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In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12–0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.

This work was conducted in the environmental immunotoxicology laboratory at Institut Armand Frappier (IAF-INRS). Financial support has been provided by the Canada Research Chair in Environmental Immunotoxicology (Dr. Michel Fournier) and Collège Doctoral International de l'Université européenne de Bretagne. This study was also supported by the INTERREG IV program (DIESE): 50 % of the Ph.D. grant was obtained by the first author, for the development of immune markers in ecotoxicology.; International audience; A multibiomarker approach was developed on juvenile Atlantic tomcod (Microgadus tomcod) to evaluate the pertinence of this approach for low-cost screening assessment of the environmental quality of various coastal sites within estuaries. Several biometric indices and biomarkers (ethoxyresorufin-O-deethylase (EROD) activity, metallothionein concentration, and immune responses) were investigated on immature and maturing tomcods (≤ 31 months) collected in four environmentally contrasted sites in the St. Lawrence Estuary (SLE). Simultaneous examination of various age classes provides the opportunity to detect short-term responses in sensitive young-of-the-year fish (e.g., EROD induction) and longer-time effects associated with chronic exposure and bioaccumulation (e.g., metallothionein induction). Principal component analysis was helpful to discriminate between responses possibly related to contaminant exposure (EROD, metallothionein) and responses that could be affected by upstream-downstream gradient (immune response, biometric indices). Measurement of a battery of biomarkers in young tomcods at several sites along the shore of the SLE is a low-cost screening investigation useful to identify hot spots requiring further investigation with chemical analysis and additional reference sites.

Metal pollution causes disturbances at various levels of biological organization in most species. Important physiological functions could be affected in the exposed individuals and among the main physiological functions, immunity may provide one (or more) effector(s) whose expression can be directly affected by a metal exposure in various macroinvertebrates. Protein expressions were studied in order to test them as molecular biomarkers of metal exposure in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, transcriptionally controlled tumor protein, protein kinase C, ubiquitin and cyclophilin-A. The level of expression of each gene was analysed in whole organism following exposures to cadmium in soil using real-time PCR. Metallothionein, transcriptionally controlled tumor protein and cyclophilin-A expression were also measured following copper exposures in soil because these genes seemed to be sensitive to copper. This work enabled to distinguish metallothionein and cyclophilin-A among the 15 selected effectors. A strong decrease of the number of transcripts was also detected for most effectors soon after the exposure to cadmium suggesting that a trade-off mechanism occurs.

International audience; Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C. aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80-90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30-50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

International audience; In an experimental food chain, Wistar rats were fed cadmium (Cd) in an inorganic (CdCl(2)) or organic (mainly associated with metallothionein from Helix aspersa snail viscera) form. After 1 month of exposure to 100 microg inorganic Cd g(-1) in food, an induction of metallothionein was observed in all target tissues. In liver, glutathione peroxidase (GSH-Px) activity decreased and alanine aminotransferase (ALAT) activity increased, suggesting that Cd causes hepatotoxicity. However, lipid peroxidation as well as catalase and caspase 3 (a marker of apoptosis) activities were not modified. At a rather low exposure (2.5 microg Cd g(-1)), metallothionein level in the kidney was found to be the most sensitive biomarker of exposure for both Cd forms. In the small intestine of rats ingesting inorganic Cd, metallothionein expression was significantly higher than that observed for rats fed organic Cd. Present results allowed proposing a simple design to assess the effect of a chemical in a trophic transfer approach.

Transfer and toxic effects of two cadmium (Cd) forms, inorganic (CdCl2 dosed rat food) or organic (contaminated snail-based rat food) were studied in Wistar rat. Cd concentrations in rat food were 0 and 2.5 μg Cd g−1 for both inorganic and organic forms and a high concentration of 100 μg Cd g−1 was also tested for the inorganic form. Rats were exposed for four weeks to contaminated food. Both forms of Cd were bioavailable to rats, with a percentage of transfer from food to rats of around 1% for all contaminated groups. Cd concentrations in rat tissues increased with increasing Cd concentrations in the food. Rats fed with organic form of Cd accumulated significantly more Cd in the main organ for Cd toxicity, the kidney, than those eating the inorganic form. Survival was not affected for any rat group but a decrease in growth and food consumption was observed for the inorganic form. As a defence system against Cd toxicity, rats increased their metallothionein (MT) synthesis at the highest Cd concentration in the target organs (kidney, liver and small intestine) and even did the same at low Cd concentrations (2.5 μg Cd g−1) in the kidney. At this low Cd concentration, MT induction was lower in the small intestine of rats ingesting organic Cd than those ingesting inorganic Cd. Bioavailability of organic and inorganic forms of Cd was similar, but subsequent Cd distribution within organs was different. This quantification of the trophic transfer of both inorganic and organic forms of a toxicant is a basis for a better assessment of the fate and effects of chemicals in food webs. Keywords: Bioavailability, Ecotoxicology, Metallothioneins

