Phospholipase A2 Enzyme Activity Research Articles

A study of the correlation between phospholipase A2 enzyme activity and anti-complement antibodies in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs or organ systems. SLE and one of its most common complications, lupus nephritis (LN), are characterized by chronic inflammation due to the secretion of pro-inflammatory molecules and a tissue deposition of immune complexes formed by autoantibodies and their target antigens, such as double-stranded DNA, other nuclear antigens, and complement proteins. Increased activity of phospholipase A2 enzymes (PLA2) like calcium-independent PLA2 (iPLA2), secretory PLA2 (sPLA2), and cellular PLA2 (cPLA2) has a crucial role in the maintenance of the inflammatory process during the course of SLE. This study aimed to evaluate PLA2 activity and identify the dominant type of PLA2 enzymes in a cohort of Bulgarian patients with SLE and LN. Additionally, we investigated whether the increased PLA2 activity was correlated with the presence of autoantibodies specific to the complement proteins C3 and factor H.

Phospholipase A2 activity is required for immune defense of European (Apis mellifera) and Asian (Apis cerana) honeybees against American foulbrood pathogen, Paenibacillus larvae.

Honeybees require an efficient immune system to defend against microbial pathogens. The American foulbrood pathogen, Paenibacillus larvae, is lethal to honeybees and one of the main causes of colony collapse. This study investigated the immune responses of Apis mellifera and Apis cerana honeybees against the bacterial pathogen P. larvae. Both species of honeybee larvae exhibited significant mortality even at 102 103 cfu/mL of P. larvae by diet-feeding, although A. mellifera appeared to be more tolerant to the bacterial pathogen than A. cerana. Upon bacterial infection, the two honeybee species expressed both cellular and humoral immune responses. Hemocytes of both species exhibited characteristic spreading behaviors, accompanied by cytoskeletal extension along with F-actin growth, and formed nodules. Larvae of both species also expressed an antimicrobial peptide called apolipophorin III (ApoLpIII) in response to bacterial infection. However, these immune responses were significantly suppressed by a specific inhibitor to phospholipase A2 (PLA2). Each honeybee genome encodes four PLA2 genes (PLA2A ~ PLA2D), representing four orthologous combinations between the two species. In response to P. larvae infection, both species significantly up-regulated PLA2 enzyme activities and the expression of all four PLA2 genes. To determine the roles of the four PLA2s in the immune responses, RNA interference (RNAi) was performed by injecting gene-specific double stranded RNAs (dsRNAs). All four RNAi treatments significantly suppressed the immune responses, and specific inhibition of the two secretory PLA2s (PLA2A and PLA2B) potently suppressed nodule formation and ApoLpIII expression. These results demonstrate the cellular and humoral immune responses of A. mellifera and A. cerana against P. larvae. This study suggests that eicosanoids play a crucial role in mediating common immune responses in two closely related honeybees.

Evaluation of Saxifraga stolonifera phenolic extracts as a potential antivenom against Deinagkistrodon acutus venom: In vitro and in vivo studies

