Cell Injury (Drake)

Question 1

B1W4Drake-Celltissue/injury-1

Apoptosis

Answer 1

Programmed cell death (physiological or pathological)

Single cells

Requires ATP

No loss of membrane integrity

Little release of cell contents (apart from extrusion-planned)

Little inflammation

Question 2

B1W4Drake-Celltissue/injury-1

Causes of Necrosis

Answer 2

(1) ischemia
(2) mitochondrial poisoning
(3) membrane damage agents (ROS, bacterial enzymes)

Question 3

B1W4Drake-Celltissue/injury-1

Sequence of events for cell death in ischemia

Answer 3

1) Decreased blood flow
2) Decreased oxygen
3) Decreased ATP
4) Decreased Na/K pump (increased cytoplasmic Na+ and Ca++)
5) Loss of mitochondrial electron transport function
6) Loss of inner mitochondrial membrane integrity
7) Lysosomes degrade cell constituents

PROCESS IS REVERSIBLE UNTIL LOSS OF INNER MITOCHONDRIAL MEMBRANE INTEGRITY

Question 4

B1W4Drake-Celltissue/injury-1

Consequences of ischemia

Answer 4

Decreased oxidative phosphorylation –> decreased ATP –>

(1) ER swelling Cell swelling (decreased Na+ pump activity)
(2) clumping of nuclear chromatin
(3) decreased protein synthesis and increased lipid deposition

Question 5

B1W4Drake-Celltissue/injury-1

Role of increased cytosolic Calcium in cell injury

Answer 5

activation of cellular enzymes–>

(1) membrane damage (due to phospholipases and proteases)
(2) nuclear damage (due to endonucleases)
(3) decreased ATP (due to ATPase activity)

Question 6

B1W4Drake-Celltissue/injury-1

Clinical conditions leading to necrosis

Answer 6

Ischemia and hypoxia

Ischemia reperfusion injury

Inflammation and infections

Chemical injury

Question 7

B1W4Drake-Celltissue/injury-1

Stages of apoptosis

Answer 7

1. Signaling stage - Initiation (generate activated initiator caspase from procaspases)
2. Effector stage - Death pathway engaged (caspase cascade generates effector caspases)
3. Terminal stage - Cell death. Morphologic changes. (endonucleases digest DNA, cytoskeleton broken apart)
4. Phagocytosis of apoptotic cell

Question 8

Necrosis

Answer 8

Occurs in many adjacent cells

Passive response

Does not require ATP

Disruption of organalle fxn

Release of cell contents

Strong inflammatory rxn

Always pathological

Question 9

What are all initiators of necrosis associated with?

Answer 9

Decreased ATP

Question 10

How can you tell if a tissue is undergoing necrosis?

Answer 10

Released intracellular proteins enter the blood and are measured as markers of tissue damage (eg liver enzymes; cardiac enzymes and proteins).

Question 11

Where does the intrinsic pathway of apoptosis occur?

Answer 11

Mitochondria

Question 12

Molecules involved in intrinsic pathway of apoptosis

Answer 12

1) Bcl2 (regulator): sits on membrane of mitochondria and binds Bax/Bac to keep it from forming mitochondrial pores; is anti-apoptotic
2) Bax/Bac (effector): forms pore in mitochondrial outer membrane; is pro-apoptotic
3) BH3 (sensor): when cell wants to undergo apoptosis, BH3 binds to Bcl2, freeing Bax/Bac to form mitochondrial pores; is pro-apoptotic

Question 13

After mitochondrial pore is formed in intrinisc pathway of apoptosis, what happens?

Answer 13

Cytochrome C and other proteins released into cytoplasm –> initiates formation of caspase complex called apoptosome –> caspase pathway that leads to apoptosis

Question 14

Extrinsic pathway of apoptosis

Answer 14

Fas death receptor present on all cells binds Fas ligand on killer T cell –> aggregate –> adaptor protein FADD attracts procaspase (forming DISC complex) –> initiate effector caspase cascade –> apoptosis

Question 15

Terminal stage of apoptosis

Answer 15

Executioner caspases: activate endonucleases (DNA fragmentation), degrade microfilaments (cytoplasmic blebbing), proteolyse lamin (nuclear blebbing)

Transglutaminase inside cell does protein cross-linking

Question 16

Why doesn’t apoptosis result in inflammation?

