Presentation
# 2
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6th Internet World Congress for Biomedical Sciences |
S. Dhakshinamoorthy(1), D. Bloom(2), Anil Jaiswal(3)
(1)(2)Department of Pharmacology. Baylor College of Medicine. - Houston. United States
(3)Baylor College of Medicine - Houston. United States
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Fig. 6. Effect of overexpression of Nrf1 and Nrf2 along with c-Jun on NQO1 gene hARE-, GST Ya gene ARE and g-GCS gene ARE-mediated CAT gene in Hep-G2 cells. The Hep-G2 cells were co-transfected with five micrograms of reporter plasmid hARE-tk-CAT, GST Ya ARE-tk-CAT or g-GCS ARE-tk-CAT and five micrograms of expression plasmids LNCX (vector alone), LNCX-Nrf1, LNCX-Nrf2, and LNCX-c-Jun individually and in the combinations as shown. LNCX-Nrf1R and LNCX-Nrf2R contained Nrf1 and Nrf2 in reverse orientation. These plasmids upon transfection in Hep-G2 cells did not increase the concentration of Nrf1 and Nrf2. On the other hand, LNCX-Nrf1C and LNCX-Nrf2C contained Nrf1 and Nrf2 cDNA in correct orientation. These plasmids upon transfection in Hep-G2 cells resulted in overexpression of Nrf1 and Nrf2. Five micrograms of RSV-b-galactosidase plasmid was included in each case as internal control of transfection efficiency. The total amount of DNA for transfection in each case was normalized to 20 micrograms with LNCX (vector alone plasmid). Forty-eight hours after transfection, the cells were analyzed for b-galactosidasae and CAT activities. The acetylated 14C-chloramphenicol in the upper spots on TLC plates were cut out, counted and used to calculate CAT activity. The various results are presented as mean ± S.E. of five independent transfection experiments.
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Fig. 7. Alignment of basic and lecine zipper regions of Nrf1 and Nrf2 with Jun (c-Jun, Jun-B and Jun-D) proteins. The conserved cysteine in basic region and leucines in leucine zipper region are shown in bold letters. Nrf1 contains two mutated leucines (leucine # 4 and 5) and Nrf2 contains one mutated leucine (leucine # 4).
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Fig. 8. Role of c-Fos and Fra-1 in repression of ARE-mediated NQO1 and GST Ya genes expression. A. Hep-G2 cells were co-transfected with 10 mg of reporter plasmid hARE-tk-CAT and different concentration of c-Fos expression plasmid LNCX-c-Fos. Five mg of RSV-b-galactosidase plasmid was included in each case as internal control of transfection efficiency. Forty eight hours after transfection, the cells were analyzed for b-galactosidase and CAT activities. B. & C. c-Fos -/- mice were purchased from Jackson Laboratories (ME). The mice were sacrificed and their organs removed and analysed for NQO1 and GST Ya proteins by Western blotting and probing with respective antibodies and activity by standard procedures. NQO1 activities are expressed as mean (± SD), in nmoles of 2,6-dichlorophenolindophenol reduced/min./mg protein. The GST YA activities are expressed as mean (± SD), in nmoles of conjugation of 1-chloro2,4-dinitrobenzene/min./mg protein.
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Fig. 9. Amino acid sequence of INrf2. The cysteines are shown in bold letters.
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| Fig. 10. Hypothetical model showing the mechanism of signal transduction from xenobiotics and antioxidants to Nrf and Jun proteins that bind to ARE and activate the NQO1 gene expression. ARE, antioxidant response element, Nrf1 and Nrf2, NF-E2 related factors, c-Jun, early response proto-oncogene, NQO1, NAD(P)H:quinone oxidoreductase1 |
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