Catalytic Activity Of Phospholipase A2 Research Articles

A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling.

Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.

Four phospholipase A2 genes encoded in the western flower thrips genome and their functional differentiation in mediating development and immunity

Eicosanoids are synthesized from phospholipids by the catalytic activity of phospholipase A2 (PLA2). Even though several PLA2s are encoded in the genome of different insect species, their physiological functions are not clearly discriminated. This study identified four PLA2 genes encoded in the western flower thrips, Frankliniella occidentalis. Two PLA2s (Fo-PLA2C and Fo-PLA2D) are predicted to be secretory while the other two PLA2s (Fo-PLA2A and Fo-PLA2B) are intracellular. All four PLA2 genes were expressed in all developmental stages, of which Fo-PLA2B and Fo-PLA2C were highly expressed in larvae while Fo-PLA2A and Fo-PLA2D were highly expressed in adults. Their expressions in different tissues were also detected by fluorescence in situ hybridization. All four PLA2s were detected in the larval and adult intestines and the ovary. Feeding double-stranded RNAs specific to the PLA2 genes specifically suppressed the target transcript levels. Individual RNA interference (RNAi) treatments led to significant developmental retardation, especially in the treatments specific to Fo-PLA2B and Fo-PLA2D. The RNAi treatments also showed that Fo-PLA2B and Fo-PLA2C expressions were required for the induction of immune-associated genes, while Fo-PLA2A and Fo-PLA2D expressions were required for ovary development. These results suggest that four PLA2s are associated with different physiological processes by their unique catalytic activities and expression patterns.

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.

Singlet-Oxygen-Induced Phospholipase A2 Inhibition: A Major Role for Interfacial Tryptophan Dioxidation.

Several studies have revealed that various diseases such as cancer have been associated with elevated phospholipase A2 (PLA2) activity. Therefore, the regulation of PLA2 catalytic activity is undoubtedly vital. In this study, effective inactivation of PLA2 due to reactive species produced from cold physical plasma as a source to model oxidative stress is reported. We found singlet oxygen to be the most relevant active agent in PLA2 inhibition. A more detailed analysis of the plasma‐treated PLA2 identified tryptophan 128 as a hot spot, rich in double oxidation. The significant dioxidation of this interfacial tryptophan resulted in an N‐formylkynurenine product via the oxidative opening of the tryptophan indole ring. Molecular dynamics simulation indicated that the efficient interactions between the tryptophan residue and phospholipids are eliminated following tryptophan dioxidation. As interfacial tryptophan residues are predominantly involved in the attaching of membrane enzymes to the bilayers, tryptophan dioxidation and indole ring opening leads to the loss of essential interactions for enzyme binding and, consequently, enzyme inactivation.

Insights into the mechanism of inhibition of phospholipase A2 by resveratrol: An extensive molecular dynamics simulation and binding free energy calculation

Phospholipase A2 (PLA2) is one of the enzymes involved in the development of cardiovascular diseases, vascular inflammation, risk of heart attacks, and strokes. This enzyme is responsible for catalyzing the hydrolytic cleavage of ester bonds of phospholipids in the biological pathway of inflammation. To prevent the undesired hydrolysis of phospholipids, the catalytic activity of PLA2 needs to be blocked. Resveratrol is a plant-derived polyphenol inhibitor, proven to have anti-inflammatory properties. However, there is still substantial ambiguity about its inhibitory function. The present study uncovers a detailed molecular mechanism behind the resveratrol action in inhibition of PLA2, by applying and comparing two 200-ns molecular dynamics simulations. The results of structural analyses revealed that the binding of resveratrol to PLA2 reduces the content of β-sheets and increases a 5-helix to PLA2 structure, producing more folding and stability in protein. In the active site, the resveratrol is placed between the N-terminal α-helix and the newly formed 5-helix through the hydrophobic interactions with ILE19 and LEU3 residues, as well as the hydrogen bond interactions. These interactions play the role of a network at the entrance of the enzyme active site and prevent the penetration of water molecules into the PLA2 cavity. A high occupancy hydrogen bonding has been identified between SER23 of the protein and hydroxyl group of resveratrol. Furthermore, the estimation of binding free energy verified the binding affinity of resveratrol is thermodynamically sufficient to be stably bounded to PLA2. It also proved that the van der Waals interactions, particularly hydrophobic interactions, have the most significant role in PLA2-resveratrol binding and stability. Overall, our results provide useful information on the stepwise mechanism of the inhibition of PLA2 enzyme by resveratrol, as a target for improving the pharmacological applications.

