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MOLECULAR BIOLOGY OF
APOPTOSIS
Definition
Apo: apart
Ptosis: fallen
Shedding of leaves from tress
During embriogenesis ------ occurs as
PCD Post-embrional life------- as apoptosis
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apoptosis
Apoptosis is used as a synonymous forPCD but PCD is physiological death, occursonly during embriogenesis.
It is a functional death and it is a good
mechanism to eliminate wasted, useless,unwanted, or crippled cells!
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Thymus
Prostate
Endometrium
Adrenal cortex
Lymphoid cells
Neurons are all subject to apoptosis
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Why have we developed such a self-
destructive system?
A. PCD allows a constant selection for thefittest cell in a colony
Every cell carries the molecular machinery
to do PCD! Cells that are sensitive to extracellular
signals will survive, cell that cannotcompete with their more vital sisters willundergo apoptosis.
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Ischemia
XRT
Toxins
Chemicals
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PCD machinery is silent until signals arriveto start PCD:
Signals:
damage to DNA
Activation of membrane receptors.Ligands are: peptides, cytokines, ATP,
ROS etc Deprivation of specific signals of GFs,
hormones or survival signals for apoptosis
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PHYSIOLOGICAL VS
PATHOLOGICAL CELL DEATH
Necrosis
Apoptosis
MOLECULAR PATHWAY OF APOPTOSIS
The initiating phase by signals
External that trigger receptors on the plasmamembrane.
Intracellular alterations
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Fas receptor?
Receptors for growth factors, cytokinesand hormones
Membrane alterations cause apoptosis.
Q. What kind of membrane alterations ??
A. Phospholipid redistributions, changes in
membrane charge, carbohydrate andsurface markers.
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Decision phase
Final decision " to live or die"
The final decision depends on expression ofseveral proto-oncogenes, called as Bcll-2 genefamily (B-Cell Leukemia Lymphoma)
Bcl-2 gene product protects B lymphocytes, Tcells against apoptosis induced by: Drugs
XRT
Heat shock
Oxidative stress
What are nuclear changes?
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MEASUREMENT OF APOPTOSIS:
TECHNIQUES BASED ONMORPHOLOGICAL CHANGES
Light microscopy
Electron microscopy
TECHNIQUES BASED ON DNAFRAGMENTATION
Measurement of endonuclease activity
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MEASUREMENT OF APOPTOSIS
TECHNIQUES BASED ON MEMBRANEALTERATIONS
Measurement of dye exclusion
TECHNIQUES BASED ON CYTOPLASMICCHANGES
Changes in intracellular enzyme activity
Measurement of calcium influx
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APOPTOSIS AND ILLNESS
APOPTOSIS AND OXIDATIVE STRESS
Background and introduction
Promotion of apoptosis by oxidative
stress Modulation of apoptosis by oxidative
stress
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Background and Intro:
Ox-stress can cause apoptotic or necrotic celldeath.
This section well talk about ways in which Ox-Stress can intersect apoptotic pathways.
ROS may accumulate due to toxic insults or
normal metabolic processes
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Cell shrinkage, chromatin condensation,internucleosomal DNA fragmentation andformation of apoptotic bodies are all
characteristic features of apoptosis. Several protease families have been
implicated in apoptosis the mostprominent being CASPASES
Caspase is aspartic acid-specific cysteineprotease which is found in zymogens almost inall cells!
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3 models of caspase action is proposed.
TNF receptor mediated
No receptor involvement
cytotoxic cell activation of caspase
Regardless of the mechanism, uponactivation caspases cleave many proteins
and finally DNA.
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A role for Ox-Stress in apoptosis has beenclarified by many scientists.
Promotion of apoptosis by OX-Stress:
Proposed mechanisms:
Fig
Modulation of apoptosis by oxidative
stress
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Apoptotic cell death can be switched tonecrosis during oxidative stress by 2mechanisms:
Inactivation of caspases due to oxidationof their active site thiol group by oxidantsor S-nitrosylation.
Decrease in ATP due to failure ofmitochondrial energy production byoxidants (Table 30.1).
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NO can also have dual effects onapoptosis
NO is reactive, unstable free radical gas
that can easily cross cell membranes. L-Arg------ NO
Low NO: Neurotransmitter, regulator in
vasodilation and platelet aggregation. High NO: Cytotoxicity
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NO may also mediate apoptosis:
Macrophages
Beta cell line
Thymocytes
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How???
Formation of iron-nitrosyl complexes withFeS-containing enzymes: This leads to
impairment of mitochondrial function -ATP depletion.
NO may directly damage DNA-mutagenesis
Generation of OONO- Apoptosis NO may inactivate several antioxidant
enzymes (CAT, GPx, SOD etc)
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NO exposure or iNOS activation mayinhibit apoptosis in
Lymphocytes
Endothelial cells
Neurons
Hepatocytes
Kidney cels
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How?? Direct inhibition of caspase (S-nitrosylation of the
active site Cys) R-S-NO is important component of signal
transduction cascades. S-nitrosylation can regulate many proteins: Enzymes Ion channels G-proteins Transcription factors NO may act as a modular switch to control
protein function via SH groups.
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For example, S-nitrosylation was shown to occurin:
Papain protease Calpain
NF-KB AP-1 These are all implicated in the regulation of
apoptosis. NO inhibition of caspase is reversible. Pro-caspase-3 was recently shown to be S-
nitrosylated on its catalytic site Cys (Cys 163). Nitrosylation/denitrosylation- may serve asa
regulatory mechanism just like.?