3D printing. Two biodegradable PLA polymers are investigated, characterized by different colors (one black and the other transparent). The study starts with the examination of each polymeric material and measurements of its main thermal properties.

  13. Efficient Conversion of Phenylpyruvic Acid to Phenyllactic Acid by Using Whole Cells of Bacillus coagulans SDM

    PubMed Central

    Zheng, Zhaojuan; Ma, Cuiqing; Gao, Chao; Li, Fengsong; Qin, Jiayang; Zhang, Haiwei; Wang, Kai; Xu, Ping

    2011-01-01

    Background Phenyllactic acid (PLA), a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA) into PLA are equivocal. Methodology/Principal Findings A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l−1) and high productivity (2.3 g l−1 h−1) under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA. Conclusions/Significance Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering. PMID:21533054

  14. PLA realizations for VLSI state machines

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Whitaker, S.; Maki, G.; Liu, K.

    1990-01-01

    A major problem associated with state assignment procedures for VLSI controllers is obtaining an assignment that produces minimal or near minimal logic. The key item in Programmable Logic Array (PLA) area minimization is the number of unique product terms required by the design equations. This paper presents a state assignment algorithm for minimizing the number of product terms required to implement a finite state machine using a PLA. Partition algebra with predecessor state information is used to derive a near optimal state assignment. A maximum bound on the number of product terms required can be obtained by inspecting the predecessor state information. The state assignment algorithm presented is much simpler than existing procedures and leads to the same number of product terms or less. An area-efficient PLA structure implemented in a 1.0 micron CMOS process is presented along with a summary of the performance for a controller implemented using this design procedure.

  15. On the Use of PLA-PHB Blends for Sustainable Food Packaging Applications

    PubMed Central

    Arrieta, Marina Patricia; Samper, María Dolores; Aldas, Miguel; López, Juan

    2017-01-01

    Poly(lactic acid) (PLA) is the most used biopolymer for food packaging applications. Several strategies have been made to improve PLA properties for extending its applications in the packaging field. Melt blending approaches are gaining considerable interest since they are easy, cost-effective and readily available processing technologies at the industrial level. With a similar melting temperature and high crystallinity, poly(hydroxybutyrate) (PHB) represents a good candidate to blend with PLA. The ability of PHB to act as a nucleating agent for PLA improves its mechanical resistance and barrier performance. With the dual objective to improve PLAPHB processing performance and to obtain stretchable materials, plasticizers are frequently added. Current trends to enhance PLA-PHB miscibility are focused on the development of composite and nanocomposites. PLA-PHB blends are also interesting for the controlled release of active compounds in the development of active packaging systems. This review explains the most relevant processing aspects of PLA-PHB based blends such as the influence of polymers molecular weight, the PLA-PHB composition as well as the thermal stability. It also summarizes the recent developments in PLA-PHB formulations with an emphasis on their performance with interest in the sustainable food packaging field. PLA-PHB blends shows highly promising perspectives for the replacement of traditional petrochemical based polymers currently used for food packaging. PMID:28850102

  16. On the Use of PLA-PHB Blends for Sustainable Food Packaging Applications.

    PubMed

    Arrieta, Marina Patricia; Samper, María Dolores; Aldas, Miguel; López, Juan

    2017-08-29

    Poly(lactic acid) (PLA) is the most used biopolymer for food packaging applications. Several strategies have been made to improve PLA properties for extending its applications in the packaging field. Melt blending approaches are gaining considerable interest since they are easy, cost-effective and readily available processing technologies at the industrial level. With a similar melting temperature and high crystallinity, poly(hydroxybutyrate) (PHB) represents a good candidate to blend with PLA. The ability of PHB to act as a nucleating agent for PLA improves its mechanical resistance and barrier performance. With the dual objective to improve PLAPHB processing performance and to obtain stretchable materials, plasticizers are frequently added. Current trends to enhance PLA-PHB miscibility are focused on the development of composite and nanocomposites. PLA-PHB blends are also interesting for the controlled release of active compounds in the development of active packaging systems. This review explains the most relevant processing aspects of PLA-PHB based blends such as the influence of polymers molecular weight, the PLA-PHB composition as well as the thermal stability. It also summarizes the recent developments in PLA-PHB formulations with an emphasis on their performance with interest in the sustainable food packaging field. PLA-PHB blends shows highly promising perspectives for the replacement of traditional petrochemical based polymers currently used for food packaging.