A non-venomous sPLA2 of a lepidopteran insect: Its physiological functions in development and immunity

Eicosanoids are oxygenated C20 polyunsaturated fatty acids that mediate various physiological processes in insects. Eicosanoid biosynthesis begins with a C20 precursor, arachidonic acid (5,8,11,14-eicosatetraenoic acid: AA). AA is usually released from phospholipids at sn-2 position by catalytic activity of phospholipase A2 (PLA2). Although various PLA2s classified into 16 gene families (= Groups) are known in various biological systems, few PLA2s are known in insects. Only two PLA2s involved in intracellular calcium independent PLA2 (iPLA2) group have been identified in lepidopteran insects with well known eicosanoid physiology. This study reports the first secretory PLA2 (sPLA2) in lepidopteran insects. A partial open reading frame (ORF) of PLA2 was obtained by interrogating Spodoptera exigua transcriptome. Subsequent 3′-RACE resulted in a full ORF (Se-sPLA2A) encoding 194 amino acid sequence containing signal peptide, calcium-binding domain, and catalytic site. Phylogenetic analysis indicated that Se-sPLA2A was clustered with other Group III sPLA2s. Se-sPLA2A was expressed in most larval instars except late last instar. Its expression was inducible by immune challenge and juvenile hormone analog injection. RNA interference of Se-sPLA2A significantly suppressed cellular immunity and impaired larval development. These results suggest that non-venomous sPLA2 plays a crucial role in immune and developmental processes in S. exigua, a lepidopteran insect.

Single Lipid Vesicle Assay for Characterizing Single-Enzyme Kinetics of Phospholipid Hydrolysis in a Complex Biological Fluid

Imaging of individual lipid vesicles is used to track single-enzyme kinetics of phospholipid hydrolysis. The method is employed to quantify the catalytic activity of phospholipase A2 (PLA2) in both pure and complex biological fluids. The measurements are demonstrated to offer a subpicomolar limit of detection (LOD) of human secretory PLA2 (sPLA2) in up to 1000-fold-diluted cerebrospinal fluid (CSF). An additional new feature provided by the single-enzyme sensitivity is that information about both relative concentration variations of active sPLA2 in CSF and the specific enzymatic activity can be simultaneously obtained. When CSF samples from healthy controls and individuals diagnosed with Alzheimer's disease (AD) are analyzed, the specific enzymatic activity is found to be preserved within 7% in the different CSF samples whereas the enzyme concentration differs by up to 56%. This suggests that the previously reported difference in PLA2 activity in CSF samples from healthy and AD individuals originates from differences in the PLA2 expression level rather than from the enzyme activity. Conventional ensemble averaging methods used to probe sPLA2 activity do not allow one to obtain such information. Together with an improvement in the LOD of at least 1 order of magnitude compared to that of conventional assays, this suggests that the method will become useful in furthering our understanding of the role of PLA2 in health and disease and in detecting the pharmacodynamic effects of PLA2-targeting drug candidates.

Interaction of characteristic structural elements of persimmon tannin with Chinese cobra PLA2

To more fully understand the mechanism by which persimmon tannin (PT) inhibited phospholipase A2 (PLA2) and the structural requirements of PT for the inhibition, the interactions between PLA2 and seven characteristic structural elements of PT including epigallocatechin-3-gallate (EGCG), myricetin, epicatechin-3-gallate (ECG), epicatechin-3-gallate-(4β → 8, 2β → O → 7)-epicatechin-3-gallate (A-type ECG dimer), epigallocatechin-3-gallate-(4β → 8, 2β → O → 7)-epigallocatechin-3-gallate (A-type EGCG dimer), epicatechin-(4β → 8, 2β → O → 7)-epicatechin (A-type EC dimer) and epicatechin-(4β → 8)-epicatechin (B-type EC dimer) were studied by enzymatic and spectroscopic methods. Molecular docking was also used to explore the possible residues involved in the interactions. The results revealed that A-type EGCG dimer and A-type ECG dimer showed higher inhibitory effects on the catalytic activity of PLA2 than monomers and B-type dimer. They induced greater conformational changes in PLA2 than other structural elements. In addition, molecular docking studies revealed that expect for lysine residues, other residues such as Trp18, Try27, Gly29, His47 and Tyr63 were involved in the interactions. We propose that A-type EGCG and ECG dimer units may be structural requirements for the interaction between PT and PLA2. Our data provide an additional structural basis for anti-PLA2 activity of persimmon tannin.

