Phospholipase A2 Enzyme Activity Research Articles

Quantum dot–NBD–liposome luminescent probes for monitoring phospholipase A2 activity

In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot-fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C6-HPC (2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn-glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A2 as low as 0.0075 U mL(-1) and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A2 inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid).

Comparison of total protein and phospholipase A2 levels in individual coralsnake venoms

Studies of differences or changes in venom protein levels or enzymatic activities have significance only if contrasted to the normal variations between individual snakes. This study involves the analysis and comparison of venom from 13 individual Texas coralsnakes (Micrurus tener tener) in order to detect differences in the volume, total protein concentration, electrophoretic profile, and PLA2 enzyme activity. A significant inverse correlation between venom volume and total protein concentration was found. Although the 13 venoms were indistinguishable from their electrophoretic protein profiles, phospholipase A2 enzymatic activities varied considerably.

Transglutaminase 2 and phospholipase A2 interactions in the inflammatory response in human Thp-1 monocytes

Several experimental approaches have demonstrated that transglutaminase 2 (TG2) increased activity is involved in monocyte activation and inflammatory response. Preliminary results also demonstrate a TG-mediated post-translational modification of phospholipase A2 (PLA2), which catalyzes the release of arachidonic acid from its lipid storage sites. The control of PLA2-mediated production of eicosanoids has been found to be of great benefit for inflammatory disease treatment. However, the identification of the mechanisms of PLA2 activation is a very complex issue, because of the presence of multiple PLA2 forms. The aim of this study was to characterize the interactions between TG2 and sPLA2 in LPS-stimulated THP-1 cells, which were treated with TPA to induce early differentiated macrophage-type model. We demonstrated that increases in TG2 enzyme activity and protein expression may be considered an early event in monocyte/macrophage activation by LPS. Under these conditions, TG2 protein was co-immunoprecipitated with PLA2 by monoclonal antibody directed against the secretory form of the enzyme (sPLA2-V). Concomitantly, the PLA2 enzyme activity increased in TPA-treated cells exposed to LPS; these high levels of enzyme activity were significant reduced by R283, a site-specific inhibitor of TG2. Moreover, confocal laser scanning microscopy analysis of double-immunostained cytochemical specimens confirmed a co-localization of BAPA-labeled proteins and sPLA2-V in LPS-treated cells. These findings give evidence of a complex TG2/sPLA2-V, suggesting the possibility that sPLA2-V is a substrate for TG2. These results demonstrated that TG2 increases produced a sustained activation of PLA2 activity, suggesting a functional interaction between these enzymes in the regulation of inflammatory response.

The Determinants for the Enzyme Activity of Human Parvovirus B19 Phospholipase A2 (PLA2) and Its Influence on Cultured Cells

Human parvovirus B19 (B19V) is the causative agent of erythema infectiosum in humans. B19 infection also causes severe disease manifestations, such as chronic anemia in immunocompromised patients, aplastic crisis in patients with a high turnover rate of red blood cells, and hydrops fetalis in pregnant women. Although a secreted phospholipase A2 (PLA2) motif has been identified in the unique region of the B19V minor capsid protein VP1(VP1u), the determinants for its enzyme activity and its influences on host cells are not well understood. The purpose of this study was to investigate the contribution of the PLA2 motif and other regions of the VP1u to the PLA2 activity, to determine the cellular localization of the VP1u protein, and to examine the effects of VP1u on cellular cytokines. We found that in addition to the critical conserved and non-conserved amino acids within the VP1u PLA2 motif, amino acid residues outside the VP1u PLA2 motif are also important for the PLA2 activity. VP1u and various mutants all revealed a nucleo-cytoplasmic distribution. UT7-Epo cells treated with prokaryotic expressed VP1u or mutant proteins with PLA2 activity released a large amount of free fatty acid (FFA), and the cell morphological change occurred dramatically. However, neither free fatty acid nor cell morphology change occurred for cells treated with the mutants without PLA2 activity. The wild type and the VP1u mutants with the PLA2 activity also activated TNF-α promoter and upregulated the transcription activity of NF-κB in transfected cells. In addition, we found that the amino acids outside the PLA2 domain are critical for the viral PLA2 activity, and that these tested VP1u mutants did not affect the localization of the VP1u protein.

