Materials and MethodsSetting up Cultures• 1 mL of each protozoan was taken from a previous culture and

added to their specific growth media.• Volvox was grown in 50mL of soil water• Prorocentrum was grown in 25mL of seawater and 25mL of

soil water• Porphyridium was grown in 50mL of seawater

Growth Curve• The growth curve was constructed using two different cultures

from each genera. The cultures used were inoculated on 3/30/09 and 4/17/09

• 10 µl from each culture was put onto a Hemacytometer. All 4 quadrants were counted. That number was divided by 4 and then multiplied by 10,000 to determine cells/mL

Hydrogen Peroxide Treatment• 10 million cells of Prorocentrum and Porphyridium and 3mL of

Volvox were used• The cells were centrifuged to form a pellet • They were then each resuspended in 1mL of their specific

growth media• Depending on the amount of hydrogen peroxide that was

going to be used, that is how much of the resuspended culture was removed and then replaced with that amount of hydrogen peroxide

The treatments used were:

These treatments sat for 24 hours at room temperature under growth lights in the lab

DNA Isolation• DNA was isolated from Qiagen’s DNeasy tissue Handbook

using the protocol for Cultured Animal Cells

Gel Electrophoresis • 20ul of the DNA was separated on a 2% gel at 80volts for 45

minutes.• The gel was stained with SYBR Green overnight to visualize

the DNA

AcknowledgmentsThis independent project was executed for BIO 336 under the supervision of Dr. Michael Bumbulis.

Discussion• The DNeasy isolation procedure designed for Cultured Animal

Cells is effective in isolating DNA from Volvox, Prorocentrum, and Porphyridium

• All three species degrade their DNA in the presence of H2O2

• Porphyridium and Volvox degrade their DNA at H2O2 concentrations < 4 mM

• Prorocentrum is tolerant of H2O2 at 4 mM but degrades its DNA at concentrations > 10 mM

• Testing Volvox and Porphyridium at lower concentrations of H2O2 should deterimine the maximum tolerance of these species

• All three species are possible candidates for future research to determine if they are potential model organisms for apoptosis research.

Vince Carcioppolo, Denise Roth, Bridget Abed, and Katie HertzelDepartment of Biology, Baldwin-Wallace College, Berea, Ohio

Literature cited

The Effects of Hydrogen Peroxide (H2O2) on Volvox, Prorocentrum, and Porphyridium

Results

3 4 5 6 7 14 21 220

500000

1000000

1500000

2000000

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3500000

Porphyridium Growth Curve

Culture 4/17Culture 3/30

Day

Num

ber

of C

ells

(Per

mL)

3 4 5 6 7 14 22 230

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1000000

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Prorocentrum Growth Curve

Culture 4/17

Culture 3/30

Day

Nu

mb

er o

f C

ells

(P

er m

L)

10 mM H2O2 exposure for 24 hours

Control

Porphyridium

100x100x

Volvox

10 mM H2O2 exposure for 24 hoursControl

100x 100x

Prorocentrum

Control 10 mM H2O2 exposure for 24 hours

100x100x

Porphyridium

4C 10 40 C 4 10 40

VolvoxC 4 10 40 80

Prorocentrum

C= no H2O2; numbers = concentration (mM) of H202 exposurefor 24 hours. Gel Stained with SYBR Green

All multicellular eukaryotes undergo apoptosis to regulate cell homeostasis and to dispose of cells with damaged DNA. When DNA damage occurs, proapoptotic proteins are released from the mitochondria triggering precursor enzymes known as caspases to cleave key cellular components (Lawen, 2003). The purpose of this experiment is to determine if three genera of protozoa also undergo this process when their DNA is damaged. Porphyridium, Prorocentrum, and Volvox were exposed to varying concentrations of hydrogen peroxide. After exposure, DNA will be isolated from the cells and compared to the DNA of untreated cells. If the DNA is fragmented it will suggest that these organisms are capable of carrying out the intrinsic pathway of apoptosis. Fragmented DNA will appear as either a streaking or laddering pattern when separated via gel electrophoresis. Cell membranes from all three genera appeared disrupted under 100x magnification after 24 hours of exposure of 10 milli Molar(mM) hydrogen peroxide. Both Porphyridium and Volvox degraded their DNA beyond detection at <4 mM; Prorocentrum showed no change to its DNA at 4 mM but showed fragmentation at 10 mM and above.

Abstract

Apoptosis can be triggered extrinsically though receptors on the cell membrane or intrinsically through a series of steps initiated by DNA damage. Both the intrinsic and extrinsic pathways of apoptosis operate through activation of enzyme precursors called caspases. A caspase contains a large and small subunit which cleave upon activation (Jékely, 2009). DNA damage or an outside signal cleave and activate initiator caspases (2, 8, 9, or 10), which cleave and activate effector caspases (3, 6, or 7). The effector caspases cleave key cellular components causing apoptosis (Lawen, 2003). The intrinsic pathway begins within the cell in response to oxidative stress or DNA damage. The activation of p53 and BCL2 proteins trigger a release of cytochrome c from the mitochondria. Once cytochrome c is in the cytoplasm it binds to another protein called Apoptotic peptidase activating factor 1 or Apaf-1 which activates and binds to the initiator caspase forming a structure called an apoptosome. This structure, which is sometimes referred to as the wheel of death activates the effector caspases to carry out apoptosis (Research Apoptosis, 2009). Once the effector caspase is activated it cleaves cellular components, killing the cell from the inside. Two important components cleaved are DNA fragmentation factor which causes condensation of the chromatin; and gelsolin, a protein associated with actin regulation, causing the membrane to bleb.

Previous research using western blotting to identify caspases indicates that Porphyridium and Volvox contain caspase 9 which is associated with DNA damage and the intrinsic pathway of apoptosis. Proteins from Prorocentrum were unable to be isolated so no predictions of its ability to undergo apoptosis could be made. If it can be shown that these protozoa contain caspases and undergo the process of apoptosis it would suggest that apoptosis is a highly conserved process which originated in unicellular organisms.

Introduction

Jékely Gáspár. The ICE family of cysteine proteases. April 2009. http://www.cryst.bbk.ac.uk/pps97/assignments/projects/jekely/caspase.htm

Lawen Alfons. Apoptosis - an introduction. BioEssays. 2003; 25:888-896.

Research Apoptosis. Apoptosis regulation and execution signaling pathways. April 2009. http://www.researchapoptosis.com/apoptosis/pathways/index

Experimental cells taken from day 14 and 21 of the 3/30 culture

Future Experiments

• Expose Volvox and Porphyridium to concentrations < 4 mM in order to determine their maximum tolerance

• Take samples from the exponential and stationary phase to determine if stage of lifecycle is important

• Expose other species of protozoa to hydrogen peroxide to determine if they are also capable of degrading their DNA in response to oxidative stress

• Develop a procedure for isolating protein from Prorocentrum and run a western blot to determine if they contain any caspases associated with the intrinsic pathway of apoptosis.

µl of 3% H2O2 H2O2 (mM) Porpyridium Prorocentrum Volvox

4.5 4 X X X

11.3 10 X X X

45 40 X X X

90 80 X