Adherence modifies the regulation of gene expression induced by interleukin-10

Interleukin-10 (IL-10) is a well known anti-inflammatory cytokine. However, we previously showed that it could present pro-inflammatory properties on human monocytes in the absence of adherence. In the present study, using macroarray technology, we analyzed the effects of IL-10 and adherence on the expression of 1050 genes in human monocytes cultured for 3 hours on plastic or Teflon(R) (to avoid adherence). Adherence alone induced specifically the expression of 12 genes and repressed that of 25 genes. In adherent monocytes, IL-10 induced the expression of 21 genes and repressed that of 50 genes. In non-adherent monocytes, IL-10 induced the expression of 45 genes and repressed that of 67. Only 3 common genes were induced while 35 common genes were repressed by IL-10 in the two culture conditions. Interestingly, we showed that IL-10 modulated conversely on Teflon(R) and plastic the expression of 16 genes, of which SOCS molecules, coproporphyrinogen oxidase, matrix metalloproteinases and complement receptor-1 (CD35). This study demonstrates that adherence has profound modulatory effects on the properties and the signaling induced by IL-10. The discovery that IL-10 can inhibit the production of coproporphyrinogen oxidase (an enzyme involved in the synthesis of heme) may shed some lights on the mechanisms of anaemia induced by IL-10. Furthermore, the inhibition of the expression of SOCS1 by IL-10 in the absence of adherence, may explain its priming effects on a subsequent LPS stimulation that we previously described.     

Mitochondrial calcium handling during ischemia-induced cell death in neurons

Mitochondria sense and shape cytosolic Ca²⁺ signals by taking up and subsequently releasing Ca²⁺ ions during physiological and pathological Ca²⁺ elevations. Sustained elevations in the mitochondrial matrix Ca²⁺ concentration are increasingly recognized as a defining feature of the intracellular cascade of lethal events that occur in neurons during cerebral ischemia. Here, we review the recently identified transport proteins that mediate the fluxes of Ca²⁺ across mitochondria and discuss the implication of the permeability transition pore in decoding the abnormally sustained mitochondrial Ca²⁺ elevations that occur during cerebral ischemia.     

Nonredundant role of Bax and Bak in Bid-mediated apoptosis

Animal models suggest that Bax and Bak play an essential role in the implementation of apoptosis and as a result can hinder tumorigenesis. We analyzed the expression of these proteins in 50 human glioblastoma multiforme (GBM) tumors. We found that all the tumors expressed Bak, while three did not express Bax. In vitro, Bax-deficient GBM (BdGBM) exhibited an important resistance to various apoptogenic stimuli (e.g., UV, staurosporine, and doxorubicin) compared to the Bax-expressing GBM (BeGBM). Using an antisense strategy, we generated Bak(-) BeGBM and Bak(-) BdGBM, which enabled us to show that the remaining sensitivity of the BdGBM to apoptosis was due to the overexpression of Bak. Bax/Bak single or double deficiency had no influence on either the clonogenicity or the growth of tumors in Swiss nude mice. Of note, Bak(-) BeGBM cells were resistant to apoptosis induced by caspase 8 (C8) but not to that induced by granzyme B (GrB). Cells lacking both Bax and Bak (i.e., Bak(-) BdGBM) were completely resistant to all stimuli including the microinjection of C8 and GrB. We show that GrB-cleaved Bid and C8-cleaved Bid differ in size and utilize preferentially Bax and Bak, respectively, to promote cytochrome c release from mitochondria. Our results suggest that Bax deficiency is compensated by an increase of the expression of Bak in GBM and show, for the first time in human cancer, that the double Bax and Bak deficiency severely impairs the apoptotic program.     

Precise physical mapping of theEscherichia coli rnb gene, encoding ribonuclease II

Ribonuclease II (encoded byrnb) is one of the two main exonucleases involved in mRNA degradation inEscherichia coli. We report the precise physical mapping ofrnb to 29 min on the chromosomal map in the vicinity ofpyrF, and clarify the genetic and physical maps of thisE. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted forrnb.     

