Mouse liver transcription profiling by array
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Dawn Applegate
PROVIDER: E-GEOD-55084 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Objective: L-type calcium channels (LTCC) homeostatically regulate calcium on a beat by beat basis, but also provide Ca that over long time scales may contribute to transcriptional regulation. We previously showed that sustained LTCC blockade (CCB) elicits LTCC remodeling in ventricular cardiac myocytes (CM). Here we hypothesize that sustained CCB has broad effects on the expression of genes involved in calcium handling. Methods and Results: Therefore, we subjected adult mice to sustained CCB for 24 hours and performed gene expression profiling. In comparison to vehicle-only control animals, 231 genes were up-regulated, and 111 genes were down-regulated by sustained LTCC blockade (p <0.01). Gene ontology analysis suggested that the CaMKIIdelta signaling pathway was up-regulated in these cells. Unexpectedly, phosphorylation of phospholamban (PLN) at threonine17 (Thr17), an index of CaMKIIdelta activity, was not changed by sustained CCB; however, the degree of phosphorylation of the neighboring PLN-Ser16 substrate site for PKA was significantly reduced by sustained CCB compared to control. Gene expression profiling suggested no change in PKA, but it showed that protein phosphatase 2A (PP2A) mRNA increased, and immunoblots demonstrated that PP2Ac-alpha protein was significantly increased by sustained CCB. Consistent with elevated PP2Ac-alpha protein expression LTCC exhibited decreased phosphorylation of the C-terminal Ser1928 PKA substrate site. Conclusions: We conclude that sustained CCB elicits a spectrum of transcriptional events, including compensatory up-regulation of LTCC and PP2Ac-alpha. Although this study is restricted to mouse, these results suggest the new hypothesis that clinically-relevant sustained LTCC blockade in humans results in changes in gene regulation in the heart. Keywords: L-type calcium channel, calcium channel blockade, verapamil Female and male ICR mice (12-14 weeks age) weighing between 25 and 30 grams were anesthetized with a ketamine/xylazine mixture ( i.p.) allowing the subcutaneous implantation of miniosmotic pumps (Alzet, model 2001). The pumps were filled with either verapamil or vehicle (0.02% ascorbic acid). Control animals carried mini-pumps with vehicle and control animals were investigated in parallel with each set of experimental animals. Mini pumps delivered verapamil at 3.6 mg/kg/day for 24 (RNA)-48 (protein) hours. After treatment animals were anesthetized and weighed. Hearts were excised, rinsed, blotted dry, weighed, and then frozen on dry ice and the stored at -80oC until studied. Animals were anesthetized and euthanized according to animal protocols approved by the University of Kentucky Institutional Animal Care and Use Committee. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85-23, revised 1996). Left ventricular free wall from female mice was rapidly excised and either snap frozen at -80oC or used immediately for RNA isolation. Three VER treated mice and 3 vehicle treated mice were used to generate RNA for microarray. Total RNA was isolated using the RNAqueous -4PCR kit (Ambion) and quantitated spectrophotometrically at 260nm. Contaminating genomic DNA was eliminated by DNase treatment (Ambion). RNA quality was assessed using the Agilent 2100 Bioanalyzer. Microarray data was obtained using the Affymetrix 430 V2 GeneChip (representing 45,101 probe sets), in accordance with the manufacturer’s specifications.
2009-12-31 | E-GEOD-8367 | biostudies-arrayexpress
Project description:Methamphetamine (METH) is a widely abused illicit amphetamine that causes acute biochemical and molecular changes in the brain. However, there are very few studies that have investigated the long-term effects of a single METH exposure in adult animals. The purpose of this study was thus to determine the consequences of a single injection of METH (10) mg/kg) on the biochemical and transcriptional effects of a second challenge injection of the drug (2.5 mg/kg) given one month later. Towards that end, we measured monoamine levels and gene expression by microarray and quantitative PCR analyses in the nucleus accumbens (NAc) of 4 groups of rats: saline-pretreated and saline-challenged (SS); saline-pretreated and METH-challenged (SM); METH-pretreated and saline-challenged (MS); and METH-pretreated and METH-challenged (MM). The rats were euthanized 2 hours after the acute challenge injection and NAc samples were harvested. The acute METH challenge caused increases in NAc DA and HVA levels in both the SM and MM groups. Microarray analyses revealed that an injection of METH (2.5 mg/kg) produced acute changes (1.8-fold; p < 0.01) in the expression of 418 (358 up-, 60 downregulated) genes including several immediate early genes (IEGs) such as Arc, c-fos, and fosB in the SM group. Several neuropeptides including Cart, Crf (Crh), and vasopressin were upregulated in that group. Injection of the higher