Exam 4 Ch 22

Question 1

Necrosis

Answer 1

Cell is damaged due to injury excess stress, heat or infection
Cell swells forming holes in the plasma membrane
It releases intracellular contents
Like enzymes, DNA, etc.
Cell may burst
Release of cell contacts can cause damage to other cells
Which can activate the inflammatory response

Question 2

What is apoptosis important in?

Answer 2

Development (Nervous system, metamorphosis, sculpting body parts, etc.)
Normal turnover of the skin and intestinal epithelium
Immune response
(Killing virus infected cells; Remove excess immune cells after an infection)

Question 3

What are the morpholoical changes in apoptosis?

Answer 3

Chromatin Condenses
DNA will fragment (200 bp fragments)
Cytoskeleton and nuclear envelope
Cell blebs and fragments
Form apoptotic bodies

Question 4

What are apoptotic bodies?

Answer 4

Membrane bound packaged cell parts, ready for phagocytosis

Question 5

What are methods to show apoptotic cells?

Answer 5

Electron microscopy, DNA “laddering” on agarose gel electrophoresis,staining nuclei with flueorescnet DNA binding dye, annexin V staining

Question 6

What is DNA “laddering” on agarose gel electrophoresis

Answer 6

In apoptosis– DNA is cut by enzymes( Cut BETWEEN nucleosomes
and results in DNA fragments in multiples of ~200 base pairs)
Shows up as “laddered” DNA in agarose gel electrophoresis
Problem: nice, but is not quantitative!

Question 7

What does staining nuclei with fluorescent DNA binding dye help you do with apoptotic cells?

Answer 7

Can count the apoptotic nuclei (quantitative)

Question 8

Phosphatidylserine (PS) head is normally on the _____ face of the membrane

Answer 8

cytosolic

Question 9

On a resting cell, what flips any phosphatidylserine from the outside to inside face?

Answer 9

Flippase enzymes

Question 10

On a resting cell, what enzymes are inactive?

Answer 10

Scramblase enzymes

Question 11

What happens to PS during apoptosis?

Answer 11

Activation of apoptosis proteases cleave and inactivates the flippases
Apoptosis proteases also cleave and ACTIVATE the scramblases
Scramblases transport any phospholipid– including PS- either way across the plasma membrane
Results in PS accumulating on the outer side of the plasma membrane
,

Question 12

What has receptors for PS? What do they do?

Answer 12

Phagocytotic macrophages. They allow macrophages to recognize and phagocytize apoptotic cells. Gives us a “label” for apoptotic cells.

Question 13

What is Annexin V staining?

Answer 13

A protein involved in blood clotting binds to phosphatidylserine (PS)
Fluorescent label Annexin V can be used to stain for apoptotic cells
Also stain DNA with fluorescent DNA-binding propidium iodide
Use a flow cytometer to count the number of fluorescent stained cells

Question 14

Analysis of V stained cells: Normal cells (viable)

Answer 14

No staining with annexin V= not apoptotic
No DNA staining with PI due to intact membrane

Question 15

Analysis of V stained cells: In Early apoptotic cells

Answer 15

Cell can stain with fluorescent Annexin V
But still no staining with PI

Question 16

Analysis of V stained cells: late apoptosis and necrosis

Answer 16

Dying cells have leaky plasma membranes
Propidium iodide leaks into the cell and stains the DNA
Cells stain with both PI and Annexin V

Question 17

What are inactive caspases?

Answer 17

Procaspases

Question 18

What are caspases. How are caspases activated?

Answer 18

They are proteases.
Proteolytic cleavage by other caspases or proteases

Question 19

When a procaspase is cleaved by a protease, what does it release? What is created?

Answer 19

Pro-domains; Creates two parts that form a heterodimer, which creates an active caspase

Question 20

What are the initators caspases?

Answer 20

Caspase 8,9,10

Question 21

The initator caspases then can activate many effector caspases, what are the effecto caspases?

Answer 21

Caspases 3 and 7

Question 22

When the initator caspases activate many effector caspases, what does this do?

Answer 22

Amplify the response

Question 23

What are the 2 ways to activate the apoptosis process?

Answer 23

Extrinsic pathway and mitochondrial pathway

Question 24

What is the extrinsic pathway?

Answer 24

One of the two ways to activate the apoptosis process
Signals are from outside of the cell
Used by the immune response killer cells to kill infected cell

Question 25

What is the mitochondria pathway?

