Urea derivatives of STI571 as inhibitors of Bcr-Abl and PDGFR kinases

The constitutively active Abl kinase activity of the Bcr-Abl oncoprotein is causative for chronic myelogenous leukemia. Urea derivatives, structurally related to the therapeutic agent STI571, have been identified, which potently inhibit the tyrosine kinase activity of recombinant Abl. In particular a dimethylamino-aniline derivative (18) inhibited c-Abl transphosphorylation with an IC(50) value of 56 nM. Although this activity was not translated into cellular activity against the constitutively activated oncogenic Bcr-Abl, a number of compounds from this series potently inhibited cellular PDGFR autophosphorylation. It was also possible to differentiate between c-Abl and PDGFR kinase inhibition, with compound 22 being selective towards Abl and 23 selective for PDGFR.     

Glomulin is predominantly expressed in vascular smooth muscle cells in the embryonic and adult mouse

Mutations in the glomulin gene result in dominantly inherited vascular lesions of the skin known as glomuvenous malformations (GVMs). These lesions are histologically distinguished by their distended vein-like channels containing characteristic 'glomus cells', which appear to be incompletely or improperly differentiated vascular smooth muscle cells (VSMCs). The function of glomulin is currently unknown. We studied glomulin expression during murine development (E9.5 days post-coitum until adulthood) by non-radioactive in situ hybridization. Glomulin was first detected at E10.5 dpc in cardiac outflow tracts. Later, it showed strong expression in VSMCs as well as a limited expression in the perichondrium. At E11.5-14.5 dpc glomulin RNA was most abundant in the walls of the large vessels. At E16.5 dpc expression was also detectable in smaller arteries and veins. The high expression of glomulin in murine vasculature suggests an important role for glomulin in blood vessel development and/or maintenance, which is supported by the vascular phenotype seen in GVM patients with mutations in this gene.     

Complementary expression patterns of retinoid acid-synthesizing and -metabolizing enzymes in pre-natal mouse inner ear structures

Retinoic acid (RA) plays a pivotal role in patterning and differentiation of the embryonic inner ear. Despite its documented effects during embryonic development, the cellular sites that synthesize or metabolize RA in the inner ear have yet to be determined. Here we describe the distribution of three synthesizing enzymes, retinaldehyde dehydrogenases 1, 2 and 3 (RALDH1, RALDH2 and RALDH3) and two catabolizing enzymes (CYP26A1 and CYP26B1) in the mouse inner ear at embryonic day 18.5 when active cell differentiation is underway. Two detection methods, radioactive and non-radioactive in situ hybridization, were employed to elucidate the tissue distribution and cellular localization of these enzymes, respectively. All of the five enzymes examined, with the exception of CYP26A1, were expressed in both vestibular and cochlear end organs. While expression of the three RALDHs was observed in various cell types, CYP26B1 expression was found only in supporting cells of the vestibular and cochlear end organs. In the cochlea, expression domains of RALDH1-3 and CYP26B1 were complementary to one another. These results reveal specific tissue- and cellular expression patterns of RA synthesizing and catabolizing enzymes in the pre-natal inner ear, and suggest that a precise control of RA concentrations in various cell types of the inner ear is achieved by the balance between RALDHs and CYP26B1 activities.     

Délocalisation de la biologie d’urgence et Assurance Qualité

La biologie délocalisée prend une importance croissante dans le fonctionnement des services traitant les urgences à l’h?pital. Elle devrait représenter une réele évolution pour la prise en charge du patient tout en améliorant l’organisation interne des unités de soins. Le biologiste occupe un r?le clé pour mener un tel projet et la mise en place d’un système d’assurance qualité adapté à cette pratique est indispensable.     

