1-Hexadecyl-2-Arachidonoylthio-2-deoxy-sn-Glycero-3-Phosphorylcholine as a Substrate for the Microtiterplate Assay of Human Cytosolic Phospholipase A2

Reynolds, Laure J.; Hughes, Lori L.; Lin, Yu; Dennis, Edward A.

doi:10.1006/abio.1994.1079

Cited by 64 publications

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“…Inhibition of ExoU PLA 2 activity by pseudolipasin A was examined using two different enzymatic assays. The first assay is based on the cleavage of arachidonyl-TEPC, resulting in the generation of a free thiol at the sn-2 position that in turn reduces DTNB, yielding a colorimetric readout (29). Since the PLA 2 activity of ExoU requires activation by an unknown eukaryotic cellular factor, each reaction was supplemented with a CHO or yeast cell extract.…”

Section: Resultsmentioning

confidence: 99%

Pseudolipasin A Is a Specific Inhibitor for Phospholipase A ₂ Activity of Pseudomonas aeruginosa Cytotoxin ExoU

et al. 2007

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A number of bacterial pathogens utilize the type III secretion pathway to deliver effector proteins directly into the host cell cytoplasm. Certain strains of Pseudomonas aeruginosa associated with acute infections express a potent cytotoxin, exoenzyme U (ExoU), that is delivered via the type III secretion pathway directly into contacting host cells. Once inside the mammalian cell, ExoU rapidly lyses the intoxicated cells via its phospholipase A 2 (PLA 2 ) activity. A high-throughput cell-based assay was developed to screen libraries of compounds for those capable of protecting cells against the cytotoxic effects of ExoU. A number of compounds were identified in this screen, including one group that blocks the intracellular activity of ExoU. In addition, these compounds specifically inhibited the PLA 2 activity of ExoU in vitro, whereas eukaryotic secreted PLA 2 and cytosolic PLA 2 were not inhibited. This novel inhibitor of ExoU-specific PLA 2 activity, named pseudolipasin A, may provide a new lead for virulence factor-based therapeutic design.Pseudomonas aeruginosa is the leading cause of hospitalacquired infections by gram-negative bacteria and is responsible for chronic infections of individuals with cystic fibrosis (15). As P. aeruginosa and other bacterial pathogens become multidrug resistant (22), there is a continual need for the identification of compounds directed at novel targets that could be developed into therapeutic agents. The ability of P. aeruginosa to cause a number of distinct infections has been attributed to its large genome, which encodes a variety of virulence factors (35). Prominent among these is the type III secretion system (TTSS), which allows the bacteria to deliver proteins directly into the host cell cytoplasm (39). ExoU is one of the toxic effector proteins delivered by the type III secretion apparatus, and its expression is associated with strains that cause acute infections (11,12). ExoU is a member of the patatin family of phospholipase A 2 (PLA 2 ), and this activity is required for cytotoxicity toward eukaryotic cells (33,34). In contrast to eukaryotic phospholipases, which remodel cellular membranes and synthesize proinflammatory secondary messengers such as arachidonic acid and leukotrienes (2, 9), ExoU PLA 2 activity primarily causes disruption of the host cytoplasmic membrane, resulting in cell lysis (24,32,33).We sought to identify compounds that inhibit type III secretion-mediated cytotoxicity by protecting tissue culture cells from infection by P. aeruginosa strains that elaborate ExoU as a sole cytotoxic effector. From a synthetic small-molecule library, we have identified compounds that protected Chinese hamster ovary (CHO) cells from the cytotoxic activity of P. aeruginosa expressing ExoU. One of the most potent compounds is pseudolipasin A (Pseudomonas phospholipase inhibitor A). Pseudolipasin A does not interfere with type III secretion in general, suggesting that the protection observed occurs downstream of the delivery of ExoU. Pseudolipasin A not only protected...

