Biomedical Engineering Reference
In-Depth Information
also exist among fungi, especially between yeasts and fi lamentous species,
due to their different morphologies. Morphological and cytological markers
are very useful for monitoring apoptosis in yeast cells, because differences
between cell populations can be quantifi ed by comparing the number of
apoptotic cells in each population (Narasimhan et al. 2001, Del Carratore et
al. 2002, Huh et al. 2002, Granot et al. 2003). The use of such markers is less
obvious in fi lamentous species for a number of reasons: fi rstly, fi lamentous
fungi are multicellular, making it diffi cult to quantify these changes.
Also, there is a lack of uniformity within the mycelium: parts of the same
colony might be of different age or at different developmental stages, and
hyphae may have different morphologies. Furthermore, many fungi are
multinucleated, and the nuclei are not always of uniform size, which makes
nuclear shrinkage a less obvious measure in some cases.
The Role of Metacaspases in Apoptosis
The initial studies on baker's yeast (
Saccharomyces cerevisiae
) demonstrated
the overproduction of the single metacaspase YCA1. As concluded from
reduced clonogenicity, this caused autocatalytic processing and resulted
in cells becoming sensitive to exogenous or aging-related oxidative stress
(Madeo et al. 2002). It was also found that overproduction of an active
protease caused greater sensitivity to exogenous stress. Madeo (2002) also
reported that a yeast strain with a
YCA1
gene (Δ
yca1
)which was disrupted
was three times less sensitive to H
2
O
2
, and ~ 5% of the cells did not suffer
from aging related cell death. Such desensitization may have been caused
by indirect effects, such as an altered protein turnover interrupting the
balance of pro-and anti-cell death mediators or it may suggest the direct
involvement of YCA1 in cell death. Levels of oxidized proteins increased
considerably in comparison to those of wild-type cells subsequent to
treatment of Δ
yca1
cells with H
2
O
2
(Khan et al. 2005). Concurrently, the
proteasome activity of Δ
yca1
cells increased and apoptosis decreased
upon H
2
O
2
treatment. The reduced capability of Δ
yca1
cells compared
with wild-type cells to deal with damaged proteins might explain the
substantial reduction in cell viability after extended culture (i.e., >30 d)
(Herker et al. 2004). However, clonogenicity assays may refl ect other
cellular states such as cell cycle arrest or metabolic defi ciencies and as a
result, the clonogenicity results therefore do not eliminate purposes of
metacaspases other than cell death involvement.
Extracts of H
2
O
2
-treated YCA1-overproducing yeast were highly active
toward the synthetic caspase substrates Val-Glu-Ile- Asp-AMC and Ile-Glu-
Thr-Asp-AMC which suggested that the YCA1 metacaspase performed as
a caspase (Madeo et al. 2002). However, such reports were later disputed
and it was shown that lysates from bacteria and H
2
O
2
-stimulated yeast