1. Apoptosis “Falling or dropping off”

2. Ughhhh….. http://focosi.immunesig.org/apoptosis.htm

5. http://en.wikipedia.org/wiki/Webbed_toes Syndactlyly (1/2500 births) http://www.indianahandcenter.com/hot_child.html

6. Too much: -Alzheimers’ -Parkinson’s -Huntington’s -AIDS -stroke Too little: -cancer -autoimmunity -persistent infections Apoptosis is a balancing act

8. http://research.yale.edu/ysm/article.jsp?articleID=357

9. Do all cells undergo apoptosis??? http://web.mala.bc.ca/simmsw/postergriffiths.htm http://www.pkdiet.com/habits.htm Bacteria Yeast All undergo their own version of programmed cell death (PCD) and exhibit physiological changes 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

10. http://www.dkfz.de/en/phd-program/projects/immunogen.html Cytotoxic drugs Irradiation

11. ProLargeSmall Asp9Asp28 Asp175 1277Caspase-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

12. http://people.bath.ac.uk/pr1cemb/Apoptosis.htm Caspase-3 Caspase-8 Caspase-9

13. http://www.dkfz.de/en/phd-program/projects/immunogen.html Cytotoxic drugs Irradiation

14. http://en.wikipedia.org/wiki/Mitochondria

15. The Bcl2 family: Members are either anti- or pro- apoptotic Other proteins besides cytochrome c are also released from the mitochondria and Include antagonists of caspase inhibitors and endonuclease G Bid and Bad are examples of “BH3” only proteins

16. http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/mito.htm

17. http://www.dkfz.de/en/phd-program/projects/immunogen.html Cytotoxic drugs Irradiation

18. 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 LN 2 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.

19. staurosporine + Jurkat Cells DEVD-pNADEVD + pNA pNA detectable at λ405 nm Caspase-3 Apoptosis is induced Cells are frozen A. B. Cells are lysed; caspase activity is measured +/- inhibitor. Don’t forget the standard Curve! Asp-Glu-Val-Asp Specificity of Caspase-3 for the substrate will be determined through use of the inhibitor DEVD-CHO

20. Staurosporine binds to the ATP binding pocket of many kinases inhibiting their activity

21. staurosporine + Jurkat Cells DEVD-pNADEVD + pNA pNA detectable at λ405 nm Caspase-3 Apoptosis is induced Cells are frozen A. B. Cells are lysed; caspase activity is measured +/- inhibitor. Don’t forget the standard Curve! Asp-Glu-Val-Asp Specificity of Caspase-3 for the substrate will be determined through use of the inhibitor DEVD-CHO

22. 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 LN 2 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.

23. CAD=Caspase Activated Deoxyribonuclease

24. http://www.actaps.com.cn/qikan/manage/htmlwenzhang/2006-3-08.htm An example of DNA laddering: Note: DNA fragmentation is a result of cleavage from multiple endonucleases

25. 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

26. http://www.nearingzero.net/screen_res/nz253.jpg