In the snake-infested mountainous regions of China, Saxifraga stolonifera [L.] Meeb is widely utilized as an immediate remedy for venomous snake bites. However, the scientific understanding of S. stolonifera's efficacy in snakebite treatment remains limited and requires further investigation. The aim of this study was to assess the inhibitory effects of Saxifraga stolonifera phenolic extracts (SSPE) on Deinagkistrodon acutus venom (DAV) and explore the potential of S. stolonifera as a valuable candidate for antivenom development. We employed our previously optimized extraction conditions to obtain SSPE. In vitro experiments utilizing diverse models were conducted to assess the inhibitory effects of the extracted phenolic compounds on DAV, specifically targeting phospholipase A2 (PLA2), proteolytic, fibrinolytic, and hyaluronidase enzymes. Furthermore, in vivo investigations were conducted to evaluate the inhibitory potential of the extracted compounds against DAV-induced hemorrhagic and edematogenic activity. To elucidate the chemical composition of the phenolic extracts, Ultra Performance Liquid Chromatography-mass spectrometry (UPLC-MS) analysis was performed. Our in vitro inhibition study showed that S. stolonifera was able to inhibit the activities of PLA2 enzyme, proteolytic enzyme, hyaluronidase and fibrinogenolytic. The median effective dose (ED50) values of SSPE for inhibiting PLA2 enzyme, proteolytic enzyme and hyaluronidase activities were 0.115 mg/mL, 0.026 mg/mL and 0.238 mg/mL, respectively. The DAV-induced hemorrhagic and edematogenic effects of the SSPE were also successfully inhibited in vivo, and the high SSPE concentration was able to completely inhibit the hemorrhage and edema. It is noteworthy that the mice suffered no harm from the high SSPE concentration. The composition analysis showed that the phenolic substances contained in SSPE are gallic acid, protocatechuic acid, chlorogenic acid, rutin, kaempferol-3-O-ɑ-L-rhamnoside, kaempferol-3-O-β-D-glucopyranoside, quercetin and kaempferol. This study provides scientific validation of the inhibitory efficacy of S. stolonifera as an emergency treatment for venomous snake bites, offering a theoretical foundation for future drug development strategies targeting snakebite.

Identification of four secretory phospholipase A2s in a lepidopteran insect, Acrolepiopsis sapporensis, and their functional association with cellular immune responses.

Eicosanoids are a group of the oxygenated C20 polyunsaturated fatty acids and play crucial roles in mediating various insect physiological processes. Catalytic activity of phospholipase A2 (PLA2) provides an initial substrate, arachidonic acid (AA), for subsequent eicosanoid biosynthesis. This study identified four different secretory PLA2 (As-PLA2A-As-PLA2D) genes encoded in the Asian onion moth, Acrolepiopsis sapporensis. A phylogenetic analysis indicated that As-PLA2A and As-PLA2D are clustered with Group III PLA2s while As-PLA2B and As-PLA2C are clustered with Group XII and Group X PLA2s, respectively. Expression levels of these PLA2 genes increased along with larval development, especially in the fat body. A bacterial immune challenge upregulated the basal expression levels of the four PLA2 genes, which resulted in significant increases of the PLA2 enzyme activity. The enzyme activity was susceptible to a calcium chelator or reducing agent, suggesting Ca2+ dependency and disulfide linkage required for the catalytic activities of the secretory type of PLA2s. In addition, the PLA2 activity was also susceptible to bromophenacyl bromide (BPB), a specific inhibitor to sPLA2, but not to intracellular PLA2 inhibitors. An addition of BPB to the immune challenge significantly prevented hemocyte-spreading behavior of A. sapporensis. BPB treatment also suppressed a cellular immune response measured by hemocyte nodule formation. However, the immunosuppression was significantly rescued by the AA addition. To determine the PLA2(s) responsible for the immunity, individual RNA interference (RNAi) treatments specific to each of the four PLA2s were performed. Injection of gene-specific double-stranded RNAs caused significant reductions in the transcript level in all four PLA2s. In all four PLA2s, the RNAi treatments prevented the cellular immune response even after the immune challenge. This study reports four secretory PLA2s encoded in A. sapporensis and their function in mediating cellular immunity.

PRDX6 Promotes Fatty Acid Oxidation via PLA2-Dependent PPARα Activation in Rats Fed High-Fat Diet.

