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cytosolic aspartate-specific proteases (CASPases) 212 cytotoxicity assays
β tubulin dimers. The 7.5-nm diameter microfilaments are
actin polymers. In addition to their interactions with myosin
filaments in muscle contraction, actin filaments may affect
movement or cell shape through polymerization and depolymerization.
Microfilaments participate in cytoplasmic
streaming, ruffling of membranes, and phagocytosis. They
may be responsible for limiting protein mobility in the cell
membrane. The proteins of the 10-nm diameter intermediate
filaments differ according to the cells in which they occur.
Vimentin intermediate filaments occur in macrophages,
lymphocytes, and endothelial cells, whereas desmin occurs in
muscle and epithelial cells containing keratin.
cytosolic aspartate-specific proteases (CASPases)
Enzymes responsible for the deliberate disassembly of a
cell into apoptotic bodies. Caspases are present as inactive
proenzymes, most of which are activated by proteolytic
cleavage. Caspase-8, caspase-9, and caspase-3 are situated
at the pivotal junctions in apoptotic pathways. Caspase-8
initiates disassembly in response to extracellular apoptosisinducing
ligands and is activated in a complex associated
with the receptor’s cytoplasmic death domains. Caspase-9
activates disassembly in response to agents or insults that
trigger release of cytochrome c from the mitochondria
and is activated when complexed with dATP, APAF-1, and
extramitochondrial cytochrome c. Caspase-3 appears to
amplify caspase-8 and caspase-9 signals into a full-fledged
commitment to disassembly. Both caspase-8 and caspase-9
can activate caspase-3 by proteolytic cleavage and caspase-3
may then cleave vital cellular proteins or activate additional
caspase by proteolytic cleavage. Many other caspases have
been described. Caspases are a group of proteases that
proteolytically disassemble the cell. Caspases are present in
healthy cells as inactive proforms. During apoptosis, most
caspases are activated by proteolytic cleavage. Caspase-9,
however, may be active without being proteolytically
cleaved. Activation is through autoproteolysis or cleavage
by other caspases. Cleavage of caspases generates a
pro-domain fragment and subunits of approximately 20 and
10kDa. Active caspases appear to be tetramers consisting of
two identical 20kDa subunits and two identical 10kDa subunits.
Detection of the 20- or 10-kDa subunit by immunoblotting
may imply activation of the caspase. Colorimetric
and fluorometric assays using fluorogenic peptide substrates
can be used to measure caspase activity in apoptotic cells.
Caspases cleave substrate proteins at the carboxyl terminus
of specific aspartates. Tetrameric peptides with fluorometric
or colorimetric groups at the carboxyl terminal have been
used to determine the Km values of caspases. Although
there is preference for peptides with a certain amino acid
(aa) sequence, the aa sequence can have some variance.
Caspases also have overlapping preferences for the tetrameric
aa sequence (i.e., the same substrates can be cleaved
by multiple caspases although one caspase may have a lower
Km). Peptides containing groups that form covalent bonds
with the cysteine residing at the active site of the caspase
are often used to inhibit caspase activities.
cytotoxic
The ability to kill cells.
cytotoxic antibody
Antibody that combines with cell surface epitopes followed
by complement fixation, which leads to cell lysis or cell
membrane injury without lysis.
cytotoxic CD8 T cells
A T lymphocyte subset that expresses the CD8 coreceptor
and recognizes peptide antigen presented in the context of
major histocompatibility complex (MHC) class I molecules.
cytotoxic cytokines
Cytokines such as TNF and Ltα that induce fatal injury of
cells.
cytotoxic drugs
Agents that kill self-replicating cells such as immunocompetent
lymphocytes. Cytotoxic drugs have been used for
anticancer therapy as well as for immunosuppression in
the treatment of transplant rejection and aberrant immune
responses. The four cytotoxic drugs commonly used for
immunosuppression include cyclophosphamide, chlorambucil,
azathioprine, and methotrexate.
cytotoxic T cells
T lymphocytes that fatally injure other cells. Most are major
histocompatibility complex (MHC) class-I-restricted, CD8 +
T lymphocytes even though CD4 + T cells may serve as killer
cells in some instances. Cytotoxic T cells are significant in
host resistance against viruses and other cytosolic pathogens.
Binding
CTL
Target
cell
Activation and
perforin release
Na +
+
H2O Osmotic
swelling
Cytotoxic T lymphocytc (CTL)-mediated target cell lysis.
Lysis
cytotoxic T lymphocyte (CTL)
A subset of antigen-specific effector T cells that play a principal
role in protection and recovery from viral infection,
mediate allograft rejection, participate in selected autoimmune
diseases, participate in protection and recovery from
selected bacterial and parasitic infections, and are active in
tumor immunity. They are CD8 + , MHC class-I-restricted,
nonproliferating, endstage effector cells. However, this
classification also includes T cells that envoke one or several
mechanisms to produce cytolysis, including perforin/
granzyme, FasL/Fas, and tumor necrosis factor α (TNF-α);
synthesize various lymphokines by T H1 and T H2 lymphocytes;
and recognize foreign antigen in the context of MHC
either class I or class II molecules.
cytotoxic T lymphocyte precursor (CTLp)
A progenitor that develops into a cytotoxic T lymphocyte
after it has reacted with antigen and inducer T cells.
cytotoxicity
The fatal injury of target cells by specific antibody and
complement or specifically sensitized cytotoxic T cells,
activated macrophages, or natural killer (NK) cells. Dye
exclusion tests are used to assay cytotoxicity produced by
specific antibody and complement. Measurement of the
release of radiolabel or other cellular constituents in the
supernatant of the reacting medium is used to determine
effector cell-mediated cytotoxicity.
cytotoxicity assays
Techniques to quantify the action of immunological effector
cells in inducing cytolysis of target cells. The cell death
K +