Reference no: EM132927560
1) What does the abbreviation MOMP stand for?
2) Draw a diagram illustrating the cascade of events initiated upon release of cytochrome c from the mitochondrion, as described in the introduction to the paper.
3) In your own words define the two proposed models for Bax and Bak - you may find it helpful to draw a diagram.
4) The authors introduce plasmids into Jurkat cells in order to suppress apoptosis in the cells in 3 fundamentally different ways. Explain the way in which apoptosis would be inhibited after transfection with the following constructs:
i) the Bcl-2 and Bcl-Xl constructs
ii) the Apaf-1 construct
iii) the XIAP and BIR1/BIR2 constructs
5) What type of cell line is Jurkat?
6) The Jurkat cells used in this experiment were fed with medium supplemented with the amino acid glutamine - indeed glutamine is routinely added to culture medium of mammalian cell lines. Why might this be?
7) Why are 3 different control plasmids (pSFFV-neo, pSUPERneo and pcDNA3-neo) used?
8) With reference to the list of antibodies used, what does "rabbit anti-Bcl-XL mean" and why do you think a clone number has been specified?
9) Why do the authors use a different anti-Bak antibody for Western blotting and the Bak activation studies?
10) Summarise the experimental data shown in Figure 1A.
11) How do the authors confirm that Bcl-2, Bcl-Xl and Apaf-1 inhibit caspase activation and apoptosis? (Figs 1B and 1C)
12) In the "experimental procedures" section, the authors neglect to say why DMSO is being used as a control in the experiments that use etoposide.
Which of the following statements is the most likely explanation as to why DMSO is being used?
Justify your choice of statement.
13) Explain the evidence to show that MOMP is inhibited in Bcl-XL and Bcl-2 overexpressing transfectants. (Figs 2 A and B)
14) Explain in your own words, the rationale for using the XIAP and the myc -BIR1/BIR2 domain constructs to investigate caspase activation.
15) What is the evidence that XIAP and myc-BIR1/BIR2 do not completely inhibit caspase activation and hence do not totally prevent apoptosis? Suggest why this might be.
16) In the proposed models, Apaf-1 activity and caspase activation are downstream of the events in the mitochodria (bak dimerisation, cyct c release). What evidence do the authors present to suggest that such models may not be entirely correct? (Figures 2 and 4)
17) What do the authors propose as an alternative model for the intrinsic pathway?
18) With reference to the speculation as to why the apoptosome is such a large complex, explain the possible advantage to the cell of the model the authors propose.
19) Who supplied the authors with the pcDNA3-XIAP and pcDNA3-Myc-BIR1/BIR2 vectors?