Nrd Ra Treatment[2] Ppt $2$ $1$

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Nrd Ra Treatment[2] Ppt $2$ $1$

  1. 1. EXPLORING THE REGULATION OF NRD-CONVERTASE EXPRESSION BY RETINOIC ACID<br />KRISHNA MOORTHY<br />SUPERVISOR: DR. ADRIAN PIEROTTI<br />
  2. 2. INTRODUCTION<br />NRD-CONVERTASE (NRDc) is a cytosolic and cell-surface metallopeptidase enzyme.<br />It is classified under Clan ME in Family M16, Subfamily A, M16.005 (merops).<br />This enzyme is present in abundant amount in endocrine tissues, in rat testis at late stages of spermatogenesis and brain cortex. <br />
  3. 3. INTRODUCTION CONTINUED….<br />The 90 amino acid acidic domain inserted close to NRDc active site differentiates it from other members of the M16 family of metalloendopeptidases.<br />NRDc acts by cleaving arginine residues on N-terminus of basic amino acid or lysine in basic pairs.<br />
  4. 4. IMPORTANCE OF ENZYME<br />Role in Alzheimer Disease, Brain Diseases, Down Syndrome, Leukemia Neoplasms and Neuroblastoma<br />
  5. 5. PREVIOUS STUDIES:<br /><ul><li>Draoui M et al (1997): When treated with retinoic acid for 2 days, SK-N-BE(2) cell lines showed an increase in the expression of NRD Convertase in the cytoplasm.
  6. 6. BE(2)-NA cell lines showed 9-fold increase in the expression of NRD Convertase within few hours to a day treatment.
  7. 7. Interestingly, NRD Convertase expression was down regulated in BE(2)-M(17) cells after 5 days treatment of Retinoic acid.</li></li></ul><li>The authors used immunochemistry and northern blotting to do this study.<br />Real time-PCR is used in this study to get more accurate results.<br />
  8. 8. OBJECTIVES:<br /><ul><li>To investigate the possible modulation of NRD-convertase expression in the presence of Retinoic acid (1 µM, 5 µM), Tamibarotene (1 µM) at different time points.</li></li></ul><li>METHODS<br />
  9. 9. Figure 1. Samples of SK-N-BE cells of Actin and NRDc on gel electrophoresis<br />
  10. 10.
  11. 11. Figure 2. Restriction digest of Actin and NRDc on gel electrophoresis<br />
  12. 12. STD OF ACTIN<br /> CONCENTRATION OF ACTIN<br />
  13. 13. STD OF NRD<br />CONCENTRATION OF NRD<br />
  14. 14. ACTIN IN COMBO WITH NRD<br />CONCENTRATION OF ACTIN<br />
  15. 15. NRD in combo<br />CONCENTRATION OF NRD<br />
  16. 16. CELL TISSUE CULTURE<br />TREATMENTS<br /> 1µM TAMBIROTENE<br /> 1µM RETINOIC ACID<br /> 5µM RETINOIC ACID<br /> TIMES<br /> 6 HOURS<br /> 24 HOURS<br /> 48 HOURS<br /> MEASURE Ct NRD Ct ACT in same tube<br /> Calculate ∆Ct = CtNRD-CtACT<br /> Calculate ∆∆Ct = ∆CtControl-∆CtTreatment<br /> ∆∆Ct = 1 = 2 fold change<br />
  17. 17. ∆∆Ct of NRDc level<br />
  18. 18. FOLD CHANGE OF NRDc LEVEL AFTER DRUG TREATMENT <br />FOLD CHANGE OF NRDc LEVEL AFTER<br />
  19. 19. BAR CHART REPRESENTATION OF FOLD CHANGE OF NRDc LEVEL<br />
  20. 20. Conclusion:<br />As presence of retinoic acid causes changes in expression of NRD levels in human SK B NE cells, it can be concluded that retinoic acid plays an important role in pathogenesis of brain diseases.<br />The results is not similar with previous works. This may be due to small number of samples treated. To get more accurate result this experiment must be repeated with more number of treatment.<br />

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