International audience; IL-6 SNP at position -174 is associated with age-related diseases characterized by an impaired Zn status. This polymorphism seems also relevant in regulating the expression of proteins, such as Metallothioneins (MT), involved in the modulation of Zn homeostasis. Since high IL-6 levels in elderly induce hypozinchemia, the IL-6-174 SNP may be useful to identify old subjects who are at risk for Zn deficiency. The objectives of this study are: (1) to choose old subjects who effectively need Zn supplementation and (2) to study the effect of Zn supplementation on Zn, immune and psychological status in genetically selected subjects. For this purpose, a baseline study comprising 895 healthy old subjects recruited in Central-Northern and Southern European Countries was carried out by evaluating their dietary intake, psychological and immune parameters as well as their Zn status. A Zn supplementation trial was performed in 110 old subjects selected on the basis of their plasma Zn levels and IL-6 SNP. After correcting for age and Zn intake, C- carriers displayed higher MT and lower levels of several parameters related to zinc status (plasma Zn, erythrocyte Zn and NO-induced release of Zn in PBMC) than C+ carriers. Better NK cell cytotoxicity and psychological functions (PSS, MMSE) were also found in C+ than C- carriers strictly related to the zinc status. However, independently by the polymorphism, all subjects with plasma zinc < or = 10.5microM showed the worst immune response and psychological functions. Supplementation was carried out in C+ and C- carriers with stable low plasma zinc levels ( < or =10.5microM at baseline and at 1 year follow-up) and in C- carriers with unstable plasma zinc (< or =10.5microM at baseline and >10.5microM at 1 year follow-up). C+ carriers with plasma zinc >10.5microM were not supplemented because showing the best immune and psychological conditions. After 48+/-2 days of supplementation with 10mg/day of Zn-aspartate, the NO-induced release of Zn, erythrocyte Zn and NK cell cytotoxicity increased in all groups selected for supplementation, including C- with unstable plasma zinc. In conclusion, the sole assessment of plasma Zn level is not reliable to exclude C- carriers from Zn supplementation. A possible explanation for the conflicting data on the identification of IL-6-174G as a "risk allele" based on different dietary intake in the studied population is also suggested.

International audience; A mitochondrial matrix copper ligand (CuL) complex, conserved in mammalian cells, is the likely source of copper for assembly of cytochrome c oxidase (CcO) and superoxide dismutase 1 (Sod1) within the intermembrane space (IMS) in yeast. Targeting the copper-binding proteins human Sod1 and Crs5 to the mitochondrial matrix results in growth impairment on non-fermentable medium caused by decreased levels of CcO. This effect is reversed by copper supplementation. Matrix-targeted Crs5 diminished Sod1 protein within the IMS and impaired activity of an inner membrane tethered human Sod1. Copper binding by the matrix-targeted proteins attenuates levels of the CuL complex without affecting total mitochondrial copper. These data suggest that attenuation of the matrix CuL complex via heterologous competitors limits available copper for metallation of CcO and Sod1 within the IMS. The ligand also exists in the cytoplasm in an apparent metal-free state.

International audience; Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38alpha mitogen-activated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.

International audience; The present study reports on the metallothionein expression in the hydrothermal vent mussel Bathymodiolus thermophilus. Metallothioneins (MT) are proteins involved in intracellular metal regulation and conserved throughout the animal kingdom. The hydrothermal vent environment presents peculiarities (high levels of sulfides and metals, low pH, anoxia) that may have driven associated species to develop original evolutionary ways to face these extreme living conditions. Mussels were exposed to different metal solutions at the atmospheric pressure. The MT mRNA levels and MT contents were measured in gills and mantles of each exposed mussel. The intracellular metal distribution was estimated in fractions obtained after the centrifugation of tissue homogenates. A few of the tested metals (Ag, Cu, Cd, Hg and Zn) were able to significantly induce MT mRNA levels. Silver was the only one that produced a significant increase of the MT protein level in both mantle and gills. The gills always presented higher MT protein levels than the mantle did, while their MT mRNA levels were similar. Our data show that MT mRNA and MT protein levels do not follow a clear relationship in the gills and mantle of B. thermophilus and we assume that a post-transcriptional control occurs in these mussels.

