Dependent Phospholipase A2 Research Articles

Novel mechanisms to enhance omega‐3 brain metabolism with APOE4

AbstractBackgroundNeuroprotective nutritional interventions involving greater consumption of polyunsaturated fats (Mediterranean diet, fish consumption or DHA supplements) are less effective in APOE4 carriers with dementia compared to non‐carriers. We recently demonstrated greater activation of calcium dependent phospholipase A2 (cPLA2) in APOE4 compared with non‐APOE4 using both postmortem human brains and in brain tissues from APOE4 animal models. Activation of cPLA2 is associated with lower omega‐3 brain levels and lower measures of blood‐brain barrier (BBB) integrity as assessed using 7T DCE MRI to derive contrast transfer values (K trans).MethodHere, we present new data on aging (18 months) old APOE mouse models on a low DHA diet, APOE4‐TR mice treated with LPS and APOE knock out mice as a model of lower BBB integrity. All models were treated with the brain penetrant cPLA2 inhibitor ASB14780 at 10 mg/kg IP daily over 8 weeks in the aging model and acutely (7 days) in the younger LPS and APOE KO models.ResultcPLA2 inhibition was associated with increased brain omega‐3 levels in both the aging APOE4 model and in the younger APOE4 mice treated acutely with LPS. In the aging APOE4 mouse model, cPLA2 inhibition associated with better performance on novel object recognition, improved synaptic markers, and lower microglial activation. In the APOE KO model, treatment was associated with improved measures of BBB with lower K trans following treatment (Figure).ConclusionThe results here demonstrate how neuroinflammation associated with APOE4 depletes brain omega‐3s via activation of cPLA2 and how reducing cPLA2 activity can rescue this phenotype. The development of specific and potent brain penetrant cPLA2 inhibitors promises to enhance the brain effects of omega‐3 enriched diets by limiting the effects of neuroinflammation on the aging APOE4 brain.

Lipidome Profiling of APOE4 AD brains implicated unresolved neuroinflammation involving polyunsaturated fatty acid metabolism

AbstractBackgroundThe Apolipoprotein E4 (APOE4) allele has been associated with an accentuated response to brain inflammation and increases the risk of AD dementia progression. Among inflammation signaling pathways, eicosanoids play a prominent role in neurodegeneration.MethodThis study compared measures of the eicosanoid lipidome from patients with AD dementia compared to age and sex‐matched controls with no cognitive impairment (NCI), stratified by APOE genotypes using postmortem brain tissues from the Religious Order Study (n = 42).ResultTargeted lipidomic analysis by mass spectrometry of the dorsolateral prefrontal cortex demonstrated lower levels of omega‐3 fatty acids eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), and DHA‐derived neuroprotectin D1 (NPD‐1) in persons with lower global cognitive function, independent of age, sex and education years. APOE4 was associated with greater pro‐inflammatory lipids (e.g., PGD2, PGE2, 12‐HHT, LTB4 and 5‐HETE) and pro‐resolving lipids (eg. lipoxins, resolvins and EETs) in patients with dementia (Figure 1). Pathway analysis implicates greater activation of calcium dependent phospholipase A2 (cPLA2), 5‐lipoxygenase (5‐LOX) and soluble epoxide hydrolase (sEH) underlying the observed lipidomic profile.ConclusionThese results confirm a chronic state of unresolved inflammation in the AD dementia brain that is accentuated in carriers of the APOE4 allele and identify novel therapeutic targets.

Uncovering mechanisms of brain inflammation in Alzheimer's disease with APOE4: Application of single cell-type lipidomics.

A chronic state of unresolved inflammation in Alzheimer's disease (AD) is intrinsically involved with the remodeling of brain lipids. This review highlights the effect of carrying the apolipoprotein E ε4 allele (APOE4) on various brain cell types in promoting an unresolved inflammatory state. Among its pleotropic effects on brain lipids, we focus on APOE4's activation of Ca2+ -dependent phospholipase A2 (cPLA2) and its effects on arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid signaling cascades in the brain. During the process of neurodegeneration, various brain cell types, such as astrocytes, microglia, and neurons, together with the neurovascular unit, develop distinct inflammatory phenotypes that impact their functions and have characteristic lipidomic fingerprints. We propose that lipidomic phenotyping of single cell-types harvested from brains differing by age, sex, disease severity stage, and dietary and genetic backgrounds can be employed to probe mechanisms of neurodegeneration. A better understanding of the brain cellular inflammatory/lipidomic response promises to guide the development of nutritional and drug interventions for AD dementia.

