Project description:We compared the gene expression profiles of prostate carcinoma cells, LNCaP, treated or not with an inhibitor of RNA Polymerase II (RNAP II), alpha-amanitin. These experiments pointed to a fraction of intronic messages that seems to be insensitive to alpha-amanitin or up-regulated in cells with blocked RNAP II transcription. Keywords: RNA-polymerase II inhibition experiments
Project description:Background and Aims: To verify the protective effect of Ezetimibe, an sodium taurocholate co-transporting polypeptide (NTCP) inhibitor, on α-amanitin poisoning in vitro and in vivo by inhibiting NTCP to prevent α-amanitin into hepatocytes. Approach and Results: In animal experiments, the survival rate was significantly improved in the treatment group. The pathomorphological characteristics of liver and kidney in the treatment group were significantly improved. In cell experiments,The cell viability of the treatment group was significantly improved, and the expression of NTCP in the treatment group was significantly decreased by immunofluorescence. In molecular docking simulations, we demonstrated the potential of NTCP to bind Ezetimibe and α-amanitin, respectively. Transcriptomics in high-throughput sequencing was used to detect the differential metabolic genes between α-amanitin poisoning group and the treatment group, and signal pathway enrichment was used to analyze the significantly different signal pathways. Conclusions: Ezetimibe, as an inhibitor of NTCP, can reduce the entry of α-amanitin into hepatocytes to play a protective role and improve the cell viability and survival rate of mice.
Project description:In order to identify RBMS1-dependent changes in RNA stability, RBMS1 levels in SW480 breast cancer cells were stably knocked-down using short-hairpin RNAs. The cells were then treated with alpha-amanitin to inhibit transcription, RNA was isolated at 0 and 9 hours post-alpha-amanitin treatment, and the samples were transcriptomically profiled.
Project description:In order to identify MBNL1-dependent changes in MBNL1 target transcript stability, MBNL1 levels in MDA-MB-231 breast cancer cells were stably knocked-down using short-hairpin RNAs. The cells were then treated with alpha-amanitin to inhibit transcription, RNA was isolated at 0 and 9 hours post-alpha-amanitin treatment, and the samples were transcriptomically profiled.
Project description:Gene expression profile in CS1AN deficient and CSBwt restored cell lines after 24 hours of UV or alphe-amanitin treatment (only for restored). The comaprison of expression profile between 0 and 24 hours revealed continouse suppresion of transcription upon UV treatment in CS1AN cell line and alpha-amanitin treated CS1AN CSBwt restored cells.
Project description:The “death cap”, Amanita phalloides, is the world’s most poisonous mushroom, responsible for 90% of mushroom-related fatalities. The most fatal component of the death cap is α-amanitin(AMA). Despite its lethal effect, the exact mechanisms of how α-amanitin poisons humans remain unclear, leading to no specific antidote available for treatment. Here, we performed a genome-wide CRISPR loss-of-function screen to identify genes and pathways involved in AMA cytotoxicity.
Project description:To investigate the effect of RPB1 CTD length on transcription dynamics, six mutant U2OS cell lines expressing tagged versions of α-amanitin resistant RPB1 with CTDs of varying lengths (Dendra2-RPB1-25R (D25), Dendra2-RPB1-52R (D52), Dendra2-RPB1-70R (D70), HaloTag-RPB1-25R (H25), HaloTag-RPB1-52R (H52), and HaloTag-RPB1-70R (H70)) were grown in α-amanitin to degrade endogenous RPB1, then their RNA was sequenced at single-cell resolution using a droplet-based system.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Gene expression profile in CS1AN deficient and CSBwt restored cell lines after 24 hours of UV or alphe-amanitin treatment (only for restored). The comaprison of expression profile between 0 and 24 hours revealed continouse suppresion of transcription upon UV treatment in CS1AN cell line and alpha-amanitin treated CS1AN CSBwt restored cells. Gene expression profiles were obtained in duplicates for un-treated (assigned as 0h time point) and treated (assigned as 24h time point) cells.