Answer 16

No release of cytokines

Apoptotic cells are rapidly cleared

Plasma membrane is preserved

Apoptotic cells secrete inhibitors of inflammation

Question 17

Diseases that result from apoptosis (both decreased and increased apoptosis)?

Answer 17

Decreased: cancer, viral infection (herpes), autoimmune (lupus)

Increased: Alzheimers, Type I diabetes, organ atrophy, ischemia (myocardial infarction, stroke)

Question 18

Reactive Oxygen Species (ROS)

Answer 18

Molecules that can generate free radicals and/or are already free radicals

Question 19

How are cells exposed to ROS?

Answer 19

1) Normal oxidative phosphorylation
2) Activation of certain receptors
3) Chemicals from environment
4) Metabolites of drugs and environmental agents
5) Impaired function of defense enzymes/mechanisms

Question 20

ROS negative effects on proteins

Answer 20

1) Modification of SH groups (S-S crosslinking)
2) Adduct formation with lysine, tyrosine, cysteine
3) Protein unfolding
4) Protein scission (splitting apart)

Question 21

ROS negative effects on lipids

Answer 21

1) Changes in membrane fluidity (damage membrane)
2) Production of receptor ligands
3) Become antigenic
4) Lipid ROS propagates ROS formation (BAD! Due to double bonds of fatty acids)
5) Fatty acid oxidation
6) Scission of lipids (measure malondialdehyde to see if this is happening)
7) Cross-linking lipids

Question 22

ROS negative effects on DNA

Answer 22

1) Base mutations/changes (usually by adding hydroxyl radical; some lead to apoptosis)
2) Chromosome breaks

Question 23

Glutathione (GSH)

Answer 23

Very important antioxidant (a metabolite)

Turns H₂O₂ –> H₂O (in general quenches oxygen free radicals)

HIGH ratio of reduced to oxidized glutathione (GSH:GSSG) tells you that there’s no cellular oxidative stress

Question 24

Glutathione peroxidase

Answer 24

Enzyme that catalyzes the reaction of Glutathione with H2O2 to turn it into H2O (also at the same time turns GSH to GSSG)

Question 25

Glutathione reductase

Answer 25

Enzyme that catalyzes the reaction where GSSG reduces NADPH to NADP+ (at the same time turns GSSG to GSH)

Question 26

Fixing lipid free radicals: Vitamin E (tocopherol) and Vitamin C (ascorbic acid)

Answer 26

Antioxidants (metabolites) Vitamin E in plasma membrane finds lipid free radicals and gets rid of them but turns into a free radical itself! Vitamin C in water soluble compartment picks up radical from vitamin E Other enzymes get rid of Vitamin C's free radical

Question 27

Enzymes to get superoxide to H2O

Answer 27

Superoxide dismutase (SOD): superoxide (O2) --\> H2O2 Catalase: H2O2 --\> H2O

Question 28

Heme oxygenase

Answer 28

Controls free iron in the process of heme catabolism (Fe2+ can generate hydroxyl radical!)

Question 29

Fixing protein free radicals that create damaged or unfolded proteins

Answer 29

1) Chapreone synthesis: they help re-fold proteins 2) Ubiquitin: tag misfolded proteins and degrade 3) Unfolded protein response (UPR): turn on tx of genes to respond to ROS stress (glutathione, folding proteins, etc) and turn off translation of unnecessary proteins

Question 30

Fixing DNA free radicals that cause DNA damage

Answer 30

Just repair DNA as usual! Ends in inhibition of cell cycle or apoptosis if damage is severe

Question 31

How do cells detect ROS via Nrf2?

Answer 31

Nrf2 is a TF that binds antioxidant response element (ARE) and increases expression of things to get rid of ROS (GSH, proteasome, anti-inflammatory) Normally, Nrf2 constantly made and degraded Nrf2 is normally bound to Keap1 (sensory molecule) which promotes degradation by ubiquitin ROS prevents Keap1 from binding Nrf2 (ROS oxidizes thiols on Keap1) so Nrf2 increases

Question 32

How do cells detect hypoxia via HIF?

Answer 32

HIFa and HIFb complex is TF that initiates tx of genes to restore O2, energy homeostasis, OR initiate apoptosis Normally, HIF constantly made and degraded HIFa normally has PHD (sensory protein) add OH, which promotes degradation by ubiquitin ROS inactivates PHD so it can no longer tag HIFa with OH for degradation, so HIF increases