The Short-Term Opening of Cyclosporin A-Independent Palmitate/Sr2+-Induced Pore Can Underlie Ion Efflux in the Oscillatory Mode of Functioning of Rat Liver Mitochondria.

Mitochondria are capable of synchronized oscillations in many variables, but the underlying mechanisms are still unclear. In this study, we demonstrated that rat liver mitochondria, when exposed to a pulse of Sr2+ ions in the presence of valinomycin (a potassium ionophore) and cyclosporin A (a specific inhibitor of the permeability transition pore complex) under hypotonia, showed prolonged oscillations in K+ and Sr2+ fluxes, membrane potential, pH, matrix volume, rates of oxygen consumption and H2O2 formation. The dynamic changes in the rate of H2O2 production were in a reciprocal relationship with the respiration rate and in a direct relationship with the mitochondrial membrane potential and other indicators studied. The pre-incubation of mitochondria with Ca2+(Sr2+)-dependent phospholipase A2 inhibitors considerably suppressed the accumulation of free fatty acids, including palmitic and stearic acids, and all spontaneous Sr2+-induced cyclic changes. These data suggest that the mechanism of ion efflux from mitochondria is related to the opening of short-living pores, which can be caused by the formation of complexes between Sr2+(Ca2+) and endogenous long-chain saturated fatty acids (mainly, palmitic acid) that accumulate due to the activation of phospholipase A2 by the ions. A possible role for transient palmitate/Ca2+(Sr2+)-induced pores in the maintenance of ion homeostasis and the prevention of calcium overload in mitochondria under pathophysiological conditions is discussed.

Lipids and brain inflammation in APOE4-associated dementia.

To highlight recent developments in studying mechanisms by which the apolipoprotein E4 (APOE4) allele affects the metabolism of brain lipids and predisposes the brain to inflammation and Alzheimer's disease (AD) dementia. APOE4 activates Ca2+ dependent phospholipase A2 (cPLA2) leading to changes in arachidonic acid (AA), eicosapentaenoic acid and docosahexaenoic acid signaling cascades in the brain. Among these changes, the increased conversion of AA to eicosanoids associates with sustained and unresolved chronic brain inflammation. The effects of APOE4 on the brain differ by age, disease stage, nutritional status and can be uncovered by brain imaging studies of brain fatty acid uptake. Reducing cPLA2 expression in the dementia brain presents a viable strategy that awaits to be tested. Fatty acid brain imaging techniques can clarify how changes to brain polyunsaturated fatty acid metabolism during the various phases of AD and guide the development of small molecules to mitigate brain inflammation.

Moderate blood alcohol and brain neurovulnerability: Selective depletion of calcium-independent phospholipase A2, omega-3 docosahexaenoic acid, and its synaptamide derivative as a potential harbinger of deficits in anti-inflammatory reserve.

Repetitive, highly elevated blood alcohol (ethanol) concentrations (BACs) of 350 to 450mg/dl over several days cause brain neurodegeneration and coincident neuroinflammation in adult rats localized in the hippocampus (HC), temporal cortex (especially the entorhinal cortex; ECX), and olfactory bulb (OB). The profuse neuroinflammation involves microgliosis, increased proinflammatory cytokines, and elevations of Ca+2 -dependent phospholipase A2 (cPLA2) and secretory PLA2 (sPLA2), which both mobilize proinflammatory ω-6 arachidonic acid (ARA). In contrast, Ca+2 -independent PLA2 (iPLA2) and anti-inflammatory ω-3 docosahexaenoic acid (DHA), a polyunsaturated fatty acid regulated primarily by iPLA2, are diminished. Furthermore, supplemented DHA exerts neuroprotection. Given uncertainties about the possible effects of lower circulating BACs that are common occurring during short- term binges, we examined how moderate BACs affected the above inflammatory events, and the impact of supplemented DHA. Young adult male rats sustaining upper-moderate BACs (~150mg/dl) from once-daily alcohol intubations were sacrificed with appropriate controls after 1week. The HC, ECX and OB were quantitatively examined using immunoblotting, neurodegeneration staining, and lipidomics assays. Whereas neurodegeneration, increases in cPLA2 IVA, sPLA2 IIA, and ARA, and microglial activation were not detected, the HC and ECX regions demonstrated significantly reduced iPLA2 levels. Levels of DHA and synaptamide, its anti-inflammatory N-docosahexaenoylethanolamide derivative, also were lower in HC, and DHA supplementation prevented the iPLA2 decrements in HC. Additionally, adult mice maintaining upper-moderate BACs from limited alcohol binges had reduced midbrain iPLA2 levels. The apparently selective depletion by moderate BACs of the metabolically linked anti-inflammatory triad of hippocampal iPLA2, DHA, and synaptamide, and of iPLA2 in the ECX, potentially indicates an unappreciated deficit in brain anti-inflammatory reserve that may be a harbinger of regional neurovulnerability.