Aims: Nonalcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease globally, which is defined as an excess accumulation of fat caused by the imbalance of lipogenesis and lipid catabolism. Recently, increasing evidence suggests that peroxiredoxin 6 (PRDX6) is involved in the pathogenesis and progression of NAFLD. However, little is known regarding its role in liver lipid catabolism. Results: We found that PRDX6 level was significantly increased in liver tissues after high-fat diet (HFD) treatment. PRDX6 knockout (KO) exacerbated HFD-induced hepatic steatosis. PRDX6 KO did not affect messenger RNA (mRNA) and protein levels of peroxisome proliferator-activated receptor alpha (PPARα). However, PRDX6 KO decreased the mRNA and protein levels of carnitine palmitoyltransferase-1alpha (CPT-1α) and acyl-CoA oxidase 1 (ACOX1), the target genes of PPARα. PRDX6 KO also did not activate AMP-activated protein kinase (AMPK)α/proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), the upstream signal of PPARα. However, PRDX6 KO reduces the levels of PPARα activators, the oxidized fatty acids (9- and 13-hydroxyoctadecadienoic acid) in HFD rats. More interestingly, PRDX6 promoted the production of oxidized fatty acids by hydrolyzing oxidized low-density lipoprotein (Ox-LDL), which depends on its phospholipase A2 (PLA2) activity. PRDX6 mutation on its PLA2 and its competitive phospholipase inhibitor inhibited the production of the oxidized fatty acids as well as the activation of PPARα. Furthermore, PRDX6 overexpression enhanced the transcriptional activation of PPARα. Innovation and Conclusion: This study elucidates for the first time the role of PLA2 enzyme activity of PRDX6 in fatty acid oxidation and reveals a novel mechanism of PRDX6 involved in liver steatosis. Antioxid. Redox Signal. 38, 1184-1200.

Functional mass spectrometry imaging maps phospholipase-A2 enzyme activity during osteoarthritis progression.

Background: Enzymes are central components of many physiological processes, and changes in enzyme activity are linked to numerous disease states, including osteoarthritis (OA). Assessing changes in enzyme function can be challenging because of difficulties in separating affected tissue areas that result in the homogenisation of healthy and diseased cells. Direct correlation between spatially-resolved enzyme distribution(s) and diseased cells/tissues can thus lead to advances in our understanding of OA pathophysiology. Herein, we present a method that uses mass spectrometry imaging (MSI) to visualise the distribution of lipase enzymes and their downstream lipid products in fresh bone and cartilage tissue sections. Immunohistostaining of adjacent tissue sections was then used to identify OA cells/tissues, which were then statistically correlated with molecular-level images. Methods: MSI was used to image lipase enzymes, their substrates, and their metabolic products to validate enzymatic activity and correlate to OA regions determined by immunohistochemistry (IHC). Based on the modified Mankin score, six non-OA and OA patient-matched osteochondral samples were analysed by matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). Due to the involvement of phospholipase A2 (PLA2) in inflammatory pathways, explant tissues were treated with IL-1β to mimic inflammation observed in OA. Bovine explant tissues were then subject to MSI methods to observe the spatial distribution of PLA2. Results: Compared with non-OA samples, OA samples showed an elevated level of multiple arachidonic acid (AA)-containing phospholipids (P < 0.001), in which the elevation in the surface and deep layer cartilage of OA tissues is correlated to elevated PLA2 activity (P < 0.001). Bovine explant tissues treated with IL-1β to mimic OA pathophysiology validated these results and displayed elevated PLA2 levels in OA mimic samples relative to the controls (P < 0.001). It was established that the PLA2G2A isoform specifically was responsible for PLA2 enzyme activity changes in OA tissues (P < 0.001). Conclusion: Our results present a reliable method for imaging enzyme dynamics in OA cartilage, which sets up the foundation for future spatial enzyme dynamics in the OA field. We demonstrated that OA patients exhibit increased expression of PLA2G2A at the superficial and deep cartilage zone that degrades cartilage differently at the spatial level. A tissue-specific PLA2G2A precision inhibition may be the potential target for OA.

Cysteine-Encapsulated Liposome for Investigating Biomolecular Interactions at Lipid Membranes.

