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In vivo effects of iron-overload on asialoglycoprotein receptor (ASGP-R) transcription
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Transcript of In vivo effects of iron-overload on asialoglycoprotein receptor (ASGP-R) transcription
In vivoIn vivo effects of effects of iron-overload on iron-overload on
asialoglycoprotein asialoglycoprotein receptor (ASGP-R) receptor (ASGP-R)
transcriptiontranscriptionByBy
Giovanni CastorGiovanni Castor
5/05/2004 California State University Long Beach
Background information on Background information on ASGP-RASGP-R
Discovered in the 1970s by Ashwell and Discovered in the 1970s by Ashwell and MorellMorell
5/05/2004 California State University Long Beach
Background information on Background information on ASGP-RASGP-R
Discovered in the 1970s by Ashwell and Discovered in the 1970s by Ashwell and MorellMorell
First reported mammalian lectinFirst reported mammalian lectin
5/05/2004 California State University Long Beach
Background information on Background information on ASGP-RASGP-R
Discovered in the 1970s by Ashwell and Discovered in the 1970s by Ashwell and MorellMorell
First reported mammalian lectinFirst reported mammalian lectin
C-type lectin in mammalian liversC-type lectin in mammalian livers
5/05/2004 California State University Long Beach
Structure of ASGP-RStructure of ASGP-R Composed of major Composed of major
and minor subunitsand minor subunits
5/05/2004 California State University Long Beach
Structure of ASGP-RStructure of ASGP-R Composed of major Composed of major
and minor subunitsand minor subunits Humans (2 subunits)Humans (2 subunits)
5/05/2004 California State University Long Beach
Structure of ASGP-RStructure of ASGP-R Composed of major Composed of major
and minor subunitsand minor subunits Humans (2 subunits)Humans (2 subunits) Rats (3 subunits)Rats (3 subunits)
5/05/2004 California State University Long Beach
Structure of ASGP-RStructure of ASGP-R Composed of major Composed of major
and minor subunitsand minor subunits Humans (2 subunits)Humans (2 subunits) Rats (3 subunits)Rats (3 subunits) 80% homology in 80% homology in
major subunitsmajor subunits
5/05/2004 California State University Long Beach
Structure of ASGP-RStructure of ASGP-R Composed of major Composed of major
and minor subunitsand minor subunits Humans (2 subunits)Humans (2 subunits) Rats (3 subunits)Rats (3 subunits) 80% homology in 80% homology in
major subunitsmajor subunits 58% homology in 58% homology in
minor subunitsminor subunits
5/05/2004 California State University Long Beach
Structure of ASGP-RStructure of ASGP-R Composed of major Composed of major
and minor subunitsand minor subunits Humans (2 subunits)Humans (2 subunits) Rats (3 subunits)Rats (3 subunits) 80% homology in 80% homology in
major subunitsmajor subunits 58% homology in 58% homology in
minor subunitsminor subunits
5/05/2004 California State University Long Beach
Known functions of Known functions of ASGP-RASGP-R
Recognizes glycoproteins Recognizes glycoproteins with a terminal galactose with a terminal galactose (Gal) or N-(Gal) or N-acetylgalactosamine (Gal-acetylgalactosamine (Gal-NAc) residues on the N-NAc) residues on the N-terminus of the terminus of the carbohydrate chaincarbohydrate chain
5/05/2004 California State University Long Beach
Known functions of Known functions of ASGP-RASGP-R
Recognizes glycoproteins Recognizes glycoproteins with a terminal galactose with a terminal galactose (Gal) or N-(Gal) or N-acetylgalactosamine (Gal-acetylgalactosamine (Gal-NAc) residues on the N-NAc) residues on the N-terminus of the terminus of the carbohydrate chaincarbohydrate chain
5/05/2004 California State University Long Beach
Known functions of Known functions of ASGP-RASGP-R
Recognizes glycoproteins Recognizes glycoproteins with a terminal galactose with a terminal galactose (Gal) or N-(Gal) or N-acetylgalactosamine (Gal-acetylgalactosamine (Gal-NAc) residues on the N-NAc) residues on the N-terminus of the terminus of the carbohydrate chaincarbohydrate chain
Investigators have found Investigators have found that ASGP-R can bind that ASGP-R can bind other ligandsother ligands
5/05/2004 California State University Long Beach
Significance & Significance & ImplicationsImplications
Over 35 years have passed since the Over 35 years have passed since the discovery of ASGP-R and the true discovery of ASGP-R and the true in in vivovivo functions have not been functions have not been discovereddiscovered
5/05/2004 California State University Long Beach
Significance & Significance & ImplicationsImplications
Over 35 years have passed since the Over 35 years have passed since the discovery of ASGP-R and the true discovery of ASGP-R and the true in in vivovivo functions have not been functions have not been discovereddiscovered
Selective targeting of Selective targeting of chemotherapeutic agents and chemotherapeutic agents and foreign genesforeign genes
5/05/2004 California State University Long Beach
Significance & Significance & ImplicationsImplications
Over 35 years have passed since the Over 35 years have passed since the discovery of ASGP-R and the true discovery of ASGP-R and the true in in vivovivo functions have not been functions have not been discovereddiscovered
Selective targeting of Selective targeting of chemotherapeutic agents and chemotherapeutic agents and foreign genesforeign genes
Implicated as a site of hepatitis B Implicated as a site of hepatitis B virus uptakevirus uptake
5/05/2004 California State University Long Beach
Preliminary StudyPreliminary Study
It was found that iron-overload of It was found that iron-overload of rats increased translation of ASGP-R rats increased translation of ASGP-R by 2-3 fold as compared to control by 2-3 fold as compared to control ratsrats
5/05/2004 California State University Long Beach
5/05/2004 California State University Long Beach
5/05/2004 California State University Long Beach
Question and HypothesisQuestion and Hypothesis
Does Does in vivoin vivo iron-overloading of rats iron-overloading of rats alter the transcription of ASGP-R?alter the transcription of ASGP-R?