dose of METH (10 mg/kg) did not influence monoamine levels but caused changes in 510 (345 up-, 165 downregulated) transcripts measured one month later (MS group). This list included Cart and Crf but not any IEGs. The MM group showed changes in 774 (572 up-, 202 downregulated) genes that also included vasopressin, Cart, and Crf whose expression was enhanced by the second METH injection. Quantitative PCR confirmed the METH-induced changes in the expression of the neuropeptides. We also confirmed the METH-induced potentiation of Crf expression in the METH-pretreated group. These observations suggest that a single injection of a moderate dose of METH can produce long-lasting changes in gene expression in the rodent NAc. The long-term increases in Crf expression also suggest that a single injection of a moderate METH dose can cause prolonged dysregulation in the endogenous stress system in the rat brain. 24 samples. SS (4), SM (6), MS (7), MM (7)
2013-06-30 | E-GEOD-46717 | biostudies-arrayexpress
Project description:In contrast to the acute METH-induced transcriptional changes, chronic METH administration produces differential changes in IEG responses and blunts the striatal effects of a single METH injection (McCoy et al., 2011). These observations suggested that chromic METH might have caused changes in the molecular machinery that controls the acute effects of the drug. Gene transcription is regulated by complex interactions of transcription factors with regulatory elements [14,15]. During resting states, DNA is compacted in a way that interferes with the binding of transcription factors whereas DNA becomes more accessible during activation of cells by various stimuli [16]. DNA is indeed packaged into chromatin whose fundamental subunit, the nucleosome, is made of 4 core histones, histones H2A, H2B, H3, and H4 that form an octomer (2 of each histone) surrounded by 146 bp of DNA [17]. The N-tails of histones possess lysine residues that can be reversibly acetylated or deacetylated by several histone acetyltransferases (HATs) or by histone deacetylases (HDACs), respectively [18,19]. These changes promote alterations in gene expression by modifying chromatin conformation and enabling or inhibiting recruitment of regulatory factors onto DNA sequences [20]. Herein, we report that the acute, but not the chronic, transcriptional effects of METH are mediated, for the most part, by increased DNA binding of histone H4 acetylated at lysine 5 (H4K5ac). These results suggest that other factors, including histone and/or DNA methylation, might play a more important role in mediating the molecular effects of chronic METH exposure. 18 samples pooled as SS (4), SM (3), MS (3), MM (4), INPUT (4): SS Saline pretreatment then saline treatment SM Saline pretreatment then METH treatment MS METH pretreatment then saline treatment MM METH pretreatment then METH treatment untreated
2013-02-28 | E-GEOD-42776 | biostudies-arrayexpress
Project description:Inflammatory bowel disease (IBD) is a condition characterized by severe intestinal inflammation and immune cell activation. The severity of the disease can be mitigated by compounds which activate peroxisome proliferator-activated receptor gamma (PPAR gamma), a receptor present widely in tissues involved in IBD pathogenesis. Our objective was to assess the affect of macrophage-specific deficiency of PPAR gamma on peripheral and colonic immune populations and colonic gene expression in experimental IBD. Macrophage-specific PPAR gamma-deficient mice (PPAR gamma flfl Lysozyme M Cre+) and control (PPAR gamma flfl Lysozyme M Cre-) littermates were treated with 2.5% dextran sodium sulfate (DSS) for 7 days. Disease activity was recorded daily and immune cell populations in the blood, spleen, mesenteric lymph nodes (MLN), and lamina propria were examined by flow cytometry. Colonic gene expression was assessed by real time PCR and microarray analyses. Our findings show that macrophage PPAR r-deficiency significantly exacerbates DSS inflammation. CD4+CD25+FoxP3+ regulatory T cells (T-regs) were significantly reduced in Cre+ mice, and MLN macrophages and CD40 expression were enhanced. There were significant differences in the number colonic macrophages between Cre+ and Cre- mice, but those from Cre+ mice expressed more CD40, Ly6C, and TLR-4. PPAR r-deficiency also increased the percent of CD8+ T cells in the lamina propria and enhanced colonic interferon gamma expression. Our findings indicate that macrophage PPAR gamma deficiency augments the severity of DSS colitis by reducing peripheral T-regs and increasing colonic macrophage activation and T cell inflammation. RNA from 3 PPAR gamma-deficient mice (PPAR gamma flfl; Lysozyme M Cre+) and 3 control (PPAR gamma flfl; Lysozyme M Cre-) littermates was processed and labeled according to the standard target labeling protocols. The samples were hybridized, stained, and scanned per standard Affymetrix protocols at the Virginia Bioinformatics Institute (VBI) core laboratory on Mouse 430 2.0 expression arrays (Affymetrix Inc., Santa Clara, CA).