The development of a strategy to investigate interfacial phenomena at lipid membranes is practically useful because most essential biomolecular interactions occur at cell membranes. In this study, a colorimetric method based on cysteine-encapsulated liposomes was examined using gold nanoparticles as a probe to provide a platform to report an enzymatic activity at lipid membranes. The cysteine-encapsulated liposomes were prepared with varying ratios of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol through the hydration of lipid films and extrusions in the presence of cysteine. The size, composition, and stability of resulting liposomes were analyzed by scanning electron microscopy (SEM), dynamic light scattering (DLS), nuclear magnetic resonance (NMR) spectroscopy, and UV-vis spectrophotometry. The results showed that the increased cholesterol content improved the stability of liposomes, and the liposomes were formulated with 60 mol % cholesterol for the subsequent experiments. Triton X-100 was tested to disrupt the lipid membranes to release the encapsulated cysteine from the liposomes. Cysteine can induce the aggregation of gold nanoparticles accompanying a color change, and the colorimetric response of gold nanoparticles to the released cysteine was investigated in various media. Except in buffer solutions at around pH 5, the cysteine-encapsulated liposomes showed the color change of gold nanoparticles only after being incubated with Triton X-100. Finally, the cysteine-encapsulated liposomal platform was tested to report the enzymatic activity of phospholipase A2 that hydrolyzes phospholipids in the membrane. The hydrolysis of phospholipids triggered the release of cysteine from the liposomes, and the released cysteine was successfully detected by monitoring the distinct red-to-blue color change of gold nanoparticles. The presence of phospholipase A2 was also confirmed by the appearance of a peak around 690 nm in the UV-vis spectra, which is caused by the cysteine-induced aggregation of gold nanoparticles. The results demonstrated that the cysteine-encapsulated liposome has the potential to be used to investigate biological interactions occurring at lipid membranes.

Dengue NS1 induces phospholipase A2 enzyme activity, prostaglandins, and inflammatory cytokines in monocytes

IntroductionDengue virus (DENV) NS1 is a non-structural secretory protein associated with severe disease and known to cause vascular leak leading to dengue haemorrhagic fever (DHF). As phospholipases A2 (PLA2) enzymes, platelet activating factor, and leukotrienes are elevated in dengue, we sought to investigate whether NS1 potentially contributes to disease pathogenesis by inducing PLA2s. MethodsTHP-1 cells and primary human monocytes of healthy adults (n = 6) were co-cultured with DENV1 NS1, LPS and media alone. The latter two were used as positive and negative controls. The cell culture supernatants and lysates were harvested at 12 and 24 h and the activity of secretory and cytoplasmic PLA2, prostaglandins (PGE2 and PGD2) were measured by ELISA and cytokines levels were measured using a magnetic Luminex assay. Expression of PLA2G4A, PLA2G2A, PLA2G5, PLA2G10, PLA2G7, GAPDH, NLRP3 and DDX58 genes were assessed using quantitative RT-PCR. ResultscPLA2 (p = 0.005), sPLA2 (p = 0.04), PGE2 metabolite (p = 0.02) and PGD2 metabolite (p = 0.04) levels were significantly higher at 12 h in monocytes co-cultured with NS1. Levels of IP-10 (p = 0.005) and IL-10 (p = 0.009) was significantly higher at 24 h, whereas IFNα level was significantly higher (p = 0.013) only at 12 h. IL-1β (p = 0.028 and p = 0.031) and TNFα (p = 0.007 and p = 0.011) showed significantly higher levels at both time points. At 12 h significant upregulation of PLA2G4A (p < 0.0001) was seen, whereas PLA2G7 (p = <0.0001), NLRP3 (p = 0.0009) and DDX58 (p = 0.0056) were significantly downregulated. This pattern changed at 24 h with PLA2G4A (p = 0.0069) showing a marked downregulation and PLA2G7, DDX58 and NLRP3 showing an upregulation, although not significant. ConclusionDengue NS1 induces the production of PLA2 enzymes, prostaglandins and inflammatory cytokines from primary human monocytes, which could play a role in vascular leak in dengue.

