Ginseng-derived patatin-related phospholipase PgpPLAIIIß alters plant growth and lignification of xylem in hybrid poplars.

Patatin-liked phospholipase A (pPLAs) are major lipid acyl hydrolases that participate in various biological functions in plant growth and development. Previously, a ginseng-derived pPLAIII homolog was reported to reduce lignin content in Arabidopsis. This led us to evaluate its possible usefulness as a biomass source in wood plant. Herein, we report that there are six members in the pPLAIII gene family in poplar. Overexpression of pPLAIIIß derived from ginseng resulted in a reduced plant height with radially expanded stem growth in hybrid poplars. Compared with the wild type (WT), the chlorophyll content was increased in the overexpression poplar lines, whereas the leaf size was smaller. The secondary cell wall structure in overexpression lines was also altered, exhibiting reduced lignification in the xylem. Two transcription factors, MYB92 and MYB152, which control lignin biosynthesis, were downregulated in the overexpression lines. The middle xylem of the overexpression line showed heavy thickening, making it thicker than the other xylem parts and the WT xylem, which rather could have been contributed by the presence of more cellulose in the selected surface area. Taken together, the results suggest that PgpPLAIIIß plays a role not only in cell elongation patterns, but also in determining the secondary cell wall composition.

Patatin-related phospholipase pPLAIIIδ increases seed oil content with long-chain fatty acids in Arabidopsis.

The release of fatty acids from membrane lipids has been implicated in various metabolic and physiological processes, but in many cases, the enzymes involved and their functions in plants remain unclear. Patatin-related phospholipase As (pPLAs) constitute a major family of acyl-hydrolyzing enzymes in plants. Here, we show that pPLAIIIδ promotes the production of triacylglycerols with 20- and 22-carbon fatty acids in Arabidopsis (Arabidopsis thaliana). Of the four pPLAIIIs (α, ß, γ, δ), only pPLAIIIδ gene knockout results in a decrease in seed oil content, and pPLAIIIδ is most highly expressed in developing embryos. The overexpression of pPLAIIIδ increases the content of triacylglycerol and 20- and 22-carbon fatty acids in seeds with a corresponding decrease in 18-carbon fatty acids. Several genes in the glycerolipid biosynthetic pathways are up-regulated in pPLAIIIδ-overexpressing siliques. pPLAIIIδ hydrolyzes phosphatidylcholine and also acyl-coenzyme A to release fatty acids. pPLAIIIδ-overexpressing plants have a lower level, whereas pPLAIIIδ knockout plants have a higher level, of acyl-coenzyme A than the wild type. Whereas seed yield decreases in transgenic plants that ubiquitously overexpress pPLAIIIδ, seed-specific overexpression of pPLAIIIδ increases seed oil content without any detrimental effect on overall seed yield. These results indicate that pPLAIIIδ-mediated phospholipid turnover plays a role in fatty acid remodeling and glycerolipid production.

Double positivity to bee and wasp venom: improved diagnostic procedure by recombinant allergen-based IgE testing and basophil activation test including data about cross-reactive carbohydrate determinants.

BACKGROUND: Specific IgE (sIgE) antibodies to both bee and wasp venom can be due to a sensitivity to both insect venoms or due to cross-reactive carbohydrate determinants (CCDs). OBJECTIVE: Investigating whether a basophil activation test (BAT) with both venoms as well as with bromelain and horseradish peroxidase (HRP) or recombinant allergen-based IgE testing can improve the diagnostic procedure. METHODS: Twenty-two Hymenoptera-venom allergic patients with sIgE antibodies to both bee and wasp venom were studied. sIgE antibodies to MUXF3 CCD, bromelain, HRP, rApi m 1, and rVes v 5 were determined, and a BAT (Flow2 CAST) with venom extracts, bromelain, and HRP was performed. Further recombinant allergen-based IgE testing was done by using an ELISA, if required. The reactivity of basophils was calculated from the insect venom concentration at half-maximum stimulation. RESULTS: Double positivity/double negativity/single positivity to rApi m 1 and rVes v 5 was seen in 12/1/9 patients. Further recombinant allergen-based IgE testing in the last ones revealed positive results to the other venom in all cases except one. BAT was double positive/double negative/single positive in 6/2/14 patients. Four patients with negative results in sIgE antibodies to CCDs had positive results in BAT. BAT with bromelain/HRP showed a sensitivity of 50%/81% and a specificity of 91%/90%. CONCLUSION: Component-resolved IgE testing elucidates the pattern of double positivity, showing a majority of true double sensitizations independent of CCD sensitization. BAT seems to add more information about the culprit insect even if the true clinical relevance of BAT is not completely determined because of ethical limitations on diagnostic sting challenges. BAT with HRP is a good method to determine sensitivity to CCDs.

