Dasatinib, imatinib and staurosporine capture compounds - Complementary tools for the profiling of kinases by Capture Compound Mass Spectrometry (CCMS)

Capture Compound Mass Spectrometry (CCMS) is a platform technology for the functional isolation of subproteomes. Here we report the synthesis of two new kinase Capture Compounds (CCs) based on the tyrosine-kinase specific inhibitors dasatinib and imatinib and compare their interaction profiles to that of our previously reported staurosporine-CCs. CCs are tri-functional molecules: they comprise a sorting function (e.g. the small molecule or drug of interest) which interacts with target proteins, a photo-activatable reactivity function to covalently trap the interacting proteins, and a sorting function to isolate the CC-protein conjugates from complex biological samples for protein identification by liquid chromatography/mass spectrometry (LC-MS/MS). We present data of CCMS experiments from human HepG2 cells and compare the profiles of the kinases isolated with dasatinib, imatinib and staurosporine CC, respectively. Dasatinib and imatinib have a more selective kinase binding profile than staurosporine. Moreover, the new CCs allow isolation and identification of additional kinases, complementing the staurosporine CC. The family of kinase CCs will be a valuable tool for the proteomic profiling of this important protein class. Besides sets of expected kinases we identified additional specific interactors; these off-targets may be of relevance in the view of the pharmacological profile of dasatinib and imatinib.     

Dynamic changes in brain functional connectivity during concurrent dual-task performance

This study investigated the spatial, spectral, temporal and functional proprieties of functional brain connections involved in the concurrent execution of unrelated visual perception and working memory tasks. Electroencephalography data was analysed using a novel data-driven approach assessing source coherence at the whole-brain level. Three connections in the beta-band (18-24 Hz) and one in the gamma-band (30-40 Hz) were modulated by dual-task performance. Beta-coherence increased within two dorsofrontal-occipital connections in dual-task conditions compared to the single-task condition, with the highest coherence seen during low working memory load trials. In contrast, beta-coherence in a prefrontal-occipital functional connection and gamma-coherence in an inferior frontal-occipitoparietal connection was not affected by the addition of the second task and only showed elevated coherence under high working memory load. Analysis of coherence as a function of time suggested that the dorsofrontal-occipital beta-connections were relevant to working memory maintenance, while the prefrontal-occipital beta-connection and the inferior frontal-occipitoparietal gamma-connection were involved in top-down control of concurrent visual processing. The fact that increased coherence in the gamma-connection, from low to high working memory load, was negatively correlated with faster reaction time on the perception task supports this interpretation. Together, these results demonstrate that dual-task demands trigger non-linear changes in functional interactions between frontal-executive and occipitoparietal-perceptual cortices.     

BMI not WHR modulates BOLD fMRI responses in a sub-cortical reward network when participants judge the attractiveness of human female bodies

In perceptual terms, the human body is a complex 3d shape which has to be interpreted by the observer to judge its attractiveness. Both body mass and shape have been suggested as strong predictors of female attractiveness. Normally body mass and shape co-vary, and it is difficult to differentiate their separate effects. A recent study suggested that altering body mass does not modulate activity in the reward mechanisms of the brain, but shape does. However, using computer generated female body-shaped greyscale images, based on a Principal Component Analysis of female bodies, we were able to construct images which covary with real female body mass (indexed with BMI) and not with body shape (indexed with WHR), and vice versa. Twelve observers (6 male and 6 female) rated these images for attractiveness during an fMRI study. The attractiveness ratings were correlated with changes in BMI and not WHR. Our primary fMRI results demonstrated that in addition to activation in higher visual areas (such as the extrastriate body area), changing BMI also modulated activity in the caudate nucleus, and other parts of the brain reward system. This shows that BMI, not WHR, modulates reward mechanisms in the brain and we infer that this may have important implications for judgements of ideal body size in eating disordered individuals.     

Multiple insulin degrading enzyme variants alter in vitro reporter gene expression

The insulin degrading enzyme (IDE) variant, v311 (rs6583817), is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma β-amyloid (Aβ), decreased risk for Alzheimer''s disease (AD) and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA) and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957), for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2) successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p = 0.04) but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5′ to the promoter (1.18 fold-change, p = 0.006) and 3′ to the reporter gene (1.29 fold change, p = 0.003), an effect contributed to by four variants (v10, v315, v154 and v311). Since IDE mediates Aβ degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.     