VEGF receptor-1 involvement in pericyte loss induced byEscherichia coliin anin vitromodel of blood brain barrier

The key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood-brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co-culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co-culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2 ) enzyme activities. In supernatants of E. coli-stimulated co-cultures, an increase in prostaglandins (PGE2) and VEGF production in comparison with untreated co-cultures were found. Incubation with E. coli in presence of AACOCF3 or BEL caused a decrease of PGE2 and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR-1 but not VEGFR-2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR-1, but not VEGFR-2 blockade, or in presence of AACOCF3 or BEL, elevated TEER values, reduced permeability and BRPC loss were found. These data suggest that VEGFR-1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention.

A catalytically-inactive snake venom Lys49 phospholipase A2 homolog induces expression of cyclooxygenase-2 and production of prostaglandins through selected signaling pathways in macrophages

The effects of a snake venom Lys-49 phospholipase A2 (PLA2) homolog named MT-II, devoid of enzymatic activity, on the biosynthesis of prostaglandins and protein expression of cyclooxygenase-2 (COX-2) and signaling pathways involved were evaluated in mouse macrophages in culture and in peritoneal cells ex vivo. Stimulation of macrophages with MT-II leads to production of prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) and protein expression of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of cytosolic PLA2 (cPLA2), but not Ca2+ independent PLA2 (iPLA2) reduced release of PGD2 and PGE2 and expression of COX-2 induced by MT-II. Inhibition of nuclear factor κB (NF-κB) significantly reduced MT-II-induced PGE2, but not PGD2 production and COX-2 expression. Inhibitors of either protein kinase C (PKC), protein tyrosine kinase (PTK), or extracellular signal-regulated kinase (ERK) pathways abrogated MT-II-induced NF-κB activation and reduced COX-2 expression and PGE2 release, whereas the p38 mitogen-activated protein kinase (MAPK) inhibitor reduced MT-II-induced COX-2 expression and PGD2 production. Inhibition of phosphatidylinositol-3-kinase (PI3K) pathway abrogated MT-II-induced NF-κB activation, but affected neither prostaglandins production nor COX-2 expression. MT-II-induced production of PGD2 and PGE2 and COX-2 expression were also observed in vivo after intraperitoneal injection into mice. Collectively, our data demonstrate that a catalytically-inactive PLA2 homolog is capable of inducing prostaglandins biosynthesis and COX-2 expression in macrophages in both in vitro and in vivo models, indicating that the enzymatic activity of PLA2 is not necessary to trigger these effects. MT-II-activated NF-κB, cPLA2 and distinct protein kinases are the principal steps involved in these cellular events.

Changes in group II phospholipase A2 gene expression in rat heart during sepsis

BackgroundThis study was undertaken to investigate alterations of group II phospholipase A2 (PLA2) gene expression and its underlying mechanism in rat heart during different phases of sepsis. Materials and methodsSepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups, control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. PLA2 enzyme activity, group II PLA2 protein level, messenger RNA (mRNA) abundance, transcription rate, and half-life were measured. ResultsPLA2 activity was decreased by 29% during early sepsis but it was increased by 49% during late sepsis. Group II PLA2 protein level was decreased by 27% during early sepsis but it was increased by 35.3% during late sepsis. Group II PLA2 mRNA was decreased by 21% during early sepsis but it was increased by 141% during late sepsis. The transcription rate of group II PLA2 mRNA was reduced by 25% during early sepsis but it was elevated by 67% during late sepsis. The half-life of group II PLA2 mRNA remained unaltered during early and late phases of sepsis. ConclusionsThese results demonstrate that PLA2 activity, group II PLA2 protein level, the mRNA abundance, and transcription rate were concurrently underexpressed during early sepsis, while they were overexpressed during late sepsis, with no change in the degradation of gene transcript. These data indicate that the biphasic changes in group II PLA2 gene expression are regulated transcriptionally during sepsis.

Synthesis of tocopheryl succinate phospholipid conjugates and monitoring of phospholipase A2 activity

Tocopheryl succinates (TOSs) are, in contrast to tocopherols, highly cytotoxic against many cancer cells. In this study the enzyme activity of secretory phospholipase A2 towards various succinate-phospholipid conjugates has been investigated. The synthesis of six novel phospholipids is described, including two TOS phospholipids conjugates. The studies revealed that the TOS conjugates are poor substrates for the enzyme whereas the phospholipids with alkyl and phenyl succinate moieties were hydrolyzed by the enzyme to a high extent.