5/05/2004 California State University Long Beach
Question and HypothesisQuestion and Hypothesis
Does Does in vivoin vivo iron-overloading of rats iron-overloading of rats alter the transcription of ASGP-R?alter the transcription of ASGP-R?
We hypothesize that the iron-We hypothesize that the iron-overload will cause an increase in overload will cause an increase in transcription of all subunits of ASGP-transcription of all subunits of ASGP-RR
5/05/2004 California State University Long Beach
Testing our hypothesisTesting our hypothesis Subjects: male Subjects: male
Sprague-Dawley rats Sprague-Dawley rats (AWB: 339001)(AWB: 339001)
5/05/2004 California State University Long Beach
Testing our hypothesisTesting our hypothesis Subjects: male Subjects: male
Sprague-Dawley rats Sprague-Dawley rats (AWB: 339001)(AWB: 339001)
5/05/2004 California State University Long Beach
Testing our hypothesisTesting our hypothesis Subjects: male Subjects: male
Sprague-Dawley rats Sprague-Dawley rats (AWB: 339001)(AWB: 339001)
Iron overload will be Iron overload will be induced by injection induced by injection of iron-dextran of iron-dextran
5/05/2004 California State University Long Beach
Testing our hypothesisTesting our hypothesis Subjects: male Subjects: male
Sprague-Dawley rats Sprague-Dawley rats (AWB: 339001)(AWB: 339001)
Iron overload will be Iron overload will be induced by injection induced by injection of iron-dextran of iron-dextran
2 Stages2 Stages
5/05/2004 California State University Long Beach
Stage 1: Creation of Stage 1: Creation of cDNA probescDNA probes
Isolation of total RNAIsolation of total RNA
5/05/2004 California State University Long Beach
Stage 1: Creation of Stage 1: Creation of cDNA probescDNA probes
Isolation of total RNAIsolation of total RNA RT-PCRRT-PCR
5/05/2004 California State University Long Beach
Stage 1: Creation of Stage 1: Creation of cDNA probescDNA probes
Isolation of total RNAIsolation of total RNA RT-PCRRT-PCR Cloning of PCR products via Cloning of PCR products via
plasmidsplasmids
5/05/2004 California State University Long Beach
Stage 1: Creation of Stage 1: Creation of cDNA probescDNA probes
Isolation of total RNAIsolation of total RNA RT-PCRRT-PCR Cloning of PCR products via Cloning of PCR products via
plasmidsplasmids PCR amplication of probe cDNA and PCR amplication of probe cDNA and
isotopic labeling of probeisotopic labeling of probe
5/05/2004 California State University Long Beach
Stage 2: Northern Blot Stage 2: Northern Blot analysisanalysis
Identification of increase or Identification of increase or decrease of ASGP-R mRNAdecrease of ASGP-R mRNA
5/05/2004 California State University Long Beach
Expected ResultsExpected Results
Anticipate data to support our Anticipate data to support our hypothesishypothesis
5/05/2004 California State University Long Beach
Expected ResultsExpected Results
Anticipate data to support our Anticipate data to support our hypothesishypothesis
Distinct differences in our Northern Distinct differences in our Northern BlotsBlots
5/05/2004 California State University Long Beach
Expected ResultsExpected Results
5/05/2004 California State University Long Beach
Concluding StatementConcluding Statement
The The in vivoin vivo function of ASGP-R has function of ASGP-R has yet to be determinedyet to be determined
5/05/2004 California State University Long Beach
Concluding StatementConcluding Statement
The The in vivoin vivo function of ASGP-R has function of ASGP-R has yet to be determinedyet to be determined
Cell surface lectins have been Cell surface lectins have been implicated in bacterial and viral implicated in bacterial and viral infectioninfection
5/05/2004 California State University Long Beach
Concluding StatementConcluding Statement
The The in vivoin vivo function of ASGP-R has function of ASGP-R has yet to be determinedyet to be determined
Cell surface lectins have been Cell surface lectins have been implicated in bacterial and viral implicated in bacterial and viral infectioninfection
Lead to treatments of diseasesLead to treatments of diseases
5/05/2004 California State University Long Beach
AcknowledgementsAcknowledgements
Howard Hughes Medical InstituteHoward Hughes Medical Institute California State University Long California State University Long
BeachBeach Dr. Douglas McAbeeDr. Douglas McAbee Dr. A.Z. MasonDr. A.Z. Mason