Analysis cyanobacteria

In addition to detection of toxicity in samples containing cyanobacteria and/or their toxins (i.e. screening), quantification and identification of the toxins present are necessary on occasions. Physicochemical methods of toxin analysis fulfil both these roles, often requiring a comparison of the test sample with purified... 

A powerful tool now employed is that of diode array detection (DAD). This function allows peaks detected by UV to be scanned, and provides a spectral profile for each suspected microcystin. Microcystins have characteristic absorption profiles in the wavelength range 200-300 nm, and these can be used as an indication of identity without the concomitant use of purified microcystin standards for all variants. A HPLC-DAD analytical method has also been devised for measurement of intracellular and extracellular microcystins in water samples containing cyanobacteria. This method involves filtration of the cyanobacteria from the water sample. The cyanobacterial cells present on the filter are extracted with methanol and analysed by HPLC. The filtered water is subjected to solid-phase clean-up using C g cartridges, before elution with methanol and then HPLC analysis. 

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

In contrast, SIMCA uses principal components analysis to model object classes in the reduced number of dimensions. It calculates multidimensional boxes of varying size and shape to represent the class categories. Unknown samples are classified according to their Euclidean space proximity to the nearest multidimensional box. Kansiz et al. used both KNN and SIMCA for classification of cyanobacteria based on Fourier transform infrared spectroscopy (FTIR).44... 

O. Schmitz, G. Boison, R. Hilscher, B. Hundeshagen, W. Zimmer, F. Lottspeich, H. Bothe (1995) Molecular biological analysis of a bidirectional hydrogenase from cyanobacteria. European Journal of Biochemistry, 233 266-276... 

The main framework is made up of five key modules for chemical library editing, enumeration, conversion, visualization, and analysis. The operations of these functionalities are accomplished by the various applications at the resource layer. For the purpose of illustration, the compound calothrixin B, a secondary metabolite isolated from the Calothrix cyanobacteria (11-13), is used as the scaffold molecule with the variable functional groups Rw] attached (Fig. 18.1). The calothrixins are redox-active natural products which display potent antimalarial and anticancer properties and thus there is interest in probing the physical as well as biological profiles of their derivatives (14). In this exercise, six functional groups have been selected as the building blocks (Table 18.1). 

C.J. Ward, K.A. Beattie, E.Y.C. Lee and G.A. Codd, Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria comparisons with high-performance liquid chromatography analysis for microcystins, FEMS Microbiol. Lett., 153 (1997) 465-473. 

C. Edwards, L.A. Lawton, K.A. Beattie, G.A. Codd, S. Pleasance and G.J. Dear, Analysis of microcystins from cyanobacteria by liquid... 

Fig. 21.1. Construction of the PP2A inhibition-based biosensor and appbcation to the electrochemical analysis of MC-LR standard solutions and real cyanobacteria cell samples.

Aquatic single cell organisms with a size range of about 0.02-200 pm in diameter occur in nature in concentrations that range from about 102 to 107 per ml. They include viruses, bacteria, cyanobacteria (formerly known as blue-green algae), autotrophic phytoplankton (unicellular plants), and heterotrophic zooplankton (unicellular animals). The analysis of aquatic organisms by flow cytometry presents... 

Harada K, Kondo F, Lawton L (1999) Laboratory analysis of cyanotoxins. In Chorus I, Barthram J (eds) Toxic cyanobacteria in water a guide to their public health consequences, monitoring and management. World Health Organization New York, NY, pp 368 105... 

As to the enzymes of PolyP metabolism in photosynthetic bacteria, polyphosphate kinase activity was revealed in the cyanobacteria Anacystis nidulans (Vaillancourt el al., 1978) and Oscillatoria redekei (Zaiss, 1985). The mutant in this enzyme had no PolyP granules observable by electron microscopy (Vaillancourt el al., 1978). The alignment analysis revealed the genes encoding putative polyphosphate kinase (ppkl and ppk.2) in several genomes of cyanobacteria (Zhang el al., 2002) (see Table 6.1 above). 

Abdel-Basset, R., and Bader, K. P. 1998. Physiological analysis of the hydrogen gas exchange in cyanobacteria. J. Photochem. Photobiol. B Biol. 43, 146-151. 

Boison, G., Bothe, H., and Schmitz, O. 2000. Transcriptional analysis of hydrogenase genes in the cyanobacteria Anacystis nidulans and Anabaena variabilis monitored by RT-PCR. Curr. Microbiol. 40 315-321. 

A combination of PPIA and microcystin immunoassay was proposed by Carmichael et al. (1999) to indicate the potential toxicity of a bloom sample and the concentration of the microcystins. A combined assay, consistent with this principle, was developed by Metcalf et al. (2001) this includes preexposure of the sample to microcystin antibodies, to make microcystins/nodularins that are present biounavailable to the subsequent addition of protein phosphatase enzyme, before assaying for protein phosphatase inhibitoiy activity. The resulting assay, termed the colorimetric immunoprotein phosphatase inhibition ass (CIPPIA), was found to be specific for microcystins and nodularins since the microcystin antibodies protect the protein phosphatase from inhibition by the toxins. Complete protection from inhibition of protein phospliatase by the antibodies indicates that the inhibition of the protein phosphatase in the sample was due to the cyanobacterial toxins. These colorimetric assays showed a good correlation with the HPLC analysis of extracts cyanobacteria. Immunoassays can also be combined with physicochemical methods such as HPLC (Zeck 2001b). In this case, the HPLC method separates the microcystins according to their hydrophobicity and the resulting fractions are analyzed by immunoassay. 

An ELISA test using monoclonal antibodies against microcystin-LR has been used by Ueno (1996b) to analyze the microcy stin concentration in environmental samples from ponds, lakes, reservoirs, and rivers in Japan, Thailand, Germany, and Portugal. Although microcystins are mainly associated with freshwater cyanobacteria, these results are relevant to the analysis of microcystins and particularly nodularins in brackish water environments. 

Meriluoto, lA.O., and Eriksson, IE., 1988. Rapid analysis of peptide toxins in cyanobacteria J Chromatog 438 93-99. 

Gehrig H, Schiissler A, Kluge M Geosiphon pyriforme, a fungus forming endocytobiosis with Nostoc (Cyanobacteria), is an ancestral member of the Glomales Evidence by SSU rRNA analysis. J Mol Evol 1996 43 71-81. 

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