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Fisher R.A.,Tulane University | Melton S.J.,Tulane University | Blake D.A.,Tulane University | Blake D.A.,Tulane Xavier Center for Bioenvironmental Research
International Journal of Environmental Analytical Chemistry | Year: 2011

A prototype submersible immunosensor with autonomous sampling characteristics has been designed and fabricated in conjunction with Sapidyne Instruments Inc. The watertight instrument is battery-powered and internally controlled; the internal controller can interface with an external computer for modification of the experimental parameters and review of results. An environmental sample is collected from the external space via a motor driven syringe such that displacement of the motor arm corresponds to a specific intake volume. Assay reagents, buffer and fluorescently labelled antibody, stored in bags within the sensor, are drawn into the syringe after the environmental sample and mixed. The final solution containing the environmental analyte and labelled antibody then passes over a flow/observation cell containing rigid 98-micron beads coated with the analyte of interest. The sensor continuously monitors the fluorescence across the flow cell and the difference in signal from the beginning to the end of the run can be converted to an estimate of analyte concentration. After optimisation steps that included selection of the fluorophore and bead support, the sensor could mix preloaded reagents and autonomously develop a standard curve for two different analytes: caffeine, a marker for untreated sewage, and hexavalent uranium, which contaminates the groundwater in the vicinity of uranium mining and processing sites. The coefficient of variation was near 15% for all concentrations examined. The minimum levels of detection for caffeine and hexavalent uranium in this assay system were 60 and 241 pM, respectively. Spike and recovery assays showed that the sensor was able to accurately predict the concentration of both analytes within the linear region of the calibration curve. Analysis of real environmental samples contaminated with uranium showed good agreement between the sensor and a standard analytical method, thus demonstrating the suitability and versatility of the submersible immunosensor as a field instrument. © 2011 Taylor & Francis.

Bratton M.R.,Tulane University | Bratton M.R.,Tulane Xavier Center for Bioenvironmental Research | Duong B.N.,University of Illinois at Chicago | Elliott S.,Tulane Xavier Center for Bioenvironmental Research | And 9 more authors.
International Journal of Oncology | Year: 2010

Both estrogen, through the estrogen receptor (ER), and growth factors, through the phosphatidylinositol-3-kinase (PI3K)-AKT pathway, have been shown to independently promote cell survival. Here, we investigated the role of ER/PI3K-AKT crosstalk in the regulation of cell survival in MCF-7 breast carcinoma cells. The ER inhibitor ICI 182,780 was used to determine the requirement of the ER for estrogen in the suppression of tumor necrosis factor-α (TNFα) induced apoptosis. Gene reporter assays and Western blot analyses were used to determine the involvement of the pro-survival factor Bcl-2 and the coactivator GRIP1 in this survival crosstalk. We demonstrated that an intact ER signaling pathway was required for estrogen to suppress apoptosis induced by TNFα. Our gene reporter assays revealed that ERα, not ERβ, was targeted by AKT, resulting in transcriptional potentiation of the full-length Bcl-2 promoter, ultimately leading to increased Bcl-2 protein levels. AKT targeted both activation function (AF) domains of the ERα for maximal induction of Bcl-2 reporter activity, although the AF-II domain was predominately targeted. In addition, AKT also caused an upregulation of GRIP1 protein levels. Finally, AKT and GRIP1 cooperated to increase Bcl-2 protein expression to a greater level than either factor alone. Collectively, our study suggests a role for ER/PI3K-AKT crosstalk in cell survival and documents the ability of AKT to regulate Bcl-2 expression via differential activation of ERα and ERβ as well as regulation of GRIP1.

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