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James Madison University Biology Department (Biosymposium)

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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?.

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slide1

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
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
slide3

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
slide5

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
slide10

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
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
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
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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