AIM： To assess the effects of obstructive cholestasis on a wider range of gene expression using microarray technology. METHODS： Male C57BI_/6J mice underwent common bile duct ligation （BDL） and were matched with p...AIM： To assess the effects of obstructive cholestasis on a wider range of gene expression using microarray technology. METHODS： Male C57BI_/6J mice underwent common bile duct ligation （BDL） and were matched with pairfed sham-operated controls. After 7 d, the animals were sacrificed and total RNA was isolated from livers and kidneys. Equal amounts of RNA from each tissue were pooled for each group and hybridized to Affymetrix GeneChip^MG-U74Av2 containing a total of 12488 probe sets. Data analysis was performed using GeneSpring 6.0 software. Northern analysis and immunofluorescence were used for validation. RESULTS： In sham-operated and BDL mice, 44 and 50% of 12488 genes were expressed in livers, whereas 49 and 51% were expressed in kidneys, respectively. Seven days after BDL, 265 liver and 112 kidney genes with GeneOntology annotation were up-regulated and 113 liver and 36 kidney genes were down-regulated in comparison with sham-operated controls. Many genes were commonly regulated in both tissues and metabolism-related genes represented the largest functional group. CONCLUSION： Following BDL, microarray analysis reveals a broad range of gene alterations in both liver and kidney.展开更多
Neuronal nicotinic acetylcholine receptors （nAChRs） containing Gt4 and 132 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affin- ity. These nAChRs are invol...Neuronal nicotinic acetylcholine receptors （nAChRs） containing Gt4 and 132 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affin- ity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between a4β2-containing （a4β2） nAChRs and other proteins remains limited. In this study, we identified proteins that inter- act with ct4β2 nAChRs in a gene-dose dependent pattern by immunopurifying β2 nAChRs from mice that differ in ct4 and β2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation （iTRAQ）. Reduced expression of either the a4 or the β2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-imrnunoprecipitated with these nAChRs. Furthermore, stratified lin- ear regression analysis indicated that levels of 17 proteins was correlated significantly with expres- sion of at4β2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a β2 nAChR interactome and describe a novel technique used to discover potential targets for pharma- cological manipulation of a4β2 nAChRs and their downstream signaling mechanisms.展开更多
We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database（YPED） that is used by investigators at more than 300 institutions worldwide. YPED ...We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database（YPED） that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a singlelaboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography–tandem mass spectrometry（LC–MS/MS） database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring（MRM）/selective reaction monitoring（SRM） assay development. We have linked YPED＇s database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results.展开更多
基金Supported by the USPHS grants DK 25636 (J. L. B.)the Yale Liver Center Cellular and Molecular Physiology and Morphology Cores (P30-34989)the Deutsche Forschungsgemeinschaft Grant DE 872/1-1 (G. U. D.)
文摘AIM： To assess the effects of obstructive cholestasis on a wider range of gene expression using microarray technology. METHODS： Male C57BI_/6J mice underwent common bile duct ligation （BDL） and were matched with pairfed sham-operated controls. After 7 d, the animals were sacrificed and total RNA was isolated from livers and kidneys. Equal amounts of RNA from each tissue were pooled for each group and hybridized to Affymetrix GeneChip^MG-U74Av2 containing a total of 12488 probe sets. Data analysis was performed using GeneSpring 6.0 software. Northern analysis and immunofluorescence were used for validation. RESULTS： In sham-operated and BDL mice, 44 and 50% of 12488 genes were expressed in livers, whereas 49 and 51% were expressed in kidneys, respectively. Seven days after BDL, 265 liver and 112 kidney genes with GeneOntology annotation were up-regulated and 113 liver and 36 kidney genes were down-regulated in comparison with sham-operated controls. Many genes were commonly regulated in both tissues and metabolism-related genes represented the largest functional group. CONCLUSION： Following BDL, microarray analysis reveals a broad range of gene alterations in both liver and kidney.
基金supported by the National Institutes of Health (NIH) [Grant No. DA14241, DA018343 (to NIDA Proteomics Center at Yale University) and UL1 RR024139 (to Yale Clinical and Translational Science Award)]supported by NIH (Grant No. T32 MH014276)+1 种基金JML was supported by NIH (Grant No. NS11323)MJM and SRG were supported by NIH (Grant No. DA003194 and DA015663)
文摘Neuronal nicotinic acetylcholine receptors （nAChRs） containing Gt4 and 132 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affin- ity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between a4β2-containing （a4β2） nAChRs and other proteins remains limited. In this study, we identified proteins that inter- act with ct4β2 nAChRs in a gene-dose dependent pattern by immunopurifying β2 nAChRs from mice that differ in ct4 and β2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation （iTRAQ）. Reduced expression of either the a4 or the β2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-imrnunoprecipitated with these nAChRs. Furthermore, stratified lin- ear regression analysis indicated that levels of 17 proteins was correlated significantly with expres- sion of at4β2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a β2 nAChR interactome and describe a novel technique used to discover potential targets for pharma- cological manipulation of a4β2 nAChRs and their downstream signaling mechanisms.
基金supported in part by the National Institutes of Health of the United States(Grant Nos.UL1 RR024139 to Yale Clinical and Translational Science Award,1S10OD018034-01 to 6500 QTrap Mass Spectrometer for Yale University,1S10RR026707-01 to 5500QTrap Mass Spectrometer for Yale University,P30DA018343 to Yale/NIDA Neuroproteomics Center and NIDDK-K01DK089006 awarded to JR)
文摘We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database（YPED） that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a singlelaboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography–tandem mass spectrometry（LC–MS/MS） database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring（MRM）/selective reaction monitoring（SRM） assay development. We have linked YPED＇s database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results.