Cytoplasmic Phospholipase A2 Is Essential in GPVI Signaling Initiated Procoagulant Platelet Formation

Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemostasis by supporting active tenase and prothrombinase complexes on their phosphatidylserine (PSer) externalized surface. When platelets are stimulated with a GPVI agonist, such as convulxin (CVX) or collagen related peptide (CRP), both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant phenotype. Activation of phospholipase A2 (PLA2) is an essential prerequisite for GPVI stimulated thromboxane A2 (TXA2) generation that induces paracrine platelet activation and aggregation. Active PLA2 cleaves the arachidonic acid from the second carbon group of glycerol within the phospholipid membrane. The resultant arachidonic acid, upon downstream enzymatic modification, is modified into TXA2, leukotrienes, and other metabolites. Inhibitors of cyclooxygenases (i.e. aspirin), which prevent arachidonic acid conversion to TXA2 are widely used clinically as antiplatelet agents. Here we investigate the role of PLA2 and its metabolites in inducing GPVI-induced PP formation.Human platelets (106/mL) were activated in the presence of three distinct PLA2 inhibitors: non-specific (PLA2; oleyloxyethyl phosphorylcholine, 1 μM), and specific to inducible (iPLA2; bromoenol lactone, 1 μM) and Ca2+-dependent cytosolic PLA2 (cPLA2; PACOCF3, 1 μM). Examination of platelets in these dilute conditions facilitates both the assessment of autocrine effects of PLA2 and its metabolites and flow cytometry analysis. Platelets were stimulated with different concentrations of GPVI agonists (CRP and CVX), stained (Annexin V for PSer, PAC1 for integrin activation and P-Selectin to assess granule release) and fixed. Additionally pharmacologic inhibitors of the TXA2 receptor (SQ29548, 0.5 μM) and lipoxygenases were utilized.As expected, PP formation in this setting was GPVI signaling and mPTP mediated, as dasatinib (SRC kinase inhibitor, 1 μM) and cyclosporin A (cyclophilin D inhibitor, 5 μM) completely inhibited PP formation. Analysis of platelet activation by flow cytometry demonstrated no effect of PLA2 inhibition on either platelet aggregatory ability or granule release. In contrast, procoagulant platelet formation was significantly decreased in the presence of the non-specific PLA2 inhibitor oleyloxyethyl phosphorylcholine (~59% decrease) and the specific cPLA2 inhibitor PACOCF3 (~51% decrease). Specific iPLA2 inhibition had no effect on any aspect of platelet activation. These results indicate that cPLA2-mediated events are required for autocrine induction of procoagulant platelet formation by GPVI. To begin to interrogate the downstream effectors mediating cPLA2's effects PP formation was examined in the presence of inhibitors of TXA2-mediated platelet activation (SQ29548) and lipoxygenase inhibitors, specifically zileuton, caffeic acid and 8,11,14-eicosatriynoate (all at 1 μM). Inhibition of either of these metabolite-mediated pathways had no effect on GPVI-induced PP formation. In contrast to TXA2's lack of effect on autocrine PP formation, platelet accretion on collagen, presumably mediated by paracrine stimulation of the TXA2 receptor, was decreased by 70% in the presence of SQ29548.These studies identify a novel pathway, mediated through activation of cPLA2, that is essential for GPVI induced PP formation. This cPLA2-mediated pathway is not mediated through the TXA2 receptor, and is distinct from that required for TXA2-mediated paracrine activation of platelet aggregation. Consistent with the findings presented here, previous studies have demonstrated minimal effect of cyclooxygenase inhibition on procoagulant platelet formation. Thus, our studies identify a novel aspirin-insensitive pathway mediating PP formation, and suggest a novel pharmacologic target that could be used for the treatment of procoagulant platelet related pathologic thromboses without affecting proaggregatory phenotype. DisclosuresNo relevant conflicts of interest to declare.

