دورية أكاديمية
A Sensitive, Cell-Based Assay for Measuring Low-Level Biological Activity of α-Amanitin.
العنوان: | A Sensitive, Cell-Based Assay for Measuring Low-Level Biological Activity of α-Amanitin. |
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المؤلفون: | Rasooly R; Foodborne Toxin Detection & Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA., Do P; Foodborne Toxin Detection & Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA., He X; Foodborne Toxin Detection & Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA., Hernlem B; Foodborne Toxin Detection & Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, USA. |
المصدر: | International journal of molecular sciences [Int J Mol Sci] 2023 Nov 16; Vol. 24 (22). Date of Electronic Publication: 2023 Nov 16. |
نوع المنشور: | Journal Article |
اللغة: | English |
بيانات الدورية: | Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE |
أسماء مطبوعة: | Original Publication: Basel, Switzerland : MDPI, [2000- |
مواضيع طبية MeSH: | Alpha-Amanitin*/pharmacology , RNA Polymerase II*, Animals ; Chlorocebus aethiops ; Humans ; Vero Cells ; HEK293 Cells ; Amanita |
مستخلص: | α-Amanitin is one of the primary toxins produced by the poisonous mushroom genus, Amanita . Because it is odorless and tasteless, it is an important cause of death from the consumption of misidentified mushrooms. To study the thermal stability of α-amanitin, novel cell-based assays were developed to measure the toxin's activity, based on the inhibition of RNA polymerase II by α-amanitin. First, an MTT-formazan cell viability assay was used to measure the biological activity of α-amanitin through the inhibition of cellular activity. This method can detect 10 μg/mL of α-amanitin in a time-dependent manner. Second, a more sensitive quantitative PCR approach was developed to examine its inhibition of viral replication. The new RT-qPCR assay enabled the detection of 100 ng/mL. At this level, α-amanitin still significantly reduced adenovirus transcription. Third, a simpler GFP expression-based assay was developed with an equal sensitivity to the RT-qPCR assay. With this assay, aqueous α-amanitin heated at 90 °C for 16 h or treated in the microwave for 3 min retained its biological activity when tested in HEK293 cells, but a slight reduction was observed when tested in Vero cells. Beyond detecting the activity of α-amanitin, the new method has a potential application for detecting the activity of other toxins that are RNA polymerase inhibitors. |
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فهرسة مساهمة: | Keywords: GFP; MTT; adenovirus; cell-based assays; food safety; qPCR; α-amanitin |
المشرفين على المادة: | 0 (Alpha-Amanitin) EC 2.7.7.- (RNA Polymerase II) |
تواريخ الأحداث: | Date Created: 20231125 Date Completed: 20231127 Latest Revision: 20231127 |
رمز التحديث: | 20231215 |
مُعرف محوري في PubMed: | PMC10671307 |
DOI: | 10.3390/ijms242216402 |
PMID: | 38003593 |
قاعدة البيانات: | MEDLINE |
تدمد: | 1422-0067 |
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DOI: | 10.3390/ijms242216402 |