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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
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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
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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
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Chemical Reaction of NO
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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
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What upregulates NOS I in neurodegenerative conditions?
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NGF upregulates NOS I and c-Jun C-Jun
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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.
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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
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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
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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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THE END
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