Influence of exogenous abscisic acid on germination and plantlet conversion frequencies of hybrid larch somatic embryos (Larix × leptoeuropaea)

The effect of exogenous abscisic acid, provided to somatic embryos during the maturation step, on endogenous abscisic acid and its main conjugated form (abscisic acid glucose ester), germination and conversion frequencies is presented in this paper. Abscisic acid measurements were obtained after a methanolic extraction, a fractionation through high performance liquid chromatography, quantitation with an immunoassay and identification of the quantitated compound using gas chromatography-mass spectrometry. Results show that endogenous abscisic acid and abscisic acid glucose ester levels are clearly correlated with the exogenous abscisic acid concentration provided to the embryos. Maturation was clearly enhanced by exogenous abscisic acid, but no correlation was found between abscisic acid concentration and germination frequency. Conversely, development of the aerial part of the germinated somatic embryos was dependent upon the abscisic acid concentration in the culture medium and results suggest that this dependence could be related to the endogenous abscisic acid content.     

Molecular dynamics of the FixJ receiver domain: movement of the beta4-alpha4 loop correlates with the in and out flip of Phe101

FixJ is a two-domain response regulator involved in nitrogen fixation in Sinorhizobium meliloti. Recent X-ray characterization of both the native (unphosphorylated) and the active (phosphorylated) states of the protein identify conformational changes of the beta4-alpha4 loop and the conserved residue Phe101 as the key switches in activation. These structures also allowed investigation of the transition between conformations of this two-component regulatory receiver domain by molecular dynamics simulations. The path for the conformational change was studied with a distance constraint directing the system from one state to the other. The simulations provide evidence for a correlation between the conformation of the beta4-alpha4 loop and the orientation of the residue Phe101. A model presenting the sequence of events during the activation/deactivation process is discussed.     

Genetics of VEGF serum variation in human isolated populations of cilento: importance of VEGF polymorphisms

Vascular Endothelial Growth Factor (VEGF) is the main player in angiogenesis. Because of its crucial role in this process, the study of the genetic factors controlling VEGF variability may be of particular interest for many angiogenesis-associated diseases. Although some polymorphisms in the VEGF gene have been associated with a susceptibility to several disorders, no genome-wide search on VEGF serum levels has been reported so far. We carried out a genome-wide linkage analysis in three isolated populations and we detected a strong linkage between VEGF serum levels and the 6p21.1 VEGF region in all samples. A new locus on chromosome 3p26.3 significantly linked to VEGF serum levels was also detected in a combined population sample. A sequencing of the gene followed by an association study identified three common single nucleotide polymorphisms (SNPs) influencing VEGF serum levels in one population (Campora), two already reported in the literature (rs3025039, rs25648) and one new signal (rs3025020). A fourth SNP (rs41282644) was found to affect VEGF serum levels in another population (Cardile). All the identified SNPs contribute to the related population linkages (35% of the linkage explained in Campora and 15% in Cardile). Interestingly, none of the SNPs influencing VEGF serum levels in one population was found to be associated in the two other populations. These results allow us to exclude the hypothesis that the common variants located in the exons, intron-exon junctions, promoter and regulative regions of the VEGF gene may have a causal effect on the VEGF variation. The data support the alternative hypothesis of a multiple rare variant model, possibly consisting in distinct variants in different populations, influencing VEGF serum levels.     

Description and mapping of the resistance of DBA/2 mice to TNF-induced lethal shock

In our search for genes that inhibit the inflammatory effects of TNF without diminishing its antitumor capacities we found that, compared with C57BL/6 mice, DBA/2 mice exhibit a dominant resistance to TNF-induced lethality. Tumor-bearing (C57BL/6 x DBA/2)(BXD)F(1) mice completely survived an otherwise lethal TNF/IFN-gamma-antitumor therapy with complete regression of the tumor. This was not the case for C57BL/6 mice. Genetic linkage analysis revealed that TNF resistance is linked to a major locus on distal chromosome 6 and a minor locus on chromosome 17. Compared with littermate controls, chromosome substitution mice carrying a DBA/2 chromosome 6 in a C57BL/6 background were significantly protected against TNF and TNF/IFN-gamma, albeit less so than DBA/2 mice. Definition of a critical region of 13 Mb on chromosome 6 was the highest mapping resolution obtained. Further analysis of candidate genes may provide a powerful tool to control TNF-induced pathologies in humans.