Comparison of statistical cluster methods in electrophoretic protein pattern analysis.

Standard electrophoresis methods were used in the qualitative and quantitative protein analyses of cerebrospinal fluid (CSF). Disc electrophoresis was carried out for detection of oligoclonal IgG bands in cerebrospinal fluid on polyacrylamide gel. Pairs of CSF and serum were taken from 30 patients, mainly with multiple sclerosis and other central nervous system dysfunctions, polyradiculoneuritis, known as Guillain-Barre syndrome, encephalitis, paraproteinemia, and analyzed. ImageMaster 1D Elite and GelPro specialized software packages were used for fast accurate image and gel analysis. The results obtained from different hierarchic cluster analysis methods were compared. In some cases, despite substantial similarities between electropherograms, different cluster methods produced different dendrograms. Therefore, the cluster analysis should be used cautiously. It offers only additional diagnostic information on the inflammatory conditions of the central nervous system.

Bioluminescence determination of enzyme activity of firefly luciferase in the presence of pesticides.

Firefly luciferase (EC 1.13.12.5) (FL) is the key enzyme in the firefly bioluminescence method (FB), which is widely used to determine the viability of living cells. The FB method can also be applied to monitoring the influence of different pollutants, such as pesticides. Firefly luciferase is a hydrophobic enzyme and its activity depends on the type of solvent, pH and substances present in the reaction mixture. The influence of three aromatic pesticides, including fenoxaprop-p-ethyl (I), diclofop-methyl (II) and metsulfuron methyl (III), on the enzyme activity was indirectly evaluated through the measurement of emitted light in the bioluminescence reaction, expressed in relative luminescence units (RLU). The reaction mixture used in the bioluminescence measurements consisted of: Tris buffer (pH 7.75), adenosine triphosphate (ATP) and ATP monitoring reagent, where FL is present. Ethanol-water solutions of each pesticide were then added at concentrations of 2.4 x 10(-4)-2.4 x 10(-8) mol/L. The FL activity inhibition factors (FL In%) were determined. The FL activity was maximally inhibited in the presence of all pesticides under study at a concentration of 2.4 x 10(-4) mol/L and was lowered by about 15-26% for pesticide I at concentrations of 2.4 x 10(-5)-2.4 x 10(-8) mol/L, whereas pesticides II and III, applied in the same concentration range, showed smaller FL inhibition values (5.3-20%). The pesticide degradation products (obtained after a 1 month period), measured in the same experimental conditions, in most cases exhibited a much less inhibitory effect on the enzyme activity than the corresponding initial pesticide.