Effect of E54 mutation of human secreted phospholipase A2 GIIE on substrate selectivity / 生物工程学报

Human secreted phospholipase A2 GIIE (hGIIE) is involved in inflammation and lipid metabolism due to its ability of hydrolyzing phospholipids. To reveal the mechanism of substrate head-group selectivity, we analyzed the effect of mutation of hGIIE on its activity and selectivity. hGIIE structural analysis showed that E54 might be related to its substrate head-group selectivity. According to the sequence alignment, E54 was mutated to alanine, phenylalanine, and lysine. Mutated genes were cloned and expressed in Pichia pastoris X33, and the enzymes with mutations were purified with 90% purity by ion exchange and molecular size exclusion chromatography. The enzymatic activities were determined by isothermal microthermal titration method. The Km of mutant E54K towards 1,2-dihexyl phosphate glycerol decreased by 0.39-fold compared with that of wild type hGIIE (WT), and the Km of E54F towards 1,2-dihexanoyl-sn-glycero-3-phosphocholine increased by 1.93-fold than that of WT. The affinity of mutant proteins with phospholipid substrate was significantly changed, indicating that E54 plays an important role in the substrate head-group selectivity of hGIIE.

Tracking of secretory phospholipase A2 enzyme activity levels from childhood to adulthood: a 21-year cohort / Monitoramento dos níveis de atividade da enzima fosfolipase A2 secretória da infância à vida adulta: uma coorte de 21 anos

Abstract Objective: Secretory 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. Method: Prospective 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. Results: sPLA2 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. Conclusions: Childhood 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.

Resumo Objetivo: A atividade da enzima fosfolipase A2 secretória (sPLA2) é um possível biomarcador inflamatório de doença cardiovascular. Examinamos o monitoramento, ou a persistência, dos níveis de atividade da enzima sPLA2 da infância à vida adulta e identificamos fatores possivelmente modificáveis que afetam o monitoramento. Método: Coorte prospectiva de 1.735 crianças (45% do sexo feminino) cujos níveis de atividade da enzima sPLA2 no soro e outros fatores de risco para doença cardiovascular foram medidos em 1980 e acompanhados até 2011. Resultados: Atividade da enzima sPLA2 monitorada da infância à vida adulta para indivíduos do sexo masculino (r = 0,39) e sexo feminino (r = 0,45). Aqueles que diminuíram seus índices de massa corporal com relação a seus pares foram mais propensos à redução dos níveis elevados de sPLA2 na infância do que a manter níveis persistentemente elevados de sPLA2 na infância e vida adulta. Aqueles que consumiram menos frutas e ganharam mais índice de massa corporal com relação a seus pares, que começaram a fumar ou foram fumantes persistentes entre a infância e vida adulta foram mais propensos a desenvolver níveis de sPLA2 elevados do que aqueles com níveis de sPLA2 não elevados persistentes. Conclusões: Os níveis de atividade da enzima sPLA2 na infância estão associados aos níveis de sPLA2 na vida adulta, 21 anos mais tarde. As mudanças saudáveis nos fatores de risco modificáveis que ocorrem entre a infância e a vida adulta podem evitar que as crianças desenvolvam níveis elevados de sPLA2 na vida adulta.

High omega-3: omega-6 fatty acids ratio increases fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in human ectopic endometrial cells