Future perspective: high-throughput construction of new ultrasensitive cytokine and virion liquid chips for high-throughput screening (HTS) of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases.

Pathogen–host cell interactions play an important role in many human infectious and inflammatory diseases. Several pathogens, including Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tb), and even the recent 2019 novel coronavirus (2019-nCoV), can cause serious breathing and brain disorders, tissue injury and inflammation, leading to high rates of mortality and resulting in great loss to human physical and mental health as well as the global economy. These infectious diseases exploit the microbial and host factors to induce serious inflammatory and immunological symptoms. Thus the development of anti-inflammatory drugs targeting bacterial/viral infection is an urgent need. In previous studies, YojI-IFNAR2, YojI-IL10RA, YojI-NRP1,YojI-SIGLEC7, and YojI-MC4R membrane–protein interactions were found to mediate E. coli invasion of the blood–brain barrier (BBB), which activated the downstream anti-inflammatory proteins NACHT, LRR and PYD domains-containing protein 2(NLRP2), using a proteomic chip conjugated with cell immunofluorescence labeling. However, the studies of pathogen (bacteria/virus)–host cell interactions mediated by membrane protein interactions did not extend their principles to broad biomedical applications such as 2019-nCoV infectious disease therapy. The first part of this feature article presents in-depth analysis of the cross-talk of cellular anti-inflammatory transduction signaling among interferon membrane protein receptor II (IFNAR2), interleukin-10 receptor subunit alpha (IL-10RA), NLRP2 and [Ca2+]-dependent phospholipase A2 (PLA2G5), based on experimental results and important published studies, which lays a theoretical foundation for the high-throughput construction of the cytokine and virion solution chip. The paper then moves on to the construction of the novel GPCR recombinant herpes virion chip and virion nano-oscillators for profiling membrane protein functions, which drove the idea of constructing the new recombinant virion and cytokine liquid chips for HTS of leading drugs. Due to the different structural properties of GPCR, IFNAR2, ACE2 and Spike of 2019-nCoV, their ligands will either bind the extracellular domain of IFNAR2/ACE2/Spike or the specific loops of the GPCR on the envelope of the recombinant herpes virions to induce dynamic charge distribution changes that lead to the variable electron transition for detection. Taken together, the combined overview of two of the most innovative and exciting developments in the immunoinflammatory field provides new insight into high-throughput construction of ultrasensitive cytokine and virion liquid chips for HTS of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases including infectious diseases, acute or chronic inflammation (acute gouty arthritis or rheumatoid arthritis), cardiovascular disease, atheromatosis, diabetes, obesity, tissue injury and tumors. It has significant value in the prevention and treatment of these serious and painful diseases. Graphical abstract

Sleep Deprivation Selectively Down-Regulates Astrocytic 5-HT2B Receptors and Triggers Depressive-Like Behaviors via Stimulating P2X7 Receptors in Mice.

Chronic loss of sleep damages health and disturbs the quality of life. Long-lasting sleep deprivation (SD) as well as sleep abnormalities are substantial risk factors for major depressive disorder, although the underlying mechanisms are not clear. Here, we showed that chronic SD in mice promotes a gradual elevation of extracellular ATP, which activates astroglial P2X7 receptors (P2X7Rs). Activated P2X7Rs, in turn, selectively down-regulated the expression of 5-HT2B receptors (5-HT2BRs) in astrocytes. Stimulation of P2X7Rs induced by SD selectively suppressed the phosphorylation of AKT and FoxO3a in astrocytes, but not in neurons. The over-expression of FoxO3a in astrocytes inhibited the expression of 5-HT2BRs. Down-regulation of 5-HT2BsRs instigated by SD suppressed the activation of STAT3 and relieved the inhibition of Ca2+-dependent phospholipase A2. This latter cascade promoted the release of arachidonic acid and prostaglandin E2. The depression-like behaviors induced by SD were alleviated in P2X7R-KO mice. Our study reveals the mechanism underlying chronic SD-induced depression-like behaviors and suggests 5-HT2BRs as a key target for exploring therapeutic strategies aimed at the depression evoked by sleep disorders.

