Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
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(10 8 bacterias ml -1 ) and used to <strong>de</strong>termine the response of the phagocytes to the fish<br />
pathog<strong>en</strong>. As a positive control phorbol myristate acetate (PMA, Sigma) (1 μg ml -1 ) was<br />
used to stimu<strong>la</strong>te the respiratory burst activity of non-infected phagocytes (data not<br />
shown). The specificity of the reaction was tested by adding superoxi<strong>de</strong> dismutase<br />
(SOD) (300 I.U. per well) to some wells containing PMA-stimu<strong>la</strong>ted phagocytes (data<br />
not shown). After incubation, cells were fixed in 70% methanol and the reduced<br />
formazan within phagocytes was solubilised by adding 120 μl 2M KOH and 140 μl<br />
dimethyl sulfoxi<strong>de</strong> (DMSO, Sigma). Finally, absorbance was read at 630 nm in a<br />
multiscan spectrophotometer (UV-1601 Spectrophotometer, Whitakker Bioproducts).<br />
In the P. cru<strong>en</strong>tum feeding experim<strong>en</strong>ts, the effect of oral administration of the<br />
alga or Sanostim was <strong>de</strong>termined on phagocyte mono<strong>la</strong>yers as <strong>de</strong>scribed above, but<br />
algal extracts or bacteria were not ad<strong>de</strong>d to the wells.<br />
2.8. Statistical analysis<br />
Results are expressed as the stimu<strong>la</strong>tion in<strong>de</strong>x (mean + standard error, SE),<br />
obtained by dividing each sample value by the mean control value. Values higher than 1<br />
reflect an increase and lower than 1 a <strong>de</strong>crease in each parameter compared to the<br />
control. Data were statistically analysed by one-way analysis of variance (ANOVA) and<br />
Tukey’s comparison of means using SPSS for Windows. Differ<strong>en</strong>ces were consi<strong>de</strong>red<br />
statistically significant wh<strong>en</strong> P< 0.05.<br />
3. Results<br />
3.1. In vitro assays<br />
Results have be<strong>en</strong> expressed as the ratio of the absorbance at 630 nm of treated<br />
phagocytes to the absorbance of non-treated phagocytes. The treatm<strong>en</strong>t of phagocytes<br />
consisted of incubation with aqueous (Figure 1a) or ethanolic (Figure 1b) algal extracts<br />
or β-glucan (Figure 1c), in the pres<strong>en</strong>ce or abs<strong>en</strong>ce of P. damse<strong>la</strong>e subsp. piscicida.<br />
Therefore the data repres<strong>en</strong>ted in the Figures are the result of a corre<strong>la</strong>tion betwe<strong>en</strong><br />
phagocytes incubated with the algal extract or β-glucan, with or without bacteria, and<br />
phagocytes incubated without the pot<strong>en</strong>tial immunostimu<strong>la</strong>nt (algal extract or β-glucan).<br />
8