- 著者
- Yuta Fujii Yutaka Kodama
- 出版者
- 日本植物細胞分子生物学会
- 雑誌
- Plant Biotechnology (ISSN:13424580)
- 巻号頁・発行日
- vol.32, no.1, pp.81-87, 2015-03-25 (Released:2015-04-04)
- 参考文献数
- 31
- 被引用文献数
- 3 22
Green fluorescent protein (GFP) was discovered from the jellyfish Aequorea victoria, and several improvements have been carried out to change its physicochemical properties. The resulting improved GFP variants have been used as reporter proteins for bioimaging techniques in various research fields including plant science. Almost all GFP variants were developed using Escherichia coli to improve fluorescence properties in mammalian cells, but the impact in other organisms such as plant cells remains to be determined. In this study, we performed comparative analysis of four improved GFP variants, GFP-S65T, eGFP, frGFP and sfGFP, with reference to the fluorescence intensity in Arabidopsis protoplasts, and found that sfGFP is the brightest. Using non-fluorescent fragments from the GFP variants, we also conducted bimolecular fluorescence complementation (BiFC) assays to find appropriate fragment pairs of GFP-based BiFC for visualization of protein–protein interactions in living plant cells. Our observations revealed that the brightest is the sfGFP-based BiFC. Further, as an evaluation method for the sfGFP-based BiFC, a BiFC competition assay was successfully completed for the first time in planta. The present study provides useful information for selection and improvement of the GFP molecule and its application to BiFC technology in plants.
- 著者
- Tomoaki Tochitani Akihito Yamashita Izumi Matsumoto Mami Kouchi Yuta Fujii Toru Yamada Izuru Miyawaki
- 出版者
- The Japanese Society of Toxicology
- 雑誌
- The Journal of Toxicological Sciences (ISSN:03881350)
- 巻号頁・発行日
- vol.44, no.9, pp.575-584, 2019 (Released:2019-09-02)
- 参考文献数
- 26
- 被引用文献数
- 2
The adrenal gland is the most common toxicological target of drugs within the endocrine system, and inhibition of adrenal steroidogenesis can be fatal in humans. However, methods to evaluate the adrenal toxicity are limited. The aim of the present study was to verify the usefulness of simultaneous measurement of blood levels of multiple adrenal steroids, including precursors, as a method to evaluate drug effects on adrenal steroidogenesis in cynomolgus monkeys. With this aim, physiological and drug-induced changes in blood levels of adrenal steroids, including cortisol, aldosterone, androgen, and their precursors were examined. First, for physiological changes, intraday and interday changes in blood steroid levels were examined in male and female cynomolgus monkeys. The animals showed circadian changes in steroid levels that are similar to those in humans, while interday changes were relatively small in males. Next, using males, changes in blood steroid levels induced by ketoconazole and metyrapone were examined, which suppress adrenal steroidogenesis via inhibition of CYP enzymes. Consistent with rats and humans, both ketoconazole and metyrapone increased the deoxycorticosterone and deoxycortisol levels, probably via CYP11B1 inhibition, and the increase was observed earlier and with greater dynamic range than the changes in cortisol level. Changes in other steroid levels reflecting the drug mechanisms were also observed. In conclusion, this study showed that in cynomolgus monkeys, simultaneous measurement of blood levels of adrenal steroids, including precursors, can be a valuable method to sensitively evaluate drug effects on adrenal steroidogenesis and to investigate the underlying mechanisms.