Endometriosis, a common chronic inflammatory disorder, is defined by the atypical growth of endometrium- like tissue outside of the uterus. Secretory phospholipase A2 group Ha [sPLA2-IIa] and fatty acid binding protein4 [FABP4] play several important roles in the inflammatory diseases, Due to reported potential anti-inflammatory effects of omega-3 and omega-6 fatty acids, the purpose of the present study was to investigate the effects of omega-3 and omega-6 polyunsaturated fatty acids [PUFAs] on fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in cultured endometrial cells. Ectopic and eutopic endometrial tissues obtained from 15 women were snap frozen. After thawing and tissue digestion, primary mixed stromal f and endometrial epithelial cell culture was performed for 8 days in culture mediums supplemented with normal and high ratios of omega-3 and omega-6 PUFA. sPLA2-IIa in the J culture medium and FABP4 level was determined using enzyme immuno assay [EIA] technique. Within ectopic endometrial cells group, the level of cellular FABP4 and extracellular sPLA2-IIa were remarkably increased under high omega-3 PUFA exposure compared with control condition [p=0.014 and p=0.04 respectively]. Omega-3 PUFAs may increase the level of cellular FABP4 and extracellular sPLA2-IIa in ectopic endometrial cells, since sPLAIIa and FABP4 may affect endometriosis via several mechanisms, more relevant studies are encouraged to know the potential effect of increased cellular FABP4 and extracellular sPLA2-IIa on endometriosis

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

Moxifloxacin Ameliorates Oleic Acid-induced Acute Lung Injury by Modulation of Neutrophilic Oxidative Stress in Rats / 결핵및호흡기질환

BACKGROUND: Based on the known immunoregulatory functions of moxifloxacin on phagocytes, the therapeutic effect of moxifloxacin on oleic acid (OA)-induced acute lung injury (ALI) was investigated. METHODS: Moxifloxacin (10 mg/kg) was given to male Sprague-Dawley rats that had been given oleic acid (OA, 30 microliter) intravenously. Five hours after OA injection, parameters demonstrating ALI were assessed to measure the effects of moxifloxacin on acute lung injury. RESULTS: The pathological findings of OA-induced ALI's was diminished by moxifloxacin. Through ultrastructural and CeCl3 EM histochemistry, moxifloxacin was confirmed to be effective in decreasing oxidative stress in the lung as well. Indices of ALI, such as lung weight/body weight ratio, protein content in bronchoalveolar lavage fluid, and lung myeloperoxidase were decreased by moxifloxacin. In diaminobenzidine immunohistochemistry, fluorescent immunohistochemistry, and Western blotting of the lung, moxifloxacin had decreased the enhanced expression of secretory phospholipase A2 (sPLA2) by OA. CONCLUSION: We concluded that moxifloxacin was effective in lessening acute inflammatory pulmonary edema caused by OA, by inhibiting the neutrophilic respiratory burst, which was initiated by the activation of sPLA2.

The Effects of Moxifloxacin in Endotoxin-induced Acute Lung Injury / 대한흉부외과학회지

BACKGROUND: The pathophysiology of acute respiratory distress syndrome with sepsis is acute lung injury (ALI) that's' caused by endotoxin (LPS). We evaluate effects of moxifloxacin on LPS-induced ALI in a rat model. MATERIAL AND METHOD: The rats were divided into 3 groups as the control group (C), the LPS insult group (L), and the LPS+moxifloxacin treated group (L-M). ALI was induced by endotracheal instillation of E.coli LPS, then moxifloxacin was given in 30 minutes. Five hours later, we checked the lung weight/body weight ratio(the L/BW ratio), the protein & neutrophils in the bronchoalveolar lavage fluid (BALF), the myeloperoxidase (MPO) activity & the malondialdehyde (MDA) content, the expressions of cytosolic and secretory phospholipase A2 (c, sPLA2), and the morphology of the lung with using a light microscope. RESULT: The L/BW ratio, the protein content and the neutrophil count in the BALF, and the MPO activity and the MDA content in lung were significantly increased in group L compared to group C, and these factors were markedly decreased in group L-M compare to group L. The cPLA2 expression and the sPLA2 expression were increased in group L and the cPLA2 expression was decreased in group L-M. Yet the sPLA2 expression was not changed in group L-M. Morphologically, many inflammatory findings were observed in group L, but not in group L-M. CONCLUSION: Many of the inflammatory changes of ALI that were caused by LPS insult were ameliorated by moxifloxacin treatment.

Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes.</p><p><b>METHODS</b>Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used.</p><p><b>RESULTS</b>Although sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality.</p><p><b>CONCLUSION</b>The ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality.</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.