Recombinant allergen-based IgE testing to distinguish bee and wasp allergy.

BACKGROUND: The identification of the disease-causing insect in venom allergy is often difficult. OBJECTIVE: To establish recombinant allergen-based IgE tests to diagnose bee and yellow jacket wasp allergy. METHODS: Sera from patients with bee and/or wasp allergy (n = 43) and patients with pollen allergy with false-positive IgE serology to venom extracts were tested for IgE reactivity in allergen extract-based tests or with purified allergens, including nonglycosylated Escherichia coli-expressed recombinant (r) Api m 1, rApi m 2, rVes v 5, and insect cell-expressed, glycosylated rApi m 2 as well as 2 natural plant glycoproteins (Phl p 4, bromelain). RESULTS: The patients with venom allergy could be diagnosed with a combination of E coli-expressed rApi m 1, rApi m 2, and rVes v 5 whereas patients with pollen allergy remained negative. For a group of 29 patients for whom the sensitizing venom could not be identified with natural allergen extracts, testing with nonglycosylated allergens allowed identification of the sensitizing venom. Recombinant nonglycosylated allergens also allowed definition of the sensitizing venom for those 14 patients who had reacted either with bee or wasp venom extracts. By IgE inhibition studies, it is shown that glycosylated Api m 2 contains carbohydrate epitopes that cross-react with natural Api m 1, Ves v 2, natural Phl p 4, and bromelain, thus identifying cross-reactive structures responsible for serologic false-positive test results or double-positivity to bee and wasp extracts. CONCLUSION: Nonglycosylated recombinant bee and wasp venom allergens allow the identification of patients with bee and wasp allergy and should facilitate accurate prescription of venom immunotherapy.

Chronic NMDA administration to rats up-regulates frontal cortex cytosolic phospholipase A2 and its transcription factor, activator protein-2.

Excessive N-methyl-D-aspartate (NMDA) signaling is thought to contribute to bipolar disorder symptoms. Lithium and carbamazepine, effective against bipolar mania, are reported in rats to reduce brain transcription of an arachidonic acid selective calcium-dependent cytosolic phospholipase A(2) (cPLA(2)), as well as expression of one of its transcription factors, activator protein (AP)-2. In this study, we determined if chronic administration of NMDA (25 mg/kg i.p.) to rats would increase brain cPLA(2) and AP-2 expression, as these antimanic drugs are known to down-regulate excessive NMDA signaling. Administration of a daily subconvulsive dose of NMDA to rats for 21 days decreased frontal cortex NMDA receptor (NR)-1 and NR-3A subunits and increased cPLA(2) activity, phosphorylation, protein, and mRNA levels. The activity and protein levels of secretory phospholipase A(2) or calcium-independent phospholipase A(2) were not changed significantly. Chronic NMDA also increased the DNA-binding activity of AP-2 and the protein levels of its alpha and beta subunits. These changes were absent following acute (3 h earlier) NMDA administration. The changes, opposite to those found following chronic lithium or carbamazepine, are consistent with up-regulated arachidonic acid release due to excessive NR signaling and may be a contributing factor to bipolar mania.

Inhibition of calcium-independent phospholipase A2 suppresses proliferation and tumorigenicity of ovarian carcinoma cells.

