کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
7557425 | 1491338 | 2016 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays
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کلمات کلیدی
RT–qPCRPXRFCSG6PDHTCDDTRISpregnane-X-receptorDMEMAHRPBSBSA - BSADMSO - DMSODulbecco's modified Eagle's medium - Medal of Eagle اصلاح شده Dulbeccobovine serum albumin - آلبومین سرم گاوIxazomib - ایگزازیمیبInhibition - بازداریReporter gene assay - بررسی ژن خبرنگارBortezomib - بورتِـزومیبanalysis of variance - تحلیل واریانسANOVA - تحلیل واریانس Analysis of varianceTris(hydroxymethyl)aminomethane - تریس (هیدروکسی متیل) آمینومتانDimethyl sulfoxide - دیمتیل سولفواکسیدfetal calf serum - سرم گوساله جنینFirefly luciferase - لوسیفراز فیرفیلیPhosphate-buffered saline - محلول نمک فسفات با خاصیت بافریCarfilzomib - کارفیلزیمیبglucose-6-phosphate dehydrogenase - گلوکز 6-فسفات دهیدروژنازaryl hydrocarbon receptor - گیرنده آرویل هیدروکربن
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays](/preview/png/7557425.png)
چکیده انگلیسی
Firefly luciferase-based reporter gene assays are the most commonly used assays to investigate the transcriptional regulation of gene expression. However, direct interaction of tested compounds with the firefly luciferase leading to altered enzymatic activity may lead to misinterpretation of experimental data. When investigating the proteasome inhibitors bortezomib, carfilzomib, and ixazomib, we observed increased luminescence for bortezomib and ixazomib, but not for carfilzomib, in a pregnane-X-receptor (PXR) reporter gene assay, which was inconsistent with the mRNA expression levels of the main PXR target gene CYP3A4. To further scrutinize this phenomenon, we performed experiments with constitutively expressed firefly luciferase and demonstrated that the increase in cellular firefly luciferase activity is independent from PXR activation or CYP3A4 promoter. Using cell-free assays with recombinant firefly luciferase enzyme, we made the counterintuitive observation that firefly luciferase activity is inhibited by bortezomib and ixazomib in a reversible and competitive manner. This inhibition stabilizes the firefly luciferase enzyme against proteolytic degradation (e.g., toward trypsin), thereby increasing its half-life with subsequent enhancement of total cellular luminescence that eventually mimicked PXR-driven luciferase induction. These data show that particular compounds can strikingly interfere with firefly luciferase and once more illustrate the importance of careful interpretation of data obtained from luciferase-based assays.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 509, 15 September 2016, Pages 124-129
Journal: Analytical Biochemistry - Volume 509, 15 September 2016, Pages 124-129
نویسندگان
Jonas Philipp Becker, Jannick Robert Clemens, Dirk Theile, Johanna Weiss,