Animals ; Cardiovascular System ; chemistry ; Female ; Group II Phospholipases A2 ; blood ; Group IV Phospholipases A2 ; blood ; Group V Phospholipases A2 ; blood ; Group X Phospholipases A2 ; blood ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

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.
Animals ; Enzyme-Linked Immunosorbent Assay ; Fatty Acids ; Group II Phospholipases A2* ; HEK293 Cells ; Inflammation ; Interleukin-6* ; Macrophages ; Mice ; Phospholipases A2, Secretory ; Phospholipids ; Plasma ; Plasmids ; RNA, Messenger ; Tumor Necrosis Factor-alpha*

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.
Humans ; Kinetics ; Mutation ; Phospholipases A2, Secretory ; Phospholipids ; Saccharomycetales ; Substrate Specificity

Infantile neuroaxonal dystrophy (INAD) is a rare neurodegenerative disease. Two boys aged 3 years and 4 years and 2 months respectively, were admitted to the hospital due to delayed mental and motor development. There were no abnormalities at birth, and both children had low muscle strength and tension on admission. One child was not able to stand alone and had impaired vision. Electromyography showed neurogenic damage, and head MRI revealed cerebellar atrophy. High-throughput sequencing revealed compound heterozygous mutations in the PLA2G6 gene in the two children. The mutations (IVS11-1G>T and c.1984C>G) in one child were new mutations, and immunohistochemistry showed a reduction in the protein expression of PLAG6 in the muscular tissue of this child. INAD has the main clinical manifestations of psychomotor developmental regression and cerebellar atrophy. High-throughput sequencing can help with clinical diagnosis.
Child, Preschool ; Group VI Phospholipases A2 ; genetics ; Humans ; Magnetic Resonance Imaging ; Male ; Mutation ; Neuroaxonal Dystrophies ; genetics ; Neurodegenerative Diseases ; genetics

No abstract available.
Phosphatidylcholines* ; Phospholipases A2* ; Phospholipases*

PURPOSE: In human subjects and animal models with acute and chronic lung injury, the bioactive lysophosphatidylcholine (LPC) is elevated in lung lining fluids. The increased LPC can promote an inflammatory microenvironment resulting in lung injury. Furthermore, pathological lung conditions are associated with upregulated phospholipase A2 (PLA2), the predominant enzyme producing LPC in tissues by hydrolysis of phosphatidylcholine. However, the lung cell populations responsible for increases of LPC have yet to be systematically characterized. The goal was to investigate the LPC generation by bronchial epithelial cells in response to pathological mediators and determine the major LPC species produced. METHODS: Primary human bronchial epithelial cells (NHBE) were challenged by vascular endothelial growth factor (VEGF) for 1 or 6 h, and condition medium and cells collected for quantification of predominant LPC species by high performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). The cells were analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for PLA2. The direct effects of LPC in inducing inflammatory activities on NHBE were assessed by transepithelial resistance as well as expression of interleukin-8 (IL-8) and matrix metalloproteinase-1 (MMP-1). RESULTS: VEGF stimulation of NHBE for 1 or 6 h, significantly increased concentrations of LPC16:0, LPC18:0, and LPC18:1 in condition medium compared to control. The sPLA2-selective inhibitor (oleyloxyethyl phosphorylcholine) inhibited the VEGF-induced release of LPC16:0 and LPC18:1 and PLA2 activity. In contrast, NHBE stimulated with TNF did not induce LPC release. VEGF did not increase mRNA of PLA2 subtypes sPLA2-X, sPLA2-XIIa, cPLA2-IVa, and iPLA2-VI. Exogenous LPC treatment increased expression of IL-8 and MMP-1, and reduced the transepithelial resistance in NHBE. CONCLUSIONS: Our findings indicate that VEGF-stimulated bronchial epithelial cells are a key source of extracellular LPCs, which can function as an autocrine mediator with potential to induce airway epithelial inflammatory injury.
Epithelial Cells* ; Group X Phospholipases A2 ; Humans ; Hydrolysis ; Interleukin-8 ; Lung ; Lung Injury ; Lysophosphatidylcholines* ; Mass Spectrometry ; Matrix Metalloproteinase 1 ; Models, Animal ; Phosphatidylcholines ; Phospholipases A2 ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger ; Vascular Endothelial Growth Factor A

OBJECTIVETo investigate the clinical symptoms and potential mutations in the PLA2G6 gene for a child with infantile neuroaxonal dystrophy.

METHODSClinical data of the patient was collected. The coding regions of PLA2G6 gene was subjected to Sanger sequencing using blood DNA from the patient and her parents.

RESULTSThe patient has presented with psychomotor regression and hypotonia, followed by development of tetraparesis. A novel homozygous mutation G68A in the PLA2G6 gene was found by DNA sequencing, while her parents were both heterozygous carriers.

CONCLUSIONThe psychomotor regression and tetraparesis of the patient was caused by infantile neuroaxonal dystrophy due to a novel homozygous mutation in the PLA2G6 gene, which was inherited from her parents.

Adult ; Base Sequence ; Brain ; diagnostic imaging ; Child, Preschool ; DNA Mutational Analysis ; Female ; Group VI Phospholipases A2 ; genetics ; Homozygote ; Humans ; Magnetic Resonance Imaging ; Male ; Molecular Sequence Data ; Mutation ; Neuroaxonal Dystrophies ; diagnostic imaging ; genetics ; Radiography

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.
Acute Lung Injury ; Animals ; Aza Compounds ; Bronchoalveolar Lavage Fluid ; Cytosol ; Light ; Lung ; Malondialdehyde ; Neutrophils ; Peroxidase ; Phospholipases A2, Secretory ; Quinolines ; Rats ; Respiratory Distress Syndrome, Adult ; Sepsis

OBJECTIVETo 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.

METHODSA 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.

RESULTSThe 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.

CONCLUSIONElevated serum sPLA2 level is a risk factor for CHD.

Adult ; Aged ; Aged, 80 and over ; C-Reactive Protein ; metabolism ; Coronary Angiography ; Coronary Disease ; blood ; diagnostic imaging ; Female ; Group II Phospholipases A2 ; Humans ; Interleukin-8 ; blood ; Lysophospholipids ; blood ; Male ; Middle Aged ; Phospholipases A ; blood ; Phospholipases A2

No abstract available.
Macrophages* ; Phospholipases A2* ; Phospholipases* ; Tumor Necrosis Factor-alpha*