2011-11-12 | E-GEOD-23421 | biostudies-arrayexpress
Project description:How proliferative and inhibitory cell signals integrate to control liver regeneration remains poorly understood. A screen for antiproliferative factors repressed after liver injury identified Tob1, a member of the PC3/BTG1 family of mitoinhibitory molecules as a target for further evaluation. Tob1 protein decreases after 2/3 hepatectomy in mice secondary to post-transcriptional mechanisms. Deletion of Tob1 increases hepatocyte proliferation and accelerates restoration of liver mass after hepatectomy. Down-regulation of Tob1 is required for normal liver regeneration and Tob1 controls hepatocyte proliferation in a dose-dependent fashion. Tob1 associates directly with both Caf1 and the Cyclin/cdk complex to modulate kinase activity. In addition, Tob1 has significant effects on the transcription of critical cell cycle components, including E2F targets and genes involved in p53 signaling. These studies provide direct evidence that levels of an inhibitory factor control the rate of liver regeneration. Our data identify Tob1 as a crucial check-point molecule that acts by by modulating levels and activity of cell cycle proteins. Samples from wild type and Tob1 null mice as normal adults and 24 hours after 2/3 hepatectomy are used. Each experimental point used data from two chips, each having a different set of three pooled animals. In summary we had 8 chips: 2 for WT time 0, 2 for WT time 24, 2 for KO time 0, 2 for KO time 24.
2010-05-28 | E-GEOD-21836 | biostudies-arrayexpress
Project description:Glaucoma is not only the second leading cause of blindness worldwide, but patient numbers will also increase significantly in the coming years due to aging societies. Glaucoma is a progressive optic neuropathy with changes at the optic nerve head, gradual retinal ganglion cell (RGC) death, and visual field loss [1]. Unfortunately, glaucoma can remain asymptomatic until it is rather far progressed, hence about 10-50 % of patients are unaware they suffer from this disease. High intraocular pressure (IOP) is the main risk factor; however, normal-tension glaucoma (NTG) occurs in patients with physiological IOP [2]. This form account for about 30 % of glaucoma patients [3]. Furthermore, sustained IOP lowering can slow down, but does not completely stop disease progression in patients. [4, 5]. Additionally, the administration of topical glaucoma medications can lead to side effects, such as ocular irritation, decreasing the compliance of patients. These facts emphasize the importance of discovering new treatment strategies. The exact pathogenesis for glaucoma is still unknown, but several factors are considered to be involved (fig.1). The most prominent factor is an elevated IOP, which can cause blockade of axonal protein transport at the lamina cribrosa, causing an initial axonal damage and RGC death by trophic insufficiency. In addition, ischemic/hypoxic damage [6], astrocyte and glia cell alterations, and excessive stimulation of the glutamatergic system [7] are discussed as possible pathomechanisms. Moreover, an involvement of the immune system is considered [8-10]. In glaucoma patients, up- and downregulations in the systemic and ocular antibody profile were detected [11-13]. Also, antibody deposits were observed in glaucomatous retinae [14]. It is likely that a combination of several pathogenic factors/mechanisms increases the possibility of developing glaucoma. For the investigation of pathomechanisms and novel therapies, it is necessary to have suitable models that allow such screening. To investigate whether antibodies detected in glaucoma patients are part of glaucoma pathogenesis or a result of disease progression, the experimental autoimmune glaucoma (EAG) animal model was established. This animal model is based on the fact that autoantibodies against S100B, a small calcium binding protein expressed in glia cells, were found to be increased in glaucoma patients [15]. In this model, an immunization with S100B led to a significant loss of RGCs after 28 days and a fast degeneration of optic nerves [20]. The IOP in this model was not altered. Therefore, we were able to mimic the effects seen in NTG patients, an IOP-independent glaucomatous degeneration, by immunizing rats with S100B. S100B is a calcium-binding protein. In the central nervous system (CNS), it is mainly expressed by glial cells such as oligodendrocytes, Schwann cells, ependymal cells, retinal Müller cells and astrocytes [16]. S100B regulates and maintains the homeostasis of the important second messenger calcium and is therefore involved in many cell activities, such as signal transduction, cell differentiation, regulation of cell motility, transcription and cell cycle processes [17, 18]. Extracellularly, S100B can act as a signal molecule and bind receptors such as the receptor for advanced glycation end products (RAGE). In high concentrations, S100B can have negative effects and lead to cell death. For example, the binding of RAGE can induce the activation of microglia cells leading to a release of proinflammatory cytokines to an excessive extent [19]. Furthermore, there seems to be a link between S100B and neuronal diseases. In glaucoma patients, high antibody titers against S100B were found [20]. To investigate the effect of S100B in glaucoma more precisely, the immunization with S100B leads to a loss of retinal ganglion cells (RGCs) without an elevation of the IOP after 28 days in the EAG model [21, 22]. The goal of the present study was to detect the retinal biomarkers in EAG animals 7 and 14 days after immunization with S100B. To detect these new markers in disease development, a label free quantitative mass spectrometry-based approach will be used.
