Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis

Mitochondria are complex organelles, and their proteomics analysis requires a combination of techniques. The emphasis in this chapter is made first on mitochondria preparation from cultured mammalian cells, then on the separation of the mitochondrial proteins with two-dimensional electrophoresis (2DE), showing some adjustment over the classical techniques to improve resolution of the mitochondrial proteins. This covers both the protein solubilization, the electrophoretic part per se, and the protein detection on the gels, which makes the interface with the protein identification part relying on mass spectrometry

Gene induction during differentiation of human monocytes into dendritic cells: an integrated study at the RNA and protein levels

Changes in gene expression occurring during differentiation of human monocytes into dendritic cells were studied at the RNA and protein levels. These studies showed the induction of several gene classes corresponding to various biological functions. These functions encompass antigen processing and presentation, cytoskeleton, cell signalling and signal transduction, but also an increase in mitochondrial function and in the protein synthesis machinery, including some, but not all, chaperones. These changes put in perspective the events occurring during this differentiation process. On a more technical point, it appears that the studies carried out at the RNA and protein levels are highly complementary.Comment: website publisher: http://www.springerlink.com/content/ha0d2c351qhjhjdm

Effects of nanoparticles on murine macrophages

Metallic nanoparticles are more and more widely used in an increasing number of applications. Consequently, they are more and more present in the environment, and the risk that they may represent for human health must be evaluated. This requires to increase our knowledge of the cellular responses to nanoparticles. In this context, macrophages appear as an attractive system. They play a major role in eliminating foreign matter, e.g. pathogens or infectious agents, by phagocytosis and inflammatory responses, and are thus highly likely to react to nanoparticles. We have decided to study their responses to nanoparticles by a combination of classical and wide-scope approaches such as proteomics. The long term goal of this study is the better understanding of the responses of macrophages to nanoparticles, and thus to help to assess their possible impact on human health. We chose as a model system bone marrow-derived macrophages and studied the effect of commonly used nanoparticles such as TiO2 and Cu. Classical responses of macrophage were characterized and proteomic approaches based on 2D gels of whole cell extracts were used. Preliminary proteomic data resulting from whole cell extracts showed different effects for TiO2-NPs and Cu-NPs. Modifications of the expression of several proteins involved in different pathways such as, for example, signal transduction, endosome-lysosome pathway, Krebs cycle, oxidative stress response have been underscored. These first results validate our proteomics approach and open a new wide field of investigation for NPs impact on macrophagesComment: Nanosafe2010: International Conference on Safe Production and Use of Nanomaterials 16-18 November 2010, Grenoble, France, Grenoble : France (2010

Silver staining of proteins in polyacrylamide gels

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. It is compatible with downstream processing, such as mass spectrometry analysis after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 d after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks

Acute WNT signalling activation perturbs differentiation within the adult stomach and rapidly leads to tumour formation

A role for WNT signalling in gastric carcinogenesis has been suggested due to two major observations. First, patients with germline mutations in adenomatous polyposis coli (APC) are susceptible to stomach polyps and second, in gastric cancer, WNT activation confers a poor prognosis. However, the functional significance of deregulated WNT signalling in gastric homoeostasis and cancer is still unclear. In this study we have addressed this by investigating the immediate effects of WNT signalling activation within the stomach epithelium. We have specifically activated the WNT signalling pathway within the mouse adult gastric epithelium via deletion of either glycogen synthase kinase 3 (GSK3) or APC or via expression of a constitutively active β-catenin protein. WNT pathway deregulation dramatically affects stomach homoeostasis at very short latencies. In the corpus, there is rapid loss of parietal cells with fundic gland polyp (FGP) formation and adenomatous change, which are similar to those observed in familial adenomatous polyposis. In the antrum, adenomas occur from 4 days post-WNT activation. Taken together, these data show a pivotal role for WNT signalling in gastric homoeostasis, FGP formation and adenomagenesis. Loss of the parietal cell population and corresponding FGP formation, an early event in gastric carcinogenesis, as well as antral adenoma formation are immediate effects of nuclear β-catenin translocation and WNT target gene expression. Furthermore, our inducible murine model will permit a better understanding of the molecular changes required to drive tumourigenesis in the stomach

<i>Gaia</i> Data Release 1. Summary of the astrometric, photometric, and survey properties

Context. At about 1000 days after the launch of Gaia we present the first Gaia data release, Gaia DR1, consisting of astrometry and photometry for over 1 billion sources brighter than magnitude 20.7. Aims. A summary of Gaia DR1 is presented along with illustrations of the scientific quality of the data, followed by a discussion of the limitations due to the preliminary nature of this release. Methods. The raw data collected by Gaia during the first 14 months of the mission have been processed by the Gaia Data Processing and Analysis Consortium (DPAC) and turned into an astrometric and photometric catalogue. Results. Gaia DR1 consists of three components: a primary astrometric data set which contains the positions, parallaxes, and mean proper motions for about 2 million of the brightest stars in common with the HIPPARCOS and Tycho-2 catalogues – a realisation of the Tycho-Gaia Astrometric Solution (TGAS) – and a secondary astrometric data set containing the positions for an additional 1.1 billion sources. The second component is the photometric data set, consisting of mean G-band magnitudes for all sources. The G-band light curves and the characteristics of ∼3000 Cepheid and RR-Lyrae stars, observed at high cadence around the south ecliptic pole, form the third component. For the primary astrometric data set the typical uncertainty is about 0.3 mas for the positions and parallaxes, and about 1 mas yr−1 for the proper motions. A systematic component of ∼0.3 mas should be added to the parallax uncertainties. For the subset of ∼94 000 HIPPARCOS stars in the primary data set, the proper motions are much more precise at about 0.06 mas yr−1. For the secondary astrometric data set, the typical uncertainty of the positions is ∼10 mas. The median uncertainties on the mean G-band magnitudes range from the mmag level to ∼0.03 mag over the magnitude range 5 to 20.7. Conclusions. Gaia DR1 is an important milestone ahead of the next Gaia data release, which will feature five-parameter astrometry for all sources. Extensive validation shows that Gaia DR1 represents a major advance in the mapping of the heavens and the availability of basic stellar data that underpin observational astrophysics. Nevertheless, the very preliminary nature of this first Gaia data release does lead to a number of important limitations to the data quality which should be carefully considered before drawing conclusions from the data

EXTL3 mutations cause skeletal dysplasia, immune deficiency, and developmental delay.

We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2-mediated STAT5 phosphorylation in patients' lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development

Two-dimensional SDS-PAGE fractionation of biological samples for biomarker discovery

Two-dimensional electrophoresis is still a very valuable tool in proteomics, due to its reproducibility and its ability to analyze complete proteins. However, due to its sensitivity to dynamic range issues, its most suitable use in the frame of biomarker discovery is not on very complex fluids such as plasma, but rather on more proximal, simpler fluids such as CSF, urine, or secretome samples. Here, we describe the complete workflow for the analysis of such dilute samples by two-dimensional electrophoresis, starting from sample concentration, then the two-dimensional electrophoresis step per se, ending with the protein detection by fluorescence