First published online November 17, 2005
Journal of Experimental Biology 208, 4427-4436 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01924
Acute thermal stress and various heavy metals induce tissue-specific pro- or anti-apoptotic events via the p38-MAPK signal transduction pathway in Mytilus galloprovincialis (Lam.)
Erene Kefaloyianni,
Eleni Gourgou,
Vanessa Ferle,
Efstathios Kotsakis,
Catherine Gaitanaki and
Isidoros Beis*
Department of Animal and Human Physiology, School of Biology, Faculty
of Sciences, University of Athens, Panepistimioupolis, Athens 157 84,
Greece
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Fig. 1. Effect of various heavy metals on the p38-MAPK phosphorylation in M.
galloprovincialis mantle tissue. (A) Phospho-p38-MAPK was detected in
extracts (100 µg of protein) from control animals (C) or animals treated
with either 1 µmol l–1 CuCl2 (top panel), 50
µmol l–1 ZnCl2 (middle panel) or 1 µmol
l–1 CdCl2 (bottom panel) for the times indicated.
Western blots shown are representative of four to six independent experiments.
The molecular mass markers (kDa) are shown to the right of the panel. (B)
Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Results
are means ± S.E.M. for four to six
independent experiments performed with similar findings.
 P<0.001, *P<0.05,
**P<0.01 versus control value.
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Fig. 2. Effect of SB203580 on the mantle tissue p38-MAPK activation by 1 µmol
l–1 CuCl2. SB203580 (1 µmol
l–1) was added to normal seawater and, after a 15 min
equilibration period of the animals, it was present throughout the experiment.
Phosphorylated (A, top panel) and total (A, bottom panel) p38-MAPK levels were
assayed in mantle tissue extracts (100 µg of protein) from control animals,
as well as from animals treated with 1 µmol l–1
CuCl2 in the absence or presence of the inhibitor. Western blots
shown are representative of four to six independent experiments performed with
similar findings. The molecular mass markers (kDa) are shown to the right of
the panel. (B) Densitometric analysis of phospho-p38-MAPK bands by laser
scanning. Results are means ± S.E.M.
for four independent experiments. P<0.001
versus control value.
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Fig. 3. Effect of various heavy metals on the p38-MAPK phosphorylation in M.
galloprovincialis gill tissue. (A) Phospho-p38-MAPK was detected in
extracts (100 µg of protein) from control animals (C) or animals treated
with either 1 µmol l–1 CuCl2 (top panel), 50
µmol l–1 ZnCl2 (middle panel) or 1 µmol
l–1 CdCl2 (bottom panel) for the times indicated.
Western blots shown are representative of four to six independent experiments.
The molecular mass markers (kDa) are shown to the right of the panel. (B)
Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Results
are means ± S.E.M. for four to six
independent experiments performed with similar findings.
P<0.001, **P<0.01 versus
control value.
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Fig. 4. Time course of the effect of thermal stress and synergistic effect of
Cu2+ combined with hyperthermia (30°C) upon p38-MAPK
phosphorylation in M. galloprovincialis mantle tissue. Phosphorylated
p38-MAPK was detected in extracts (100 µg of protein) from control animals
maintained at 15°C (Con) or animals maintained at 4°C (A, top panel)
or 30°C (A, bottom panel) for the indicated times. The molecular mass
markers (kDa) are shown to the right of the panel. (C) Phosphorylated p38-MAPK
(top panel) or total p38-MAPK (bottom panel) levels were detected in extracts
(100 µg of protein) from control animals maintained at 15°C and animals
maintained at 30°C for 30 min, either in the absence or presence of 1
µmol l–1 CuCl2. Western blots shown are
representative of four independent experiments. (B,D) Densitometric analysis
of phospho-p38-MAPK bands by laser scanning. Results are means ±
S.E.M. for four independent experiments
performed with similar results. P<0.001,
*P<0.05, **P<0.01 versus control value.
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Fig. 5. DNA fragmentation and caspase-3 activation in the mantle and gill tissues
from M. galloprovincialis specimens treated with various heavy
metals. (A) (Left panel) DNA fragmentation induced by 1 µmol
l–1 CuCl2 for 30 min, in the absence or presence
of 1 µmol l–1 SB203580, or 50 µmol l–1
ZnCl2 for 30 min in the mantle tissue. (Right panel) DNA
fragmentation induced by 1 µmol l–1 CuCl2 for
30 min, 50 µmol l–1 ZnCl2 for 30 min or 1
µmol l–1 CdCl2 for 60 min in the gill tissue.
Gels shown are representative of three independent experiments performed with
similar results. (B) Specimens (four animals per group) were incubated in
normal seawater (controls) or treated with either 1 µmol
l–1 CuCl2 in the absence or presence of 1 µmol
l–1 SB203580, 50 µmol l–1
ZnCl2 or 1 µmol l–1 CdCl2 (for 30,
30 or 60 min, for each heavy metal, respectively). Endogenous full-length
pro-caspase-3 and large active fragments of caspase-3 were detected using a
specific rabbit monoclonal antibody in extracts (100 µg of protein) from
mantle (top panel) and gill (bottom panel) tissues. Western blots shown are
representative of four independent experiments performed with similar results.
n.s., non specific.
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Fig. 6. Induction of Hsp70 expression by thermal stress and/or 1 µmol
l–1 CuCl2 in M. galloprovincialis gill
tissue. (A) Hsp70 was detected in extracts (100 µg of protein) from gill
tissue of animals incubated at 30°C for 30 min either in the absence or
presence of 1 µmol l–1 CuCl2 or at 15°C
with 1 µmol l–1 CuCl2 in the absence or
presence of 1 µmol l–1 SB203580 for 30 min. (B)
Densitometric analysis of Hsp70 bands by laser scanning. Results are means
± S.E.M. for four independent
experiments performed with similar results. The molecular mass markers (kDa)
are shown to the right of the panel. P<0.001,
**P<0.01 versus control value.
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© The Company of Biologists Ltd 2005
