Auto-protective redox buffering systems in stimulated macrophages

BACKGROUND: Macrophages, upon encounter with micro-organisms or stimulated by cytokines, produce various effector molecules aimed at destroying the foreign agents and protecting the organism. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are front line molecules exerting strong cytotoxic activities against micro-organisms and many cells, including macrophages themselves. Using cells of the murine macrophage cell line (RAW 264.7) stimulated in vitro with lipopolysaccharide (LPS) and/or interferon (IFN-γ), which induce strong endogenous NO production, we examined by which mechanisms a fraction of activated macrophages protect themselves from nitrosative stress and manage to escape destruction? RESULTS: We observed that survivors (10–50% depending on the experiments) had acquired a resistant phenotype being capable to survive when further exposed in vitro to an apoptosis inducing dose of the NO donor compound DETA-NO. These cells expressed an increased steady-state levels of Mn SOD, CuZn SOD and catalase mRNA (130–200%), together with an increased activity of the corresponding enzymes. Intracellular concentration of glutathione was also increased (× 3.5 fold at 6 hours, still maintained × 5.2 fold at 48 hours). Neither mRNA for glutathione peroxydase, γ-glutamylcysteine synthase and glutathione reductase, nor thioredoxine and thioredoxine reductase, were significantly modified. Additional experiments in which RAW 264.7 cells were stimulated with LPS and/or IFN-γ in the presence of relatively specific inhibitors of both Mn and Cu/Zn SOD, aminotriazol (ATZ) catalase inhibitor and buthionine sulfoximine (BSO) glutathione inhibitor, showed that inhibiting LPS-induced up-regulation of intracellular redox buffering systems also prevented acquisition of the resistant phenotype. CONCLUSIONS: Our data suggest a direct causal relationship between survival of a fraction of macrophages and a up-regulation of key sets of auto-protective intracellular redox buffering systems, occurring simultaneously with modulation of expression of apoptotic molecules of the Bcl(2)-Bcl-(XL)/Bax-Bad family

Cell cycle features of primate embryonic stem cells.

International audienceUsing flow cytometry measurements combined with quantitative analysis of cell cycle kinetics, we show that rhesus monkey embryonic stem cells (ESCs) are characterized by an extremely rapid transit through the G1 phase, which accounts for 15% of the total cell cycle duration. Monkey ESCs exhibit a non-phasic expression of cyclin E, which is detected during all phases of the cell cycle, and do not growth-arrest in G1 after gamma-irradiation, reflecting the absence of a G1 checkpoint. Serum deprivation or pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) did not result in any alteration in the cell cycle distribution, indicating that ESC growth does not rely on mitogenic signals transduced by the Ras/Raf/MEK pathway. Taken together, these data indicate that rhesus monkey ESCs, like their murine counterparts, exhibit unusual cell cycle features in which cell cycle control mechanisms operating during the G1 phase are reduced or absent

InfraPhenoGrid: A scientific workflow infrastructure for Plant Phenomics on the Grid

International audiencePlant phenotyping consists in the observation of physical and biochemical traits of plant genotypes in response to environmental conditions. Challenges , in particular in context of climate change and food security, are numerous. High-throughput platforms have been introduced to observe the dynamic growth of a large number of plants in different environmental conditions. Instead of considering a few genotypes at a time (as it is the case when phenomic traits are measured manually), such platforms make it possible to use completely new kinds of approaches. However, the data sets produced by such widely instrumented platforms are huge, constantly augmenting and produced by increasingly complex experiments, reaching a point where distributed computation is mandatory to extract knowledge from data. In this paper, we introduce InfraPhenoGrid, the infrastructure we designed and deploy to efficiently manage data sets produced by the PhenoArch plant phenomics platform in the context of the French Phenome Project. Our solution consists in deploying scientific workflows on a Grid using a middle-ware to pilot workflow executions. Our approach is user-friendly in the sense that despite the intrinsic complexity of the infrastructure, running scientific workflows and understanding results obtained (using provenance information) is kept as simple as possible for end-users

Ciliary Beating Recovery in Deficient Human Airway Epithelial Cells after Lentivirus Ex Vivo Gene Therapy

Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1–deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT–PCR and western blot, respectively. Human airway epithelial cells that were DNAI1–deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease

p38 mitogen activated protein kinase controls two successive-steps during the early mesodermal commitment of embryonic stem cells.: P38 CONTROLS THE MESODERMAL COMMITMENT OF ES CELLS

