Transcription profiling of rat aortic media during aging
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Axon- GenePix4000B
ORGANISM(S): Rattus norvegicus
SUBMITTER: Sophie Nadaud
PROVIDER: E-MEXP-588 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Recombinant tissue-type plasminogen activator (tPA) is the fibrinolytic drug of choice to treat stroke patients. However, a growing body of evidence indicates that besides its beneficial thrombolytic role, tPA can also have a deleterious effect on the ischaemic brain. Although ageing influences stroke incidence, complications and outcome, age-dependent relationships between endogenous tPA and stroke injuries have not been investigated yet. Here, we report that ageing is associated with a selective lowering of brain tPA expression in the murine brain. Moreover, our results show that albumin D site-binding protein (DBP) as a key age-associated regulator of the neuronal transcription of tPA. Additionally, inhibition of DBP-mediated tPA expression confers in vitro neuroprotection. Accordingly, reduced levels of tPA in old mice are associated with smaller excitotoxic/ischaemic injuries and protection of the permeability of the neurovascular unit during cerebral ischaemia. Likewise, we provide neuroradiological evidence indicating the existence of an inverse relationship between age and the volume of the ischaemic lesion in patients with acute ischaemic stroke. Together, these results indicate that the relationship among DBP, tPA and ageing play an important role in the outcome of cerebral ischaemia. For each stage (4 months and 21 months), samples were prepared by pooling an equal amount of the 3 individual RNA. Five M-BM-5g of total RNA from each pool were reverse-transcribed in the presence of 7.5M-BM-5M random hexamers (GE Healthcare), 75M-BM-5M aminoallyl-dUTP (Sigma-Aldrich) and 100U Rtases Reverse-iT Blend (Thermo scientific) overnight at 37M-BM-0C. Aminoallyl cDNAs were then labelled with Cy5 or Cy3 mono-Reactive Dye (GE Healthcare) according to the manusfacturerM-bM-^@M-^Ys instructions. Labelled cDNAs were then purified on Qiaquick PCR prurification kit (Qiagen) and hybridized to the RNG-MRC_MM25k_EVRY microarrays (Le brigand et al., NAR, 2006) according to the RNG procedures (http://www.microarray.fr:8080/merge/index). Each pool was hybridized in duplicate dye-swap independent experiments. After hybridization, median signal and median background intensities were extracted using Genepix 4.1 software (Axon Instruments). We have first evaluated the level of expression of each gene by calculating the mean ratio M-bM-^@M-^\signal/backgroundM-bM-^@M-^] for each corresponding spot from the 2 dye-swap experiments. Genes were considered to be expressed if their mean ratio was higher or equal to 1.2. Genes with mean ratio < 1.2 in the 2 conditions were not used for further analysis. These filtered data were then submitted to VARAN (http//:www. bionet. espci.fr) for normalization by lowess fit and differential expression analysis (Golfier et al., 2004). Normalized log2ratio 21 months/4 months were then used for further statistical analysis by SAM software (Tusher et al., 2001) and t-test with a p-value equal to 5% using TIGR Multiexperiment Viewer (TM4:MeV, http://www.tm4.org/mev.html). The final list of differentially expressed genes was established by comparing the results obtained with the 3 methods.