PLA2 (phospholipase A2) enzymes play critical roles in membrane phospholipid homoeostasis and in generation of lysophospholipid growth factors. In the present study, we show that the activity of the cytosolic iPLA2 (calcium-independent PLA2), but not that of the calcium-dependent cPLA2 (cytosolic PLA2), is required for growth-factor-independent, autonomous replication of ovarian carcinoma cells. Blocking iPLA2 activity with the pharmacological inhibitor BEL (bromoenol lactone) induces cell cycle arrest in S- and G2/M-phases independently of the status of the p53 tumour suppressor. Inhibition of iPLA2 activity also leads to modest increases in apoptosis of ovarian cancer cells. The S- and G2/M-phase accumulation is accompanied by increased levels of the cell cycle regulators cyclins B and E. Interestingly, the S-phase arrest is released by supplementing the growth factors LPA (lysophosphatidic acid) or EGF (epidermal growth factor). However, inhibition of iPLA2 activity with BEL remains effective in repressing growth-factor- or serum-stimulated proliferation of ovarian cancer cells through G2/M-phase arrest. Down-regulation of iPLA2b expression with lentivirus-mediated RNA interference inhibited cell proliferation in culture and tumorigenicity of ovarian cancer cell lines in nude mice. These results indicate an essential role for iPLA2 in cell cycle progression and tumorigenesis of ovarian carcinoma cells.

Dietary n-3 PUFA deprivation alters expression of enzymes of the arachidonic and docosahexaenoic acid cascades in rat frontal cortex.

The enzymes that regulate the brain arachidonic acid (AA) cascade have been implicated in bipolar disorder and neuroinflammation. Fifteen weeks of dietary n-3 polyunsaturated fatty acid (PUFA) deprivation in rats decreases the concentration of docosahexaenoic acid (DHA) and increases its half-life within the brain. Based on this, we hypothesized that such dietary deprivation would decrease expression of enzymes responsible for the metabolic loss of DHA while increasing expression of those responsible for the metabolism of AA. Fifteen weeks of n-3 PUFA deprivation significantly decreased the activity, protein and mRNA expression of the DHA regulatory phospholipase A2 (PLA2), calcium-independent iPLA2, in rat frontal cortex. In contrast the activities, protein and mRNA levels of the AA selective calcium-dependent cytosolic phospholipase (cPLA2) and secretory sPLA2 were increased. Cyclooxygenase (COX)-1 protein but not mRNA was decreased in the n-3 PUFA-deprived rats whereas COX-2 protein and mRNA were increased. This study suggests that n-3 PUFA deprivation increases the half-live of brain DHA by downregulating iPLA2. The finding that n-3 PUFA deprivation increases cPLA2, sPLA2 and COX-2 is opposite to what has been reported after chronic administration of anti-manic agents to rats and suggests that n-3 PUFA deprivation may increase susceptibility to bipolar disorder.

RETRACTED: Chronic administration of carbamazepine down-regulates AP-2 DNA-binding activity and AP-2alpha protein expression in rat frontal cortex.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of author Stanley Rapoport, with approval from Biological Psychiatry Editor, John H. Krystal, MD. The National Institutes of Health has found that Dr. Jagadeesh S. Rao engaged in research misconduct by falsifying data in Figures 1, 3, and 5 of the aforementioned manuscript. No other authors were implicated in the data falsification

The main triglyceride-lipase from the insect fat body is an active phospholipase A(1): identification and characterization.