The IRE1α Inhibitor KIRA6 Blocks Leukotriene Biosynthesis in Human Phagocytes

The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) has been linked to inflammation and lipid mediator production. Here we report that the potent IRE1α inhibitor, KIRA6, blocks leukotriene biosynthesis in human phagocytes activated with lipopolysaccharide (LPS) plus N-formyl-methionyl-leucyl-phenylalanine (fMLP) or thapsigargin (Tg). The inhibition affects both leukotriene B4 (LTB4) and cysteinyl leukotriene (cys-LTs) production at submicromolar concentration. Macrophages made deficient of IRE1α were still sensitive to KIRA6 thus demonstrating that the compound’s effect on leukotriene production is IRE1α-independent. KIRA6 did not exhibit any direct inhibitory effect on key enzymes in the leukotriene pathway, as assessed by phospholipase A2 (PLA2), 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S) enzyme activity measurements in cell lysates. However, we find that KIRA6 dose-dependently blocks phosphorylation of p38 and ERK, mitogen-activated protein kinases (MAPKs) that have established roles in activating cytosolic PLA2α (cPLA2α) and 5-LOX. The reduction of p38 and ERK phosphorylation is associated with a decrease in cPLA2α phosphorylation and attenuated leukotriene production. Furthermore, KIRA6 inhibits p38 activity, and molecular modelling indicates that it can directly interact with the ATP-binding pocket of p38. This potent and unexpected, non-canonical effect of KIRA6 on p38 and ERK MAPKs and leukotriene biosynthesis may account for some of the immune-modulating properties of this widely used IRE1α inhibitor.

Functional, immunological characterization, and anticancer activity of BaMtx: A new Lys49- PLA2 homologue isolated from the venom of Peruvian Bothrops atrox snake (Serpentes: Viperidae)

Bothorps atrox is responsible for most of the ophidism cases in Perú. As part of the envenoming, myotoxicity is one of the most recurrent and destructive effects. In this study, a myotoxin, named BaMtx, was purified from B. atrox venom to elucidate its biological, immunological, and molecular characteristics. BaMtx was purified using CM-Sephadex-C-25 ion-exchange resin and SDS-PAGE analysis showed a unique protein band of 13 kDa or 24 kDa under reducing or non-reducing conditions, respectively. cDNA sequence codified a 122-aa mature protein with high homology with other Lys49-PLA2s; modeled structure showed a N-terminal helix, a β-wing region, and a C-terminal random coil. This protein has a poor phospholipase A2 enzymatic activity. BaMtx has myotoxic (DMM = 12.30 ± 0.95 μg) and edema-forming (DEM = 26.00 ± 1.15 μg) activities. Rabbit immunization with purified enzyme produced anti-BaMtx antibodies that reduced 50.28 ± 10.15% of myotoxic activity and showed significant cross-reactivity against B. brazili and B pictus venoms. On the other hand, BaMtx exhibits mild anti-proliferative and anti-migratory effects on breast cancer cells, affecting the ROS and NADH levels, which may reduce mitochondrial respiration. These results contribute to the understanding of B. atrox Lys49-PLA2 effects and establish the anticancer potential de BaMtx.

The Pseudomonas aeruginosa Type III Secretion System Exoenzyme Effector ExoU Induces Mitochondrial Damage in a Murine Bone Marrow-Derived Macrophage Infection Model.

ABSTRACTPseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes nosocomial pneumonia, urinary tract infections, and bacteremia. A hallmark of P. aeruginosa pathogenesis is disruption of host cell function by the type III secretion system (T3SS) and its cognate exoenzyme effectors. The T3SS effector ExoU is phospholipase A2 (PLA2) that targets the host cell plasmalemmal membrane to induce cytolysis and is an important virulence factor that mediates immune avoidance. In addition, ExoU has been shown to subvert the host inflammatory response in a noncytolytic manner. In primary bone marrow-derived macrophages (BMDMs), P. aeruginosa infection is sensed by the nucleotide-binding domain containing leucine-rich repeats-like receptor 4 (NLRC4) inflammasome, which triggers caspase-1 activation and inflammation. ExoU transiently inhibits NLRC4 inflammasome-mediated activation of caspase-1 and its downstream target, interleukin 1β (IL-1β), to suppress activation of inflammation. In the present study, we sought to identify additional noncytolytic virulence functions for ExoU and discovered an unexpected association between ExoU, host mitochondria, and NLRC4. We show that infection of BMDMs with P. aeruginosa strains expressing ExoU elicited mitochondrial oxidative stress. In addition, mitochondria and mitochondrion-associated membrane fractions enriched from infected cells exhibited evidence of autophagy activation, indicative of damage. The observation that ExoU elicited mitochondrial stress and damage suggested that ExoU may also associate with mitochondria during infection. Indeed, ExoU phospholipase A2 enzymatic activity was present in enriched mitochondria and mitochondrion-associated membrane fractions isolated from P. aeruginosa-infected BMDMs. Intriguingly, enriched mitochondria and mitochondrion-associated membrane fractions isolated from infected Nlrc4 homozygous knockout BMDMs displayed significantly lower levels of ExoU enzyme activity, suggesting that NLRC4 plays a role in the ExoU-mitochondrion association. These observations prompted us to assay enriched mitochondria and mitochondrion-associated membrane fractions for NLRC4, caspase-1, and IL-1β. NLRC4 and pro-caspase-1 were detected in enriched mitochondria and mitochondrion-associated membrane fractions isolated from noninfected BMDMs, and active caspase-1 and active IL-1β were detected in response to P. aeruginosa infection. Interestingly, ExoU inhibited mitochondrion-associated caspase-1 and IL-1β activation. The implications of ExoU-mediated effects on mitochondria and the NLRC4 inflammasome during P. aeruginosa infection are discussed.

Activation of the cytosolic calcium-independent phospholipase A2 β isoform contributes to TRPC6 externalization via release of arachidonic acid

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A2 (PLA2), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA2 in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA2 enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA2 involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA2α, cPLA2γ, iPLA2β, or iPLA2γ. Downregulation of the β isoform of iPLA2 blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.

Hemorrhagic changes and microglia activation induced by Macrovipera lebetina obtusa venom with the inhibited enzymatic activity in rat brain.

The metalloproteinases and phospholipase A2 are the main enzymes in the venom of Macrovipera lebetina obtusa that play a decisive role in the destructive and toxic effects on the organism of the prey. Metalloproteinases cause hemorrhagic damage, destroy the basement membrane of the blood vessel and disrupt the connections between endothelial cells. Phospholipase A2 causes hemolysis of erythrocytes, destroy the cell membranes, and inhibits the adhesion of platelets and so on. The state of the capillaries of the rat brain and microglia under the action of the venom with separately inhibited enzymes was investigated and compared to the action of the crude venom. Also, the toxicity LD50 of the venom of Macrovipera lebetina obtusa with the inhibited enzymatic activity was determined. The histochemical study showed that the inhibition of phospholipase A2 enzymatic activity did not significantly change the vasodestructive effect of the venoms. In case of action of a venom with inhibited enzymatic activity of metalloproteinases, low activity of microglia and less damaged capillaries were observed. The toxicity of the venom with inhibited phospholipase A2 and with inhibited metalloproteinases was respectively 1.8 and 3.7 times weaker than that of the crude venom. We can claim that both the toxicity of the venom of Macrovipera lebetina obtusa, the damaged brain vessels and the increased activity of CNS microglia are determined mainly by the action of metalloproteinases.

Inhibition of key enzymes linked to snake venom induced local tissue damage by kolaviron.

