Dependent Phospholipase Research Articles

Thermal Stabilization of a Bacterial Zn(II)-Dependent Phospholipase C through Consensus Sequence Design.

Proteins' extraordinary performance in recognition and catalysis has led to their use in a range of applications. However, proteins obtained from natural sources are oftentimes not suitable for direct use in industrial or diagnostic setups. Natural proteins, evolved to optimally perform a task in physiological conditions, usually lack the stability required to be used in harsher conditions. Therefore, the alteration of the stability of proteins is commonly pursued in protein engineering studies. Here, we achieved a substantial thermal stabilization of a bacterial Zn(II)-dependent phospholipase C by consensus sequence design. We retrieved and analyzed sequenced homologues from different sources, selecting a subset of examples for expression and characterization. A non-natural consensus sequence showed the highest stability and activity among those tested. Comparison of the stability parameters of this stabilized mutant and other natural variants bearing similar mutations allows us to pinpoint the sites most likely to be responsible for the enhancement. Point mutations in these sites alter the unfolding process of the consensus sequence. We show that the stabilized version of the protein retains full activity even in harsh oil degumming conditions, making it suitable for industrial applications.

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.

CircPLCE1 facilitates the malignant progression of colorectal cancer by repressing the SRSF2-dependent PLCE1 pre-RNA splicing.

Studies have demonstrated that circular RNAs (circRNAs) play important roles in various types of cancer; however, the mechanisms of circRNAs located in the nucleus have rarely been explored. Here, we report a novel circular RNA circPLCE1 (hsa_circ_0019230) that facilitates the malignant progression of colorectal cancer (CRC) by repressing serine/arginine‐rich splicing factor 2 (SRSF2)‐dependent phospholipase C epsilon 1 (PLCE1) pre‐RNA splicing. Quantitative real‐time polymerase chain reaction was used to determine the expression of circPLCE1 in CRC tissues and cells. Cell Counting Kit‐8, Transwell and flow cytometric assays were used to assess the role of circPLE1 in CRC cell proliferation, migration and apoptosis, respectively. An animal study was conducted to test the role of circPLCE1 in vivo. Furthermore, catRAPID and RPISeq were used to predict the possible binding proteins of circPLCE1. RNA fractionation and RNA immunoprecipitation assays were used to confirm the RNA‐protein interaction. In this study, we found that circPLCE1 was more significantly down‐regulated in CRC tissues compared with that in adjacent normal tissues. However, circPLCE1 knockdown suppressed CRC cell proliferation, migration and invasion and increased apoptosis. Nude mouse experiments showed that ectopic expression of circPLCE1 dramatically increased tumour growth in vivo. Mechanistically, circPLCE1 directly bound to the SRSF2 protein, repressing SRSF2‐dependent PLCE1 pre‐RNA splicing, resulting in the progression of CRC. Individually mutating the binding sites of circPLCE1 abolished the inhibition of PLCE1 mRNA production. Our study revealed a novel molecular mechanism in the regulation of PLCE1 and suggested a new function of circular RNA.

Dissecting Cellular Mechanisms of Long-Chain Acylcarnitines-Driven Cardiotoxicity: Disturbance of Calcium Homeostasis, Activation of Ca2+-Dependent Phospholipases, and Mitochondrial Energetics Collapse.

Long-chain acylcarnitines (LCAC) are implicated in ischemia-reperfusion (I/R)-induced myocardial injury and mitochondrial dysfunction. Yet, molecular mechanisms underlying involvement of LCAC in cardiac injury are not sufficiently studied. It is known that in cardiomyocytes, palmitoylcarnitine (PC) can induce cytosolic Ca2+ accumulation, implicating L-type calcium channels, Na+/Ca2+ exchanger, and Ca2+-release from sarcoplasmic reticulum (SR). Alternatively, PC can evoke dissipation of mitochondrial potential (ΔΨm) and mitochondrial permeability transition pore (mPTP). Here, to dissect the complex nature of PC action on Ca2+ homeostasis and oxidative phosphorylation (OXPHOS) in cardiomyocytes and mitochondria, the methods of fluorescent microscopy, perforated path-clamp, and mitochondrial assays were used. We found that LCAC in dose-dependent manner can evoke Ca2+-sparks and oscillations, long-living Ca2+ enriched microdomains, and, finally, Ca2+ overload leading to hypercontracture and cardiomyocyte death. Collectively, PC-driven cardiotoxicity involves: (I) redistribution of Ca2+ from SR to mitochondria with minimal contribution of external calcium influx; (II) irreversible inhibition of Krebs cycle and OXPHOS underlying limited mitochondrial Ca2+ buffering; (III) induction of mPTP reinforced by PC-calcium interplay; (IV) activation of Ca2+-dependent phospholipases cPLA2 and PLC. Based on the inhibitory analysis we may suggest that simultaneous inhibition of both phospholipases could be an effective strategy for protection against PC-mediated toxicity in cardiomyocytes.