The main triglyceride-lipase (TG-lipase) from the fat body of Manduca sexta has been identified as the homolog of Drosophila melanogaster CG8552. This protein is conserved among insects and also shares significant sequence similarity with vertebrate phospholipases (PLs) from the phosphatidic acid preferring-phospholipase A1 (PA-PLA(1)) family. It is shown here that the TG-lipase is also a PL. TG-lipase and PL activities copurify and are inhibited by, or resistant to, the same lipase inhibitors, indicating that both activities are catalyzed by the same enzyme and active site. The PL activity of TG-lipase corresponded to PL type A(1). The concentration dependence of lipase activity with TG and PL micellar substrates showed saturation kinetics, with apparent K(m) values of 152 +/- 11 and 7.8 +/- 1.1 muM, respectively. TG-lipase was able to hydrolyze the major phospholipid components of the lipid droplets, phosphatidylcholine and phosphatidylethanolamine. The enzyme hydrolyzes 77 molecules of TG for every molecule of PL contained in the lipid droplets. It was observed that the activation of lipolysis in vivo is accompanied by activation of the hydrolysis of phospholipids of the lipid droplets. These results suggest that the PL activity of the insect TG-lipase could be required to allow access of the lipase to TG molecules contained in the core of the lipid droplets.

BE-I-PLA2, a novel acidic phospholipase A2 from Bothrops erythromelas venom: isolation, cloning and characterization as potent anti-platelet and inductor of prostaglandin I2 release by endothelial cells.

A novel acidic Asp49 phospholipase A(2) was isolated from Bothrops erythromelas (jararaca malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a molecular weight of 13,649.57 Da as estimated by mass spectrometry. N-terminal and four internal peptides were sequenced, covering around one-third of the complete toxin sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 457 bp and encodes a protein with significant sequence similarity to many other phospholipase A(2) from snake venoms. When tested in platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the principal platelets receptors was observed. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was only partially inhibited. In human umbilical-cord veins endothelial cells, BE-I-PLA2 was neither apoptotic nor proliferative but stimulated endothelial cells to release prostaglandin I(2), suggesting an increase of its potential anti-platelet activity in vivo. Further studies are required in order to determine the exact mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregation.

DNA vaccines and allergic diseases.

1. Allergic diseases are characterized by inappropriate immune responses to common environmental antigens. The prevalence of these diseases has been increasing worldwide for reasons that are not exactly clear. 2. Current treatment is largely symptomatic. Because the initial observation that simple plasmid DNA injections resulted in in vivo protein expression and induction of adaptive immune responses to the encoded antigen, the potential of modifying the allergic immune responses by DNA vaccination so as to treat and prevent these diseases has been explored extensively. 3. In the present paper we review preclinical studies using animal models of allergic diseases, with an emphasis on DNA vaccine design, for house dust mite allergens-related allergic asthma.

Chronic fluoxetine upregulates activity, protein and mRNA levels of cytosolic phospholipase A2 in rat frontal cortex.

Chronic lithium and carbamazepine, which are effective against mania in bipolar disorder, decrease the activity of cytosolic phospholipase A(2) (cPLA(2)) and the turnover rate of arachidonic acid in phospholipids in rat brain. Assuming that stages of bipolar disorder are related to brain arachidonic acid metabolism, we hypothesized that drugs effective in depression would increase cPLA(2) activity. To test this hypothesis, adult male CDF-344 rats were administered fluoxetine (10 mg/kg intraperitoneally (i.p.) or saline (control) (i.p.) chronically for 21 days. Frontal cortex cPLA(2) protein, phosphorylated cPLA(2), activity and mRNA levels were increased after chronic fluoxetine. Transcription factors (activator protein-1, activator protein-2, glucocorticoid response element, polyoma enhancer element-3 and nuclear factor-kappa B) that are known to regulate cPLA(2) gene expression were not significantly changed by chronic fluoxetine, but nuclear AU-rich element/poly(U)-binding/degradation factor-1 RNA-stabilizing protein was increased significantly. The results suggest that chronic fluoxetine increases brain cPLA(2) gene expression post-transcriptionally by increasing cPLA(2) mRNA stabilization. Chronic fluoxetine's effect on cPLA(2) expression was opposite to the effect reported with chronic lithium or carbamazepine administration, and may be part of fluoxetine's mode of action.

Expression of phospholipase A2 group IVA (PLA2G4A) is upregulated by human chorionic gonadotropin in bovine granulosa cells of ovulatory follicles.