  17. Anti-cancer evaluation of quercetin embedded PLA nanoparticles synthesized by emulsified nanoprecipitation.

    PubMed

    Pandey, Sanjeev K; Patel, Dinesh K; Thakur, Ravi; Mishra, Durga P; Maiti, Pralay; Haldar, Chandana

    2015-04-01

    This study was carried out to synthesize quercetin (Qt) embedded poly(lactic acid) (PLA) nanoparticles (PLA-Qt) and to evaluate anti-cancer efficacy of PLA-Qt by using human breast cancer cells. PLA-Qt were synthesized by using novel emulsified nanoprecipitation technique with varying dimension of 32 ± 8 to 152 ± 9 nm of PLA-Qt with 62 ± 3% (w/w) entrapment efficiency by varying the concentration of polymer, emulsifier, drug and preparation temperature. The dimension of PLA-Qt was measured through transmission electron microscopy indicating larger particle size at higher concentration of PLA. The release rate of Qt from PLA-Qt was found to be more sustained for larger particle dimension (152 ± 9 nm) as compared to smaller particle dimension (32 ± 8 nm). Interaction between Qt and PLA was verified through spectroscopic and calorimetric methods. Delayed diffusion and stronger interaction in PLA-Qt caused the sustained delivery of Qt from the polymer matrix. In vitro cytotoxicity study indicate the killing of ∼ 50% breast cancer cells in two days at 100 μg/ml of drug concentration while the ∼ 40% destruction of cells require 5 days for PLA-Qt (46 ± 6 nm; 20mg/ml of PLA). Thus our results propose anticancer efficacy of PLA-Qt nanoparticles in terms of its sustained release kinetics revealing novel vehicle for the treatment of cancer. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. 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.

  19. Dual-Enzyme Characteristics of Polyvinylpyrrolidone-Capped Iridium Nanoparticles and Their Cellular Protective Effect against H2O2-Induced Oxidative Damage.

    PubMed

    Su, Hua; Liu, Dan-Dan; Zhao, Meng; Hu, Wei-Liang; Xue, Shan-Shan; Cao, Qian; Le, Xue-Yi; Ji, Liang-Nian; Mao, Zong-Wan

    2015-04-22

    Polyvinylpyrrolidone-stabilized iridium nanoparticles (PVP-IrNPs), synthesized by the facile alcoholic reduction method using abundantly available PVP as protecting agents, were first reported as enzyme mimics showing intrinsic catalase- and peroxidase-like activities. The preparation procedure was much easier and more importantly, kinetic studies found that the catalytic activity of PVP-IrNPs was comparable to previously reported platinum nanoparticles. Transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) characterization indicated that PVP-IrNPs had the average size of approximately 1.5 nm and mainly consisted of Ir(0) chemical state. The mechanism of PVP-IrNPs' dual-enzyme activities was investigated using XPS, Electron spin resonance (ESR) and cytochrome C-based electron transfer methods. The catalase-like activity was related to the formation of oxidized species Ir(0)@IrO2 upon reaction with H2O2. The peroxidase-like activity originated from their ability acting as electron transfer mediators during the catalysis cycle, without the production of hydroxyl radicals. Interestingly, the protective effect of PVP-IrNPs against H2O2-induced cellular oxidative damage was investigated in an A549 lung cancer cell model and PVP-IrNPs displayed excellent biocompatibility and antioxidant activity. Upon pretreatment of cells with PVP-IrNPs, the intracellular reactive oxygen species (ROS) level in response to H2O2 was decreased and the cell viability increased. This work will facilitate studies on the mechanism and biomedical application of nanomaterials-based enzyme mimic.

  20. The CO 2 permeability and mixed gas CO 2/H 2 selectivity of membranes composed of CO 2-philic polymers

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

    Barillas, Mary Katharine; Enick, Robert M.; O’Brien, Michael

    2011-04-01

    The objective of this work was to design polymeric membranes that have very high CO 2 permeability and high mixed gas selectivity toward CO 2 rather than hydrogen. Therefore the membranes were based on "CO 2-philic" polymers that exhibit thermodynamically favorable Lewis acid:Lewis base and hydrogen bonding interactions with CO 2. CO 2-philic polymers that are solid at ambient temperature include polyfluoroacrylate (PFA); polyvinyl acetate (PVAc); and amorphous polylactic acid (PLA). Literature CO 2 permeability values for PVAc and PLA are disappointingly low. The cast PFA membranes from this study had low permeabilities (45 barrers at 25º C) and verymore » low CO 2/H 2 selectivity of 1.4. CO 2-philic polymers that are liquid at ambient conditions include polyethylene glycol (PEG), polypropylene glycol (PPG), polybutylene glycol with a linear -((CH 2) 4O)-repeat unit (i.e., polytetramethylene ether glycol (PTMEG)), polybutylene glycol (PBG) with a branched repeat unit, perfluoropolyether (PFPE), poly(dimethyl siloxane) (PDMS), and polyacetoxy oxetane (PAO). A small compound, glycerol triacetate (GTA) was also considered because it is similar in chemical structure to a trimer of PVAc. These liquids were tested as supported liquid membranes (SLM) and also (with the exception of PAD and GTA) as rubbery, crosslinked materials. Mixed gas permeability was measured using equimolar mixtures of CO 2 and H 2 feed streams at one atmosphere total pressure in steady-state flux experiments over the 298-423 K temperature range. The most promising SLMs were those composed of PEG, PTMEG, GTA, and PDMS. For example, at 37º C the PEG-, PTMEG-, GTA- and PDMS-based SLMs exhibited CO 2/H 2 selectivity values of ~11, 9, 9, and 3.5, respectively, and CO 2 permeability values of ~800, 900, 1900, and 2000 barrers, respectively. Crosslinked versions of the PEG, PTMEG and PDMS membranes at 37º C exhibited selectivity values of ~5, 6, and 3.5, respectively, and CO 2 permeability values of