An association between the BanI polymorphism of the PLA2G4A gene for calcium-dependent phospholipase A2 and plasma glucose levels among females with schizophrenia

Abnormal glucose and lipid metabolism may be associated with altered cytosolic Ca2+-dependent phospholipase A2 (cPLA2) signaling in patients with schizophrenia. The relationship between schizophrenia and the functional BanI polymorphism (rs10798059 variant, A/G polymorphism) of the PLA2G4A gene for cPLA2 has been extensively investigated. We previously reported that it can influence several clinical features of schizophrenia, and it was shown to contribute to schizophrenia risk in several population studies. We performed PCR/RFLP genotyping of 263 Croatian patients (males/females: 139/124) to investigate the relationship between the BanI polymorphism and fasting plasma glucose and lipid levels in patients with schizophrenia. Our results indicate that the BanI polymorphic variant contributes significantly to plasma glucose levels in female patients. Females carrying the PLA2G4A-G allele (PLA2G4A-GG homozygous and PLA2G4A-AG heterozygous) presented with lower glucose levels than PLA2G4A-AA homozygous carriers, and the PLA2G4A genotype contributed approximately 6% of plasma glucose level variability in this group of patients.

Inhibitory effects of thromboxane A2 generation by ginsenoside Ro due to attenuation of cytosolic phospholipase A2 phosphorylation and arachidonic acid release

BackgroundThromboxane A2 (TXA2) induces platelet aggregation and promotes thrombus formation. Although ginsenoside Ro (G-Ro) from Panax ginseng is known to exhibit a Ca2+-antagonistic antiplatelet effect, whether it inhibits Ca2+-dependent cytosolic phospholipase A2 (cPLA2α) activity to prevent the release of arachidonic acid (AA), a TXA2 precursor, is unknown. In this study, we attempted to identify the mechanism underlying G-Ro-mediated TXA2 inhibition. MethodsWe investigated whether G-Ro attenuates TXA2 production and its associated molecules, such as cyclooxygenase-1 (COX-1), TXA2 synthase (TXAS), cPLA2α, mitogen-activated protein kinases, and AA. To assay COX-1 and TXAS, we used microsomal fraction of platelets. ResultsG-Ro reduced TXA2 production by inhibiting AA release. It acted by decreasing the phosphorylation of cPLA2α, p38-mitogen-activated protein kinase, and c-Jun N-terminal kinase1, rather than by inhibiting COX-1 and TXAS in thrombin-activated human platelets. ConclusionG-Ro inhibits AA release to attenuate TXA2 production, which may counteract TXA2-associated thrombosis.

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

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

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

Phospholipase A2 (PLA2) catalyzes the position-specific hydrolysis of fatty acids linked to the sn-2 position of phospholipids (PLs). PLA2s make up a very large superfamily, with more than known 15 groups, classified into secretory PLA2 (sPLA2), Ca2+-dependent cellular PLA2 (sPLA2) and Ca2+-independent cellular PLA2 (iPLA2). Only a few insect sPLA2s, expressed in venom glands and immune tissues, have been characterized at the molecular level. This study aimed to test our hypothesis that insects express iPLA2, using the beet armyworm, Spodoptera exigua, our model insect. Substantial PLA2 activities under calcium-free condition were recorded in several larval tissue preparations. The PLA2 activity was significantly reduced in reactions conducted in the presence of a specific iPLA2 inhibitor, bromoenol lactone (BEL). Analysis of a S. exigua hemocyte transcriptome identified a candidate iPLA2 gene (SeiPLA2-A). The open reading frame encoded 816 amino acid residues with a predicted molecular weight of 90.5 kDa and 6.15 pI value. Our phylogenetic analysis clustered SeiPLA2-A with the other vertebrate iPLA2s. SeiPLA2-A was expressed in all tissues we examined, including hemocytes, fat body, midgut, salivary glands, Malpighian tubules and epidermis. Heterologous expression in Sf9 cells indicated that SeiPLA2-A was localized in cytoplasm and exhibited significant PLA2 activity, which was independent of Ca2+ and inhibited by BEL. RNA interference (RNAi) of SeiPLA2-A using its specific dsRNA in the fifth instar larvae significantly suppressed iPLA2 expression and enzyme activity. dsSeiPLA2-A-treated larvae exhibited significant loss of cellular immune response, measured as nodule formation in response to bacterial challenge, and extended larval-to-pupal developmental time. These results support our hypothesis, showing that SeiPLA2-A predicted from the transcriptome analysis catalyzes hydrolysis of fatty acids from cellular PLs and plays crucial physiological roles in insect immunity and larval growth.