International audience; The molecular response to hypoxia stress in aquatic invertebrates remains relatively unknown. In this study, we investigated the response of the Pacific oyster Crassostrea gigas to hypoxia under experimental conditions and focused on the analysis of the differential expression patterns of specific genes associated with hypoxia response. A suppression subtractive hybridization method was used to identify specific hypoxia up- and downregulated genes, in gills, mantle and digestive gland, after 7-10 days and 24 days of exposure. This method revealed 616 different sequences corresponding to 12 major physiological functions. The expression of eight potentially regulated genes was analysed by RT-PCR in different tissues at different sampling times over the time course of hypoxia. These genes are implicated in different physiological pathways such as respiration (carbonic anhydrase), carbohydrate metabolism (glycogen phosphorylase), lipid metabolism (delta-9 desaturase), oxidative metabolism and the immune system (glutathione peroxidase), protein regulation (BTF3, transcription factor), nucleic acid regulation (myc homologue), metal sequestration (putative metallothionein) and stress response (heat shock protein 70). Stress proteins (metallothioneins and heat shock proteins) were also quantified. This study contributes to the characterization of many potential genetic markers that could be used in future environmental monitoring, and could lead to explore new mechanisms of stress tolerance in marine mollusc species.

Because oxidative stress is involved in arterial hypertension, impairment of hepatic antioxidant defences could develop in the course of this disease. Metallothionein (MT), an antioxidant protein, is present in high rates in the liver. The aim of this study was to investigate the effect of a mineralocorticoid-salt treatment on blood pressure, hepatic antioxidant enzyme activities, and cardiac MT levels in transgenic MT null mice compared with control mice to further clarify the role of MT during the experimental development of arterial hypertension. Control and transgenic MT –/– mice were submitted to an 8-week mineralocorticoid-salt treatment. Hepatic glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase activities and cardiac MT and mineral levels were measured. Mineralocorticoid-salt treatment induced an increase in blood pressure in both transgenic MT –/– and control mice that was associated with an impairment of liver antioxidant status. MT deficiency was associated with modifications of hepatic antioxidant enzyme activities and with a decrease in cardiac iron levels. Adaptive processes of antioxidant systems may explain the absence of an effect of metallothionein deficiency on the development of mineralocorticoid-salt hypertension. The interactions that occur between the in vivo antioxidant systems probably produce a complex regulation of the oxidative balance and consequently prevent antioxidant deficiency.Key words: hepatic antioxidant enzymes, metallothionein, transgenic mice, DOCA-salt hypertension.

The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [35S]GTPgammaS incorporation. The EGFPhMOR protein had the expected molecular weight (70kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken.

DNA cross-linking agents such as mitomycin C (MMC) and cisplatin are used as chemotherapeutic agents in cancer treatment. However, the molecular mechanism underlying their antitumor activity is not entirely clear. Critical steps in cytotoxicity toward cross-linking agents can involve DNA repair efficiency, inhibition of replication, cell-cycle checkpoints, regulation, and induction of apoptosis. The complexity of the mechanisms of the mammalian cell defense against cross-linking agents is reflected by the existence of many complementation groups identified in rodent cells that are specifically sensitive to MMC. We recently showed that increased induction of apoptosis contributes to the MMC sensitivity of the group represented by the V-H4 hamster mutant cell line. In this study, through the analyses of a substractive library, we discovered that sensitive V-H4 cells display a 40-fold increase of steady-state expression of metallothionein II (MT-II) mRNA compared with resistant parental V79 cells. Down-regulation of MT-II by antisense oligonucleotides partially restores MMC resistance in V-H4 cells, indicating that MT-II overexpression is directly involved in MMC hypersensitivity of these cells. MTs have been reported to regulate the activation of NF-kappaB, one of the key proteins that modulates the apoptotic response. Here we found that NF-kappaB activation by MMC is impaired in V-H4 cells and is partially restored following down-regulation of MT-II by antisense oligonucleotides. All these data suggest that the overexpression of MT-II in V-H4 cells impairs NF-kappaB activation by MMC, resulting in decreased cell survival and enhanced induction of apoptosis.

International audience; The PML-RAR alpha hybrid protein generated by the t(15;17) translocation in acute promyelocytic leukemia (APL) is thought to play a central role in the oncogenic process. However, analysis of the oncogenic activity of the fusion protein in tissue culture assays or in mice has been hampered by its apparent toxicity in multiple murine cells. To circumvent this problem, we generated an inducible line of transgenic mice, MT135, in which the expression of PML-RAR alpha is driven by the metallothionein promoter. After 5 days zinc stimulation, 27 out of 54 mice developed hepatic preneoplasia and neoplasia including foci of basophilic hepatocytes, dysplasia and carcinoma with a significantly higher incidence of lesions in females than in males. The rapid onset of liver pathologies was dependent on overexpression of the transgene since it was not detected in noninduced transgenic animals of the same age. The PML-RAR alpha protein was always present in altered tissues at much higher levels than in the surrounding normal liver tissues. In addition, overexpression of PML-RAR alpha resulted in a strong proliferative response in the hepatocytes. We conclude that overexpression of PML-RAR alpha deregulates cell proliferation and can induce tumorigenic changes in vivo.