Phospholipase A2 in human ascitic fluid. Purification, characterization and immunochemical detection.
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ABSTRACT:
SUBMITTER: Kortesuo PT
PROVIDER: S-EPMC1151477 | biostudies-other | 1991 Aug
REPOSITORIES: biostudies-other
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Project description:Introduction:Malignant ascites (MA) is a major cause of morbidity that occurs in 37% of ovarian cancer patients. The accumulation of MA in the peritoneal cavity due to cancer results in debilitating symptoms and extremely poor quality of life. There is an urgent unmet need to expand the understanding of MA to design effective treatment strategies, and to improve MA diagnosis. Objective:Our purpose here is to contribute to a better characterization of MA metabolic composition in ovarian cancer. Method:We determined the metabolic composition of ascitic fluids resulting from orthotopic growth of two ovarian cancer cell lines, the mouse ID8-vascular endothelial growth factor (VEGF)-Defb29 cell line and the human OVCAR3 cell line using high-resolution 1H MRS. ID8-VEGF-Defb29 tumors induce large volumes of ascites, while OVCAR3 tumors induce ascites less frequently and at smaller volumes. To better understand the factors driving the metabolic composition of the fluid, we characterized the metabolism of these ovarian cancer cells in culture by analyzing cell lysates and conditioned culture media with 1H NMR. Results:Distinct metabolite patterns were detected in ascitic fluid collected from OVCAR3 and ID8-VEGF-Defb29 tumor bearing mice that were not reflected in the corresponding cell culture or conditioned medium. Conclusion:High-resolution 1H NMR metabolic markers of MA can be used to improve characterization and diagnosis of MA. Metabolic characterization of MA can provide new insights into how MA fluid supports cancer cell growth and resistance to treatment, and has the potential to identify metabolic targeting strategies to reduce or eliminate the formation of MA.
| S-EPMC5804489 | biostudies-other
Project description:A rapid and highly sensitive point-of-care (PoC) lateral flow assay for phospholipase A2 (PLA2) is demonstrated in serum through the enzyme-triggered release of a new class of biotinylated multiarmed polymers from a liposome substrate. Signal from the enzyme activity is generated by the adhesion of polystreptavidin-coated gold nanoparticle networks to the lateral flow device, which leads to the appearance of a red test line due to the localized surface plasmon resonance effect of the gold. The use of a liposome as the enzyme substrate and multivalent linkers to link the nanoparticles leads to amplification of the signal, as the cleavage of a small amount of lipids is able to release a large amount of polymer linker and adhesion of an even larger amount of gold nanoparticles. By optimizing the molecular weight and multivalency of these biotinylated polymer linkers, the sensitivity of the device can be tuned to enable naked-eye detection of 1 nM human PLA2 in serum within 10 min. This high sensitivity enabled the correct diagnosis of pancreatitis in diseased clinical samples against a set of healthy controls using PLA2 activity in a point-of-care device for the first time.
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Project description:Our aim is to study selected cerebrospinal fluid (CSF) glycerophospholipids (GP) that are important in brain pathophysiology. We recruited cognitively healthy (CH), minimally cognitively impaired (MCI), and late onset Alzheimer's disease (LOAD) study participants and collected their CSF. After fractionation into nanometer particles (NP) and supernatant fluids (SF), we studied the lipid composition of these compartments. LC-MS/MS studies reveal that both CSF fractions from CH subjects have N-acyl phosphatidylethanolamine, 1-radyl-2-acyl-sn-glycerophosphoethanolamine (PE), 1-radyl-2-acyl-sn-glycerophosphocholine (PC), 1,2-diacyl-sn-glycerophosphoserine (PS), platelet-activating factor-like lipids, and lysophosphatidylcholine (LPC). In the NP fraction, GPs are enriched with a mixture of saturated, monounsaturated, and polyunsaturated fatty acid species, while PE and PS in the SF fractions are enriched with PUFA-containing molecular species. PC, PE, and PS levels in CSF fractions decrease progressively in participants from CH to MCI, and then to LOAD. Whereas most PC species decrease equally in LOAD, plasmalogen species account for most of the decrease in PE. A significant increase in the LPC-to-PC ratio and PLA2 activity accompanies the GP decrease in LOAD. These studies reveal that CSF supernatant fluid and nanometer particles have different GP composition, and that PLA2 activity accounts for altered GPs in these fractions as neurodegeneration progresses.
