James Madison University Biology Department (Biosymposium) Presented By Linda A. Yu Date: April 21, 2001 Prepared 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?

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.

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