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.

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.

Propofol Inhibits Phosphorylation of N -methyl-d-aspartate Receptor NR1 Subunits in Neurons

2006 ◽  
Vol 104 (4) ◽  
pp. 763-769 ◽  
Author(s):  
Seth Kingston ◽  
Limin Mao ◽  
Lu Yang ◽  
Anish Arora ◽  
Eugene E. Fibuch ◽  
...  
Keyword(s):  
Glutamate Receptors ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Cortical Neurons ◽  
Protein Phosphatase 2A ◽  
Ionotropic Glutamate Receptors ◽  
Dependent Manner ◽  
Calyculin A ◽  
General Anesthetic ◽  
Phosphatase 2A

Background Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-D-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation. Methods The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons. Results Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist-induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits. Conclusions Together, these data indicate an inhibitory effect of a general anesthetic propofol on NMDAR NR1 subunit phosphorylation in neurons. This inhibition was mediated through a signaling mechanism involving activation of protein phosphatase 2A.

Protein Phosphatase 2A Inactivates Bcl2’s Antiapoptotic Function by Dephosphorylation and Up-Regulation of Bcl2-p53 Binding.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1436-1436
Author(s):  
Xingming Deng ◽  
Fengqin Gao ◽  
Tammy Flagg ◽  
W. Stratford May
Keyword(s):  
Cell Death ◽  
Cell Survival ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Apoptotic Cell ◽  
Protein Phosphatase 2A ◽  
Survival Function ◽  
Phosphorylation Site ◽  
Apoptotic Cell Death ◽  
Phosphatase 2A

Abstract DNA damage-induced p53/Bcl2 interaction at the outer mitochondrial membranes results in a Bcl2 conformational change and loss of its antiapoptotic function. Our data now indicate that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor, okadaic acid (10 nM), or specific disruption of PP2A activity by the expression of SV40 small tumor antigen enhances Bcl2 phosphorylation and suppresses the cisplatin-stimulated Bcl2-p53 interaction in association with prolonged cell survival. By contrast, C2-ceramide, a potent PP2A activator, reduces Bcl2 phosphorylation and increases Bcl2-p53 binding and promotes apoptotic cell death, suggesting that PP2A may function as a physiological regulator of Bcl2 by, at least in part, affecting its association with p53. Overexpression of the PP2A catalytic subunit (PP2A/C) suppresses Bcl2 phosphorylation in association with increased p53-Bcl2 binding and apoptotic cell death. By contrast, specific depletion of PP2A/C by RNA interference enhances Bcl2 phosphorylation, suppresses p53-Bcl2 interaction and prolongs cell survival. Purified PP2A can directly enhance the formation of the p53-Bcl2 complex in vitro in an okadaic acid-sensitive manner, supporting a direct mechanism. Importantly, PP2A directly interacts with Bcl2 at its BH4 domain which may function as the PP2A ‘docking site’ to potentially ‘bridge’ PP2A to the flexible loop domain which contains the physiological serine 70 phosphorylation site. Thus, PP2A may provide a double whammy to Bcl2’s survival function by both dephosphorylating and enhancing p53-Bcl2 binding. Therapeutically stimulating Bcl2 dephosphorylation and/or increasing Bcl2/p53 binding by activating PP2A may represent an efficient and novel antineoplastic approach.

scholarly journals A protein phosphatase 2A from Fagus sylvatica is regulated by GA3and okadaic acid in seeds and related to the transition from dormancy to germination

2006 ◽  
Vol 128 (1) ◽  
pp. 153-162
Author(s):  
Mary Paz González-García ◽  
Dolores Rodríguez ◽  
Carlos Nicolás ◽  
Gregorio Nicolás ◽  
Oscar Lorenzo
Keyword(s):  
Fagus Sylvatica ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Phosphatase 2A

scholarly journals Acute regulation of renal Na+/H+ exchanger NHE3 by dopamine: role of protein phosphatase 2A

2010 ◽  
Vol 298 (5) ◽  
pp. F1205-F1213 ◽  
Author(s):  
I. Alexandru Bobulescu ◽  
Henry Quiñones ◽  
Serge M. Gisler ◽  
Francesca Di Sole ◽  
Ming-Chang Hu ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Simian Virus 40 ◽  
Receptor Activation ◽  
T Antigen ◽  
Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56δ (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, coimmunoprecipitation, blot overlay, and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small T antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition.

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