Prostaglandins are required for the ovulatory process, and their biosynthesis depends on the initial release of arachidonic acid from membrane phospholipids. We hypothesized that phospholipase A2 group IVA (PLA2G4A) expression is upregulated in granulosa cells (GC) at ovulation. We have characterized bovine PLA2G4A cDNA, and investigated its spatiotemporal regulation at the mRNA and protein levels in hCG-induced ovulatory follicles and in vitro, using forskolin-stimulated GC. Regulation of PLA2G4A mRNA expression was studied in GC obtained from bovine follicles collected at different developmental stages: small follicles (2-4 mm), dominant follicles at Day 5 (D5) of the estrous cycle, ovulatory follicles 24 h following injection of hCG, and corpus luteum at D5. PLA2G4A mRNA increased by 14-fold in GC of hCG-stimulated versus dominant follicles (P < 0.0001). Follicular walls obtained from ovulatory follicles recovered at 0, 6, 12, 18, and 24 h post-hCG injection showed an initial 16-fold increase in PLA2G4A transcript at 12 h that reached a 45-fold increase at 24 h, as compared to 0 h (P < 0.0001). Immunoblots of GC extracts showed an initial induction of the PLA2G4A protein at 18 h post-hCG, reaching a maximum at 24 h. Immunohistochemistry observations showed that PLA2G4A signal was mainly observed in mural GC compared to antral GC in hCG-stimulated follicles. Stimulation of cultured bovine GC with 10 microM of forskolin caused an increase in PLA2G4A mRNA and protein. Ovulation is associated with an LH/hCG-dependent induction of PLA2G4A in GC via the adenylyl cyclase/cAMP pathway.

Intracellular pH signals in the induction of secondary pathways--the case of Eschscholzia californica.

Transient peaks of the cytoplasmic pH are essential elements in a number of signal cascades that activate environmental responses or developmental processes in plant cells but little is known about the mechanisms of their generation. In many plant cells, elicitation of the hypersensitive response is preceded by a perturbation of the ionic balance at the plasma membrane including the inhibition of the proton pump and the influx of H+ from the apoplast. A basically different mechanism of cytoplasmic acidification that is fed by vacuolar protons has been discovered in cell suspensions of the California Poppy (Eschscholzia californica). These cells react to a yeast glycoprotein elicitor with the overproduction of benzophenanthridine alkaloids. Low elicitor concentrations trigger the biosynthesis of these phytoalexins without invoking elements of the hypersensitive response. Accumulated data support the existence of a signal path that includes the following steps: Links between the above events that connect them within a distinct signal path are substantiated by the phenotypes of transformed cell lines that either display lowered Galpha levels due to antisense transformation or express Galpha-binding antibodies in the cytoplasm. All of these cell lines lack the elicitor-activation of PLA2 and of vacuolar proton fluxes and show an impaired phytoalexin response to low elicitor concentrations. High elicitor concentrations trigger alkaloid biosynthesis via an increase of jasmonate at a pH-independent signal path.

Crystal structure of a heterodimer of phospholipase A2 from Naja naja sagittifera at 2.3 A resolution reveals the presence of a new PLA2-like protein with a novel cys 32-Cys 49 disulphide bridge with a bound sugar at the substrate-binding site.

The crystal structure of the phospholipase A2 (PLA2) heterodimer from Naja naja sagittifera reveals the presence of a new PLA2-like protein with eight disulphide bridges. The heterodimer is formed between a commonly observed group I PLA2 having seven characteristic disulfide bonds and a novel PLA2-like protein (Cys-PLA2) containing two extra cysteines at two highly conserved sites (positions 32 and 49) of structural and functional importance. The crystals of the heterodimer belong to tetragonal space group P41212 with cell dimensions, a = b = 77.7 A and c = 68.4 A corresponding to a solvent content of 33%, which is one of the lowest values observed so far in the PLA2 crystals. The structure has been solved with molecular replacement method and refined to a final R value of 21.6% [Rfree = 25.6%]. The electron density revealed the presence of cysteines 32 and 49 that are covalently linked to give rise to an eighth disulphide bridge in the PLA2-like monomer. A non-protein high-quality electron density was also observed at the substrate-binding site in the PLA2-like protein that has been interpreted as N-acetylglucosamine. The overall tertiary folds of the two monomers are similar having all features of PLA2-type folding. A zinc ion is detected at the interface of the heterodimer with fivefold coordination while another zinc ion was found on the surface of Cys-PLA2 with sixfold coordination. The conformations of the calcium-binding loops of both monomers are significantly different from each other as well as from those in other group I PLA2s. The N-acetylglucosamine molecule is favorably placed in the substrate-binding site of Cys-PLA2 and forms five hydrogen bonds and several van der Waals interactions with protein atoms, thus indicating a strong affinity. It also provides clue of the possible mechanism of sugar recognition by PLA2 and PLA2-like proteins. The formation of heterodimer seems to have been induced by zinc ion.

