Highly Toxic Microcystis aeruginosa Strain, Isolated from So Paulo-Brazil, Produce Hepatotoxins and Paralytic Shellfish Poison Neurotoxins

While evaluating several laboratory-cultured cyanobacteria strains for the presence of paralytic shellfish poison neurotoxins, the hydrophilic extract of Microcystis aeruginosa strain SPC777-isolated from Billings`s reservoir, So Paulo, Brazil-was found to exhibit lethal neurotoxic effect in mouse bioassay. The in vivo test showed symptoms that unambiguously were those produced by PSP. In order to identify the presence of neurotoxins, cells were lyophilized, and the extracts were analyzed by HPLC-FLD and HPLC-MS. HPLC-FLD analysis revealed four main Gonyautoxins: GTX4(47.6%), GTX2(29.5%), GTX1(21.9%), and GTX3(1.0%). HPLC-MS analysis, on other hand, confirmed both epimers, with positive Zwitterions M(+) 395.9 m/z for GTX3/GTX2 and M(+) 411 m/z for GTX4/GTX1 epimers. The hepatotoxins (Microcystins) were also evaluated by ELISA and HPLC-MS analyses. Positive immunoreaction was observed by ELISA assay. Alongside, the HPLC-MS analyses revealed the presence of [l-ser(7)] MCYST-RR. The N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA was chosen as the target sequence to detect the presence of the mcy gene cluster. PCR amplification of the NMT domain, using the genomic DNA of the SPC777 strain and the MSF/MSR primer set, resulted in the expected 1,369 bp product. The phylogenetic analyses grouped the NMT sequence with the NMT sequences of other known Microcystis with high bootstrap support. The taxonomical position of M. aeruginosa SPC777 was confirmed by a detailed morphological description and a phylogenetic analysis of 16S rRNA gene sequence. Therefore, co-production of PSP neurotoxins and microcystins by an isolated M. aeruginosa strain is hereby reported for the first time.

Synthesis of Cyclic Guanidine Intermediates of Anatoxin-a(s) in Both Racemic and Enantiomerically Pure Forms

The alkyl chain of anatoxin-a(s) (cyclic guanidines), which can be used as an intermediate in the total synthesis of anatoxin-a(s), was synthesized in both racemic and enantiomerically pure forms. These enantiomerically pure cyclic compounds can be used as chiral inductors in some reactions. The two racemic routes disclosed herein have the advantages of high overall yield and mild reaction conditions. Both routes proceed through an intermediate 2,3-diaminoacid - an important synthetic scaffold - with good yields. Furthermore, the N,N-dimethyl-2(tosylimino)imidazolidine-4-carboxamide might be obtained from 2-(tosylimino)imidazolidine-4-carboxylic acid followed by selective reduction of the carbonyl functionality. All synthesized compounds were analyzed by mass spectrometry and (1)H NMR and (13)C NMR spectroscopy.

Methods for detection of anatoxin-a(s) by liquid chromatography coupled to electrospray ionization-tandem mass spectrometry

Anatoxin-a(s) is a potent irreversible inhibitor of the enzyme acetylcholinesterase with a unique N-hydroxyguanidine methylphosphate ester chemical structure. Determination of this toxin in environmental samples is hampered by the lack of specific methods for its detection. Using the toxic strain of Anabaena lemmermani PH-160 B as positive control, the fragmentation characteristics of anatoxin-a(s) under collision-induced dissociation conditions have been investigated and new LC-MS/MS methods proposed. Recommended ion transitions for correct detection of this toxin are 253 > 58, 253 > 159, 235 > 98 and 235 > 96. Chromatographic separation is better achieved under HILIC conditions employing a ZIC-HILIC column. This method was used to confirm for the first time the production of anatoxin-a(s) by strains of Anabaena oumiana ITEP-025 and ITEP-026. Considering no standard solutions are commercially available, our results will be of significant use for the correct identification of this toxin by LC-MS/MS. (C) 2009 Elsevier Ltd. All rights reserved.