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

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

Abstract 479: The effects of sarcophine-diol (SD) on melanoma B16F10 cell line

Abstract Sarcophine diol (SD) is a non-toxic semi-synthetic derivative of sarcophine, a marine natural product. Previously, we studied the chemopreventive effects of SD on non-melanoma tumor development using both animal and cellular models. In this current study, we used the mouse melanoma B16F10 cell line to investigate the possible effects of SD on melanoma cell development. We found that SD at a 250 µM concentrations inhibits cell duplication. Melanoma cells treated with SD do not form the contractile ring, a multi-protein complex involved in cell division. As a result of cytoskeleton modification, cells treated with SD do not show the ability to carry out de novo synthesis of DNA, and their DNA show a high degree of fragmentation compared to untreated controls. To explore the molecular mechanism(s) involved in the disassembly of the contractile machinery, our studies show that SD treatment inhibits cell membrane permeability for small molecular mass compounds (e.g. ethidium bromide which is used as an indicator for cell permeability to Ca2+ ions). These findings are consistent with the observation that SD increases cleavage of phospholipase A2 (PLA2), resulting in low PLA2 enzymatic activity. These findings are also consistent with the observation that SD, in a time- and concentration-dependent manner, inhibits the expression level of the cyclooxygenase-2 (Cox-2). As a result of the decline in membrane permeability in SD treated cells and the negative effect of SD on the protein level of phospholipase C (PLC), which both affect the activity of contractile machinery, the non-dividing cells undergo apoptosis. The latter notion is supported by findings that SD inhibits the expression of STAT-3 (signal transducer and activator of transcription protein), and cyclin D1 (an activator of cyclin-dependent kinase 4, Cdk4). SD treatment also enhances the cellular level of the tumor suppressing protein 53 (p53) and stimulates cleavage of the nuclear poly(ADP-ribose) polymerase (cleaved-PARP). SD also enhances the cellular level of cleaved-Caspase-3, -8, -9 and stimulates enzymatic activities of Caspase-3, -8 and -9. All these findings, in addition to inhibition of cell viability, suggest that SD most likely inhibits melanoma cell growth by arresting the cell-division cycle in a Go quiescent phase and simultaneously activates apoptosis via extrinsic and intrinsic pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 479. doi:1538-7445.AM2012-479

INHIBITORY EFFECTS OF PLANT COMPOUNDS ON ENZYMATIC AND ACTIVITIES INDUCED BY A SNAKE PHOSPHOLIPASE A<SUB>2</SUB>

Polyphenolic compounds have shown to inhibit toxic effects induced by snake venom proteins. In this work, we demonstrate that gallic acid, ferulic acid, caffeic acid, propylgallate and epigallocatechingallate inhibit the enzymatic activity of a phospholipase A2 (PLA2), using egg yolk as substrate. The IC50 values are between 0.38 – 3.93 mM. These polyphenolic compounds also inhibit the PLA2 enzymatic activity when synthetic substrate is used. Furthermore, these compounds decreased the cyotoxic effect induced by a myotoxic PLA2; specifically, epigallocatechin gallate exhibited the best inhibitory capacity with 90.92 ± 0.82%, while ferulic acid showed the lowest inhibitory activity with 30.96 ± 1.42%. Molecular docking studies were performed in order to determine the possible modes of action of phenolic compounds. All polyphenols showed hydrogen bonds with an active site of enzyme; moreover, epigallocatechingallate presented the strongest binding compared with the other compounds. Additionally, a preliminary structure-activity relationship analysis showed a correlation between the IC50 and the topological polar surface area of each compound (p = 0.0491, r = -0.8079 (-0.9878 to -0.2593)), which indicates the surface area required for each molecule to bind with the majority of the enzyme. Furthermore, our results show that propylgallate and epigallocatechingallate are two novel natural products with anti-myotoxic potential. The topical application of these plant polyphenols at the bite site could lead to prevent myotoxicity; however, further in vivo studies would be necessary to confirm the in vitro results.