Molecular cloning of two toxic phospholipases A2 from the crown-of-thorns starfish Acanthaster planci venom.

The full-length cDNAs encoding two toxic phospholipases A2 (AP-PLA2-I and -II) from the crown-of-thorns starfish Acanthaster planci venom were individually cloned by RT-PCR, 3'RACE and 5'RACE. In common with both AP-PLA2s, the precursor protein is composed of a signal peptide, a propeptide and a mature protein (136 and 135 residues for AP-PLA2-I and -II, respectively). The four motifs (Ca2+-binding loop, Ca2+-binding site, active site and catalytic network) characteristic of groups I and II PLA2s are well conserved in both AP-PLA2s. In addition to this, the presence of the elapid and pancreatic loops and the involvement of a propeptide in the precursors suggested that AP-PLA2s are highly analogous to the group IB PLA2s. However, when compared to the amino acid sequence of bovine pancreatic PLA2, the representative group IB PLA2, AP-PLA2s require some amino acid insertions and deletions in the region 76-100, as previously observed for the starfish Asterina pectinifera PLA2s. Furthermore, the phylogenetic tree made clearly demonstrated that AP-PLA2s and A. pectinifera PLA2s are distinguishable from the group IB PLA2s as well as other PLA2s, being classified into a new group.

Molecular cloning, expression, and characterization of secretory phospholipase A2 in tobacco.

Phospholipase A2 (PLA2) activity was investigated in various tissues of tobacco (Nicotiana tabacum). PLA2 activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA2 activity in the flower appears to have originated from both Ca2+-dependent and -independent PLA2. A cDNA clone for protein with homology to animal secretory PLA2 (sPLA2), denoted as Nt PLA2, was isolated from the tobacco flower. The cDNA of Nt PLA2 encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence for mature Nt PLA2 contains 12 cysteines, a Ca2+ binding loop, and a catalytic domain that are commonly conserved in animal sPLA2. The Nt PLA2 mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA2 was expressed as a fusion protein with thioredoxin in Escherichia coli. From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then the recombinant mature Nt PLA2 was purified by ion exchange chromatography. It was discovered that the purified Nt PLA2 essentially requires Ca2+, for the enzyme activity when the activity was determined using mixed-micellar phospholipid substrates with sodium cholate. The optimal activity of Nt PLA2 was at pH 8-10 when PC was used as a substrate.

Effect of retinoic acid on gene expression in human conjunctival epithelium: secretory phospholipase A2 mediates retinoic acid induction of MUC16.

PURPOSE: How vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. This study sought to identify vitamin A-responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial (HCjE) cell line grown with all-trans-retinoic acid (RA). The analysis showed that secretory phospholipase A(2) group IIA (sPLA(2)-IIA) was the gene most upregulated by RA, followed by the membrane-associated mucin MUC16 at a later time point. Since eicosanoids, the product of arachidonic acid generated by the PLA(2) family, have been shown to increase mucin production, this study sought to determine whether sPLA(2) mediates the RA induction of MUC16. METHODS: HCjE cells were cultured with or without RA for 3, 6, 24, and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips and analyzed using commercial software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA(2) is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad-spectrum PLA(2) inhibitor aristolochic acid (ArA) or the specific sPLA(2)-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis. RESULTS: After RA addition, 28 transcripts were upregulated and 6 downregulated by more than twofold (P < 0.01) at both 3 and 6 hours (early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours (late phase). Group IIA sPLA(2), significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA(2) upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA(2)-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced (P < 0.01). CONCLUSIONS: The RA-associated upregulation of membrane-associated mucin MUC16 at late phase appears to be through sPLA(2)-IIA. Upregulation of this hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A facilitates maintenance of the wet-surfaced phenotype on the ocular surface.

