scholarly journals PP2A Inhibition Assay Using Recombinant Enzyme for Rapid Detection of Okadaic Acid and Its Analogs in Shellfish

Toxins ◽  
2010 ◽  
Vol 2 (1) ◽  
pp. 195-204 ◽  
Author(s):  
Tsuyoshi Ikehara ◽  
Shihoko Imamura ◽  
Atsushi Yoshino ◽  
Takeshi Yasumoto
Keyword(s):  
Correlation Coefficient ◽  
Digestive Gland ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Rapid Detection ◽  
Inhibition Assay ◽  
Shellfish Poisoning ◽  
Diarrhetic Shellfish Poisoning ◽  
Ic50 Values ◽  
Phosphatase 2A

Okadaic acid and its analogs (OAs) responsible for diarrhetic shellfish poisoning (DSP) strongly inhibit protein phosphatase 2A (PP2A) and thus are quantifiable by measuring the extent of the enzyme inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant human PP2A (rhPP2Ac) for use in a microplate OA assay. OA, dinophysistoxin-1(DTX1), and hydrolyzate of 7-O-palmitoyl-OA strongly inhibited rhPP2Ac activity with IC50 values of 0.095, 0.104, and 0.135 nM, respectively. The limits of detection and quantitation for OA in the digestive gland of scallops and mussels were 0.0348 μg/g and 0.0611 μg/g respectively, and, when converted to the whole meat basis, are well below the regulation level proposed by EU (0.16 μg/g whole meat). A good correlation with LC-MS data was demonstrated, the correlation coefficient being 0.996 with the regression slope of 1.097.

scholarly journals Specification of the Okadaic Acid Equivalent for Okadaic Acid, Dinophysistoxin-1, and Dinophysistoxin-2 Based on Protein Phosphatase 2A Inhibition and Cytotoxicity Assays Using Neuro 2A Cell Line

2021 ◽  
Vol 9 (10) ◽  
pp. 1140
Author(s):  
Tsuyoshi Ikehara ◽  
Kazuya Chikanishi ◽  
Naomasa Oshiro
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Shellfish Poisoning ◽  
Diarrhetic Shellfish Poisoning ◽  
Oral Toxicity ◽  
Cytotoxicity Assays ◽  
The Core ◽  
Phosphatase 2A ◽  
Core Activity

Diarrhetic shellfish poisoning (DSP) is a globally occurring disease threatening public health and trade. The causative toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1), and dinophysistoxin-2 (DTX2) are collectively called OAs, and are quantified using the LC-MS/MS method. The hazardous effect of total OAs is expressed as the sum of OA equivalents defined for respective OAs based on mouse lethality, produced by either intraperitoneal (OAip) or oral administration (OAor). OAs are potent inhibitors of protein phosphatase 2A (PP2A) and are cytotoxic, necessitating expansion of the concept of OA equivalents to all relevant bioactivities. In this study, we determined OA equivalents for respective OA members in PP2A inhibition and cytotoxicity assays. To secure result credibility, we used certified OAs, reference materials, and PP2A produced using genetic engineering. The relative ratio of the OA equivalents determined by PP2A inhibition assays for OA, DTX1, and DTX2 were 1.0:1.6:0.3, while the ratio determined using the cytotoxicity assays indicated 1.0:1.5:0.5. OA equivalents showed a similar tendency in the PP2A inhibition and cytotoxicity assays, and matched better with oral toxicity data than intraperitoneal toxicity in mice. The PP2A inhibition assay, which measures the core activity of the OAs, suggested a higher OA equivalent for DTX1 than that currently used.

A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels

Toxicon ◽  
1996 ◽  
Vol 34 (7) ◽  
pp. 743-752 ◽  
Author(s):  
Aurelia Tubaro ◽  
Chiara Florio ◽  
Elena Luxich ◽  
Silvio Sosa ◽  
Roberto Della Loggia ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Inhibition Assay ◽  
Phosphatase 2A

scholarly journals Evaluation of protein phosphatase 2A (PP2A) inhibition assay for rapid detection of DSP toxins in scallop

2017 ◽  
Vol 83 (3) ◽  
pp. 367-372 ◽  
Author(s):  
TSUYOSHI IKEHARA ◽  
TSUBASA KINOSHITA ◽  
AYAKA KUROKAWA ◽  
SHIHOKO NAKASHIMA ◽  
KIMIHIKO MAEKAWA ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Rapid Detection ◽  
Protein Phosphatase 2A ◽  
Inhibition Assay ◽  
Phosphatase 2A ◽  
Dsp Toxins

A protein phosphatase 2A (PP2A) inhibition assay using a recombinant enzyme for rapid detection of microcystins