Docking study of ligands into the colchicine binding site of tubulin

Cancer is a major cause of mortality in developed countries, following only cardiovascular diseases. Death of cancerous cells can be achieved by stopping mitosis and the antimitotic class of drugs formed by the spindle poisons can be used for this purpose. Their role is to disorganize the mitotic spindle by targeting its main constituent, the microtubules, themselves made of heterodimers of alpha and beta-tubulin. They disrupt the dynamics of the microtubules either by stabilizing them, as do paclitaxel or epothilones, or destabilizing them, as do colchicine. The binding site of colchicine seems to lie between the two units of the tubulin dimer. Here, we report on the characterization of this site by the docking of a series of reference compounds, and the subsequent docking of ligands prepared in our laboratory.     

Preparation and in vivo toxicity study of solid lipid microparticles as carrier for pulmonary administration

The purpose of this research was to investigate the effects of processing conditions on the characteristics of solid lipid microparticles (SLM) with a potential application as carriers for pulmonary administration. Compritol (5.0% wt/wt) SLM dispersions were prepared by rotor-stator homogenization, at different surfactant concentrations and emulsification times. The SLM were characterized, in terms of morphology and size, after lyophilization and sterilization by autoclaving process. In vivo assessment was carried out in rats by intratracheal instillation of either placebo or SLM dispersion, and by bronchoalveolar lavage for cytological analysis. Mean particle size of 4 to 5 μm was achieved using 0.3% and 0.4% (wt/wt) of emulsifier (Poloxamer 188) and emulsification times of 2 and 5 minutes. The particles showed spherical shape and smooth surface. The morphology of microparticles, the size, and the size distribution were not substantially modified after lyophilization and sterilization. Total cell counts showed no significant differences between placebo and SLM 0.5% or 2.5% groups. Regarding cytology, percentage of polymorphonuclear neutrophils and macrophages did not significantly differ between groups. These results suggest that a single intratracheal administration of the SLMs does not induce a significant inflammatory airway response in rats and that the SLMs might be a potential carrier for encapsulated drug via the pulmonary route.     

In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and gamma-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.     

A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells

Homologous gene targeting is the ultimate tool for reverse genetics, but its use is often limited by low efficiency. In a number of recent studies, site- specific DNA double-strand breaks (DSBs) have been used to induce efficient gene targeting. Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. In this study, we present two novel, chimeric meganucleases, derived from homing endonucleases. The first one is able to induce recombination in yeast and mammalian cells, whereas the second cleaves a novel (chosen) DNA target site. These results are a first step toward the generation of custom endonucleases for the purpose of targeted genome engineering.     

Horizontal Gene Transfer of “Prototype” Nramp in Bacteria

Eukaryotic Nramp genes encode divalent metal ion permeases important for nutrition and resistance to microbial infection. Bacterial homologs encode proton-dependent transporters of manganese (MntH), and other divalent metal ions. Bacterial MntH were classified in three homology groups (A, B, C) and MntH C further subdivided in C, C, C. The proteins from C. tepidum (MntH B) and E. faecalis (MntH C1, 2), divergent in sequence and hydropathy profile, conferred increased metal sensitivity when expressed in E. coli, suggesting conservation of divalent metal transport function in MntH B and C. Several genomic evidence suggest horizontal gene transfer (HGT) of mntH C genes: (i) The enterobacteria Wigglesworthia mntH C gene is linked to an Asn t-RNA, and its sequence most conserved with Gram positive bacteria homologs; (ii) all the C genes identified in oral streptococcaceae are associated with different potentially mobile DNA elements; (iii) Lactococcus lactis and Burkholderia mallei genomes contain an mntH gene prematurely terminated and a novel full-length mntH C gene; (iv) remarkable sequence relatedness between the unicellular alga C. reinhardtii prototype Nramp and some MntH C (e.g., Nostoc spp., Listeria spp.) suggests HGT between Eukarya and Bacteria. Other prototype Nramp genes (intronless, encoding proteins strongly conserved with MntH A and B proteins) identified in invertebrates represent a possible source for transfer of Nramp genes toward opportunistic bacteria. This study demonstrates complex evolution of MntH in Bacteria. It is proposed that prototype Nramp are ancestors of bacterial MntH C proteins, which could facilitate bacterial infection. Equally contributing authors (Etienne Richer and Pascal Courville).