Snakebite envenoming is an important public health problem that threatens the lives of healthy individuals especially in many tropical countries like Nigeria. Antivenins, the only efficient approach for snakebite envenoming, are limited in their efficacy in the neutralization of local tissue damage. Snake venom phospholipase A2 (PLA2), protease, hyaluronidase and l-amino acid oxidase (LAAO) are the major hydrolytic enzymes involve in local tissue damage. Therefore, this study evaluates the inhibitory effect of kolaviron (KV) against Naja n. nigricollis (NNN) snake venom hydrolytic enzymes involved in local tissue damage. Kolaviron was evaluated for its ability to inhibit the hydrolytic enzyme activities of NNN venom phospholipase A2 (PLA2), protease, hyaluronidase and l-amino acidoxidase (LAAO). Present study also deals with the neutralization of NNN venom enzyme(s) induced complications such as myotoxic, edemic, hemolytic and procoagulant effects. Kolaviron inhibited the PLA2, protease, hyaluronidase and LAAO enzyme activities of NNN venom in adose-dependent manner. Furthermore, myotoxic, edemic, hemolytic and procoagulant effects induced by NNN venom enzyme were neutralized significantly (p<0.05) when different doses of KV were pre-incubated with venom before assays. These findings clearly present kolaviron as apotent inhibitor against NNN venom hydrolytic enzymes involved in local tissue damage and may act by either forming an inhibitor-enzyme complex that restricts the substrate availability to the enzyme or direct binding to the enzyme active site that affects the enzyme activity thereby mitigating venom-induced toxicity.

Phospholipase A2 way to hydrolysis: Dint formation, hydrophobic mismatch, and lipid exclusion

Phospholipase A2 (PLA2) exerts a wide range of biological effects and attracts a lot of attention of researchers. Two sites are involved in manifestation of PLA2 enzymatic activity: catalytic site responsible for substrate binding and fatty acid cleavage from the sn-2 position of a glycerophospholipid, and interface binding site (IBS) responsible for the protein binding to lipid membrane. IBS is formed by positively charged and hydrophobic amino acids on the outer surface of the protein molecule. Understanding the mechanism of PLA2 interaction with the lipid membrane is the most challenging step in biochemistry of this enzyme. We used a combination of experimental and computer simulation techniques to clarify molecular details of bee venom PLA2 interaction with lipid bilayers formed by palmitoyloleoylphosphatidylcholine or dipalmitoylphosphatidylcholine. We found that after initial enzyme contact with the membrane, a network of hydrogen bonds was formed. This led to deformation of the interacting leaflet and dint formation. The bilayer response to the deformation depended on its phase state. In a gel-phase bilayer, diffusion of lipids is restricted therefore chain melting occurred in both leaflets of the bilayer. In the case of a fluid-phase bilayer, lateral diffusion is possible, and lipid polar head groups were excluded from the contact area. As a result, the bilayer became thinner and a large hydrophobic area was formed. We assume that relative ability of a bilayer to come through lipid redistribution process defines the rate of initial stages of the catalysis.

Characterization of plasma secretory phospholipase A2 activity in the prairie rattlesnake (Crotalus viridis)

BackgroundPlasma phospholipase A2 (PLA2) enzyme activity is a key component of innate immunity in most vertebrates. We evaluated circulating secreted PLA2 activity of prairie rattlesnakes (Crotalus viridis) by incubation of plasma with bacteria labeled with fluorescent membrane lipids.ResultsIncubation of bacteria with increasing volumes of plasma resulted in volume-dependent lysis of fatty acids from bacterial membranes. The activity was rapid, with substantial activity recorded after only 5 min of incubation with labeled bacteria, and a linear response for 20 min. In addition, the lysis activity was temperature-dependent, increasing activities from 5 to 20 °C, peak activities at 25–30 °C, and then decreasing activities from 35 to 40 °C. Furthermore, the activity was inhibited in a concentration-dependent manner by p-bromophenacyl bromide, a specific inhibitor of PLA2 activity, which indicated that the observed activities were due to the presence of PLA2 in the plasma of C. viridis.ConclusionsThis study represents the first description of secretory PLA2 activity in the plasma of a snake. Our study shows that in addition to being an important component of snake venom, PLA2 enzymes play an important role in the snake’s immune response.

