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Expression Data from Sod2-/- and Sod2+/+ Mouse Erythroblasts.

ABSTRACT: The mitochondrial superoxide dismutase (SOD2) is a major antioxidant protein which detoxifies superoxide anion radicals generated by mitochondrial respiration (Weisiger and Fridovich, J. Biol. Chem. 1973). We designed a model of oxidative stress-induced anemia caused by SOD2-deficiency (Friedman et al. J. Exp. Med. 2001). Our previous work showed that mice reconstituted with SOD2-deficient hematopoietic stem cells develop an anemia with striking similarity to human sideroblastic anemia (SA) (Friedman et al. Blood 2004; Martin et al. Exp Hematol 2005). Our overall goal was to define early events in the pathogenesis of SOD2-deficiency SA and, in particular, to identify genes involved in the response of erythroid progenitors to oxidative stress. We compared gene expression of sorted TER-119+ CD71+ erythroblasts from SOD2-/- ('KO') versus Sod2+/+ ('WT') hematopoietic stem cell recipients using cDNA microarrays. Samples used in this study were re-hybridized with GLYCOv2 oligonucleotide arrays. GLYCOv2 oligonucleotide arrays are custom Affymetrix GeneChip arrays (Affymetrix, Santa Clara, CA) designed for the Functional Glycomics Gateway, collaboration between the Consortium for Functional Glycomics (CFG, http://www.functionalglycomics.org/) and Nature Publishing Group. A complete description of the array can be found at http://www.functionalglycomics.org/static/consortium/resources/resourcecoree.shtml . Hybridization results and quality control details can be found at https://www.functionalglycomics.org/glycomics/publicdata/microarray.jsp , by clicking on the ‘Raw Data’ icon link for microarray experiment ‘Jeff Friedman 1: Sod2 KO anemic mice’. Briefly but importantly, one Sod2-/- sample, which was prepared separately, and whose GAPDH 3'/5' ratio was off range, was legitimately removed from the analysis of the GLYCOv2 and 430 2.0 arrays. Keywords: Genotype comparison, genetic modification

ORGANISM(S): Mus musculus

PROVIDER: GSE8726 | GEO | 2008/08/06

SECONDARY ACCESSION(S): PRJNA101979

REPOSITORIES: GEO

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Project description:The mitochondrial superoxide dismutase (SOD2) is a major antioxidant protein which detoxifies superoxide anion radicals generated by mitochondrial respiration (Weisiger and Fridovich, J. Biol. Chem. 1973). We designed a model of oxidative stress-induced anemia caused by SOD2-deficiency (Friedman et al. J. Exp. Med. 2001). Our previous work showed that mice reconstituted with SOD2-deficient hematopoietic stem cells develop an anemia with striking similarity to human sideroblastic anemia (SA) (Friedman et al. Blood 2004; Martin et al. Exp Hematol 2005). Our overall goal was to define early events in the pathogenesis of SOD2-deficiency SA and, in particular, to identify genes involved in the response of erythroid progenitors to oxidative stress. We compared gene expression of sorted TER-119+ CD71+ erythroblasts from SOD2-/- ('KO') versus Sod2+/+ ('WT') hematopoietic stem cell recipients using cDNA microarrays. Samples used in this study were re-hybridized with GLYCOv2 oligonucleotide arrays. GLYCOv2 oligonucleotide arrays are custom Affymetrix GeneChip arrays (Affymetrix, Santa Clara, CA) designed for the Functional Glycomics Gateway, collaboration between the Consortium for Functional Glycomics (CFG, http://www.functionalglycomics.org/) and Nature Publishing Group. A complete description of the array can be found at http://www.functionalglycomics.org/static/consortium/resources/resourcecoree.shtml . Hybridization results and quality control details can be found at https://www.functionalglycomics.org/glycomics/publicdata/microarray.jsp , by clicking on the âRaw Dataâ icon link for microarray experiment âJeff Friedman 1: Sod2 KO anemic miceâ. Briefly but importantly, one Sod2-/- sample, which was prepared separately, and whose GAPDH 3'/5' ratio was off range, was legitimately removed from the analysis of the GLYCOv2 and 430 2.0 arrays. Experiment Overall Design: We sorted Sod2-/- and Sod2+/+ size-matched mouse erythroblasts based on the expression of two developmental surface markers, CD71 and TER-119. Total RNA from 4 biological replicates per genotype were extracted and hybridized on 8 Affymetrix Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA). After quality controls (see scan protocol), analysis was performed with GeneSifter (VizX Labs, Seattle, WA) using 4 Sod2+/+ (control samples, WT) and 3 Sod2-/- (KO) replicates.

