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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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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


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



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


What upregulates nos i in neurodegenerative conditions
What upregulates NOS I in neurodegenerative conditions?


Ngf upregulates nos i and c jun
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
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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