A bioactive flavan-3-ol from the stem bark of Neocarya macrophylla

Antisnake venom activity of the ethylacetate and n-butanol soluble fractions of the methanol stem bark extract of Neocarya macrophylla were evaluated against Naja nigricollis venom. There was maximum protection to mice against venom-induced action after administration of the pre-incubated mixture of the venom and the fractions compared with the venom only. The fractions were also able to significantly inhibit phospholipase A2 enzyme activity. Based on the above findings, the ethylacetate fraction was subjected to purification using column chromatography and gel filtration; the procedures led to the isolation of a flavan-3-ol, catechin. The structure of the compound was identified based on chemical test, NMR analysis (1D- & 2D-NMR) and comparison with existing data in the literature. The catechin have demonstrated significant (p<0.05) antisnake venom activity in vitro with 100% survival rate; it was also found to be 42.0% effective in combating the hydrolytic action of N. nigricollis PLA2 enzyme. Catechin significantly (p<0.05) reduced the nociceptive response induced by formalin in both phases and there was remarkable inhibition of formalin-induced inflammation at the 1st, 2nd and 3rd hours, though the effect was not statistically significant compared to the control. The compound, catechin could serve as a lead in the development of novel antisnake venoms, analgesics and anti-inflammatory agents.

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus.

Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

Mouse Models of Genetically Altered Peroxiredoxin 6.

Peroxiredoxin 6 (Prdx6) has been shown to have three enzymatic activities: peroxidase, phospholipase A2 (PLA2) and acyl transferase. The peroxidase activity is unusual, as it is capable of reducing phospholipid hydroperoxides (as well as hydrogen peroxide and short chain organic peroxides). Knockout and overexpressing mice have been produced that demonstrate the effect that eliminating or overproducing Prdx6 has on the animals’ physiology. In addition, mutations in various amino acids of Prdx6 have been identified that interfere with different enzymatic functions as well as protein transport. These mutations were originally characterized biochemically; subsequently, several knock-in mouse strains have been produced, each containing one mutation. These mice include the S32T knock-in that affects protein transport, the C47S knock-in that inactivates the peroxidase enzymatic activity, the D140A knock-in that inactivates the PLA2 enzymatic activity and the H26A knock-in that inactivates the peroxidase and blocks binding to phospholipids. This review summarizes the properties of these mice based upon studies conducted with the knockout, overexpressing and knock-in mice and the effect of the genetic changes on the biochemistry and physiology of these mice. The availability of these mice is also briefly discussed.

Tracking of secretory phospholipase A2 enzyme activity levels from childhood to adulthood: a 21‐year cohort

ObjectiveSecretory phospholipase A2 (sPLA2) enzyme activity is a potential inflammatory biomarker for cardiovascular disease. We examined the tracking, or persistence, of sPLA2 enzyme activity levels from childhood to adulthood, and identify potentially modifiable factors affecting tracking. MethodProspective cohort of 1735 children (45% females) who had serum sPLA2 enzyme activity levels and other cardiovascular disease risk factors measured in 1980 that were followed‐up in 2001. ResultssPLA2 activity tracked from childhood to adulthood for males (r=0.39) and females (r=0.45). Those who decreased body mass index relative to their peers were more likely to resolve elevated childhood sPLA2 levels than have persistent elevated sPLA2 levels in childhood and adulthood. Those who consumed less fruit, and gained more body mass index relative to their peers, began smoking or were a persistent smoker between childhood and adulthood were more likely to develop incident elevated sPLA2 levels than those with persistent not elevated sPLA2 levels. ConclusionsChildhood sPLA2 enzyme activity levels associate with adult sPLA2 levels 21 years later. Healthful changes in modifiable risk factors that occur between childhood and adulthood might prevent children from developing elevated sPLA2 levels in adulthood.