Characterization of a cDNA encoding Arabidopsis secretory phospholipase A2-alpha, an enzyme that generates bioactive lysophospholipids and free fatty acids.

Phospholipase A2s (PLA2s) are enzymes that liberate lysophospholipids and free fatty acids (FFAs) from membrane phospholipids in response to hormones and other external stimuli. This report describes the cloning and functional characterization of a PLA2 cDNA from Arabidopsis thaliana, AtsPLA2-alpha, which represents one of four secretory PLA2 (sPLA2) genes in Arabidopsis. The encoded protein is 148-amino acid polypeptide and is predicted to contain a 20-amino acid signal peptide at its amino terminus. The predicted mature form (Mr=14,169) of AtsPLA2-alpha exhibited approximately 5 times the specific activity of its pre-processed form. Different from animal sPLA2s, AtsPLA2-alpha showed a significant preference for the acyl group linoleic acid over palmitic acid in phospholipid hydrolysis. Like some animal sPLA2s, however, it has a slight preference for phosphatidylethanolamine over phosphatidylcholine as the substrate. The specific activity of AtsPLA2-alpha continuously increased as the Ca2+ concentration was increased to 10 mM, and the optimal pH range was very broad and biphasic between 6 and 11. AtsPLA2-alpha transcript was detected at low levels in roots, stems, leaves, and flowers but not in siliques.

Natural disruption of group 2 phospholipase A2 gene protects against choline-deficient ethionine-supplemented diet-induced acute pancreatitis and lung injury.

OBJECTIVES: Group 2 phospholipase A2 (PLA2) plays an important role in the pathogenesis of multiple organ failure associated with acute pancreatitis. C57 BL/6J mice are natural group 2 PLA2 knockout mice lacking group 2 PLA2 mRNA. To clarify the role of group 2 PLA2 in the exacerbation of acute pancreatitis, we studied the biologic and histologic alterations in choline-deficient and ethionine-supplemented (CDE) diet-induced pancreatitis in group 2 PLA2-deficient C57 BL/6J mice and compared them with those in wild-type mice. METHODS: Female C57 BL/6J mice weighing 20 to 22 g were fed a CDE diet for 3 days to induce pancreatitis. Female C3H/HEJ mice were used as controls. Mice were killed on days 1, 2, and 3 after the onset of the CDE diet. The severity of pancreatitis was evaluated by survival rate, plasma PLA2 activity, serum amylase level, histologic changes in the pancreas and lung, and myeloperoxidase activity in the lung. RESULTS: The survival rate of C57 BL/6J mice was 100% up to day 3 after the onset of the CDE diet, whereas that of the control mice was 42% on day 3. Plasma PLA2 activity in control mice increased on day 3 but did not increase in C57 BL/6J mice. Serum amylase activity on day 3 in C57 BL/6J mice was 15,480 +/- 3036 SU/dL, which was significantly lower than that in the control mice (43,760 +/- 8657 SU/dL, P < 0.01). Histologic changes in the pancreas of C57 BL/6J mice were markedly milder than in control mice. The degree of alveolar membrane thickening and infiltration of inflammatory cells in the lung of C57 BL/6J mice were overtly less than those of the controls. Myeloperoxidase activity in the lung of C57 BL/6J mice was lower, albeit insignificant, than in C3H/HEJ mice. CONCLUSIONS: Natural disruption of the group 2 PLA2 gene protects against CDE diet-induced acute pancreatitis and associated lung injury. These findings support the view that group 2 PLA2 is one of the factors in the exacerbation of severe acute pancreatitis.