![Page 1: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/1.jpg)
James Madison UniversityBiology Department (Biosymposium)
Presented ByLinda A. Yu
Date: April 21, 2001Prepared for: Dr. Terrie Rife
Transcription Factors involved in the Upregulation of NOS I transcription by Phorbal Myristic Acid
![Page 2: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/2.jpg)
What do all of these conditions have in common?
Stroke Spinal Cord Injury Alzheimer’s Disease Parkinson’s Disease Huntington’s Disease ALS (Lou Gehrig’s Disease) Mad Cow Disease
![Page 3: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/3.jpg)
Overview
• Introduction of NO and NOS• Importance of NOS I• Relevance of PMA (Protein kinase C pathway)• Procedures• Results and Conclusions• Improvement for the future
![Page 4: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/4.jpg)
Chemical Reaction of NO
![Page 5: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/5.jpg)
What does NO do?
• Nitric Oxide– Intracellular messenger - Dichotomous effect
-Small amount: beneficial -long-term memory-synapse formation-neuronal plasticity-prevents cell death
- Large quantities: detrimental
-induces cell death
![Page 6: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/6.jpg)
![Page 7: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/7.jpg)
What upregulates NOS I in neurodegenerative
conditions?
![Page 8: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/8.jpg)
c-JUN
0
20
40
60
80
100
120
140
0
5
10
15
20
25
30
35
0 20 40 60 80 100 120 140 160
NOS I
c-JUN Expression NOS I Expression
Days after Nerve Transection
c-JUN and NOS I Expression Are Upregulated with Similar Patterns Following Nerve Transection
![Page 9: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/9.jpg)
NGF upregulates NOS I and c-Jun
C-Jun
![Page 10: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/10.jpg)
Protein Kinase C pathway
•Protein Kinase C: activated by a chemical phorbal ester: phorbol myristic acid (PMA)
•Serine/threonine kinase•Maximally activated in the presence of DAG (diacylglycerol) and calcium ions: mitogen activated protein (MAP) kinases, pkA and pkC.•Involved in the signal transduction pathways initiated by hormones, growth factors and neurotransmitters. (NGF pathway).•NGF pathway: signal cascades involved in upregulation of NOS I.•Transcription factors whose expression upregulated: -two leucine zipper proteins: c-fos and c-jun; AP1, AP2, and NFk-B.
![Page 11: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/11.jpg)
Procedures Pheochromcytoma cells - PC12 cells derived from the
adrenal gland of rats were grown in a monolayer and grown on a plate until 70-80% confluency.
Treatments: - DMSO
- No treatment -PMA in DMSO (6 hours) -PMA in DMSO (8 hours)
Assay for Changes in NOS I Expression by RT-PCR
![Page 12: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/12.jpg)
Procedures (cont’d) RNA isolation using Trizol Method.
RNA concentration measured : spectrophotometer.
RT-PCR (Reverse transcriptase PCR): to detect changes in gene expression.
PCRed cDNA in an 2% agarose gel:
Beta-actin:control NOS1
![Page 13: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/13.jpg)
For the Future….
If NOS I is upregulated following PMA treatment
Test to find the specific NOS I promoter that is upregulated by PMA
Isolate some of the transcription factors that are upregulated in the pkC pathway.
If NOS I is not upregulated following PMA treatment Try to find other activation sites in the NGF pathway to see
which transcription factors are upregulated when NOS I is upregulated.
Do further experiments on eNOS and iNOS.
![Page 14: James Madison University Biology Department (Biosymposium)](https://reader035.vdocuments.net/reader035/viewer/2022070418/56815923550346895dc64d13/html5/thumbnails/14.jpg)
THE END