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James Madison University Biology Department (Biosymposium)

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?.

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James Madison University Biology Department (Biosymposium)

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  1. 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

  2. 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

  3. 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

  4. Chemical Reaction of NO

  5. What does NO do? • Nitric Oxide • Intracellularmessenger • - Dichotomous effect • -Small amount: beneficial • -long-term memory • -synapse formation • -neuronal plasticity • -prevents cell death • - Large quantities: detrimental • -induces cell death

  6. What upregulates NOS I in neurodegenerative conditions?

  7. NGF upregulates NOS I and c-Jun C-Jun

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. THE END

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