The interaction of a polymeric persimmon proanthocyanidin fraction with Chinese cobra PLA2 and BSA

To elucidate the anti-venom mechanism of persimmon tannin, the interaction between a polymeric persimmon proanthocyanidin fraction (PT40) and phospholipase A2 (PLA2) or bovine serum albumin (BSA) were studied using a competitive binding assay and spectroscopic methods including Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), and resonance light scattering (RLS) spectroscopy. The results revealed that PT40 has a higher affinity for PLA2 than for BSA at physiological pH and induced greater conformational changes in PLA2 than in BSA. PT40 covalently bound to PLA2 in a reaction probably involving Lys residues. We propose that the high affinity of PT40 for PLA2 and the covalent modification of PLA2 by PT40 may be responsible for the ability of the tannin to irreversibly inhibit PLA2 catalytic activity, to prevent edema, and to neutralize the lethality of Chinese cobra PLA2in vivo.

Chemical investigation of lipids of a marine sponge Spirastrella vagabunda

Sponges are among the simplest of multicellular animals and are often described as the most primitive of all. Study of sponge lipids is as important as other secondary metabolites and has been well documented in a series of extensive reviews by Berge and Barnathan, Rod kina, etc. [1, 2]. Regular research into lipids of sponges began in the 1970s [3] and continues till today because they have the greatest diversity of fatty acids, sterols etc. Studies on fatty acids and sterols as lipid components of sponges and other organisms have been elaborately taken up by a number of investigators [4, 5]. There are only a few investigations on this species, Spirastrella vagabunda. The aqueous extract of this species has shown high phospholipase A2 catalytic activity [6]. The bacteria isolated from this sponge produced acetylcholinesterase, an essential enzyme [7], but no investigation on the chemistry of this species has been done so far. This paper presents a study of the fatty acid profile and sterol composition of the total lipid of S. vagabunda for the first time (Table 1). A significant number of fatty acids were identified by GC-MS. The fatty acids were characterized by linear saturated fatty acids, monobranched saturated fatty acids, polybranched saturated fatty acid, monounsaturated fatty acids, and polyunsaturated fatty acids. The percentage of straight-chain fatty acids was 28.22% of the total fatty acids. Generally, among the saturated fatty acids, C16:0 and C18:0 dominated in most of the sponges. In the present investigation, the main saturated fatty acids are also C16:0 (6.37%) and C18:0 (5.77%). It is interesting that long-chain saturated fatty acids like C23:0, C24:0, C25:0, and C28:0 are present in good quantity in S. vagabunda. S. vagabunda contains the highest percentage of monomethyl branched fatty acids, i.e., 45.5% of the total fatty acid content, as in the marine sponge species Geodia gibberosa, which also contains 40–50% of branched fatty acids [8]. Among these branched fatty acids the amount of 11-methyloctadecanoic acid was significantly high, i.e., 13.31% of the total FA. A long-chain branched fatty acid, i.e., br-24-methylhexacosanoic acid, is also present in good quantity (8.97%). The identification of 11-methyl-18:0 and 24-methyl-26:0 (ante-iso) acids was finally confirmed by comparing their key mass fragments (11-methyl-18:0: m/z 312 M+, 269, 213, 185, 143; 24-methyl-26:0: m/z 424 M+, 381, 339, 199, 266, 143) obtained by GC-MS analysis with their literature data. The presence of saturated isoand ante-iso acids (branched fatty acids) is known to have a bacterial origin [9, 10]. So, the presence of symbiotic bacteria cannot be excluded. 3,7,11-Trimethyldodecanoic acid, a very rarely found polymethyl branched fatty acid, which was earlier identified from the marine sponge Xestospongia muta [11], was reported to be present in S. vagabunda, which is an important finding. The identification of 3,7,11-trimethyldodecanoic acid was confirmed by the key mass fragments at m/z 101 and at m/z 171, which indicate the methyl branching at 3 and 7 position [11]. Generally in various sponge species, the percentage of monoenes varies from 2% to 50% of the total FA content. But in the present investigation, only one monoenic acid C18:1 (2.42%) is identified. C18:1 (9) and C18:1 (11) are the principal isomers of C18 monoenes in sponges [2]. The GC-MS spectrum of the C18:1 showed important mass fragments at m/z 296 (M+), 264, 221, 180, 123, 109, and 96, and by comparing with the literature data, the monoenic acid is identified as C18:1 (9). The polyunsaturated FA of this species is represented by one diene and one trienoic acids with total content of 17.91%.

