apoptosis “falling or dropping off”
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Apoptosis “Falling or dropping off”. Ughhhh…. http://focosi.immunesig.org/apoptosis.htm. Syndactlyly (1/2500 births). http://en.wikipedia.org/wiki/Webbed_toes. http://www.indianahandcenter.com/hot_child.html. Apoptosis is a balancing act. Too much: -Alzheimers’ -Parkinson’s - PowerPoint PPT PresentationTRANSCRIPT
Apoptosis“Falling or dropping off”
Ughhhh…..
http://focosi.immunesig.org/apoptosis.htm
http://en.wikipedia.org/wiki/Webbed_toes
Syndactlyly (1/2500 births)
http://www.indianahandcenter.com/hot_child.html
Too much: -Alzheimers’ -Parkinson’s -Huntington’s -AIDS -stroke
Too little: -cancer -autoimmunity -persistent infections
Apoptosis is a balancing act
http://research.yale.edu/ysm/article.jsp?articleID=357
Do all cells undergo apoptosis???
http://web.mala.bc.ca/simmsw/postergriffiths.htm http://www.pkdiet.com/habits.htm
BacteriaYeast
•All undergo their own version of programmed cell death (PCD) and exhibit physiologicalchanges similar to apoptosis in mammalian cells. Both plants and yeast have “caspase-like” proteins while to date, bacteria do not.
http://www.cbs.dtu.dk/staff/dave/roanoke/bio101ch06.htm
Plant
http://www.dkfz.de/en/phd-program/projects/immunogen.html
Cytotoxic drugsIrradiation
Pro Large Small
Asp9 Asp28 Asp175
1 277Caspase-3
Caspases..the central players:
•The caspase family is highly conserved; at least 14 members (11 of these are found in mammals
•We will only be talking about Caspase-8, Caspase-9, and Caspase-3 in this class
•Classified as cysteine aspartate specific proteases due to the required cysteine in the active site and the fact that they cleave after an aspartate residue
•In order to be active, the precursor caspase (a zymogen) must be processed by cleavage of the prodomain/large subunit and the large subunit/small subunit
•Caspases can be classified as “initiator” or “effector”
•Substrates of caspases include cell structural components, regulatory proteins, and inhibitors of apoptosis
http://people.bath.ac.uk/pr1cemb/Apoptosis.htm
Caspase-3
Caspase-8
Caspase-9
http://www.dkfz.de/en/phd-program/projects/immunogen.html
Cytotoxic drugsIrradiation
http://en.wikipedia.org/wiki/Mitochondria
The Bcl2 family: Members are either anti- or pro- apoptotic
Other proteins besides cytochrome c are also released from the mitochondria andInclude antagonists of caspase inhibitors and endonuclease G
Bid and Bad are examples of “BH3” only proteins
http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/mito.htm
http://www.dkfz.de/en/phd-program/projects/immunogen.html
Cytotoxic drugsIrradiation
So, what are we doing this week?
A. Caspase assay: used to detect the enzymatic activity of Caspase-3 as an indicator of apoptosis
You will be inducing apoptosis (w/Staurosporine) today, freezing them in LN2 and doing the assay on Day 2
B. TUNEL assay: used to measure DNA fragmentation characteristic of cells which have initiated apoptosis
You will use cells that have already been induced with Staurosporine to perform the assay, but will not look at your slides until Day 2.
staurosporine
+ Jurkat Cells
DEVD-pNA DEVD + pNApNA detectable at λ405 nm
Caspase-3
Apoptosis is inducedCells are frozen
A.
B. Cells are lysed; caspase activity is measured +/- inhibitor. Don’t forget the standardCurve!
Asp-Glu-Val-Asp
Specificity of Caspase-3 for the substrate will be determined through use ofthe inhibitor DEVD-CHO
Figure 2 Overview of PDK1Figure 2 Overview of PDK1--kinase domain bound to staurosporine kinase domain bound to staurosporine
Biochemical Journal www.biochemj.org Biochem.Biochemical Journal www.biochemj.org Biochem. J. (2003) 375, 255J. (2003) 375, 255--262 262
Staurosporine binds to the ATP binding pocket of many kinases inhibiting their activity
staurosporine
+ Jurkat Cells
DEVD-pNA DEVD + pNApNA detectable at λ405 nm
Caspase-3
Apoptosis is inducedCells are frozen
A.
B. Cells are lysed; caspase activity is measured +/- inhibitor. Don’t forget the standardCurve!
Asp-Glu-Val-Asp
Specificity of Caspase-3 for the substrate will be determined through use ofthe inhibitor DEVD-CHO
So, what are we doing this week?
A. Caspase assay: used to detect the enzymatic activity of Caspase-3 as an indicator of apoptosis
You will be inducing apoptosis (w/Staurosporine) today, freezing them in LN2 and doing the assay on Day 2
B. TUNEL assay: used to measure DNA fragmentation characteristic of cells which have initiated apoptosis
You will use cells that have already been induced with Staurosporine to perform the assay, but will not look at your slides until Day 2.
CAD=Caspase Activated Deoxyribonuclease
http://www.actaps.com.cn/qikan/manage/htmlwenzhang/2006-3-08.htm
An example of DNA laddering:
Note: DNA fragmentation isa result of cleavage from multiple endonucleases
TUNEL assay
Fluorescein-12-dUTP
http://www.emdbiosciences.com/html/cbc/Apoptosis_Resource_changes_in_nucleus.htm
Visualized with fluorescence Microscope at ~520 nm
(TdT-mediated dUTP Nick-End Labeling)TdT=Terminal deoxynucleotidyl transferase
http://www.nearingzero.net/screen_res/nz253.jpg