دورية أكاديمية
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A Sensitive, Cell-Based Assay for Measuring Low-Level Biological Activity of α-Amanitin.

التفاصيل البيبلوغرافية
العنوان: A Sensitive, Cell-Based Assay for Measuring Low-Level Biological Activity of α-Amanitin.
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
الوصف
تدمد:1422-0067
DOI:10.3390/ijms242216402