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.

Osmotic activation of a Ca2+-dependent phospholipase C pathway that regulates ∆N TRPV1-mediated currents in rat supraoptic neurons.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus regulate body fluid balance by releasing the hormones vasopressin (VP) and oxytocin (OT) in an osmolality‐dependent manner. Elevations of external osmolality increase MNC firing and hormone release. MNC osmosensitivity is largely due to activation of a mechanosensitive non‐selective cation current that responds to osmotically‐evoked changes in MNC volume and is mediated by an N‐terminal variant of the TRPV1 channel (∆N TRPV1). We report a novel mechanism by which increases in osmolality may modulate ∆N TRPV1‐mediated currents and thus influence MNC electrical behaviour. We showed previously that acute elevations of external osmolality activate the enzyme phospholipase C (PLC) in isolated MNCs. We now show that the osmotic activation of PLC has a time course and dose‐dependence that is consistent with a role in MNC osmosensitivity and that it contributes to the osmotically‐evoked increase in non‐selective cation current in MNCs through a protein kinase C‐dependent pathway. We furthermore show that the mechanism of osmotic activation of PLC requires an increase in internal Ca2+ that depends on influx through L‐type Ca2+ channels. Our data therefore suggest that MNCs possess an osmotically‐activated Ca2+‐dependent PLC that contributes to the osmotic activation of ∆N TRPV1 and may therefore be important in MNC osmosensitivity and in central osmoregulation.

Activation of the sigma receptor 1 modulates AMPA receptor-mediated light-evoked excitatory postsynaptic currents in rat retinal ganglion cells

Sigma receptor (σR), a unique receptor family, is classified into three subtypes: σR1, σR2 and σR3. It was previously shown that σR1 activation induced by 1μM SKF10047 (SKF) suppressed N-methyl-d-aspartate (NMDA) receptor-mediated responses of rat retinal ganglion cells (GCs) and the suppression was mediated by a distinct Ca2+-dependent phospholipase C (PLC)–protein kinase C (PKC) pathway. In the present work, using whole-cell patch-clamp techniques in rat retinal slice preparations, we further demonstrate that SKF of higher dosage (50μM) significantly suppressed AMPA receptor (AMPAR)-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) of retinal ON-type GCs (ON GCs), and the effect was reversed by the σR1 antagonist BD1047, suggesting the involvement of σR1. The SKF (50μM) effect was unlikely due to a change in glutamate release from bipolar cells, as suggested by the unaltered paired-pulse ratio (PPR) of AMPAR-mediated EPSCs of ON GCs. SKF (50μM) did not change L-EPSCs of ON GCs when the G protein inhibitor GDP-β-S or the protein kinase G (PKG) inhibitor KT5823 was intracellularly infused. Calcium imaging further revealed that SKF (50μM) did not change intracellular calcium concentration in GCs and persisted to suppress L-EPSCs when intracellular calcium was chelated by BAPTA. The SKF (50μM) effect was intact when protein kinase A (PKA) and phosphatidylinostiol (PI)-PLC signaling pathways were both blocked. We conclude that the SKF (50μM) effect is Ca2+-independent, PKG-dependent, but not involving PKA, PI-PLC pathways.

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.

A new era of secreted phospholipase A2

A new era of secreted phospholipase A₂

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.

CsrA impacts survival of Yersinia enterocolitica by affecting a myriad of physiological activities.

BackgroundA previous study identified a Yersinia enterocolitica transposon mutant, GY448, that was unable to export the flagellar type three secretion system (T3SS)-dependent phospholipase, YplA. This strain was also deficient for motility and unable to form colonies on Lauria-Bertani agar medium. Preliminary analysis suggested it carried a mutation in csrA. CsrA in Escherichia coli is an RNA-binding protein that is involved in specific post-transcriptional regulation of a myriad of physiological activities. This study investigated how CsrA affects expression of the flagellar regulatory cascade that controls YplA export and motility. It also explored the effect of csrA mutation on Y. enterocolitica in response to conditions that cue physiological changes important for growth in environments found both in nature and the laboratory.ResultsThe precise location of the transposon insertion in GMY448 was mapped within csrA. Genetic complementation restored disruptions in motility and the YplA export phenotype (Yex), which confirmed this mutation disrupted CsrA function. Mutation of csrA affected expression of yplA and flagellar genes involved in flagellar T3SS dependent export and motility by altering expression of the master regulators flhDC. Mutation of csrA also resulted in increased sensitivity of Y. enterocolitica to various osmolytes, temperatures and antibiotics.ConclusionsThe results of this study reveal unique aspects of how CsrA functions in Y. enterocolitica to control its physiology. This provides perspective on how the Csr system is susceptible to adaptation to particular environments and bacterial lifestyles.

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.