Question

5) This image shows data from two regions of the body. One is mature brain from adult mice. The other is brain from embryonic mice. 75 (4pts) Describe one way these researchers could have tested their cells for the a. presence of apoptosis. 50 P-Bcl-2 Bcl-2 (4pts) In the bar graph if mature brain is shown in white, while embryonic brain is b. Cyto C (Mito.frac.) shown in Grey. Based on your knowledge of why cells undergo apoptosis why do you think their results make sense? Cyto C (Cyto. frac.) c. (4pts) Based on this image, what is the effect of apoptosis on Bcl-2? Why is it important that they have both the BCL-2 gel and the P-Bcl2 gel rather then just showing P-Bcl2? d. (4pts) In the next experiment they isolated a mitochondrial fraction from their cells (Mito. frac.) and a cytoplasmic fraction-lacking all mitochondria (Ctyo. frac.) They tested both of these fractions for Cytochrome C. Based on your understanding of apoptosis fill in the data that you would expect for this result, and describe your reasoning for this result below. % Cell Death

Answer 1

a. Apoptosis is a programmed cell death while necrosis is accidental death.Apoptosis happens because of activation and cleavage of some enzymes like caspases.When there is stress or pathogen attack or in the development stage of neuronal system, inactive caspases become mature caspases and they activate inflammasome which leads to activation of autophagy which leads to cell death by lysosmes.

So,researchers could perform apoptosis study using caspase-1,caspase-3 and caspase-8(apotosis markers) and reactive oxygen species study(ROS) estimation using Flowcytometry(Can study stages of apoptosis like early stage,late stage), RTPCR,western blotting and DCFDA assay. If apoptosis is there ROS will be more and caspase levels will be more and cell death in late stage(flowcytometry) will be found.

b.

Cell death study can be performed using MTT/XTT (Mitochondrial reductase enzyme presented in live cells reduce MTT reagent to formazan).The represented results make sense, because in the embryonic development stage tissue regeneratin takes place so apotosis will be more. Neuronal cells undergo apoptosis when the new cells forms to remove neuronal debris. Similarly matured neuronal cell death is less and takes more time than developing embryonic cells because of the their completely developed structure.

c. BCL2 is anti apoptotic marker which prevents apoptosis by inhibiting mitochondrial membrane damage leading the release of cytochrome c further caspase activation. In the image attached, although bcl2 expression is same but phosporylated bcl2 is more in embriyoinc brain.Because bcl2 phoshorylates to p-bcl2 because of growtf factors which play role in tissue regeneration in embryonic brain. So measuring phoshorylated form of bcl2 is important to study the mechanism of apoptosis.

d. When cells undergoes apoptosis mitochondrial membrane damages and more cytochome c release which activates caspases. Hence cyto c will be more in embriyonic mouse brain than adult mouse brain as more apoptosis is there in embryonic mouse brain than adult mouse brain

so in Cyto fractions --- == (= indicates more and -- indicates less)

MIto fractions   ==    --