Estimation of the microcystin content in cyanobacterial field samples from German lakes using the colorimetric protein-phosphatase inhibition assay and RP-HPLC

1999 ◽  
Vol 14 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Birgit Wirsing ◽  
Thomas Flury ◽  
Claudia Wiedner ◽  
Uwe Neumann ◽  
Jürgen Weckesser
Keyword(s):  
Protein Phosphatase ◽  
Inhibition Assay ◽  
Rp Hplc ◽  
Field Samples ◽  
Protein Phosphatase Inhibition ◽  
Phosphatase Inhibition

scholarly journals Detection of Toxigenicity by a Probe for the Microcystin Synthetase A Gene (mcyA) of the Cyanobacterial Genus Microcystis: Comparison of Toxicities with 16S rRNA and Phycocyanin Operon (Phycocyanin Intergenic Spacer) Phylogenies

2001 ◽  
Vol 67 (6) ◽  
pp. 2810-2818 ◽  
Author(s):  
Daniel Tillett ◽  
Dorothy L. Parker ◽  
Brett A. Neilan
Keyword(s):  
16S Rrna ◽  
Protein Phosphatase ◽  
Intergenic Spacer ◽  
Rrna Gene ◽  
Monophyletic Cluster ◽  
Microcystin Synthetase ◽  
Field Samples ◽  
Protein Phosphatase Inhibition ◽  
Phosphatase Inhibition ◽  
Cyanobacterial Genus

ABSTRACT The relationship between toxigenicity and phylogeny within the cyanobacterial genus Microcystis is unclear. To investigate this issue, we have designed PCR primers for theN-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA and have probed 37Microcystis sp. cultures as well as several field samples. The NMT region was present in all 18 laboratory strains that gave positive reactions in the protein phosphatase inhibition assay for microcystin but was absent in 17 nontoxic strains. Two other nontoxic strains, one of which had previously been reported to produce microcystin, possessed the NMT region. Detection of NMT-specific DNA in field samples corresponded to periods of toxicity as assessed by protein phosphatase inhibition. The Microcystis strains formed a monophyletic cluster based on 16S rRNA gene sequences but comprised two groups with respect to phycocyanin intergenic spacer (PC-IGS) sequences. Toxic and nontoxic strains appeared to be erratically distributed within the PC-IGS and 16S rRNA trees. Sequence analysis of the NMT domain revealed two coherent groups. The genomic region immediately downstream of the mcyABC cluster in all 20 NMT-positive strains contained an open reading frame of unknown function (uma1) at a conserved distance frommcyC. All nontoxic strains also containeduma1, which is not cotranscribed withmcyABC. The consistent linkage of mcyC touma1 suggests that mcyC has not been frequently transferred into nontoxic strains via any mechanism involving insertion at random chromosomal locations. These results are discussed with respect to various mechanisms that could explain the patchy distribution of toxigenicity among the variousMicrocystis clades.

scholarly journals Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases

Marine Drugs ◽  
2016 ◽  
Vol 14 (3) ◽  
pp. 54 ◽  
Author(s):  
Caroline Moore ◽  
Jeanette Juan ◽  
Yanping Lin ◽  
Cynthia Gaskill ◽  
Birgit Puschner
Keyword(s):  
Protein Phosphatase ◽  
Inhibition Assay ◽  
Protein Phosphatase Inhibition ◽  
Phosphatase Inhibition

scholarly journals Colorimetric Immuno-Protein Phosphatase Inhibition Assay for Specific Detection of Microcystins and Nodularins of Cyanobacteria

2001 ◽  
Vol 67 (2) ◽  
pp. 904-909 ◽  
Author(s):  
James S. Metcalf ◽  
Steven G. Bell ◽  
Geoffrey A. Codd
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Cyclic Peptide ◽  
Calyculin A ◽  
Inhibition Assay ◽  
Specific Detection ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibition ◽  
Phosphatase Inhibition

ABSTRACT A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and nodularin detection and analysis do not provide information on the toxicity of microcystin and/or nodularin variants. Furthermore, protein phosphatase inhibition-based assays for these toxins are not specific and respond to other environmental protein phosphatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We addressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a colorimetric protein phosphatase inhibition system in a single assay, designated the colorimetric immuno-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin variants (microcystin-LR, -D-Asp3-RR, -LA, -LF, -LY, -LW, and -YR) and nodularin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and nodularin-containing laboratory strains and environmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (R 2 = 0.94, P< 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA procedure combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and nodularins.

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