Project description:Gene expression profiling was performed using Anemic Sod 2 Knockout mice and was compared to Gene expression profiles from Sod 2 WT mice. Background: Our laboratory is interested in the role of oxidative stress in aging and human disease. Currently, a major focus of our efforts is the characterization of a murine hemolytic anemia resulting from oxidative stress, a model for the human disorder Sideroblastic Anemia. This model is based upon transplantation of hematopoietic stem cells from mice that are deficient in the antioxidant protein SOD2 (Friedman J. Exp. Med. 2001 193:925). SOD2 protects against the cytotoxicity of mitochondrial superoxide produced as a byproduct of oxidative phosphorylation (Weisiger J. Biol. Chem. 1973 248:4793). In characterization of the model to date, we have focused upon RBC proteomic analysis to identify 41 proteins differentially expressed when comparing membrane and soluble RBC fractions from SOD2-/- vs. SOD2+/+ mice (Friedman et al. submitted). In addition, we have characterized the in vivo response of animals to a novel catalytic antioxidant (Euk-189) that has b! oth SOD and catalase activities. In addition to proteins involved in mitochondrial function and stress response we find a number of cell surface molecules with significant differential expression. Thrombospondin is found at higher levels in SOD2-/- red cell membrane fractions. This is an extracellular matrix glycoprotein involved in cell adhesion and migration that interacts with glycosaminoglycans (especially the heparan sulfate proteoglycan perlecan) expressed at the endothelial cell surface (Feitsma J. Biol. Chem. 2000 275:9396, Vischer Eur. J. Cell. Biol. 1997 73:332, Sun J. Biol. Chem. 1989 15:2885). Thrombospondin is likely an acquired surface molecule on our red cells, which is of interest as this molecule has been found on the surface of sickle red cells and appears to mediate abnormal adhesion of red cells to vessel wall (Hillery Blood 1996 87:4879, Sugihara Blood 1992 15:2634). CD44 is also found at higher levels in SOD2-/- membrane fractions. CD44 is an adhesion receptor for the high molecular weight polysaccharide hyaluronic acid and has been shown recently to be involved in the trafficking of stem cells to the bone marrow (Avigdor Blood January 2004). Higher expression of these molecules on the surface of SOD2-/- red cells may affect their adhesive interactions with vascular endothelium and may in part explain the early removal of these cells from the circulationóresulting in the observed hemolytic anemia. Some proteins that accumulate on the surface of SOD2 deficient red cells are likely molecular damage sensors, binding to surfaces altered by oxidative damage. The role of specific glycoproteins in such binding remains to be elucidated. Additional surface proteins from red cells show differential expression, many of which are also glycosylated. Planned Experiment: As the next step in characterization of the anemia model, we will compare the gene expression profile of red cell progenitors isolated from Sod2-/- and Sod2+/+ animals. We have defined cell-sorting conditions for isolation of erythroid progenitor cells from marrow of our mice.

Project description:Aim: To determine the effect of an AtrbohC mutation on the gene expression pattern in primary root tissue, to identify candidate genes acting downstream of AtrbohC, particularly any encoding antioxidant-related proteins, signal transduction components or proteins known to be required for normal root-hair development. Background: Root-hairs are a model system for investigating plant cell polarity. The root-hair mutant rhd2 (Schiefelbein and Somerville, 1990. Plant Cell, 2:235) has short hairs that burst at their tips, (Jones and Smirnoff, unpublished). RHD2 has been cloned and is identical to AtrbohC (L. Dolan, pers. comm.), which encodes a homologue of the superoxide-generating neutrophil respiratory burst oxidase catalytic subunit gp91phox (Torres et al., 1998. Plant J., 14:365). Superoxide rapidly dismutates to hydrogen peroxide (H2O2), suggesting that the rhd2 phenotype may result from reduced H2O2 levels in root-hair cells. Low doses of exogenous antioxidants phenocopy the rhd2 root-hair phenotype in wild-type plants (Jones and Smirnoff, unpublished) further supporting a role for H2O2 in root-hair growth. Fluorescent dyes that detect H2O2 show distinct localisation patterns in growing root-hair cells, (Jones and Smirnoff, unpublished). H2O2 may be an important second messenger in plant cell signalling with proposed roles in the development of cotton fibres (Potikha et al., 1999. Plant Physiol., 119: 849) and in ABA-induced stomatal closure (Zhang et al., 2001. Plant Physiol., 126: 1438). In cultured Arabidopsis cells H2O2 induces gene expression, including that of a gp91phox homologue, (Desikan et al., 1998. J. Exp. Bot., 49: 1767; Desikan, et al., 2000. Free Rad. Biol. Med., 28: 773; Baxter-Burrell et al., 2002. Science, 296: 2026) and activates a MAP kinase cascade (Desikan et al., 1999. J. Exp Bot., 50: 1863). cDNA microarray technology has been used previously to examine the effects of H2O2 on gene expression during oxidative stress (Desikan et al., 2001. Plant Physiol., 127: 159). We wish to investigate the effects of H2O2 on gene expression during root development using the rhd2 mutant. We are currently determining the expression pattern of RHD2. By extracting RNA from the small region of the primary root (for wild-type and rhd2 plants grown in sterile conditions) where root hairs are growing we hope to enrich for root-hair RNAs. This may reveal candidate genes that could be examined more closely at the single-cell level. This approach will provide new insights into the role of H2O2 in root-hair development. Keywords: strain_or_line_design

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Expression Data from Sod2-/- and Sod2+/+ Mouse Erythroblasts.

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