Generation of reactive oxygen species in different fractions of the coelomocytes of holothurian Eupentacta fraudatrix in response to the thermostable toxin of Yersinia pseudotuberculosis in vitro |
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| Authors: | Dolmatova L S Eliseykina M G Timchenko N F Kovaleva A L and Shitkova O A |
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| Institution: | (1) V. I. Il'ichev Pacific Oceanological Institute, 690041 Vladivostok, Russia;(2) Institute of Marine Biology, 690041 Vladivostok, Russia;(3) Research Institute of Epidemiology and Microbiology, 690087 Vladivostok, Russia |
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| Abstract: | Pure fraction (92%–95%) of phagocytes (FP) and a mixture of amoebocytes (62%) and morula cells (38%)-FPMC- of the holothurianEupentacta fraudatrix' (Holothuroidea, Dendrochirota) were obtained by using ficoll-verographine step gradient. Basal production of reactive oxygen species (ROS) in FP quantified
by using reduction of nitroblue tetrazolium (NBT) was more than twice that in FPMC. Thermostable toxin ofYersinia pseudotuberculosis (TST) at different concentrations (0.2; 0.5; 2.5 μg/ml, but not 0.1 μg/ml) stimulated NBT reduction in FPMC after 24 h incubation.
In FP, TST at concentrations of 0.1 and 0.2 μg/ml inhibited and at concentrations of 0.5 and 2.5 μg/ml stimulated NBT reduction
after 24 h incubation. Maximal effect was observed in FP and FPMC at TST concentrations of 0.5 and 0.2 μg/ml, respectively.
Addition of catalase (0.7 μg/ml) to the cells treated with TST (2.5 μg/ml) was followed by a decrease in NBT reduction compared
to that under toxin treatment alone. TST stimulated superoxide dismutase activity in concentration-dependent manner (maximum
at 0.5 μg/ml concentration in FP) after 24 h treatment, and this stimulation was prevented by a commercial catalase. Plant
lectin concanavalin A stimulated NBT reduction more than 5-fold in FPMC compared to the control. With addition of TST, lectin
stimulated ROS to lesser extent than that with lectin alone. When catalase, TST, and lectin were added into the FPMC simultaneously,
ROS increase was similar to that under lectin treatment alone. On the whole, data obtained indicated that ROS generation in
holothurian coelomocytes especially occurs in both stimulated and not stimulated phagocytes, and that changes in ROS production
by these cells may be one of the mechanisms of antibacterial protection of holothurians.
This work was financially supported by the Russian Foundation for Fundamental Research Grant (No. 00-04-48949). |
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| Keywords: | bacterial toxin coelomocytes holothurian reactive oxygen species superoxide dismutase |
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