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

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Presentation on theme: "In vivo effects of iron- overload on asialoglycoprotein receptor (ASGP-R) transcription By Giovanni Castor."— Presentation transcript:

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

2 5/05/2004 California State University Long Beach Background information on ASGP-R Discovered in the 1970s by Ashwell and Morell Discovered in the 1970s by Ashwell and Morell

3 5/05/2004 California State University Long Beach Background information on ASGP-R Discovered in the 1970s by Ashwell and Morell Discovered in the 1970s by Ashwell and Morell First reported mammalian lectin First reported mammalian lectin

4 5/05/2004 California State University Long Beach Background information on ASGP-R Discovered in the 1970s by Ashwell and Morell Discovered in the 1970s by Ashwell and Morell First reported mammalian lectin First reported mammalian lectin C-type lectin in mammalian livers C-type lectin in mammalian livers

5 5/05/2004 California State University Long Beach Structure of ASGP-R Composed of major and minor subunits Composed of major and minor subunits

6 5/05/2004 California State University Long Beach Structure of ASGP-R Composed of major and minor subunits Composed of major and minor subunits Humans (2 subunits) Humans (2 subunits)

7 5/05/2004 California State University Long Beach Structure of ASGP-R Composed of major and minor subunits Composed of major and minor subunits Humans (2 subunits) Humans (2 subunits) Rats (3 subunits) Rats (3 subunits)

8 5/05/2004 California State University Long Beach Structure of ASGP-R Composed of major and minor subunits Composed of major and minor subunits Humans (2 subunits) Humans (2 subunits) Rats (3 subunits) Rats (3 subunits) 80% homology in major subunits 80% homology in major subunits

9 5/05/2004 California State University Long Beach Structure of ASGP-R Composed of major and minor subunits Composed of major and minor subunits Humans (2 subunits) Humans (2 subunits) Rats (3 subunits) Rats (3 subunits) 80% homology in major subunits 80% homology in major subunits 58% homology in minor subunits 58% homology in minor subunits

10 5/05/2004 California State University Long Beach Structure of ASGP-R Composed of major and minor subunits Composed of major and minor subunits Humans (2 subunits) Humans (2 subunits) Rats (3 subunits) Rats (3 subunits) 80% homology in major subunits 80% homology in major subunits 58% homology in minor subunits 58% homology in minor subunits

11 5/05/2004 California State University Long Beach 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 Recognizes glycoproteins with a terminal galactose (Gal) or N- acetylgalactosamine (Gal-NAc) residues on the N-terminus of the carbohydrate chain

12 5/05/2004 California State University Long Beach 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 Recognizes glycoproteins with a terminal galactose (Gal) or N- acetylgalactosamine (Gal-NAc) residues on the N-terminus of the carbohydrate chain

13 5/05/2004 California State University Long Beach 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 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 Investigators have found that ASGP-R can bind other ligands

14 5/05/2004 California State University Long Beach Significance & Implications Over 35 years have passed since the discovery of ASGP-R and the true in vivo functions have not been discovered Over 35 years have passed since the discovery of ASGP-R and the true in vivo functions have not been discovered

15 5/05/2004 California State University Long Beach Significance & Implications Over 35 years have passed since the discovery of ASGP-R and the true in vivo functions have not been discovered 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 Selective targeting of chemotherapeutic agents and foreign genes

16 5/05/2004 California State University Long Beach Significance & Implications Over 35 years have passed since the discovery of ASGP-R and the true in vivo functions have not been discovered 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 Selective targeting of chemotherapeutic agents and foreign genes Implicated as a site of hepatitis B virus uptake Implicated as a site of hepatitis B virus uptake

17 5/05/2004 California State University Long Beach Preliminary Study It was found that iron-overload of rats increased translation of ASGP-R by 2-3 fold as compared to control rats It was found that iron-overload of rats increased translation of ASGP-R by 2-3 fold as compared to control rats

18 5/05/2004 California State University Long Beach

19 5/05/2004 California State University Long Beach

20 5/05/2004 California State University Long Beach Question and Hypothesis Does in vivo iron-overloading of rats alter the transcription of ASGP-R? Does in vivo iron-overloading of rats alter the transcription of ASGP-R?