2022-02-16 | PXD023995 | Pride
Project description:Alcoholism is a relapsing disorder associated with excessive consumption after periods of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic intoxication but are poorly characterized. Here we report identification of pathways altered during abstinence in prefrontal cortex, a brain region associated with cognitive dysfunction and damage in alcoholics. To determine the influence of genetic differences, an animal model was employed with widely divergent responses to alcohol withdrawal, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines. Mice were chronically exposed to highly intoxicating concentrations of ethanol and withdrawn, then left abstinent for 21 days. Transcriptional profiling by microarray analyses identified a total of 562 genes as significantly altered during abstinence. Hierarchical cluster analysis revealed that the transcriptional response correlated with genotype/withdrawal phenotype rather than sex. Gene Ontology category overrepresentation analysis identified thyroid hormone metabolism, glutathione metabolism, axon guidance and DNA damage response as targeted classes of genes in low response WSR mice, with acetylation and histone deacetylase complex as highly dimorphic between WSR and WSP mice. Confirmation studies in WSR mice revealed both increased neurotoxicity by histopathologic examination and elevated triidothyronine (T3) levels. Most importantly, relapse drinking was reduced by inhibition of thyroid hormone synthesis in dependent WSR mice compared to controls. These findings provide in vivo physiological and behavioral validation of the pathways identified. Combined, these results indicate a fundamentally distinct neuroadaptive response during abstinence in mice genetically selected for divergent withdrawal severity. Identification of pathways altered in abstinence may aid development of novel therapeutics for targeted treatment of relapse in abstinent alcoholics. A total of 32 microarrays were run with 4 biological replicates per treatment, line, and sex. Selection replicates (i.e. WSP-1 and WSP-2) for each treatment, line, and sex were collapsed to improve statistical power (n=4) and to facilitate in the identification of phenotype related effects and exclude selection artifacts. For comparisons, EtOH regulation was determined by comparing 4 arrays from (for example) Male WSR EtOH treated versus 4 arrays from Male WSR Air treated arrays.
2011-01-05 | E-GEOD-23165 | biostudies-arrayexpress
Project description:To understand the molecular and cellular mechanisms of pathogenesis of autosomal recessive polycystic kidney disease , we performed a microarray gene expression profiling in early stage kidneys of B6C3Fe a/a-bpck mutant and wild-type mice at postnatal day 3. Genes with over 1.5-fold expression changes in mutant kidneys compared with age matched wild-type tissues were selected for analysis. This study represents the first widespread profiling of B6C3Fe a/a-bpck mutant mouse kidney and provides a valuable platform for better understanding the molecular mechanisms of polycystic kidney disease in human. Mutational analysis was performed in accordance with the protocol of Jackson Laboratory, total RNA was extracted from kidneys of 3 days of postnatal age using a monophasic solution of phenol/guanidine isothiocyanate and TRIzol reagent (Invitrogen) according to their manual, and the samples were incubated with RNase-free DNase I (Ambion). The quality and concentration of each sample were confirmed by spectrophotometry. Affymetrix 430 2.0 arrays were used according to standard Affymetrix procedures. Data analysis was performed with dChip software (Dec.2010 version). These measurements were confirmed by Real-Time PCR.
2011-12-01 | E-GEOD-34051 | biostudies-arrayexpress
Project description:Comparison of time-series gene expression pattern in hippocampus of hypoxia tolerant or sensitive rats. At 12 time-points ({0h, 1h, 3h, 12h, 24h, 48h} x 2) after ischemia operation, microdissected CA1 regions of the two groups were respectively subjected to oligonucleotide-based microarray analysis. MIAME information can be obtained at the web link.
2007-10-30 | E-GEOD-1357 | biostudies-arrayexpress
Project description:Dilated cardiomyopathy (DCM) is a major risk factor for developing heart failure and is often associated with an increased risk for life-threatening arrhythmia. Although numerous causal genes for DCM have been identified, RNA binding motif 20 (RBM20) remains one of the few splicing factors that, when mutated or genetically ablated, leads to the development of DCM. In this study we sought to identify changes in the cardiac proteome in RBM20 deficient rat hearts using global quantitative proteomics to gain insight into the molecular mechanisms precipitating the development of DCM secondary to RBM20 loss. Our analysis identified changes in titin interacting proteins, as well as mitochondrial enzymes, implicating activation of pathological hypertrophy and mitochondrial dysfunction in DCM development in RBM20 deficient rats. Collectively, our findings provide the first look into changes in the cardiac proteome associated with genetic ablation of RBM20.
2023-05-12 | PXD033429 | Pride