Effect of Procainhydrochloride on Phospholipase A2 Catalytic Activity in Sodium Taurocholate-Induced Acute Experimental Pancreatitis in Rats

Background: In human acute pancreatitis (AP) the local anaesthetic procainhydrochloride (procain-HCl) is given intravenously for pain treatment. Procain has been shown to inhibit catalytic activity of pancreatic (group I) phospholipase A2 (PLA2) and non-pancreatic (group II) PLA2. Both enzymes are important mediators for the local and systemic inflammatory process in AP. To determine the effect of procain, we examined serum and tissue levels of both types of PLA2 activity in the experimental rodent taurocholate model of AP. Methods: In 60 rats, severe pancreatitis was induced by taurocholate. Forty rats were treated with procain-HCl intravenously at a dosage of 2 mg/kg body weight/h either at or 1 h after induction of pancreatitis. Twenty rats served as controls. We measured catalytic activities of group I and group II PLA2 in serum and tissue samples of lung and pancreas. Results: Serum group II PLA2 catalytic activity was significantly reduced 3 and 6 h after AP induction in rats treated with procain-HCl (p < 0.001) in both treatment groups. In pancreatic and lung tissue, group II PLA2 catalytic activity was significantly reduced compared with normal values (p < 0.001). Conclusion: Procain-HCl given intravenously either at or 1 h after induction of necrotizing pancreatitis significantly inhibits group II PLA2 catalytic activity in serum and tissues.

Phospholipase A2 activity of crocodile serum

Abstract The catalytic activity of phospholipase A2 (PLA2) was measured in serum samples from 32 crocodiles in Thailand and Australia by a method using 14C-oleic acid-labelled autoclaved Escherichia coli membranes as a substrate. The highest PLA2 activity was measured in the serum of Crocodylus siamensis (n = 9, mean ± SD), 13.3 ± 3.1 U/l followed by hybrid C. siamensis × C. porosus (n = 6), 10.4 ± 8.7 U/l and Crocodylus porosus (n = 17), 4.3 ± 3.0 U/l. The difference between C. siamensis and C. porosus was highly significant (P &lt; 0.001). The gender of the animals and the geographical location of the crocodile farms were not significant variables affecting serum PLA2 levels. It was concluded that PLA2 is present in crocodilian serum and may have an antimicrobial role.

Renal tubular cell injury and serum phospholipase A2 activity in acute pancreatitis.

Early diagnosis of threatening renal complication is essential for the adequate treatment of acute pancreatitis. Deposition of phospholipase A2 (PLA2) in rat renal proximal tubular cells has been observed in experimentally induced acute pancreatitis. The value of measuring the catalytic activity of PLA2 in serum as an early warning of developing renal tubular cell injury was therefore investigated in a prospective study of 31 consecutive patients suffering from acute pancreatitis. A positive correlation was found (r = 0.66, P < 0.001) between the highest serum PLA2 activity, as measured early in the course of acute pancreatitis, and the highest N-acetyl-beta-glucosaminidase (NAG):creatinine ratio in the urine. The correlation between the highest serum concentration of immunoreactive pancreatic PLA2 and the highest urinary NAG:creatinine ratio was weaker (r = 0.36, P < 0.05). These results indicate that the measurement of the catalytic activity of PLA2 in serum early in acute pancreatitis may provide a simple test for the detection of threatening renal complication.

