Chaivimol, Jittra (1995) The study of peptide toxins from freshwater cyanobacteria. PhD thesis, University of Warwick.

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Abstract

The freshwater reservoirs in the English Midlands were monitored during 1991 to 1993 for the diversity of cyanobacterial species and whether or not any of the species present produced toxins, emphasis being focused upon the toxic hepatopeptides. An HPLC analysis protocol has been developed which gives satisfactory resolution, both quantitative and qualitative, of "presumptive" cyanobacterial peptide toxins. Microcystin-LR, microcystin-RR and nodularin were used as the reference peptide toxins with detection limits of 50-100 ng under the employed conditions. Microcystis aeruginosa PCC 7806, a characterised hepatopeptide toxin producer, was taken as the reference toxic cell biomass.

The species isolated and characterised included Oscillatoria, Aphanizomenon, Anabaena, Microcystis, Nodularia and Nostoc. The Oscillatoria population present in Lower Shustoke reservoir were proved by mouse bioassay to be toxic throughout the period of evaluation. The lethal dose of cell lysate (LD₁₀₀) varied from 20 mg to 80 mg per kg body weight after intraperitoneal injection into mice. The toxic peptides from this strain were isolated and characterised by high performance liquid chromatography. There were three or four peptide toxins being expressed with spectral characteristics very close to those of the microcystins and nodularin.

Molecular weight and linearised fingerprints of reference peptide toxins were successfully analysed by FAB-MS using a Kratos MS50 and a Kratos Concept II HH four sector. The relative molecular masses of presumptive toxins were determined by matrix assisted laser desorption ionisation (MALDI) mass spectrometry to be 1023, 1022, 1043 and 981 Da.

The temporal variation of cellular expression of these presumptive toxins was monitored. There was considerable variation in the toxicity of the cellular biomass throughout the year and a corresponding variation in the cellular toxin content. The overall biomass toxicity was dependant on the ratio of the component toxins within the individual cells.

The variability in toxin expression/structure appeared to be linked to environmental factors. In this study, the concentration of hepatotoxins produced by the Oscillatoria species positively correlated with surface water temperature, cyanobacterial cell count, as well as concentration of ortho-phosphate and total phosphate while there was a negative correlation with total nitrate concentration. However, alteration in gene expression and/or the physiological status of the cells was not addressed.

Several alternatives to mouse bioassay have been examined for the determination of cyanobacterial toxicity. The protein phosphatase assay is one of the most promising procedures since phosphorylase phosphatase activity was strongly inhibited by the reference toxins when assayed against phosphorylase a substrate and crude mouse liver homogenate as sources of enzymes. Microcystin-LR is about 10 times more toxic than microcystin-RR when injected intraperitoneally into mice. However, they both have approximately the same IC₅₀ (50% inhibition of phosphorylase phosphatase activity). This difference may be due to either permeability effects or differential activation of the two microcystins in vivo. Drosophila melanogaster bioassay was shown to be a useful indicator of toxicity.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history > QH301 Biology Q Science > QP Physiology Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Peptides -- Analysis, Toxins, Cyanobacteria, Freshwater biology
Official Date:	March 1995
Dates:	Date Event March 1995 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Dow, Crawford S.
Sponsors:	Ministry of Science, Technology and Environment, Thailand
Extent:	xxiv, 291 leaves : illustrations
Language:	eng

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