Disruption of the langerin/CD207 gene abolishes Birbeck granules without a marked loss of Langerhans cell function

Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.     

An array of microfabricated pillars to study cell migration

Mechanical forces play an important role in various cellular functions, such as tumor metastasis, embryonic development or tissue formation. Cell migration involves dynamics of adhesive processes and cytoskeleton remodelling, leading to traction forces between the cells and their surrounding extracellular medium. To study these mechanical forces, a number of methods have been developed to calculate tractions at the interface between the cell and the substrate by tracking the displacements of beads or microfabricated markers embedded in continuous deformable gels. These studies have provided the first reliable estimation of the traction forces under individual migrating cells. We have developed a new force sensor made of a dense array of soft micron-size pillars microfabricated using microelectronics techniques. This approach uses elastomeric substrates that are micropatterned by using a combination of hard and soft lithography. Traction forces are determined in real time by analyzing the deflections of each micropillar with an optical microscope. Indeed, the deflection is directly proportional to the force in the linear regime of small deformations. Epithelial cells are cultured on our substrates coated with extracellular matrix protein. First, we have characterized temporal and spatial distributions of traction forces of a cellular assembly. Forces are found to depend on their relative position in the monolayer : the strongest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. Consequently, these forces are quantified and correlated with the adhesion/scattering processes of the cells.     

Genetic analysis of two bile salt hydrolase activities in Lactobacillus acidophilus NCFM

Two genes, bshA and bshB, encoding bile salt hydrolase enzymes (EC 3.5.1.24) were identified in the genome sequence of Lactobacillus acidophilus NCFM. Targeted inactivation of these genes via chromosomal insertion of an integration vector demonstrated different substrate specificities for these two enzymes.     

Simultaneous activation of PLA2 and PLC are required to promote acrosomal reaction stimulated by progesterone via G-proteins

The acrosome reaction (AR) is a special exocytotic process promoted by signal transduction pathways studied in many laboratories. Progesterone (P4) is one of the trigger molecules proposed. Upon the binding of P4 to its receptor, several molecules could be activated, including G-proteins, phospholipase A(2) (PLA(2)), and phospholipase C (PLC). The role of these molecules was analyzed in this study using the Chlortetracycline (CTC) protocol to detect and quantify the AR. Incubation of capacitated sperm cells with GTPgammas (GTPgammas, a mimetic of G-protein activation), arachidonic acid (AA, product of PLA(2) action), or phorbol ester (PMA, an activator of PLC) for 15 min increased the AR to a similar percentage as P4. Conversely, a decrease in the AR was detected when sperm cells were incubated with P4 after preincubation with: GDPbetaS (GDP, an inhibitor of G-protein activation), ONO RS-82 (ONO, an inhibitor of PLA(2)), or neomycin (Neo, an inhibitor of PLC) for 15 min. To analyze the activation sequence of G proteins, PLA(2), and PLC combinations of these mimetic/inhibitors were used during successive incubation periods. Inhibition promoted by GDP, ONO, and Neo were overcome by 15-min incubation with GTPgammas, AA, or PMA, respectively. But GTPgammas or P4 did not reverse the inhibition due to incubation with Neo and ONO. Interestingly, this dual inhibition was reverted by another 15-min incubation with AA or PMA. Results presented here could indicate that the AR triggered by P4 is driven by activation of G-proteins, that in turn activate PLA(2) and PLC simultaneously, that finally promote acrosomal exocytosis.     

Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice

We report the first endothelial lineage-specific transgenic mouse allowing live imaging at subcellular resolution. We generated an H2B-EYFP fusion protein which can be used for fluorescent labeling of nucleosomes and used it to specifically label endothelial cells in mice and in differentiating embryonic stem (ES) cells. A fusion cDNA encoding a human histone H2B tagged at its C-terminus with enhanced yellow fluorescent protein (EYFP) was expressed under the control of an Flk1 promoter and intronic enhancer. The Flk1::H2B-EYFP transgenic mice are viable and high levels of chromatin-localized reporter expression are maintained in endothelial cells of developing embryos and in adult animals upon breeding. The onset of fluorescence in differentiating ES cells and in embryos corresponds with the beginning of endothelial cell specification. These transgenic lines permit real-time imaging in normal and pathological vasculogenesis and angiogenesis to track individual cells and mitotic events at a level of detail that is unprecedented in the mouse.