Phospholipase A2 in Cnidaria

Phospholipase A2 (PLA2) is an enzyme present in snake and other venoms and body fluids. We measured PLA2 catalytic activity in tissue homogenates of 22 species representing the classes Anthozoa, Hydrozoa, Scyphozoa and Cubozoa of the phylum Cnidaria. High PLA2 levels were found in the hydrozoan fire coral Millepora sp. (median 735 U/g protein) and the stony coral Pocillopora damicornis (693 U/g) that cause skin irritation upon contact. High levels of PLA2 activity were also found in the acontia of the sea anemone Adamsia carciniopados (293 U/g). Acontia are long threads containing nematocysts and are used in defense and aggression by the animal. Tentacles of scyphozoan and cubozoan species had high PLA2 activity levels: those of the multitentacled box jellyfish Chironex fleckeri contained 184 U/g PLA2 activity. The functions of cnidarian PLA2 may include roles in the capture and digestion of prey and defense of the animal. The current observations support the idea that cnidarian PLA2 may participate in the sting site irritation and systemic envenomation syndrome resulting from contact with cnidarians.

Meconium Induces Only Localized Inflammatory Lung Injury in Piglets

Neonatal meconium aspiration often produces severe respiratory distress due to an inflammatory pulmonary injury, but the extension of this damaging reaction to the noncontaminated lung regions is still uncertain. To investigate the presence of generalized pulmonary inflammatory response, 31 anesthetized and ventilated neonatal piglets (1-3 d) were studied. Meconium (n = 16) or saline (n = 15) was instilled unilaterally into the right lung, and analysis of the lung tissue or bronchoalveolar lavage (BAL) fluid from both lungs was performed after 12 h. Meconium increased the wet/dry weight ratio, histologic tissue injury score and tissue myeloperoxidase activity as well as BAL fluid total cell count in the contaminated lung. Tumor necrosis factor-alfa concentrations in BAL fluid did not however differ significantly. Furthermore, in the meconium-instilled lungs the tissue and lavage fluid catalytic activity of phospholipase A2 (PLA2) and tissue PLA2 group-I and group-II concentrations were significantly elevated. Although BAL fluid catalytic activity of PLA2 was moderately increased also in the meconium noninstilled lung, significant inflammatory injury in this lung was absent. The results thus indicate that meconium aspiration induces severe local inflammation and lung injury, but significant generalized pulmonary inflammatory damage in the pathogenesis of meconium aspiration syndrome is unlikely.

Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression.

The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.

Catalytic activity of phospholipase A2 in blood plasma and pancreatic tissue perfusion in early experimental acute pancreatitis.

To investigate the sequence of changes in the catalytic activity of phospholipase A2 in plasma and pancreatic tissue perfusion and oxygenation in mild and severe acute pancreatitis in pigs. Twenty-four pigs were randomized into the groups of severe acute pancreatitis, mild acute pancreatitis, and controls. The pancreatic duct of eight anesthetized and mechanically ventilated pigs was cannulated, and taurocholic acid was infused into the pancreatic duct to induce severe acute pancreatitis. Eight animals received intraductal saline and developed mild acute pancreatitis. Eight pigs were cannulated only and served as controls. Central hemodynamics, arterial blood gases, and acid-base balance were stable throughout the study period in all three groups. Pancreatic tissue oxygenation decreased in pigs with severe acute pancreatitis and increased in animals with mild acute pancreatitis. The catalytic activity of phospholipase A2 in plasma remained stable, and there was no difference between the groups. Similarly, C-reactive protein values remained within the normal range during the study period in all groups. Plasma phospholipase A2 levels do not react to the changes in pancreatic tissue perfusion in the early phase of mild and severe acute pancreatitis.

Resistance of transgenic mice expressing human group II phospholipase A2 to Escherichia coli infection.

