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

English-learning one- to two-year-olds do not show a consonant bias in word learning.

Following the proposal that consonants are more involved than vowels in coding the lexicon (Nespor, Peña & Mehler, 2003), an early lexical consonant bias was found from age 1;2 in French but an equal sensitivity to consonants and vowels from 1;0 to 2;0 in English. As different tasks were used in French and English, we sought to clarify this ambiguity by using an interactive word-learning study similar to that used in French, with British-English-learning toddlers aged 1;4 and 1;11. Children were taught two CVC labels differing on either a consonant or vowel and tested on their pairing of a third object named with one of the previously taught labels, or part of them. In concert with previous research on British-English toddlers, our results provided no evidence of a general consonant bias. The language-specific mechanisms explaining the differential status for consonants and vowels in lexical development are discussed

Origin and justification of the use of the arrhenius relation to represent the reaction rate of the thermal decomposition of a solid

Degradation models are commonly used to describe the generation of combustible gases when predicting fire behavior. A model may include many sub-models, such as heat and mass transfer models, pyrolysis models or mechanical models. The pyrolysis sub-models require the definition of a decomposition mechanism and the associated reaction rates. Arrhenius-type equations are commonly used to quantify the reaction rates. Arrhenius-type equations allow the representation of chemical decomposition as a function of temperature. This representation of the reaction rate originated from the study of gas-phase reactions, but it has been extrapolated to liquid and solid decomposition. Its extension to solid degradation needs to be justified because using an Arrhenius-type formulation implies important simplifications that are potentially questionable. This study describes these simplifications and their potential consequences when it comes to the quantification of solid-phase reaction rates. Furthermore, a critical analysis of the existing thermal degradation models is presented to evaluate the implications of using an Arrhenius-type equation to quantify mass-loss rates and gaseous fuel production for fire predictions

Novel reporter systems for facile evaluation of I-SceI-mediated genome editing

Two major limitations to achieve efficient homing endonuclease-stimulated gene correction using retroviral vectors are low frequency of gene targeting and random integration of the targeting vectors. To overcome these issues, we developed a reporter system for quick and facile testing of novel strategies to promote the selection of cells that undergo targeted gene repair and to minimize the persistence of random integrations and non-homologous end-joining events. In this system, the gene target has an I-SceI site upstream of an EGFP reporter; and the repair template includes a non-functional EGFP gene, the positive selection transgene MGMTP140K tagged with mCherry, and the inducible Caspase-9 suicide gene. Using this dual fluorescent reporter system it is possible to detect properly targeted integration. Furthermore, this reporter system provides an efficient approach to enrich for gene correction events and to deplete events produced by random integration. We have also developed a second reporter system containing MGMTP140K in the integrated target locus, which allows for selection of primary cells with the integrated gene target after transplantation. This system is particularly useful for testing repair strategies in primary hematopoietic stem cells. Thus, our reporter systems should allow for more efficient gene correction with less unwanted off target effects

Androstane-3β,5α,6β,17β-tetrol tri­hydrate

The title hydrated tetrol, C19H32O4·3H2O, was synthesized by stereoselective reduction of the compound 3β,5α,6β-trihy­droxy­androstan-17-one. All rings are fused trans. The organic mol­ecules are connected head-to-tail along the c axis via O—H⋯O hydrogen bonds. Layers of water mol­ecules in the ab plane inter­connect these chains. A quantum chemical ab initio Roothan Hartree–Fock calculation of the isolated mol­ecule gives values for the mol­ecular geometry close to experimentally determined ones, apart from the C—O bond lengths, whose calculated values are significantly smaller than the measured ones, probably a consequence of the involvement of the C—OH groups in the hydrogen-bonding network

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

Seven-membered ring scaffolds for drug discovery: access to functionalised azepanes and oxepanes through diazocarbonyl chemistry

Functionalised azepane and oxepane scaffolds were prepared using diazocarbonyl chemistry and elaborated to show their potential use in library synthesis. Key dicarbonyl containing seven-membered rings were functionalised via diastereoselective Luche reduction of the ketone followed by manipulation of the ester and amine groups. Further scaffolds could be accessed by C-alkylation of the dicarbonyl compounds. In addition, an oxepane containing amino acid could be prepared via a diastereoselective enamine reduction

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