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Project description:Phospholipases A(2) (PLA(2)s) catalyze hydrolysis of fatty acids from the sn-2 position of phospholipids. Here we report the identification and characterization of a membrane-associated intracellular calcium-dependent, adipose-specific PLA(2) that we named AdPLA (adipose-specific phospholipase A(2)). We found that AdPLA was highly expressed specifically in white adipose tissue and was induced during preadipocyte differentiation into adipocytes. Clearance of AdPLA by immunoprecipitation significantly decreased PLA activity in white adipose tissue lysates but had no effect on liver lysates, where expression was hardly detectable. In characterizing AdPLA, we employed radiochemical assays with TLC analysis of the enzyme activity of lysates from COS-7 cells overexpressing AdPLA. For kinetic studies, we produced purified recombinant AdPLA for use in a lipoxidase-coupled spectrophotometric assay. AdPLA generated free fatty acid and lysophospholipid from phosphatidylcholine with a preference for hydrolysis at the sn-2 position. Although we found low but detectable lysophospholipase activity, AdPLA showed no significant activity against a variety of other lipid substrates. Calcium was found to activate AdPLA but was not essential for activity. Studies with known phospholipase inhibitors, including bromoenolactone, methyl arachidonyl fluorophosphate, AACOCF(3), 7,7-dimethyl-5,8-eicosadienoic acid, and thioetheramide, supported that AdPLA is a phospholipase. Mutational studies showed that His-23 and Cys-113 are critical for activity of AdPLA and suggested that AdPLA is likely a His/Cys PLA(2). Overall, although AdPLA is similar to other histidine phospholipases in pH and calcium dependence, AdPLA showed different characteristics in many regards, including predicted catalytic mechanism. AdPLA may therefore represent the first member of a new group of PLA(2)s, group XVI.
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Project description:Several phospholipase A2 (PLA2) activities have been identified in rat lung homogenate and shown to be important in metabolism of lung phospholipids. One PLA2 activity is Ca(2+)-independent, active in vitro at pH 4, and inhibited by a substrate analogue, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Purification of this rat lung PLA2 by approx. 550-fold was carried out by sequential column chromatographies using DE-52, Sephacryl-100, heparin-Sepharose, and phenyl-Sepharose columns. The purified activity had an acidic pH optimum, was Ca(2+)-independent, was inhibited by MJ33 in a dose-dependent manner (50% inhibition at 3 mol%), was unaffected by treatment with p-bromophenacyl bromide or mercaptoethanol, and had a unique N-terminal amino acid sequence. The apparent molecular mass was 15 kDa on gel electrophoresis and activity was recovered in part by renaturation of protein from the gel. The properties of this enzyme suggest a new class of PLA2.
| S-EPMC1137462 | biostudies-other
Project description:Phospholipases A2 (PLA2s) belong to a superfamily of enzymes responsible for hydrolysis of the sn-2 fatty acids of membrane phospholipids to release arachidonic acid. PLA2s are the rate limiting enzyme for the downstream synthesis of prostaglandins and leukotrienes that are the main mediators of inflammation. The extracellular forms of this enzyme are also called the secretary phospholipase A2 (sPLA2) and are distributed extensively in most of the tissues in the human body. Their integral role in inflammatory pathways has been the primary reason for the extensive research on this molecule. The catalytic mechanism of sPLA2 is initiated by a histidine/aspartic acid/calcium complex within the active site. Though they are known to have certain housekeeping functions, certain mutations of sPLA2 are known to be implicated in causation of certain pathologies leading to diseases such as atherosclerosis, cardiovascular diseases, benign fleck retina, neurodegeneration, and asthma. We present an overview of human sPLA2 and a comprehensive compilation of the mutations that result in various disease phenotypes. The study not only helps to have a holistic understanding of human sPLA2 mutations and their clinical implications, but is also a useful platform to initiate research pertaining to structure-function relationship of the mutations to develop effective therapies for management of these diseases.