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

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

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

ObjectiveTo study the inhibitory activity of ethanolic extract from Mimosa pigra and Ipomoea asarifolia against Trypanosoma evansi (T. evansi) calcium dependent phospholipase A2. MethodsThe calcium dependent phospholipase A2 (E C 3.1.1.4) enzyme was isolated from T. evansi and purified to electrophoretic homogeneity under non denaturing conditions. It was solubilized from T. evansi cells recovered from white albino rats which were previously inoculated by intraperitoneal injection of infected camel blood. Two indigeneous ethanolic plant extracts used locally for treatment of trypanosomiasis were tested for the inhibition of phospholipases A2. ResultsDouble reciprocal plots of the initial velocity data of the inhibition by the indigenous plant extracts revealed a noncompetitive pattern of inhibition for the Ipomoea asarifolia and a competitive inhibition for Mimosa pigra in a dose dependent fashion. The extrapolated inhibition binding constant (Ki) of these extracts were found to be 2.0×102 μg/mL and 1.12×102 μg/mL respectively. ConclusionsThe low Ki values obtained for these extracts towards this enzyme are an indication of high affinity of the extract or the active components (present in the plants) are for these enzyme and therefore, could be explored to serve as a cheap source of T. evansi PLA2 antidote and as well help in designing a novel drug with high efficiency.

Involvement of palmitate/Ca2 +(Sr2 +)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca2+-induced (Pal/Ca2+) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca2+ with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca2+ from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca2+ influx, seems to be partly due to the opening of Pal/Ca2+ pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca2+-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca2+ (Sr2+) also induced an outflow of K+ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K+/H+ exchange blocker, and the reuptake was prevented by an ATP-dependent K+ channel inhibitor. It was also shown that the addition of Sr2+ to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr2+ and K+ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca2+-dependent phospholipase A2, and a new Sr2+ cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr2+. All of the Ca2+- and Sr2+-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca2+ pores in the maintenance of cell ion homeostasis.

Misfolding of Apoprotein B-100, LDL Aggregation and 17-β -estradiol in Atherogenesis

The long quest for a missing mechanistic rationale accounting for the correlation between plasma cholesterol levels and cardiovascular disease (CVD) has been focused on various possible modifications of low density lipoprotein (LDL), turning this physiological cholesterol carrier into a damaging agent able to trigger atherogenesis and later the onset of the disease. In addition to the debated oxidized LDL (oxLDL), a modified LDL with a misfolded apoprotein B-100, called electronegative LDL(-) for its negative charge due to an increased amount of free fatty acids, is commonly present in plasma. LDL(-) is generated by the action of secretory calcium dependent phospholipase A2. LDL(-) primes LDL aggregation and amyloid formation according to mechanisms very similar to those observed in other misfolding diseases. The LDL particle aggregates recall the structure and size of the subendothelial lipid droplets described in early atherogenesis and elicit a powerful inflammatory response. The use of 17-β-estradiol (E2) confirmed that the suggested atherogenicity of LDL (-) is mostly dependent on the misfolded character of its apoprotein. E2 binding to the apoprotein of native LDL, through a specific and saturable receptor, inhibits misfolding phenomenon despite an unaffected production of LDL (-) by phospholipase A2, ultimately preventing LDL aggregation. The apoprotein misfolding in LDL(-) emerges as a possible significant trigger mechanism of atherogenesis. Potential implications for the development of novel therapeutic approaches might be hypothesized in perspective. The existing evidence is discussed and reported in this review.

Chip electrophoretic separation of highly homologous ammodytoxin isoforms: Three neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom

Ammodytoxins (Atxs), a group of Ca(2+) -dependent neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom, are mainly responsible for venom toxicity. Within the Atx group, LD50 values between three isoforms, A, B, and C are differing with AtxA exhibiting an LD50 value by an order of magnitude lower (more toxic) than the other two isoforms. This difference in toxicity justifies the necessity to prepare suitable antibodies and thus isoform separation to characterize the Atx content of Vipera ammodytes ammodytes venom is of importance. However, a high homology between the three Atx isoforms (differences in only two, respectively, three residues within the last 18 amino acids at the C-terminus, total length 122 residues) hindered the successful separation of isoforms to date. As the investigated phospholipases A2 were reported to exhibit differences in pI values, we concentrate with the current work on the separation of Atx isoforms after fluorescence labeling via chip electrophoresis on a commercially available instrument to build the basis for a fast and easy to handle screening method. In the course of our work, we were able to show that samples of AtxA, AtxB, and AtxC declared to be homogenous by standard analytical techniques consisted indeed of more than one isoform of which the relative amounts were calculated by using the newly developed method.

