Microcystins variants

In order to demonstrate the viability of the approach, protein phosphatase inhibition was first performed with the enzyme in solution and detected by colorimetric methods. Two microcystin variants, microcystin-LR and microcystin-RR, were used. Both enzymes were inhibited by these toxins, although to a different extent. The 50% inhibition coefficients (IC50) towards microcystin-LR were 0.50 and 1.40 pgL 1 (concentrations in the microtitre well) for the Upstate and the GTP enzymes, respectively. Hence, the Upstate enzyme was more sensitive. The IC50 towards microcystin-RR were 0.95 and 2.15 pgL-1 for the Upstate and the GTP enzymes, respectively. As expected, microcystin-LR was demonstrated to be a more potent inhibitor. 

Shepard et al. [167] also reported the use of TiC>2 photocatalysis for the degradation of three microcystin variants, -LR, -YR and -YA. Each of these toxins rapidly decomposed on photo catalysis and a half fife of less than 5 min was reported for each microcystin. The effect of pH was found to strongly influence the destruction of a series of microcystins including LR, RR, LW and... 

These findings show that TiC>2 photo catalysis is a very effective method for the removal of these potentially hazardous compounds from drinking water. When considering large-scale application of this process for removal of mi-crocystins from potable waters, optimisation of reaction conditions must be performed with a range of representative microcystin variants. 

One of the invariant amino acids is a unique P-amino acid called Adda (2S,3S,8S,9S)-3 amino-9 methoxy-2,6,8-trimethyl-10-phei5fldeca-4,6-dienoic acid is the most unusual structure in this group of cyanobacterial cyclic peptide toxins). A two-letter suffix (XY) is ascribed to each individual toxin to denote the two variant amino acids (Carmichael 1988). X is commonly leucine, arginine, or tyrosine. Y is arginine, alanine, or methionine. Variants of all the invarianf amino acids have now been reported, e.g., desmethyl amino acids and/or replacement of the 9-methoxy group of Adda by an acetyl moiety. Currently there are in excess of 60 variants of microcystin that have been characterized (Rinehart 1994 Sivonen and Jones 1999). Of these 60 compounds, microcystin-LR would appear to be the microcystin most commonly found in cyanobacteria. It is also common for more than one microcystin to be found in a particular strain of cyanobacterium (Namikoshi 1992 Lawton 1995). The microcystin variants may also differ in toxicity (Carmichael 1993). The literature indicates that hepatotoxic blooms ofM aeruginosa containing microcystins occur commonly worldwide. 

To improve the specific response against microcystin immimoconjugates, synthetic lipopeptides were used as adjuvants and were found to invoke a greater immune response than the use of classical adjuvants such as Freunds adjuvant. However, an ELISA for the detection of free microcystin was not developed using these antibodies. Cross-reactivities of various microcystin variants and nodn-larin with monoclonal antibodies have been found affecting to the specificity of these antibodies for the recognition of the mentioned toxins. The number of purified microcystin variants that have been tested by ELISA using monoclonal antibodies shows marked differences between methods. 

Biochemicals, Nottingham, U.K.). All are advocated by the manufacturers for the analysis of micro-cystins in water, and several microcystin variants have been tested using these systems. 

Antibodies produced against a synthetic 6E-Adda moiety were also used to develop an ELISA method, with good detection limits and good immunocross-reactivities for the microcystin variants tested (LR, RR YR LW LF desmethylRR, nodularin, microcystin desmethyl-LR (Eischer 2001). 

Studies since, on four microcystin variants, have shown large differences in the adsorption of the variants [71, 77, 78]. Surprisingly, the largest and the most hydrophihc of the toxins studied, mRR with two arginine groups in the variable positions, showed the highest adsorption on a range of PACs, contrary... 

The adsorption of microcystins has been shown to be strongly affected by NOM [78]. In this work the effect, for four microcystin variants, was shown to be a function of the DOC concentration. This is prohahly a result of the direct competitive effect, where the competitive NOM, those compounds approximately the same molecular weight as the microcystins, makes up the hulk of the NOM. Therefore the bulk characterization parameter, DOC, gives an indication of the concentration of competing compounds, where for MIB and geosmin it could not [27, 63, 69]. 

Cook, D. and Newcombe, G. (2002). Removal of microcystin variants with powdered activated achon. Water Sci. Technol. .Water Supply, 2(5-6), 201-8. 

Adda) in position 5 followed by D-glutamic acid (D-Glu) y-linked to N-methyl-dehydroalanine (Mdha) (50-52). The main stmctural variations of the more than 50 variants isolated so far, are related to mutations at the two L-amino acids labeled with X and Y in Fig. 4 (53) correspondingly, microcystin variants are named according to the residues X and Y (54). Among all the variants identified so far the most abundant and, correspondingly, the most studied is the microcystin-LR. 

Since a large number of microcystins has been already isolated and chemically characterized, mutations and modifications in the structure have allowed the identification of essential pharmacophores. The variable L-amino acid residues X and Y are most commonly Leu and Arg, respectively. But microcystin variants with both positions occupied by the basic Arg residue or by hydrophobic residues like Leu and Ala or Leu and Tyr are also produced by various Microcystis strains. While substitution of the Arg residue in position Y with hydrophobic residues or with Met(O) is without significant effect on the toxicity, replacement of the Leu residue in position X with Arg or Met(O) leads to significantly reduced toxicity (53). Variants with... 

Another variant of PP2A assay is the one reported by Isobe et al. [166] where a firefly bioluminescence system is used for the detection of protein phosphatase 2A inhibitors, in which luciferin phosphate is hydrolyzed to luciferin and inorganic phosphate by protein phosphatase 2A. The recent commercial availability of the phosphatase enzymes, which obviates the need to isolate them from animal tissues, also makes this approach very attractive. However, not all microcystins variants react with protein phosphatase enzymes to a similar extent [161,163] and the assay is sensitive to protein phosphatase inhibitors other than microcystins, such as okadaic acid, tautomycin, and calyculin A. In addition, the cyanobacterial sample itself may contain phosphatase activity that masks the presence of toxins [160]. As a consequence, the lack of specificity of the protein phosphatase inhibition assays requires that additional confirmatory analytical methods be employed for specific analysis of cyanobacterial toxins. 

There have been some attempts to enhance sensitivity using ninhydrin derivatization of the guanidine moiety on the arginine residue with fluorescence detection [183]. However, the inability to detect microcystin variants, which do not contain arginine, is a major disadvantage for this approach. Another attempt to increase the sensitivity has been the derivatization of the diene on the Adda moiety using DMEQ-TAD (4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl) ethyl] -... 

Microcystin variant (MC-XZ) Variable amino acid X Variable amino acid Z Cyanobacterial species Aqneons samples Different environmental samples Animal tissne samples Blue-green alga biomass samples ... 

Most of the available toxicological data on microcystins have been based on microcystin-LR. Other microcystin variants appear to be similar to microcystin-LR in their toxicological effects, but they differ in potency. Other factors that need to be taken into account when interpreting available data are that much of the data have been derived from experiments in rodents and rabbits using intraperitoneal injection as the route of exposure, and the extracts used in studies may consist of complex, crude mixtures of components derived from toxic algal blooms, or more or less purified and characterized components (Zhao et al., 2008, 2009). Extrapolation between routes of exposure and between the toxicities of different complex mixtures can make the comparison of data from different studies challenging. 

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