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James Madison University Biology Department (Biosymposium). Transcription Factors involved in the Upregulation of NOS I transcription by Phorbal Myristic Acid. Presented By Linda A. Yu. Date: April 21, 2001 Prepared for: Dr. Terrie Rife. What do all of these conditions have in common?. - PowerPoint PPT Presentation
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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
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
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
Chemical Reaction of NO
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
What upregulates NOS I in neurodegenerative
conditions?
c-JUN
0
20
40
60
80
100
120
140
0
5
10
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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
NGF upregulates NOS I and c-Jun
C-Jun
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.
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
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
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.
THE END