International audienceEmbryonic stem (ES) cells differentiate in vitro into all cell lineages. We previously found that the p38 mitogen activated kinase (p38MAPK) pathway controls the commitment of ES cells toward either cardiomyogenesis (p38 on) or neurogenesis (p38 off ). In this study, we show that p38α knock-out ES cells do not differentiate into cardiac, endothelial, smooth muscle, and skeletal muscle lineages. Reexpression of p38MAPK in these cells partially rescues their mesodermal differentiation defects and corrects the high level of spontaneous neurogenesis of knock-out cells. Wild-type ES cells were treated with a p38MAPK-specific inhibitor during the differentiation process. These experiments allowed us to identify 2 early independent successive p38MAPK functions in the formation of mesodermal lineages. Further, the first one correlates with the regulation of the expression of Brachyury, an essential mesodermal-specific transcription factor, by p38MAPK. In conclusion, by genetic and biochemical approaches, we demonstrate that p38MAPK activity is essential for the commitment of ES cell into cardiac, endothelial, smooth muscle, and skeletal muscle mesodermal lineages

Submicron particles in the Rhine River - I. Physico-chemical characterization

This paper describes a complete sampling, fractionation and characterization scheme for submicrometer particles isolated from the Rhine River. Decreasing particle size fractions were obtained by means of gravitational sedimentation, cascade centrifugation/ultracentrifugation and cascade filtration. These size fractions were analyzed by photon correlation spectroscopy (PCS), micro-electrophoresis (ME), transmission electron microscopy (TEM), light scattering (LS), inductively coupled plasma-atomic emission spectrometry (ICP-AES) and total organic carbon (TOC), which gave complementary results concerning the nature of the particles. The data indicated that submicron particles contribute only a small proportion of the total particle mass and volume, but an important proportion of the total particle number. Moreover, their specific surface area may be quite large. Associations of submicrometer particles with organic macromolecules and fibrils, which may have maintained such particles in suspension, were observed

Low titer lentiviral transgenesis in rodents with simian immundeficiency virus vector

Efficient production of transgenic animals using low-titer lentiviral constructs remains challenging. Here we demonstrate that microinjection of simian immundeficiency virus-derived lentiviral constructs can produce transgenic mice and rats with high efficiency even when using low-titer virus preparations

Effect of point mutations in the decoding site (C1400) region of 16S ribosomal RNA on the ability of ribosomes to carry out individual steps of protein synthesis

International audienceIn order to probe the relationship between structure and function of the ribosome, an in vitro system [Denman et al. (1988) Biochemistry (preceding paper in this issue)] was used to make a series of base changes around C1400, a residue known to be at the decoding site. Replacement of C1400 by U, A, or G, deletion of single bases at and to either side of C1400, and insertion of C or U next to C1400 were done. In a separate study, a mutant with seven extra nucleotides at the 3' end was constructed. The activity of these 11 mutants in A and P site binding and in initiation-dependent and initiation-independent peptide synthesis was analyzed. None of the base substitutions of C1400 were markedly inhibitory despite the almost complete conservation of this residue in ribosomal RNAs from a wide range of species. The insertions and deletions completely blocked initiation-dependent peptide synthesis but markedly stimulated the initiation-independent reaction. The effects on tRNA binding were variable. The extra stem and loop at the 3' end blocked initiation-dependent peptide synthesis but did not influence the other assays. The only modification to block all ribosomal function was the deletion of G1401. It appears that while the conserved and cross-linkable C1400 is not essential for function, the adjacent conserved G1401 is

Submicron particles in the rhine river - II. Comparison of field observations and model predictions

The particle size distribution in the Rhine River near Basle (Switzerland) was measured for a period of over 1 year. Peaks in the measured size distributions were consistently observed in the ranges of 100–200, 300–700 nm and 1–3 μm. The size distributions observed did not vary greatly with time or flow rate. Predictions of a classical coagulation/sedimentation model agreed well with these field observations. This agreement between model predictions and field measurements indicated that the unknown (and probably dynamic) “initial” particle distribution may have been quickly transformed, either in the river or in the interstitial soil solution, through coagulation and sedimentation into the characteristic and relatively stable shape experimentally observed