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Section: Resultsmentioning

confidence: 99%

Pseudolipasin A Is a Specific Inhibitor for Phospholipase A ₂ Activity of Pseudomonas aeruginosa Cytotoxin ExoU

et al. 2007

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“…More details about this technique are available in the ''Electronic supplementary material.'' Then, two specific high sensitivity, non-radioactive kits were used to measure sPLA2 (Assay Designs, Ann Arbor, MI, USA) and cPLA2 (Cayman Chemical Company Ann Arbor, MI, USA) activities, as previously described [23,24]. The sPLA2 kit uses hexadecanoylthio-1-ethyl phosphorylcholine as substrate, whereas arachidonoyl-thio-phosphatidylcholine is used for cPLA2, given the different substrate affinity of the two enzymes [23,24].…”

Section: Meconiummentioning

confidence: 99%

Role of distinct phospholipases A2 and their modulators in meconium aspiration syndrome in human neonates

et al. 2011

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“…Valproate down-regulates brain cyclooxygenase 691 position by PLA 2 releases free thiol, which is detected by Ellman's reagent (Reynolds et al 1994). PLA 2 activity was determined in the supernatant using a cPLA2 assay kit (Cayman Chemicals), in the presence and absence of the specific inhibitor of Ca 2+ -independent PLA 2 (iPLA 2 ), bromoenol lactone (Ackermann et al 1995), which was incubated for 15 min at 25°C at a concentration of 10 lM before the assay.…”

Section: Measurement Of Pla 2 Activitymentioning

confidence: 99%

Valproic acid down‐regulates the conversion of arachidonic acid to eicosanoids via cyclooxygenase‐1 and ‐2 in rat brain

Bosetti

Weerasinghe

Rosenberger

et al. 2003

Journal of Neurochemistry

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Sodium valproate, a mood stabilizer, when chronically administered to rats (200 mg/kg i.p. daily for 30 days) significantly reduced the brain protein levels of cyclooxygenase (COX)-1 and COX-2, without altering the mRNA levels of these enzymes. COX activity was decreased, as were the brain concentrations of 11-dehydrothromboxane B 2 and prostaglandin E 2 (PGE 2 ), metabolites of arachidonic acid (AA) produced via COX. In contrast, the brain protein level of 5-lipoxygenase and the concentration of its AA metabolite leukotriene B 4 were unchanged. In view of published evidence that lithium chloride administered chronically to rats, like chronic valproate, reduces AA turnover within brain phospholipids, and that lithium post-transcriptionally down-regulates COX-2 but not COX-1 protein level and enzyme activity, these observations suggest that mood stabilizers generally modulate the release and recycling of AA within brain phospholipids, and the conversion of AA via COX-2 to PGE 2 and related eicosanoids. If targeting this part of the 'AA cascade' accounts for their therapeutic action, non-steroidal antiinflammatory drugs or selective COX-2 inhibitors might prove effective in bipolar disorder.

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1-Hexadecyl-2-Arachidonoylthio-2-deoxy-sn-Glycero-3-Phosphorylcholine as a Substrate for the Microtiterplate Assay of Human Cytosolic Phospholipase A2

Cited by 64 publications

References 0 publications

Pseudolipasin A Is a Specific Inhibitor for Phospholipase A ₂ Activity of Pseudomonas aeruginosa Cytotoxin ExoU

Pseudolipasin A Is a Specific Inhibitor for Phospholipase A ₂ Activity of Pseudomonas aeruginosa Cytotoxin ExoU

Role of distinct phospholipases A2 and their modulators in meconium aspiration syndrome in human neonates

Valproic acid down‐regulates the conversion of arachidonic acid to eicosanoids via cyclooxygenase‐1 and ‐2 in rat brain

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1-Hexadecyl-2-Arachidonoylthio-2-deoxy-sn-Glycero-3-Phosphorylcholine as a Substrate for the Microtiterplate Assay of Human Cytosolic Phospholipase A2

Cited by 64 publications

References 0 publications

Pseudolipasin A Is a Specific Inhibitor for Phospholipase A 2 Activity of Pseudomonas aeruginosa Cytotoxin ExoU

Pseudolipasin A Is a Specific Inhibitor for Phospholipase A 2 Activity of Pseudomonas aeruginosa Cytotoxin ExoU

Role of distinct phospholipases A2 and their modulators in meconium aspiration syndrome in human neonates

Valproic acid down‐regulates the conversion of arachidonic acid to eicosanoids via cyclooxygenase‐1 and ‐2 in rat brain

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Pseudolipasin A Is a Specific Inhibitor for Phospholipase A ₂ Activity of Pseudomonas aeruginosa Cytotoxin ExoU

Pseudolipasin A Is a Specific Inhibitor for Phospholipase A ₂ Activity of Pseudomonas aeruginosa Cytotoxin ExoU