Group II phospholipase A2 (PLA2) is a newly recognized antibacterial acute-phase protein. Recently we observed that transgenic mice expressing group II PLA2 (PLA2(+) mice) were able to resist experimental Staphylococcus aureus infection by killing the bacteria, as indicated by improved survival and by the small numbers of live bacteria in their tissues (V. J. O. Laine, D. S. Grass, and T. J. Nevalainen, J. Immunol. 162:7402-7408, 1999). To establish the role of group II PLA2 in Escherichia coli infection, the host responses of PLA2(+) mice and their PLA2-deficient C57BL/6J littermates (PLA2(-) mice) were studied after intraperitoneal administration of E. coli. The levels of group II PLA2 in sera of PLA2(+) mice increased after the administration of E. coli, and the concentration of group II PLA2 correlated significantly with the catalytic activity of PLA2 in serum. PLA2(+) mice showed lower rates of mortality and less bacterial growth in peritoneal lavage fluid, blood, and spleen and liver tissues than PLA2(-) mice. Unlike the observations with staphylococcal infection, serum and peritoneal lavage fluid did not inhibit the growth of E. coli in vitro. The results indicate that expression of the group II PLA2 transgene improves the host defense of mice against E. coli infection.

Human meconium has high phospholipase A2 activity and induces cellular injury and apoptosis in piglet lungs.

Aspiration of meconium produces an inflammatory reaction resulting in necrotic changes in lung tissue. To further investigate the mechanisms of the meconium-induced early pulmonary injury, twenty 10-12-d-old piglets were studied for lung tissue ultrastructural and apoptotic changes and phospholipase A2 activity. Twelve piglets received an intratracheal bolus (3 mL/kg) of a 20-mg/mL (thin, n = 6) or 65-mg/mL (thick, n = 6) mixture of human meconium, and control piglets (n = 5) received the same amount of intratracheal saline. Three ventilated piglets with no aspiration were also studied. Pulmonary hemodynamics and systemic oxygenation were followed for 6 h after meconium or saline insufflation. In the control groups, the pulmonary tissue showed open alveolar spaces and intact vascular walls, whereas meconium administration resulted in severe pneumonitis, with alveolar spaces filled with inflammatory exudate. Meconium instillation additionally resulted in edematous changes in the vascular walls and alveolar epithelium, whereas type II pneumocytes were intact. The amount of apoptotic cells was increased, especially in the respiratory epithelium, and the catalytic activity of phospholipase A2 in lung tissue samples was significantly elevated after thick meconium instillation. This activity rise proved to be mainly because of human group I phospholipase A2, introduced by meconium. Our data thus show that aspiration of meconium leads to severe lung tissue inflammation with early ultrastructural changes in the pulmonary alveolar walls and is associated with apoptotic cell death in the epithelium, already during the first hours after the insult. These results further suggest that high phospholipase A2 activity, mainly introduced into the lungs within the meconium, may have an important role in the initiation of these alterations in neonatal lungs.

Effect of New Inhibitor of Type II Phospholipase A2 on Experimental Acute Pancreatitis in Rats

The catalytic activity of type II phospholipase A2 (PLA2) in blood has been reported to increase in acute pancreatitis and to reflect the severity of pancreatitis. In this study, we evaluated the effects of a new inhibitor of type II PLA2, S5920/LY315920Na, on trypsin-taurocholate-induced pancreatitis in rats. Hemorrhagic pancreatitis was induced by an infusion of a mixture of trypsin and taurocholate into the pancreatic duct. S5920/LY315920Na was administered subcutaneously at 0 h and 3 h after the induction of pancreatitis. Survival rates for 24 h in rats treated with 0.1 and 1 mg/kg of S5920/LY315920Na were significantly higher than that in untreated rats (71 and 86% vs. 14%). Serum levels of amylase and lipase in rats treated with 1 mg/kg of S5920/LY315920Na were significantly lower than those in untreated rats (amylase, 6,903 vs. 32,516 U/L; and lipase, 514 vs. 6,710 U/L) at the time of death or 24 h after the induction of pancreatitis. Plasma levels of S5920/LY315920Na were enough to inhibit catalytic activity of PLA2 in plasma for 9 h. A new inhibitor of type II PLA2, S5920/LY315920Na, inhibited catalytic activity of PLA2 and improved the survival rate in experimental hemorrhagic pancreatitis in rats.