Toxicon ◽  
2008 ◽  
Vol 51 (8) ◽  
pp. 1368-1373 ◽  
Author(s):  
Tsuyoshi Ikehara ◽  
Shihoko Imamura ◽  
Naomasa Oshiro ◽  
Satsuki Ikehara ◽  
Fukiko Shinjo ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Rapid Detection ◽  
Protein Phosphatase 2A ◽  
Recombinant Enzyme ◽  
Inhibition Assay ◽  
Phosphatase 2A

First Detection and Seasonal Variation of Lipophilic Toxins Okadaic Acid, Dinophysistoxin-1, and Yessotoxin in Korean Gastropods

2012 ◽  
Vol 75 (11) ◽  
pp. 2000-2006 ◽  
Author(s):  
KA JEONG LEE ◽  
JONG SOO MOK ◽  
KI CHEOL SONG ◽  
HONGSIK YU ◽  
DOO SEOG LEE ◽  
...  
Keyword(s):  
Seasonal Variation ◽  
Okadaic Acid ◽  
Public Health Problem ◽  
Shellfish Poisoning ◽  
Diarrhetic Shellfish Poisoning ◽  
Marine Animals ◽  
Liquid Chromatography Tandem Mass ◽  
Lipophilic Toxins ◽  
Digestive Glands ◽  
Pectenotoxin 2

Okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2, and yessotoxin (YTX) are classes of lipophilic toxins found in marine animals. OA and DTX1 accumulation causes diarrhetic shellfish poisoning, a worldwide public health problem. Diarrhetic shellfish poisoning has not previously been reported in gastropods, which are widely consumed in Korea. Seasonal variation in marine lipophilic toxins in gastropods was investigated using liquid chromatography–tandem mass spectrometry. Eighty specimens of Neptunea cumingii, 65 specimens of Rapana venosa, and 95 specimens of Batillus cornutus were collected at the Tongyeong fish market on the southern coast of Korea between May 2009 and December 2010. OA, DTX1, and YTX were detected in meat and digestive glands in all gastropod species studied. Pectenotoxin-2 was not found in any sample tested. Lipophilic toxins were detected in the digestive glands of gastropods; no lipophilic toxin was detected in the salivary glands of the carnivorous gastropods, N. cumingii and R. venosa. The highest concentrations of OA (21.5 ng/g) and DTX1 (8.4 ng/g) were detected in the digestive glands of R. venosa, and the maximum concentration of YTX (13.7 ng/g) was found in the digestive glands of N. cumingii. The maximum toxicities in gastropod tissues were lower than the European standard for acceptable levels. The concentrations of lipophilic toxins in carnivorous gastropods showed a high degree of seasonal variation; lipophilic toxins in carnivorous gastropods were found predominantly in spring and summer. This is the first report of the occurrence of lipophilic toxins in Korean gastropods.

Stimulatory phosphorylation of cAMP-specific PDE4D5 by contractile agonists is mediated by PKC-dependent inactivation of protein phosphatase 2A

2008 ◽  
Vol 294 (1) ◽  
pp. G327-G335 ◽  
Author(s):  
Karnam S. Murthy ◽  
Wimolpak Sriwai
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Dependent Inactivation ◽  
Rhythmic Cycles ◽  
Phosphatase 2A ◽  
Contraction And Relaxation ◽  
Camp Levels

Smooth muscle of the gut undergoes rhythmic cycles of contraction and relaxation. Various constituents in the pathways that mediate muscle contraction could act to cross-regulate cAMP or cGMP levels and terminate subsequent relaxation. We have previously shown that cAMP levels are regulated by PKA-mediated phosphorylation of cAMP-specific phosphodiesterase 3A (PDE3A) and PDE4D5; the latter is the only PDE4D isoform expressed in smooth muscle. In the present study we have elucidated a mechanism whereby cholecystokinin (CCK) and, presumably, other contractile agonists capable of activating PKC can cross-regulate cAMP levels. Forskolin stimulated PDE4D5 phosphorylation and PDE4D5 activity. CCK significantly increased forskolin-stimulated PDE4D5 phosphorylation and activity and attenuated forskolin-stimulated cAMP levels. The effect of CCK on forskolin-induced PDE4D5 phosphorylation and activity and on cAMP levels was blocked by the inhibitors of PLC or PKC and in cultured muscle cells by the expression of Gαq minigene. The effects of CCK on PDE4D5 phosphorylation, PDE4D5 activity, and cAMP levels were mimicked by low (1 nM) concentrations of okadaic acid, but not by a low (10 nM) concentration of tautomycin, suggesting involvement of PP2A. Purified catalytic subunit of PP2A but not PP1 dephosphorylated PDE4D5 in vitro. Coimmunoprecipitation studies demonstrated association of PDE4D5 with PP2A and the association was decreased by the activation of PKC. In conclusion, cAMP levels are cross-regulated by contractile agonists via a mechanism that involves PLC-β-dependent, PKC-mediated inhibition of PP2A activity that leads to increase in PDE4D5 phosphorylation and activity and inhibition of cAMP levels.

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