Abstract:
Several stresses to tissues including hyperthermia, ischemia, mechanical trauma
and heavy metals have been demonstrated to affect the regulation of a subset of the
family of heat shock proteins of70kOa (hsp70). In several organisms following some of
these traumas, the levels of hsp70 mRNA and proteins are dramatically upregulated.
However, the effects of the stress on limb and tail amputation in the newt Notophthalmus
viridescens, involving mechanical tissue damage, have not adequately been examined.
In the present study, three techniques were utilized to quantitate the levels of
hsp70 mRNA and protein in the tissues of the forelimbs and tails of newts during the
early post-traumatic events following surgical resection of these:: appendages. These
included quantitative Western blotting of proteins separated by both one and twodimensional
SDS-polyacrylamide gel electrophoresis and quantitative Northern blot
analysis of total RNA.
In tissues of both the limb and tail one hour after amputation, there were no
significant differences in the levels of hsp70 protein measured by one-dimensional SOSPAGE
followed by Western blotting, when compared to the levels measured in the
unamputated limb. A 30 minute heat shock at 35°C failed to elicit an increase in the
levels of hsp70 protein in these tissues. Further analysis using the more sensitive 20
PAGE separation of stump tissue proteins revealed that at least some of the five hsp70
isoforms of the newt may be differentially regulated in limbs and tails in response to
trauma. It appears also that amputation of the tail and limb tissues leads to slight
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elevation in the levels of HSP70 mRNA when compared to those of their respective
unstressed tissues.