P02-533 - N-3 Fatty Acid Supplementation Influences Membrane Fatty Acids and Phospholipase A 2 Activity in Patients at Risk to Develop Psychosis

IntroductionDecreased levels of polyunsaturated membrane fatty acids (PUFA) and increased activity of cytosolic phospholipase A2 (PLA2) enzymes (key regulating enzymes of membrane remodelling and PUFA availability) are supporting pillars of the “membrane phospholipids concept of schizophrenia”. Assuming that membrane PUFA profile and PLA2 activity are altered during the at risk phase of disorder and influenced by fatty acid supplementation, we investigated PUFA profiles and PLA2 activity simultaneously in ultra high-risk (UHR) subjects before and after (n-3) fatty acids supplementation.MethodIn 81 UHR patients (aged between 13 and 25 years) PUFA levels were assessed in erythrocyte membranes using gas chromatography, and cytosolic PLA2 activity was measured in blood serum using a fluorometric HPTLC-based assay. Measurements were performed before and after a 6 month interval of placebo-controlled supplementation with n-3 fatty acids.ResultsAt baseline significant associations were found between (n-9) and (n-6)-PUFA levels and psychopathology (especially in negative symptoms) assessed by the PANSS according to PACE criteria. (n-3)-PUFA supplementation caused significant changes in (n-3)- and (n-6)-PUFA levels and a significant decrease of PLA2 activity.ConclusionOur results support associations between membrane biochemistry and psychopathology (especially negative symptoms) in people at risk to develop psychosis. Supplementation of n-3 PUFA increases PUFA availability at membrane level and modulates membrane repair and remodelling processes. Assuming that PLA2 activity reflects neuronal damage, PUFA supplementation might unfold neuroprotective effects.

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

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

Synthesis and evaluation of sesquiterpene lactone inhibitors of phospholipase A2 from Bothrops jararacussu

Several sesquiterpene lactone were synthesized and their inhibitive activities on phospholipase A2 (PLA2) from Bothrops jararacussu venom were evaluated. Compounds Lac01 and Lac02 were efficient against PLA2 edema-inducing, enzymatic and myotoxic activities and it reduces around 85% of myotoxicity and around 70% of edema-inducing activity. Lac05–Lac08 presented lower efficiency in inhibiting the biological activities studied and reduce the myotoxic and edema-inducing activities around only 15%. The enzymatic activity was significantly reduced. The values of inhibition constants (KI) for Lac01 and Lac02 were approximately 740 μM, and for compounds Lac05–Lac08 the inhibition constants were approximately 7.622–9.240 μM. The enzymatic kinetic studies show that the sesquiterpene lactones inhibit PLA2 in a non-competitive manner. Some aspects of the structure–activity relationships (topologic, molecular and electronic parameters) were obtained using ab initio quantum calculations and analyzed by chemometric methods (HCA and PCA). The quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA2 (Lac01–Lac04) present lower values of highest occupied molecular orbital (HOMO) energy and molecular volume (VOL) and bigger values of hydrophobicity (LogP). These results indicate some topologic aspects of the binding site of sesquiterpene lactone derivatives and PLA2.

Metabolic Syndrome and Carotid Intima-Media Thickness in Young Adults: Roles of Apolipoprotein B, Apolipoprotein A-I, C-Reactive Protein, and Secretory Phospholipase A2: The Cardiovascular Risk in Young Finns Study

Aberrations in apolipoprotein (apo) metabolism and increased systemic inflammation associate with the metabolic syndrome (MetS) and may contribute to its atherogenicity. We examined whether the association between carotid atherosclerosis and MetS in a population of young adults is mediated by apoB and apoA-I and/or by inflammatory markers C-reactive protein and type II secretory phospholipase A2. We used cross-sectional and 6-year prospective data from the cardiovascular risk in young Finns study. In young adults (aged 24 to 39 years), apoB, C-reactive protein, and type II secretory phospholipase A2 enzyme activity were significantly higher and apoA-I lower in subjects with MetS (N=325) than in subjects without MetS (N=1858). In prospective analysis (N=1587), both MetS and high apoB predicted (P<0.0001) incident high carotid intima-media thickness, defined as carotid intima-media thickness >90th percentile and/or plaque. The association between MetS and incident high carotid intima-media thickness was attenuated by approximately 40% after adjustment with apoB. Adjustments with apoA-I, C-reactive protein, or type II secretory phospholipase A2 did not diminish the association. High levels of apoB, C-reactive protein, and type II secretory phospholipase A2 and low levels of apoA-I associate with MetS in young adults. The atherogenicity of MetS in this population assessed by incident high carotid intima-media thickness appears to be substantially mediated by elevated apoB but not inflammatory markers.

