Measurement of fluoride-induced endoplasmic reticulum stress using Gaussia luciferase.
ABSTRACT Endoplasmic reticulum (ER) stress and its consequent activation of the unfolded protein response (UPR) signaling pathway have been implicated in several pathophysiologic disorders as well as in drug resistance to treatment of tumors. Several techniques have been devised that qualitatively and quantitatively demonstrate the presence of ER stress and the activation of the UPR; however, most of these methods cannot be used to measure ER stress in real time. Here we describe the use of cells stably transduced with a secreted reporter, Gaussia luciferase (Gluc), to measure fluoride-induced ER stress. Factors that affect ER homeostasis, such as high-dose fluoride, will cause decreased Gluc secretion that can be measured as a decrease in Gluc activity in the culture medium supernatant. Gluc catalyzes the oxidative decarboxylation of coelenterazine (CTZ) to coeleneteramide, resulting in blue bioluminescence (λ(max) 485 nm). Therefore, Gluc activity can be easily quantified by mixing a small aliquot of the medium supernatant with CTZ and measuring the resulting bioluminescence in a luminometer. Among the various reporters used so far, Gluc is regarded as the most sensitive indicator of ER stress. A second advantage for using Gluc is its ability to function in a wide pH range. This is especially useful for studying fluoride-mediated toxicity as fluoride-induced stress is enhanced under acidic conditions. Since Gluc can be measured in a noninvasive manner, it has been used in several in vitro and in vivo applications. In this chapter, we detail our methodology for using Gluc to monitor fluoride-induced ER stress.
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ABSTRACT: Technological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in applied biomedical research, using selected cases. We look at the use of zebrafish embryos as disease models, taking fetal alcohol syndrome and tuberculosis as examples. We discuss advances in imaging, in culture techniques (including microfluidics), and in drug delivery (including new techniques for the robotic injection of compounds into the egg). The use of zebrafish embryos in early stages of drug safety-screening is discussed. So too are the new behavioral assays that are being adapted from rodent research for use in zebrafish embryos, and which may become relevant in validating the effects of neuroactive compounds such as anxiolytics and antidepressants. Readouts, such as morphological screening and cardiac function, are examined. There are several drawbacks in the zebrafish model. One is its very rapid development, which means that screening with zebrafish is analogous to "screening on a run-away train." Therefore, we argue that zebrafish embryos need to be precisely staged when used in acute assays, so as to ensure a consistent window of developmental exposure. We believe that zebrafish embryo screens can be used in the pre-regulatory phases of drug development, although more validation studies are needed to overcome industry scepticism. Finally, the zebrafish poses no challenge to the position of rodent models: it is complementary to them, especially in early stages of drug research.Birth Defects Research Part C Embryo Today Reviews 06/2011; 93(2):115-33. DOI:10.1002/bdrc.20206 · 3.87 Impact Factor
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ABSTRACT: An R345W mutation in fibulin-3 causes its inefficient secretion, increased intracellular steady-state levels, and the macular dystrophy, Malattia Leventinese (ML), a disease similar to age-related macular degeneration. It is unknown whether R345W causes ML through increased intracellular levels, by the secretion of a potentially aggregation-prone protein, or both. To identify small molecules that alter the secretion of fibulin-3, we developed ARPE19 retinal cell lines that inducibly express wild-type (WT) or R345W fibulin-3 fused to an enhanced Gaussia luciferase (eGLuc2). Screening of the Library of Pharmacologically Active Compounds demonstrated that these cell lines and the GLuc assay are suitable for high-throughput chemical screening. Two estrogen-related compounds enhanced fibulin-3 secretion, whereas a diverse series of small molecules reduced fibulin-3 secretion. A counterscreen identified compounds that did not substantially alter the secretion of unfused eGLuc2, demonstrating at least partial selectivity for fibulin-3. A secondary assay using untagged fibulin-3 confirmed that the top three inhibitory compounds reduced R345W fibulin-3 secretion. Interestingly, in untagged fibulin-3 studies, one compound, phorbol 12-myristate 13-acetate, reduced R345W fibulin-3 secretion while minimally enhancing WT fibulin-3 secretion, the desired activity and selectivity we sought for ML. The identified compounds could serve as tools for probing the etiology of fibulin-3-related diseases.Journal of Biomolecular Screening 12/2012; 18(6). DOI:10.1177/1087057112469405 · 2.01 Impact Factor