763C>G polymorphism of the secretory PLA2IIa gene is associated with endometriosis in Iranian women

Endometriosis is a chronic gynecological disease resulting from complex interactions between genetic, hormonal, environmental and oxidative stress and intrinsic inflammatory components. The aim of this study was to investigate the potential association of the 763C>G polymorphism in the secretory phospholipase A2 group IIa gene [PLA2G2A] with the risk of endometriosis in Iranian women. Ninety seven patients with endometriosis along with 107 women who were negative for endometriosis after laparoscopy and laparatomy, and served as the control group, were enrolled for this cross-sectional study. Samples were genotyped using the polymerase chain reaction-restriction fragment length polymorphism method. Multivariate analysis was used to examine the association between the risk of endometriosis and the 763C>G polymorphism of PLA2G2A. Genotype distributions of PLA2G2A were significantly different between patients and the controls [p<0.001, OR=0.22, 95% CI=0.21-0.39]. Correlation analysis showed that there was a significant association between the normal homozygous genotype and susceptibility to endometriosis [p<0.001]. The present study suggests that the 763C>G polymorphism of PLA2G2A plays an important role as an independent factor in the risk of endometriosis in Iranian women

Purification and characterization of a phospholipase A2-IIA from common stingray (Dasyatis pastinaca) intestine.

A phospholipase A2 belonging to IIA group secretory PLA2 was isolated and purified to homogeneity from the intestine of common stingray (Dasyatis pastinaca) using acidic treatment (pH 1.5) and ammonium sulphate precipitation methods combined with single-column ion-exchange chromatography. The purified enzyme was found to be a glycosylated monomeric protein with a molecular mass of about 14 kDa. The stingray sPLA2-IIA had optimum activity at 45°C, unlike known mammalian PLA2-IIAs, which show optimum activity at 37°C. The purified enzyme exhibited a specific activity of 290 U/mg at optimal conditions (pH 9.5 and 45°C) in the presence of 6 mM NaDC and 8 mM CaCl2 with egg yolk as substrate. The NH2-terminal sequence of the enzyme and some protein fragments obtained from its tryptic digestion were also determined. All sequences obtained were similar to those of sPLA2-IIA. The enzyme also showed good stability in the presence of organic solvents, acidic and alkaline pH media and high temperature conditions. Thus, the purified enzyme exhibited a number of unique and promising properties, making it a potential possible candidate for future applications in the treatment of phospholipid-rich industrial effluents and synthesis of useful preparations for the food production and processing industry.

Effect of polyunsaturated fatty acids on secretory phospholipase A2 type IIa in ectopic endometrial cells

Endometriosis is a common chronic inflammation which leads to infertility and chronic pelvic pain in affected women. Secretory phospholipase A2 type IIa [sPLA2IIa] is an acute phase reactant that is markedly increased in inflammatory disorders. To assess the effects of omega-3 and omega-6 polyunsaturated fatty acids [PUFAs] administration in endometrial cells culture on sPLA2IIa level and cell survival comparing homolog ectopic versus eutopic endometrial cells from endometriosis patients. In this experimental study, ectopic and eutopic endometrial tissue samples obtained from 15 endometriosis patients were immediately frozen. After thawing and tissue digestion, mixed stromal and endometrial gland cells were cultured for 8 days in three different culture media; balanced omega-3/omega-6, high omega-3 and high omega-6 PUFAs ratio. Cell survival was measured using 2, 3-bis [2-methoxy-4-nitro-5-sulfophenyl]-5-[phenylamino] carbonyl-2H- tetrazolium hydroxide [XTT] method and sPLA2IIa level assessed with ELISA technique. The sPLA2IIa level was significantly higher in the ectopic endometrial cell culture compared to the eutopic group for each of the three matched treatments [balanced, high omega-3 and high omega-6]. Also the sPLA2IIa level in the ectopic endometrial cell group was remarkably increased by each of the three PUFAs treatments compared to control condition [p<0.05, p<0.01, p<0.05 respectively]. Cell survival in the eutopic group was significantly decreased by high omega-6 culturing compared to control medium [p<0.05]. The increase in sPLA2IIa level in ectopic endometrial cells by fatty acid treatments [especially high omega-3], strengthens the hypothesis that PUFAs stimulate secretion of cytokines leading to increased sPLA2IIa level

Elevated serum secretory type II phospholipase A2 in patients with coronary heart disease / 中华心血管病杂志

