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In vivo effects of iron-overload on asialoglycoprotein receptor (ASGP-R) transcription

In vivo effects of iron-overload on asialoglycoprotein receptor (ASGP-R) transcription. By Giovanni Castor. Background information on ASGP-R. Discovered in the 1970s by Ashwell and Morell. Background information on ASGP-R. Discovered in the 1970s by Ashwell and Morell

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In vivo effects of iron-overload on asialoglycoprotein receptor (ASGP-R) transcription

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  1. In vivo effects of iron-overload on asialoglycoprotein receptor (ASGP-R) transcription By Giovanni Castor

  2. Background information on ASGP-R • Discovered in the 1970s by Ashwell and Morell California State University Long Beach

  3. Background information on ASGP-R • Discovered in the 1970s by Ashwell and Morell • First reported mammalian lectin California State University Long Beach

  4. Background information on ASGP-R • Discovered in the 1970s by Ashwell and Morell • First reported mammalian lectin • C-type lectin in mammalian livers California State University Long Beach

  5. Structure of ASGP-R • Composed of major and minor subunits California State University Long Beach

  6. Structure of ASGP-R • Composed of major and minor subunits • Humans (2 subunits) California State University Long Beach

  7. Structure of ASGP-R • Composed of major and minor subunits • Humans (2 subunits) • Rats (3 subunits) California State University Long Beach

  8. Structure of ASGP-R • Composed of major and minor subunits • Humans (2 subunits) • Rats (3 subunits) • 80% homology in major subunits California State University Long Beach

  9. Structure of ASGP-R • Composed of major and minor subunits • Humans (2 subunits) • Rats (3 subunits) • 80% homology in major subunits • 58% homology in minor subunits California State University Long Beach

  10. Structure of ASGP-R • Composed of major and minor subunits • Humans (2 subunits) • Rats (3 subunits) • 80% homology in major subunits • 58% homology in minor subunits California State University Long Beach

  11. Known functions of ASGP-R • Recognizes glycoproteins with a terminal galactose (Gal) or N-acetylgalactosamine (Gal-NAc) residues on the N-terminus of the carbohydrate chain California State University Long Beach

  12. Known functions of ASGP-R • Recognizes glycoproteins with a terminal galactose (Gal) or N-acetylgalactosamine (Gal-NAc) residues on the N-terminus of the carbohydrate chain California State University Long Beach

  13. Known functions of ASGP-R • Recognizes glycoproteins with a terminal galactose (Gal) or N-acetylgalactosamine (Gal-NAc) residues on the N-terminus of the carbohydrate chain • Investigators have found that ASGP-R can bind other ligands California State University Long Beach

  14. Significance & Implications • Over 35 years have passed since the discovery of ASGP-R and the true in vivo functions have not been discovered California State University Long Beach

  15. Significance & Implications • Over 35 years have passed since the discovery of ASGP-R and the true in vivo functions have not been discovered • Selective targeting of chemotherapeutic agents and foreign genes California State University Long Beach

  16. Significance & Implications • Over 35 years have passed since the discovery of ASGP-R and the true in vivo functions have not been discovered • Selective targeting of chemotherapeutic agents and foreign genes • Implicated as a site of hepatitis B virus uptake California State University Long Beach

  17. Preliminary Study • It was found that iron-overload of rats increased translation of ASGP-R by 2-3 fold as compared to control rats California State University Long Beach

  18. California State University Long Beach

  19. California State University Long Beach

  20. Question and Hypothesis • Does in vivo iron-overloading of rats alter the transcription of ASGP-R? California State University Long Beach

  21. Question and Hypothesis • Does in vivo iron-overloading of rats alter the transcription of ASGP-R? • We hypothesize that the iron-overload will cause an increase in transcription of all subunits of ASGP-R California State University Long Beach

  22. Testing our hypothesis • Subjects: male Sprague-Dawley rats (AWB: 339001) California State University Long Beach

  23. Testing our hypothesis • Subjects: male Sprague-Dawley rats (AWB: 339001) California State University Long Beach

  24. Testing our hypothesis • Subjects: male Sprague-Dawley rats (AWB: 339001) • Iron overload will be induced by injection of iron-dextran California State University Long Beach

  25. Testing our hypothesis • Subjects: male Sprague-Dawley rats (AWB: 339001) • Iron overload will be induced by injection of iron-dextran • 2 Stages California State University Long Beach

  26. Stage 1: Creation of cDNA probes • Isolation of total RNA California State University Long Beach

  27. Stage 1: Creation of cDNA probes • Isolation of total RNA • RT-PCR California State University Long Beach

  28. Stage 1: Creation of cDNA probes • Isolation of total RNA • RT-PCR • Cloning of PCR products via plasmids California State University Long Beach

  29. Stage 1: Creation of cDNA probes • Isolation of total RNA • RT-PCR • Cloning of PCR products via plasmids • PCR amplication of probe cDNA and isotopic labeling of probe California State University Long Beach

  30. Stage 2: Northern Blot analysis • Identification of increase or decrease of ASGP-R mRNA California State University Long Beach

  31. Expected Results • Anticipate data to support our hypothesis California State University Long Beach

  32. Expected Results • Anticipate data to support our hypothesis • Distinct differences in our Northern Blots California State University Long Beach

  33. Expected Results California State University Long Beach

  34. Concluding Statement • The in vivo function of ASGP-R has yet to be determined California State University Long Beach

  35. Concluding Statement • The in vivo function of ASGP-R has yet to be determined • Cell surface lectins have been implicated in bacterial and viral infection California State University Long Beach

  36. Concluding Statement • The in vivo function of ASGP-R has yet to be determined • Cell surface lectins have been implicated in bacterial and viral infection • Lead to treatments of diseases California State University Long Beach

  37. Acknowledgements • Howard Hughes Medical Institute • California State University Long Beach • Dr. Douglas McAbee • Dr. A.Z. Mason California State University Long Beach

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