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Project description:Nocardia veterana is a recently characterized species within the genus Nocardia, and only three human clinical isolates have been reported for this species. We describe a case of ascitic fluid infection in an immunocompromised patient due to N. veterana. To our knowledge, this is the first report of a Nocardia sp. strain from ascitic fluid and the fourth report of N. veterana isolated from human samples. Chemotaxonomic methods showed the strain to belong to the genus Nocardia, and identification to the species level was done by 16S ribosomal DNA gene sequencing. The antibiotic susceptibility profile of N. veterana is reported here for the second time. The strain was deposited in the Collection of the Pasteur Institute and in the Culture Collection of the University of Göteborg (CIP 107497 and CCUG 46576). The corresponding 16S ribosomal DNA gene sequence is available from the GenBank database under accession number AY149599. A phylogenetic analysis was conducted and showed that N. veterana was most closely related to the recently characterized species Nocardia africana rather than to Nocardia vaccinii, as previously reported.
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Project description:Mammalian secretory class V phospholipase A2 (PLA2) is a newly discovered PLA2 that is implicated in eicosanoid formation in inflammatory cells. As a first step towards understanding the structure, function and regulation of this PLA2, we constructed a bacterial expression vector for human secretory class V PLA2 (hV-PLA2), over-expressed and purified the protein, and determined its physical and kinetic properties. When compared with human class IIa enzyme (hIIa-PLA2), hV-PLA2 has several distinct properties. First, hV-PLA2 can catalyse the hydrolysis of phosphatidylcholine more effectively than hIIa-PLA2 by two orders of magnitude. Secondly, hV-PLA2 has much higher binding affinity and activity for compactly packed phosphatidylcholine bilayers than hIIa-PLA2. Finally, hV-PLA2 has much reduced thermal stability compared with hIIa-PLA2. These data suggest that hV-PLA2 is better suited than hIIa-PLA2 for acting on the outer cellular membrane and liberating arachidonic acid from membrane phospholipids. Also, the unusually low thermal stability of hV-PLA2 might contribute to tighter regulation of its activities in extracellular media.
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Project description:As saprophytes or disease causing microorganisms, fungi acquire nutrients from dead organic material or living host organisms. Lipids as structural components of cell membranes and storage compartments play an important role as energy-rich food source. In recent years, it also has become clear that lipids have a wide range of bioactive properties including signal transduction and cell to cell communication. Thus, it is not surprising that fungi possess a broad range of hydrolytic enzymes that attack neutral lipids and phospholipids. Especially during infection of a mammalian host, phospholipase A(2) (PLA(2)) enzymes released by fungi could play important roles not only for nutrient acquisition and tissue invasion, but for intricate modulation of the host's immune response. Sequencing of fungal genomes has revealed a wide range of genes encoding PLA(2) activities in fungi. We are just beginning to become aware of the significance these enzymes could have for the fungal cells and their interaction with the host.
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Project description:The phospholipase A2 superfamily of enzymes plays a significant role in the development and progression of numerous inflammatory diseases. Through their catalytic action on membrane phospholipids, phospholipases are the upstream regulators of the eicosanoid pathway releasing free fatty acids for cyclooxygenases, lipoxygenases, and cytochrome P450 enzymes which produce various well-known inflammatory mediators including leukotrienes, thromboxanes and prostaglandins. Elucidating the association of phospholipases A2 with the membrane, the extraction and binding of phospholipid substrates, and their interactions with small-molecule inhibitors is crucial for the development of new anti-inflammatory therapeutics. Studying phospholipases has been challenging because they act on the surface of cellular membranes and micelles. Multidisciplinary approaches including hydrogen/deuterium exchange mass spectrometry, molecular dynamics simulations, and other computer-aided drug design techniques have been successfully employed by our laboratory to study interactions of phospholipases with membranes, phospholipid substrates and inhibitors. This review summarizes the application of these techniques to study four human recombinant phospholipases A2.
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