Biomedical Engineering Reference
In-Depth Information
It involves a diverse array of stimuli that activate intracellular targets
and mitochondria initiated events. The extrinsic and intrinsic pathways
converge on the same execution pathway, which is initiated by the cleavage
of caspase-3 (Sharon et al. 2009).
Thus far, evidence only exists for components of an intrinsic-like
pathway in fungi. It remains unclear whether the extrinsic pathway is
altogether absent or if it is regulated by as-yet unidentifi ed proteins (Sharon
et al. 2009).
Apoptosis is defi ned by a sequence of unique morphological changes.
Cell shrinkage and chromatin condensation are the fi rst visible changes. This
is followed by extensive plasma membrane blebbing, nuclear fragmentation
and formation of apoptotic bodies, which are subsequently engulfed by
phagosomes. The process terminates with decomposition of the apoptotic
bodies within the phagosomes and complete recycling of the components.
It should be noted that although the mechanisms and morphologies of
apoptosis and necrosis differ, there is considerable overlap between the two,
and it is not always possible to distinguish apoptosis from necrosis using
conventional microscopy. Therefore, determination of apoptosis cannot
rely solely on cell morphological markers and must also be supported by
the presence of additional apoptosis-specifi c markers (Sharon et al. 2009).
Biochemical and cytological responses of apoptotic cells include the
accumulation of reactive oxygen species (ROS), activation of caspases,
DNA cleavage by specifi c endonucleases and externalization of the inward
facing phosphatidylserine in the cell's lipid bilayer (Elmore 2007). These
responses can be monitored by a variety of methods. These methods are
used to determine apoptosis. It should be noted that not all methods are
appropriate to all situations, and the choice of method must take into
consideration its relevant advantages and disadvantages.
A review by Sharon and colleagues in 2009 listed the following methods
which are widely used to determine apoptotic-like cell death in fungi:
• Accumulation of ROS due to an oxidative burst can be detected
by various oxidation-sensitive chemicals, which change their
absorbance spectrum or emit fl uorescence in the presence of ROS.
•
Cleavage of nuclear DNA by Ca
2+-
and Mg
2+-
dependent endonucleases
during apoptosis results in specifi c DNA fragmentation that can be
visualized on an agarose gel after electrophoresis. The fragments
form a 'DNA ladder' which is considered a hallmark of apoptosis.
•
DNA cleavage can also be detected by the terminal dUTP nick end-
labeling (TUNEL) method, in which a terminal transferase is used
to add fl uorescein-labeled UTP to the 3'- end of the DNA fragments,
which can then be detected by fl uorescence microscopy.