<p><b>OBJECTIVE</b>To measure the serum level of secretory type II phospholipase A2 (sPLA2) in patients with coronary heart disease and investigate the possible relationship with IL-8 and LPA.</p><p><b>METHODS</b>A total of 110 patients with acute coronary syndrome (ACS), 63 patients with stable coronary heart disease (SCHD) group and 89 non-CHD control patients were studied. Serum levels of sPLA2, IL-8, LPA and hs-CRP were measured and the correlation among these parameters was observed.</p><p><b>RESULTS</b>The levels of serum sPLA2 [(68 +/- 17) U/ml], IL-8 [(182 +/- 80) pg/ml] and LPA [(2.85 +/- 0.36) micromol/L] were significantly higher in CHD patients than those in controls [sPLA2: (55 +/- 12) U/ml; IL-8: (119 +/- 33) pg/ml; LPA: (2.34 +/- 0.36) micromol/L, all P < 0.01], and sPLA2 and IL-8 were also significantly higher in ACS patients [sPLA2: (71 +/- 18) U/ml; IL-8: (195 +/- 78) pg/ml] than those in SCHD patients [sPLA2: (63 +/- 12) U/ml; IL-8: (159 +/- 79) pg/ml, both P < 0.01]. Serum sPLA2 level was positively correlated with hs-CRP, IL-8 and LPA (r = 0.203, P = 0.007; r = 0.658, P < 0.01; r = 0.231, P = 0.005, respectively). The relative risk of having CHD is 6.248 (P < 0.01) with the sPLA2 level above 63.75 U/ml.</p><p><b>CONCLUSION</b>Elevated serum sPLA2 level is a risk factor for CHD.</p>

Effects of rhubarb on the immune substances secreted from intestine in mice / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the therapeutic mechanism of rhubarb in protecting the intestinal muco-membranous barrier in the mice.</p><p><b>METHOD</b>Bal b/c mice were divided into 2 groups, gavaged with normal saline and 10% rhubarb decoction, respectively. The animals were killed after 24 hours after the treatments. The intestinal juice was collected after intestinal lavage and centrifuged for determination of IgA, total protein, C3, high density lipoprotein, type II PLA2 activity, and content of lysozyme. At the same time, 40 mg of small intestine were incised in each mouse. Reverse transcription polymerase chain reaction (RT-PCR) and gel image analysis were performed to detect the content of the cryptdin gene expression.</p><p><b>RESULT</b>The content of IgA, total protein, the C3, lysozyme, and the type II PLA2 activity in intestinal lavaged juice exhibited the statistical differences between the two groups (P < 0.05). There were no significant difference in the ontents of HDL, cryptdin-1 and cryptdin-4 gene expression between the two groups (P > 0.05).</p><p><b>CONCLUSION</b>Rhubarb could increase secretion of several immune associated substances of the mucous membrane in normal intestine, indicating a possibility to abate the injury of intestine mucus resulted from severe stress induced by trauma, burn and shock. Through above mechanisms Rhubarb may also reduce the incidence of bacterial translocation and systemic inflammatory reaction syndrome (SIRS).</p>

The Production Mechanism of TNF-alpha and IL-6 by Group IIA Phospholipase A2 / 영남의대학술지

BACKGROUND: Secretory phospholipase A2 (sPLA2) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 (sPLA2-IIA) has been detected in the inflammatory fluids, and its plasma level increases in the inflammatory disease. This study examined the effect of sPLA2-IIA on mouse macropahges in order to investigate the potential mechanism of sPLA2-induced inflammation. MATERIALS AND METHODS: Wild type PLA2 and mutant H48Q PLA2 were purified from HEK293 cells transfected with the corresponding plasmids, and the PLA2 activities were measured using 1-palmitoyl-2-[1- (14) C]linoleoyl-3-phosphatidylethanolamine as substrates. The TNF-alpha and IL-6 released in the supernatants were determined by ELISA. In addition, the TNF-alpha and IL-6 mRNA were analyzed by RT-PCR. RESULTS: sPLA2-IIA stimulated the production of TNF-alpha and IL-6 in a dose- and time-dependent manner. In addition, the effect of sPLA2-IIA on cytokine production from the macrophage was found to be associated with the accumulation of their specific mRNA. The mRNA levels of TNF-alpha and IL-6 peaked at 2 and 6 hours in a time-dependent manner, respectively. CONCLUSION: In conclusion, the production of proinflammatory cytokine might be mediated by the binding of sPLA2-IIA to the receptors.

Comparison of the activities of wild type and mutant enhancing factor/mouse secretory phospholipase A2 proteins.

Enhancing factor (EF) protein, an isoform of secretory phospholipase A2 (PLA2), was purified as a modulator of epidermal growth factor from the small intestine of the Balb/c mouse. It was for the first time that a growth modulatory property of sPLA2 was demonstrated. Deletion mutation analysis of EF cDNA carried out in our laboratory showed that enhancing activity and phospholipase activity are two separate activities that reside in the same molecule. In order to study the specific amino acids involved in each of these activities, two site-directed mutants of EF were made and expressed in vitro. Comparison of enhancing activity as well as phospholipase A2 activity of these mutant proteins with that of wild type protein helped in identification of some of the residues important for both the activities

Sequence analysis of full length cDNA for enhancing factor/phospholipase A2.

Enhancing factor (EF) protein was initially purified as a modulator of epidermal growth factor from small intestines of mouse. The cDNA sequence, obtained by RT-PCR, revealed that EF belonged to the non-pancreatic, phospholipase A2 (PLA2) family. This was the first report of the mouse PLA2. In the present paper we report the complete cDNA sequence of EF gene, in which the 5' sequence has been obtained by RAcE-PCR. The predicted amino acid sequence was computer analysed and the putative sites for enzyme action, calcium binding and heparin binding have been identified. The complete protein sequence of EF along with 16 aligned sequences were used to infer a phylogenetic tree. From this data the mouse EF was grouped with other membrane associated PLA2 with a bootstrap value of 98% indicating that it belonged to this class.