JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

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Expression of Two Gelsolins in Response to Heavy-ions
Irradiation and Desiccation in the Sleeping
Chironomid Polypedilum vanderplanki
O. Gusev a) , T. Kikawada a) , T. Sakashita b) , T. Funayama b) , Y. Kobayashi b) and T. Okuda a)
a) Anhydrobiosis Research Unit, National Institute of Agrobiological Sciences,
b) Radiation-Applied Biology Division, QuBS, JAEA
Larvae of the sleeping chironomid Polypedilum
vanderplanki survives prolonged complete desiccation and
1, 2)
high-dose irradiations . We found earlier that both
desiccation and heavy ion irradiation cause severe DNA
damage in cells of the larvae and post-anhydrobiosis and
post-irradiation recovery of nucleic acids takes at least a few
days. During the DNA recovery, cells of the larvae do not
undergo apoptosis, but the mechanism of preventing
apoptosis is still unknown. Gelsolin is one of the potential
inhibitors of apoptosis, and a key protein for cell-motility
processes, but its activity in insects is yet to be studied 3) .
At the present study we have analyzed mRNA expression of
two members of gelsolin family in the larvae subjected to
anhydrobiosis and irradiation.
We used the doses of ion beams which were previously
found to be ID50 (median inhibitory dose) for adult
emergence (70 Gy) of the chironomid 1, 2) . The larvae were
exposed to 50 MeV 4 He (LET∞=16.2 keV/µm) ion beam
delivered from the azimuthally-varying-field cyclotron at
TIARA. Total RNA from hydrated, dehydrating,
rehydrated, and irradiated larvae was extracted using Trizol
(Invitrogen) and the RNeasy Mini Kit (Qiagen, Hilden,
Germany), and reverse transcribed using Ready-To-Go TM
T-Prime First-Strand Kit (GE Healthcare Bio-Sciences,
Piscataway, NJ). The RNA samples from dehydrating and
rehydrating larvae were named “D” and “R”, respectively,
and numbers correspond to the hours of treatment.
Real-time PCR was performed using a LightCycler ® 2.0
Real-Time PCR apparatus (Roche Diagnostics, Basel,
Switzerland) with SYBR ® Green PCR Master Mix (TaKaRa,
Ohtsu, Japan).
Two members of gelsolin family were different in
number of functional gelsolin domains: gelsolin-1 (Fig. 1)
contained three domains and gelsolin-2 (Fig. 2) had six
functional domains. Both gelsolins we up-regulated after
irradiation with 70 Gy ion beams and gelsolin-1 showed two
clear wave of expression during the anhydrobiosis cycle,
while gelsolin-2 was up-regulated only during rehydration
of the larvae. Initial, approximately 10-fold increase of
gelsolin-1 gene expression was observed in the beginning of
dehydration of the larvae (Fig. 1) and then the up-regulation
of the expression was observed during the larvae rehydration
reaching peak (>70 folds compared to control wet larvae, i.e.
0 h of D and IR) at 12 hours of rehydration.
Members of gelsolin family may play important roles in
anhydrobiosis, i.e. first increase of gelsolin-1 expression
during initial stage of dehydration related to trial of the
JAEA-Review 2010-065
organism to neutralize negative effect of molecular
crowding on cell motility in dehydrating larvae and the
second wave of expression of both gelsolins upon
rehydration might also be related to restoration of activity of
muscle proteins and general cell motility in the reviving
larvae. As they occurred after irradiation, we assume that
they are also involved in the suppression of apoptosis upon
irradiation and anhydrobiosis by blocking cytochrome-C
release from mitochondria in a same way as it was found in
mammalian cells 3) .
relative expression
100
80
60
40
20
0
0 8 24 48 1 3 12 24 48
Fig. 1 Relative expression of gelsolin-1 in the larvae of
P. vanderplanki: during desiccating (D) and
following rehydration (R), and in non-desiccated
larvae after irradiation with 70 Gy of 4 He (IR).
relative expression
- 98 -
10
5
0
Fig. 2 Relative expression of gelsolin-2 in the larvae of
P. vanderplanki during desiccating (D) and following
rehydration (R), and in non-desiccated larvae after
irradiation with 70 Gy of 4 He (IR).
References
1) Watanabe et al., Int. J. Radiat. Biol. 82 (2006) 587.
2) Watanabe et al., Int. J. Radiat. Biol. 82 (2006) 835.
3) Silacci et al., Cell. Mol. Life Sci. 61 (2004) 2614.
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