2009-08-23 | E-GEOD-9004 | biostudies-arrayexpress
Project description:Transforming growth factor-M-NM-2 (TGF-M-NM-2) signalling controls a number of cerebral functions and dysfunctions including synaptogenesis, amyloid-M-NM-2 accumulation, apoptosis and excitotoxicity. Using cultured cortical neurons prepared from either wild type or transgenic mice over-expressing a TGF-M-NM-2 responsive luciferase reporter gene (SBE-Luc), we demonstrated a progressive loss of TGF-M-NM-2 signalling during neuronal maturation and survival. Moreover, we showed that neurons exhibit increasing amounts of the serine protease HtrA1 (high temperature responsive antigen 1) and corresponding cleavage products during both in vitro neuronal maturation and brain development. In parallel of its ability to promote degradation of TGF-M-NM-21, we demonstrated that blockage of the proteolytic activity of HtrA1 leads to a restoration of TGF-M-NM-2 signalling, subsequent overexpression of the serpin type -1 plasminogen activator inhibitor (PAI-1) and neuronal death. Altogether, we propose that the balance between HtrA1 and TGF-M-NM-2 could be one of the critical events controlling both neuronal maturation and developmental survival. Keywords: HtrA1 / neuronal survival / PAI-1 / TGF-M-NM-2 signalling / tPA Total RNA were extracted from 3 cultures of 2 DIV Human neurons. For each stage, equal amounts of each RNA were pooled and 5M-BM-5g were reverse-transcribed, labelled and hybridized on pangenomic microarrays. Each pool was hybridized in duplicate dye-swap independent experiments.
2008-04-15 | E-GEOD-11162 | biostudies-arrayexpress
Project description:Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterising the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the waterflea, is a textbook example for predator induced phenotypic plastic defences including changes in life-history, behaviour and morphology. However, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages.<br><br>We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. D. magna is known to develop an array of morphological changes in the presence of T. cancriformis including changes of carapace morphology and cuticle hardening. To get a more comprehensive idea of this phenomenon, we studied four different genotypes originating from habitats with different predation history, reaching from predator-free to temporary habitats containing T. cancriformis.<br><br>We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype dependant manner. Proteins connected to genotype dependant responses were related to the cuticle, protein synthesis and calcium binding whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype dependant responses at the proteome level correlated well with local adaptation to Triops predation.<br><br>Altogether, our study provides new insights concerning genotype dependant and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.
2015-07-03 | PXD001787 | Pride
Project description:We integrated metabolome and proteome profiles of the parental cell line 143B.TK- versus ρ0, including PTM analyses such as phosphorylation and ubiquitination to characterize the impact of the absence of mtDNA for the entire cell. For quantitative proteome profiling, we used a shotgun LC-MS/MS approach including the classical SILAC labeling. For comprehensive metabolome profiling, we applied a targeted LC-MS approach, based on multiple reaction monitoring (MRM).</br></br>Our study revealed that mtDNA depletion leads to a non-uniform down-regulation of the mitochondrial energy metabolism in ρ0 cells on the proteome level. Metabolites of the TCA cycle were highly dysregulated which in turn had an impact on the amino acid levels, which were up regulated. Perturbation of the mitochondrial energy metabolism could lead to an activation of the retrograde response, indicated by sets of up-regulated signaling pathways in ρ0 cells, further supported by altered phosphorylation in signaling pathways and the cytoskeleton as well as de-ubiquitination of SLC transporters.
2016-02-08 | PXD002425 | Pride
Project description:Effect of 5.4 ppm polycyclic aromatic hydrocarbons (PAHs) and 18.2 ppm alkylphenols (APs) on gene expression in adult Zebrafish (Danio rerio) liver after 1 and 7 weeks of water-borne exposure.
2008-12-11 | E-MEXP-1685 | biostudies-arrayexpress
Project description:Four independent pools of zebrafish embryo were injected with prp2 morphants and after 24 hours post fertilization, gene expression profiles were compared to their respective controls, using microarray. A dye swap design experiment using four microarray slides were conducted.
2009-10-15 | E-MEXP-2365 | biostudies-arrayexpress
Project description:Transcription profiles of wild-type and transgenic Arabidopsis plants overexpressing the AtCPK1 gene
2011-01-07 | E-MEXP-2532 | biostudies-arrayexpress
Project description:Signaling pathways specifically induced by the oncogenic receptor Xmrk can be elucidated by comparing gene expression in cells with the activated receptor to gene expression in cells with the non-activated receptor. Mouse melanocytes transfected with an inducible version of the oncogenic receptor Xmrk, were starved for 72 h and afterwards stimulated with EGF. We compared cells stimulated for 15 minutes, 1 h, 2 h, 4 h, 8 h or 24 h, respectively, to unstimulated cells.