Lipid oxidation and fatty acid profile related to broiler breast meat color abnormalities

The aim of this work was to study the influence of lipid oxidation on broiler breast meat (Pectoralis major m) color abnormalities. There were 27.0 % more lipid oxidation in PSE in relation to normal meat and 41.0 % more in relation to DFD-like meat (p&lt;0.05). The fatty acid profile was also significantly different since the arachidonic acid (AA) fraction increased 38.6 and 70.5 % in PSE meat comparing to normal and DFD-like meats, respectively. The ratio PUFA/SFA changed in these three types of meat: 0.736, 0.713 and 0.694 for PSE, normal and DFD-like meat respectively, reflecting the highest production of polyunsaturated fatty acids in the PSE meat samples. Results indicated that phospholipase A2 enzyme activities played a relevant role towards the development of PSE-meat syndrome in a cascade of biochemical reactions promoting the formation of free radicals from AA, which ultimately damaged the muscle membrane systems.

Synaptotagmin-2 Controls Regulated Exocytosis but Not Other Secretory Responses of Mast Cells

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

Parvovirus capsid carries a phospholipase A2 enzymatic activity

Nonenveloped viruses enter target cells by endocytosis. Prisoner into intracellular vesicles, they use different mechanisms to allow liberation of their genome and its transport in replication and expression sites. The parvoviruses are small nonenveloped single-stranded DNA viruses that should enter the cell nucleus for replication. One of their capsid proteins carries a phospholipase A2 (PLA2) enzymatic activity that is necessary for infectiosity. The viral PLA2 leads to membrane lysis allowing the liberation of the nucleocapsid from vesicles and its transport to the nucleus. The PLA2 could be involved in immunopathologic phenomenon observed during B19 infection in humans.

Hemolytic and anticoagulant study of the neurotoxin vipoxin and its components—basic phospholipase A2 and an acidic inhibitor

In the present study, we demonstrate for the first time that the potent neurotoxin vipoxin from the venom of Vipera ammodytes meridionalis exhibits hemolytic and anticoagulant properties. By investigating the effects of phospholipids and calcium ions on hemolysis, we established that the phospholipase A2 (PLA2) enzyme activity is responsible for the hemolytic properties. This was confirmed by chemical modification of the PLA2 active-site histidine residue with p-bromophenacylbromide. Applying different clotting assays, we show that the PLA2 is a weakly anticoagulant enzyme, which affects intrinsic tenase complex by the hydrolysis of procoagulant phospholipids, rather than by nonenzymatic mechanisms (binding to specific coagulation factors). The whole complex--vipoxin--does not affect the coagulation system.

Lipid regulation in SZ95 sebocytes by active and inactive phospholipases A2fromBothrops moojenivenom

The scientific background for this work is based on the fact that sebocytes reduce lipid production during the skin ageing process. The relationships between ageing effects and lipid reduction on a molecular level and ways to influence them are not yet fully identified. For this reason we investigated lipid stimulation in SZ95 sebocytes by Bothrops moojeni snake venom gel filtration fractions (Botmo GF). Several Botmo GFs increased lipid synthesis in SZ95 sebocytes without apparent toxic or apoptotic effects in applied concentrations. Botmo GF 11 was further purified by RP-HPLC. Fraction Botmo GF 11-117 showed phospholipase A2 (PLA2) activity, whereas fraction Botmo GF 11-101 did not. At a concentration of 1 μg/ml Botmo GF 11-101 enhanced neutral lipid synthesis by 150% and Botmo GF 11-117 by 310% compared to untreated SZ95 sebocytes. It was supposed that sebum production is stimulated via a PPAR agonistic mechanism of free fatty acids that were released by PLA2 activity. However, the present data surprisingly indicate that lipid synthesis stimulation by Botmo GF 11-101 and Botmo GF 11-117 is independent of PLA2 enzymatic activity in Botmo GF 11 subfractions. Thus, it is suspected that both Bothrops moojeni fractions act via different mechanisms on lipid stimulation in SZ95 sebocytes.Although the exact mode of actions are not yet identified, the Botmo GF 11-101 and Botmo GF 11-117 might be interesting tools for the investigation of sebocyte lipogenesis and may be helpful to the development of therapeutic concepts for the treatment of age-related skin dryness.