21 5/05/2004 California State University Long Beach Question and Hypothesis Does in vivo iron-overloading of rats alter the transcription of ASGP-R? 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 We hypothesize that the iron-overload will cause an increase in transcription of all subunits of ASGP-R

22 5/05/2004 California State University Long Beach Testing our hypothesis Subjects: male Sprague- Dawley rats (AWB: 339001) Subjects: male Sprague- Dawley rats (AWB: 339001)

23 5/05/2004 California State University Long Beach Testing our hypothesis Subjects: male Sprague- Dawley rats (AWB: 339001) Subjects: male Sprague- Dawley rats (AWB: 339001)

24 5/05/2004 California State University Long Beach Testing our hypothesis Subjects: male Sprague- Dawley rats (AWB: 339001) Subjects: male Sprague- Dawley rats (AWB: 339001) Iron overload will be induced by injection of iron-dextran Iron overload will be induced by injection of iron-dextran

25 5/05/2004 California State University Long Beach Testing our hypothesis Subjects: male Sprague- Dawley rats (AWB: 339001) Subjects: male Sprague- Dawley rats (AWB: 339001) Iron overload will be induced by injection of iron-dextran Iron overload will be induced by injection of iron-dextran 2 Stages 2 Stages

26 5/05/2004 California State University Long Beach Stage 1: Creation of cDNA probes Isolation of total RNA Isolation of total RNA

27 5/05/2004 California State University Long Beach Stage 1: Creation of cDNA probes Isolation of total RNA Isolation of total RNA RT-PCR RT-PCR

28 5/05/2004 California State University Long Beach Stage 1: Creation of cDNA probes Isolation of total RNA Isolation of total RNA RT-PCR RT-PCR Cloning of PCR products via plasmids Cloning of PCR products via plasmids

29 5/05/2004 California State University Long Beach Stage 1: Creation of cDNA probes Isolation of total RNA Isolation of total RNA RT-PCR RT-PCR Cloning of PCR products via plasmids Cloning of PCR products via plasmids PCR amplication of probe cDNA and isotopic labeling of probe PCR amplication of probe cDNA and isotopic labeling of probe

30 5/05/2004 California State University Long Beach Stage 2: Northern Blot analysis Identification of increase or decrease of ASGP-R mRNA Identification of increase or decrease of ASGP-R mRNA

31 5/05/2004 California State University Long Beach Expected Results Anticipate data to support our hypothesis Anticipate data to support our hypothesis

32 5/05/2004 California State University Long Beach Expected Results Anticipate data to support our hypothesis Anticipate data to support our hypothesis Distinct differences in our Northern Blots Distinct differences in our Northern Blots

33 5/05/2004 California State University Long Beach Expected Results

34 5/05/2004 California State University Long Beach Concluding Statement The in vivo function of ASGP-R has yet to be determined The in vivo function of ASGP-R has yet to be determined

35 5/05/2004 California State University Long Beach Concluding Statement The in vivo function of ASGP-R has yet to be determined The in vivo function of ASGP-R has yet to be determined Cell surface lectins have been implicated in bacterial and viral infection Cell surface lectins have been implicated in bacterial and viral infection

36 5/05/2004 California State University Long Beach Concluding Statement The in vivo function of ASGP-R has yet to be determined The in vivo function of ASGP-R has yet to be determined Cell surface lectins have been implicated in bacterial and viral infection Cell surface lectins have been implicated in bacterial and viral infection Lead to treatments of diseases Lead to treatments of diseases

37 5/05/2004 California State University Long Beach Acknowledgements Howard Hughes Medical Institute Howard Hughes Medical Institute California State University Long Beach California State University Long Beach Dr. Douglas McAbee Dr. Douglas McAbee Dr. A.Z. Mason Dr. A.Z. Mason

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