2008-08-31 | E-MEXP-1311 | biostudies-arrayexpress
Project description:Expression analysis was performed in 77 Wilms tumors in order to select genes responsible for tumor development, metastasis and progression.
2009-02-05 | E-MEXP-221 | biostudies-arrayexpress
Project description:We applied a non targeted mass spectrometric assay (LCMSE), to quantify 55 abundant plasma proteins in specimens from 31 healthy volunteers. Quantitation was done by HI3 peptide measurement using a single internal standard to estimate protein concentrations. Results for 7 apolipoproteins were compared with those obtained using isotope labelled standards, while 12 proteins were compared to routine immunoassays. Blood samples were obtained from 31 healthy volunteers (17 males; 14 females) with a median age of 46 years and a range of 22-67 years. Total plasma protein concentration was assayed with a BCA-assay (20) according to the manufacturer’s protocol (Thermo). Samples were diluted tenfold in 0.1% Rapigest SF (Waters Corporation, Milford, MA), 50 mM ammonium bicarbonate and heated at 95° C for 15 min. Subsequently, plasma samples were reduced with 5 mM dithiothreitol (60° C, 30 min) and alkylated with 15 mM iodoacetamide (ambient temperature, dark, 30 min). Proteolytic digestion was performed with modified trypsin (gold grade, Promega, Madison WI) at 0.3 units/µg protein, (37° C, 20 hours) unless indicated otherwise. Following digestion, Rapigest SF was broken down by adding 1% trifluoroacetic acid (pH<2, 37 °C, 45 min). Peptide solutions were centrifuged (20,000 x g, 10 min) and supernatant was collected. Prior to analyses a MASSPREP protein digestion standard (Waters Corporation, ADH1 or ENO from Saccharomyces cerevisiae) was added for quantitation purposes. LC-MS analyses were performed using ~ 0.21 µg of the final plasma protein digest mixtures (384 times total dilution) unless indicated otherwise. LC separations of tryptic peptides were performed with a NanoAcquity system (Waters Corporation), coupled to a Synapt G2 quadrupole time of flight mass spectrometer (Waters Corporation, Manchester, UK). Accurate mass precursor and fragment ion LC-MS data were collected in data independent LCMSE mode of acquisition. Continuum LC-MS data were processed using ProteinLynx GlobalSERVER version 2.5 (PLGS 2.5, Waters Corporation). Parameter settings: digest reagent trypsin, allow 1 ‘missed cleavage’, search tolerances automatic, typically 5 ppm for precursor and 15 ppm for product ions, fixed modification cysteine carbamidomethylation, and variable modification methionine oxidation. Protein identifications were obtained searching the human SwissProt entries of an UniProt database (release 13.2). This database was modified to include N-terminal processing of proteins using protein maturation device software, with ADH1 and ENO1 of S. cerevisiae appended as internal standard to address technical variation and allow concentration determinations. Estimation of false-positive identification rates was done by searching a randomized version of the abovementioned human protein database generated within PLGS 2.5. Data were exported as csv-files for further, detailed analysis. Stringent criteria were applied for quantitation, protein identifications were only considered significant if reported in 14 or more samples. Protein false positive identification rates were estimated using the criteria mentioned above and no false positives were identified in these searches. DATASET: KC1-31: 31 healthy volunteers QC1-10: 10 times injection of a single sample to ascertain analytical variation over 9 days of measurements. 20120802_QC1-QC6: 6 pooled plasma samples worked up and analysed during a single day of measurements to ascertain Intra-assay variation. 20120427_S2,S4;20120525_S18,S19;20120626_S3,S4;20120802_QC7: 7 pooled plasma samples processed and analysed during 3 months of routine measurements to analyse Inter-assay variation 20130416_s1-s5: pooled plasma samples spiked with a QCONCATAMER for 11 apolipoproteins labelled by heavy Lysine and Arginine (+6.02 AMU) to quantify apolipoproteins by internal isotope labelled standard.
2015-10-20 | PXD000347 | Pride