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Ernestas Gaidamauskas Kanokkarn Saejueng Alvin A. Holder Subalita Bharuah Boris A. Kashemirov Debbie C. Crans Charles E. McKenna 《Journal of biological inorganic chemistry》2008,13(8):1291-1299
Metal complexation reactions can be used effectively as sensors to measure concentrations of phosphate and phosphate analogs.
Recently, a method was described for the detection of phosphate or ATP in aqueous solution based on the displacement by these
ligands of pyrocatechol violet (PV) from a putative 2:1 (Yb3+)2PV complex. We have not been able to reproduce this stoichiometry and report this work in order to correct the coordination
chemistry upon which sensor applications are based. In our work, colorimetric and spectrophotometric detection of phosphate
was confirmed qualitatively (blue PV + Yb3+; yellow + Pi); however, the sequence of visual changes on the titration of PV with 2 equiv. of Yb3+ and back titration with ATP as described previously could not be reproduced. In contrast to the linear response to Pi that was reported previously, the absorbance response at 443 or 623 nm was found to be sigmoidal using the recommended 2:1
Yb3+:PV solution (100 μM:50 μM, pH 7, HEPES). Furthermore, both continuous variation titration and molar ratio analysis (Job plot)
experiments are consistent with 1:1, not 2:1, YbPV complex stoichiometry at pH 7 in HEPES buffer, indicating that the deviation
from linearity is produced by excess Yb3+. Indeed, using a 1:1 Yb3+:PV ratio produces a linear response in ΔAbs at 443 or 623 nm on back titration with analyte (phosphate or ATP). In addition,
speciation analysis of the Yb–ATP system demonstrates that a 1:1 complex containing Yb3+ and ATP predominates in solution at μM metal ion and ATP concentrations. Paramagnetic 1H NMR spectroscopy directly establishes the formation of Yb3+–solute complexes in dilute aqueous solution. The 1:1 YbPV complex can be used for the colorimetric measurement of phosphate
and ATP concentrations from ~2 μM.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
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Charles E. McKenna (Corresponding author)Email: |
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Gupta SC Reuter S Phromnoi K Park B Hema PS Nair M Aggarwal BB 《The Journal of biological chemistry》2011,286(2):1134-1146
TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax. 相似文献
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Ernestas Gaidamauskas Boris A. Kashemirov Kanokkarn Saejueng Charles E. McKenna Debbie C. Crans 《Journal of inorganic biochemistry》2009,103(12):1652-1657
The coordination chemistry of bisphosphonates with Yb3+ was investigated to evaluate the potential of the UV-vis based detection method using the Yb3+-pyrocatechol complexation reaction as a sensor for bisphosphonates. The complexation chemistry of Yb3+ with phosphate and ATP analogs was previously described (E. Gaidamauskas, K. Saejueng, A.A. Holder, S. Bharuah, B.A. Kashemirov, D.C. Crans, C.E. McKenna, J. Biol. Inorg. Chem. 13 (2008) 1291-1299), and we here studied the complexation chemistry of bisphosphonates in this system. The spectrophotometric assay yields direct evidence for formation of a 4:3 metal to ligand complex at neutral pH. Direct evidence for Yb3+:methylenebis(phosphonate) complexes with 1:1 and 1:2 stoichiometry was also obtained by potentiometry at acidic and basic pH. Direct evidence for complex formation was obtained using 1H NMR spectroscopy although the stoichiometry was not accessed at neutral pH. Our results suggest that the spectroscopic observation of the YbPV complex can be used to conveniently measure concentrations of bisphosphonates down to 2-3 μM. 相似文献
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