Gold/Phospholipid nanoconstructs as label-free optical probes for evaluating phospholipase A2 activity

A facile, monophasic strategy, involving a cooperative solvent, dimethylformamide (DMF), to synthesize core/shell architectured gold–phospholipid hybrid nanoconstructs (PLGNPs) was presented herein. We employed the as-synthesized PLGNPs as enzymatic substrates and detecting probes, leading to the development of a novel, lag time-free quantitative assay to evaluate the activity of Ca2+-dependent phospholipase A2 (PLA2), an inflammatory protein that (i) plays a role in the pathogenesis of many inflammatory diseases, (ii) is a mediator in atherosclerosis and ischemic damage to cardiomyocytes, and (iii) has been implicated in the cause of neurodegenerative diseases. Our new bioassay exhibited high specificity, improved speed (assay time:≤20min), acceptable sensitivity, and a limit of detection (1.82nM, equivalent to 0.04unit/mL and 260ng/dL) below the cut-off value of circulating PLA2 (2.07nM, equivalent to 290ng/dL) present in serum samples collected from healthy testers. We characterized the as-obtained PLGNPs using UV–vis spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). These PLGNPs were considerably robust and biocompatible—displaying extraordinary stability against salt-induced aggregation, oxidant etching, and repetitive freeze/thaw treatment—because of the presence of their modifying interfacial thiol (1-dodecanethiol) and phospholipid [1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)] units.

Phospholipase A2, Oxidative Stress, and Neurodegeneration in Binge Ethanol-Treated Organotypic Slice Cultures of Developing Rat Brain

Brain neurodamage from chronic binge ethanol (EtOH) exposure is linked to neuroinflammation and associated oxidative stress. Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures of developing brain age, we reported that binge EtOH promotes release of a neuroinflammatory instigator, arachidonic acid (AA), concomitant with neurodegeneration, and that mepacrine, a global inhibitor of phospholipase A2 (PLA2) enzymes mobilizing AA from phospholipids, is neuroprotective. Here, we sought with binge EtOH-treated HEC cultures to establish that PLA2 activity is responsible in part for significant oxidative stress and to ascertain the PLA2 families responsible for AA release and neurodegeneration. HEC slices, prepared from 1-week-old rats and cultured 2 to 2.5 weeks, were exposed to 100 mM EtOH over 6 successive days, with 4 daytime "withdrawals" (no EtOH). Brain 3-nitrotyrosinated (3-NT)- and 4-hydroxy-2-nonenal (4-HNE)-adducted proteins, oxidative stress footprints, were immunoassayed on days 3 through 6, and mepacrine's effect was determined on day 6. The effects of specific PLA2 inhibitors on neurodegeneration (propidium iodide staining) and AA release (ELISA levels in media) in the cultures were then determined. Also, the effect of JZL184, an inhibitor of monoacylglycerol lipase (MAGL) which is reported to mobilize AA from endocannabinoids during neuroinflammatory insults, was examined. 3-NT- and 4-HNE-adducted proteins were significantly increased by the binge EtOH exposure, consistent with oxidative stress, and mepacrine prevented the increases. The PLA2 inhibitor results implicated secretory PLA2 (group II sPLA2) and to some extent Ca(2+) -independent cytosolic PLA2 (group VI iPLA2) in binge EtOH-induced neurotoxicity and in AA release, but surprisingly, Ca(2+) -dependent cytosolic PLA2 (group IV cPLA2) did not appear important. Furthermore, unlike PLA2 inhibition, MAGL inhibition failed to prevent the neurodegeneration. In these developing HEC slice cultures, pro-oxidative signaling via sPLA2 and iPLA2, but not necessarily cPLA2 or MAGL, is involved in EtOH neurotoxicity. This study provides further insights into neuroinflammatory phospholipase signaling and oxidative stress underlying binge EtOH-induced neurodegeneration in developing (adolescent age) brain in vitro.