Quantitation of p53 nuclear relocation in response to stress using a yeast functional assay: effects of irradiation and modulation by heavy metal ions.

Many regulatory proteins undergo transient nuclear relocation under physical or chemical stress. This phenomenon is, however, difficult to assess due to the lack of sensitive and standardized biological assays. Here, we describe a new quantitative nuclear relocation assay (QNR), based on expression in yeasts of chimeric proteins in which an artificial transcription factor is fused to a target protein acting as driver for relocation. This assay combines the experimental versatility of yeast with quantitation of nuclear relocation at low levels of protein expression. We have assessed the nuclear relocation of yeast Yap1 and human p53, two transcription factors that relocate to the nucleus in response to oxidative-stress and DNA damage, respectively. We show that p53 efficiently drives the relocation of the chimeric reporter in response to irradiation and that this process requires the C-terminal nuclear export signal (NES). Cd2+ and Hg2+, two metal ions inducing DNA damage as well as conformational changes in p53, have opposite effects on p53 relocation in response to DNA damage. Whereas Hg2+ effects are synergistic to DNA damage, Cd2+ inhibits relocation and sequesters p53 into the cytoplasm. These results demonstrate the effectiveness of QNR to investigate the regulation of p53 shuttling in response to stress signals including suspected environmental carcinogens.

Rapid detection and quantification of the marine toxic algae, Alexandrium minutum, using a super-paramagnetic immunochromatographic strip test.

The dinoflagellates of Alexandrium genus are known to be producers of paralytic shellfish toxins that regularly impact the shellfish aquaculture industry and fisheries. Accurate detection of Alexandrium including Alexandrium minutum is crucial for environmental monitoring and sanitary issues. In this study, we firstly developed a quantitative lateral flow immunoassay (LFIA) using super-paramagnetic nanobeads for A. minutum whole cells. This dipstick assay relies on two distinct monoclonal antibodies used in a sandwich format and directed against surface antigens of this organism. No sample preparation is required. Either frozen or live cells can be detected and quantified. The specificity and sensitivity are assessed by using phytoplankton culture and field samples spiked with a known amount of cultured A. minutum cells. This LFIA is shown to be highly specific for A. minutum and able to detect reproducibly 10(5)cells/L within 30min. The test is applied to environmental samples already characterized by light microscopy counting. No significant difference is observed between the cell densities obtained by these two methods. This handy super-paramagnetic lateral flow immnunoassay biosensor can greatly assist water quality monitoring programs as well as ecological research.

Direct and fast detection of Alexandrium minutum algae by using high frequency microbalance.

In this paper, a simple detection of a toxic algae, Alexandrium minutum, was developed using highly sensitive quartz crystal microbalance. In terms of performance, compared with other conventional analytical tools, the main interest of our immunosensor is based on a fast and direct detection of these living cells. This system requires the use of one monoclonal antibody directed against the surface antigen of A. minutum. We demonstrate that the whole living and motile algae are caught and detected. The high specificity of the biosensor is also demonstrated by testing several other dinoflagellate species. The frequency shift is correlated to the A. minutum cell concentration. This simple system is potentially promising for environmental monitoring purposes.

One step immunochromatographic assay for the rapid detection of Alexandrium minutum.

Harmful algal blooms represent a major threat to marine production, and particularly to shellfish farming. Current methods for analyzing environmental samples are tedious and time consuming because they require taxonomists and animal experiments. New rapid detection methods, such as immunoassays, are sought for alerting purposes and for the study of algal ecodynamics in their natural environment. Alexandrium minutum, which causes paralytic shellfish poisoning, occurs with increasing frequency along European coasts. We have developed a one step immunochromatographic assay which is based on the principle of immunochromatographic analysis and involves the use of two distinct monoclonal antibodies directed against surface antigens of A. minutum. The primary specific antibody was conjugated with colloidal gold, and the secondary antibody (capture reagent) is immobilized on a strip of nitrocellulose membrane. We could demonstrate that whole algae are able to diffuse without restriction in the porous material. The assay time for this qualitative but highly specific assay was less than 15 min, suitable for rapid on-site testing.