Answer 25

One of the two ways to activate the apoptosis process
Signal usually comes from inside of the cell (usually in response to injury or stress; involves the mitochondria)

Question 26

Activation of apoptosis by intracellular stimuli- In the mitochondrial pathway, cell damage or cell stress can induce cells to undergo apoptosis. Stress or damage can be:

Answer 26

Growth factor deprivation( Example: nerve growth factor protects neurons from cell death)
Loss of adherence for adherent cells
Chemotherapeutic agents
UV radiation
Oxidative stress

Question 27

What does the Bcl-2 family include?

Answer 27

Anti-apoptotic regulators (Bcl-2, Bcl-xl), these prevent apoptosis
Pro-apoptotic regulators (Bax, Bak), these induce apoptosis

Question 28

What is cytochrome-c?

Answer 28

Component of the electron-transport chain and found between in the inner membranes of the mitochondria

Question 29

How is cytochrome c kept from leaking out of the membrane?

Answer 29

Bcl-2 and Bcl-xl bind to Bax and Bak to prevent their oligomerization

Question 30

Survival signals from receptors like nerve growth factor (NGF)

Answer 30

Survival signal activate PI-3Kinase. PI-3-kinase activates protein kinase (Akt or PKB). PKB phosphorylates bad, a pro-apoptotic protein
p-Bad binds to the 14-3-3 protein (This prevents Bad from inhibiting Bcl2/Bcl-xl). Bcl-2/Bcl-xl are free to inhibit Bax

Question 31

What are factors with receptors that activate PI3K?

Answer 31

Survival signals such as:
Nerve growth factor
Many other growth factors
Integrins

Question 32

What happens if you remove the survival signal?

Answer 32

Bad is free to bind to Bcl-2/Bcl-xl
Bad inhibits bcl-2/Bcl-xl from interacting with Bax
Bax oligomerizes and forms pores
Cytochrome c is released from the mitochondria into the cytosol
Cytochrome c in the cytosol is NOT normal

Question 33

If the cytochrome c is in the cytosol, what occurs?

Answer 33

Cytochrome c binds to Apaf-1 monomer
Allows multimerization of seven apaf-1s
Results in association of TWO procaspase-9 with apaf-1 and assembly of the apotosome
7 Apaf-1-cytochrome C oligomerize
Results in association of 2 procaspase-9 with the apoptosome
Procaspase-9 binding induces conformational change that results in activation of the caspases
Active caspase-9 then cleaves and activates many effector caspase-3’s

Question 34

How does extrinsic activation of apoptosis occur?

Answer 34

Triggered from outside of the cell by activation of death receptors. Macrophages (secreting TNF) and cytotoxic T cells (CTLs; with Fas) can kill infected cells by the Extrinsic Apoptotic Pathway

Question 35

What are the members of the Tumor Necrosis Factor Receptor Family (Death receptors)?

Answer 35

TNF-R1
CD95 (Fas)
Death receptor 3,4,5 (DR3, DR4, DR5)
Nerve growth factors receptor p75 (NGFR)

Question 36

How is apoptosis induced by death receptors?

Answer 36

3 death signals
(3 molecules of TNF and
3 Fas Ligands Receptors on CTLs) will bind to 3 death receptors on the target cell (3 TNF receptors and 3 Gas receptors)
Recruits Procaspase-8
(An initiator caspase)
activates Caspase-8

Question 37

How can Caspase-8 can also activate the mitochondrial pathway of apoptosis?

Answer 37

Active Caspase-8, cleaves BID, produces t-BID. t-BID binds to and inhibits Bcl-2 which leads to release of cytochrome c and activation of caspase-9
Activates mitochondrial apoptosis pathway!

Question 38

What is the effecor mechanism of apoptosis?

Answer 38

Both caspase-8 and caspase-9 activate the effector caspase-3 (and 7)
Caspase-3 destroys key components of cellular infrastructure (Nuclear lamina (lamins), cytoskeleton (actin), this results in cell blebbing)
Caspase-3 cleaves Poly (ADP-ribose) polymerase (PARP). PARP can repair single-strand DNA nicks
Destruction of PARP helps prevent DNA repair
CAD= Caspase-activated DNase
(Normally bound to ICAD, ICAD prevents CAD activation, capase-3 cleaves ICAD, ICAD is then released from CAD,
Active CAD moves to the nucleus and cleaves DNA

Question 39

Why degrade DNA?

Answer 39

Prevents the release of intact DNA
Cells can pick up foreign DNA
200 bp is too small for most genes
Viral DNA cannot be transferred to other cell

Question 40

Why study apoptosis?

Answer 40

Insight into development
(Developmental diseases)
Cancer
(Mechanism to induce apoptosis in cancer cells and mechanisms cancers use to survive and avoid killing)
Immune diseases
Stroke, myocardial diseases, etc.
Neurodegenerative diseases