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Chemopreventive Agents
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Ergothioneine has several reported chemopreventive effects, but the specific mechanisms involved have proved challenging to identify, with research focusing mainly on antioxidant effects which help the body to eliminate free radicals associated with both cancer and heart disease. In support of this, in vitro experiments have demonstrated that ergothioneine can scavenge hydroxyl radicals (including hypochlorous acid), inhibit production of oxidants by metal ions, and may contribute to the regulation of metalloenzymes and the transport of metal ions. However, as these characteristics were evaluated in cell-free systems, their relevance to in vivo activity is uncertain. However, in cellular studies it has been shown that depletion of ergothioneine leads to augmented oxidative stress and cell death. There is also evidence that it protects water-soluble proteins from oxidative damage, and the high concentration of ergothioneine in mitochondria suggests a role in protecting this organelle from damage by reactive oxygen species that accumulate with normal oxidative metabolism. There is also some evidence that it protects cells from damage induced by UV radiation and reactive nitrogen species. Together, all these lines of evidence suggest that ergothioneine may play the role of a physiological cytoprotectant.
Translation
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
As Capecchi and Webster (1975) noted, the f2 or R17 RNA-directed protein-synthesizing system was instrumental in developing an assay to detect the release factors. The assay employed RNA from a mutant of R17 or f2, in which the seventh codon in the viral coat protein gene has mutated from CAG, which coded for glutamine, to UAG, which coded for release. In the unfractionated E. coli cell-free amino acid incorporating system, RNA from this mutant directed the synthesis of a small amino-terminal coat protein fragment, the hexapeptide fMet-Ala-Ser-Asn-Phe-Thr, which was released free of tRNA (Webster et al. 1967; Bretscher 1968). The cell-free system contained whatever factors were required for release. In order to control the release of the polypeptide chain, the synthesis had to be performed in a stepwise fashion using a partially fractionated cell-free amino acid incorporating system. By this method, a substrate for examining the mechanism of polypeptide chain termination was generated. This substrate, the 70S ribosomal-mRNA complex containing the coat protein N-terminal hexapeptide, was used to look for the E. coli supernatant factors which would mediate the release of the hexapeptide. Surprisingly, the search revealed not the expected hypothetical chain-terminating tRNA, but rather a protein which mediated release of the polypeptide chain (Capecchi 1967c). A more convenient assay for polypeptide chain termination was subsequently developed by Caskey et al. (1968). This assay followed the release of fMet from a ribosomal-AUG-fMet-tRNAf complex in response to added terminator trinucleotides.
Selective Gene De-Repression By De-Repressor RNA
Published in M. Gerald, M.D. Kolodny, Eukaryotic Gene Regulation, 2018
By contrast, assays reflecting the molecular uniqueness of the particular gene derepression might best be observed in cell-free systems of isolated nuclei, isolated chromatin, or even isolated gene loci, in which the de-repressor molecule interacts with a specific portion of the DNA genome. These assays are often blurred in cell-free systems by the lack of a characteristic biological effect, reflecting the significance of the particular gene de-repression.
Malaria transmission-blocking vaccines: wheat germ cell-free technology can accelerate vaccine development
Published in Expert Review of Vaccines, 2019
Kazutoyo Miura, Mayumi Tachibana, Eizo Takashima, Masayuki Morita, Bernard N. Kanoi, Hikaru Nagaoka, Minami Baba, Motomi Torii, Tomoko Ishino, Takafumi Tsuboi
Effective protein expression systems with their proper folding are essential for production and use of proteins in biochemical and biomedical research including malaria vaccine research. Among the different approaches used for protein synthesis, cell-free systems have gained high attention for their ability to rapidly produce proteins under controlled conditions in the post-genome era [80]. While driving the progress of cell-free protein expression, E. coli-derived expression systems have failed, in many cases, to express the proteins in full-length, or in soluble forms. Moreover, E. coli-derived systems may not properly fold proteins of the eukaryotic targets [80]. Hence, E. coli-expression systems often yield inactive, misfolded or truncated proteins including those from malaria parasites [81]. In contrast, the wheat germ cell-free protein synthesis system (WGCFS) has successfully expressed a wide range of eukaryotic proteins, including complex proteins, with good yield, indicating that WGCFS is the method of choice for production of stable, properly folded proteins and for high-throughput protein expression at various scales. The advantages of WGCFS over other protein expression systems had been discussed intensively [80,82–84]. The high performance of WGCFS has been further demonstrated in the so-called ‘human protein factory’ study [85]. The project targeted the expression of 13,364 human proteins, where 12,996 of their clones produced protein in the WGCFS (97.2%); of those, 12,682 were found in the soluble fraction. Hence, WGCFS can be a very effective high-throughput expression system for rapid preparation of malaria parasite proteins for antigen discovery, characterization, and generation of quality antibodies [86,87].
Design of artificial cells: artificial biochemical systems, their thermodynamics and kinetics properties
Published in Egyptian Journal of Basic and Applied Sciences, 2022
Adamu Yunusa Ugya, Lin Pohan, Qifeng Wang, Kamel Meguellati
Generally, artificial cells are made of three parts, namely cellular compartments, transcription and translation machinery, and genetic components. The three steps involved in the construction of artificial cells are: (1) use of synthetic modules to construct genetic circuits in vivo, (2) testing the constructed circuits in vivo, and (3) providing the transcription and translation engines in cell-free systems. The testing and optimization of newly-constructed genetic circuits illustrated the feedback loop between (1) and (2). The cell-free systems and the circuits are enclosed inside the synthetic artificial cells. The first step involved in the construction of artificial cells is the design and testing of genetic circuits such as logic gates, promoters, and different transcription factors. All the processes were conducted in cycles between in vivo and in vitro systems. The constructed circuits were tested in cell-free systems in the second step. The constructed circuits were tested to see whether their functions were affected by artificial chemical environments. Finally, the third step involved the encapsulation of the cell-free systems inside the membranes. The two major types of cell-free systems in use are whole cell extracts and protein synthesis using recombinant elements (PURE) systems. The prokaryotic or eukaryotic cytosols were used for the extraction of the whole cell by removing the natural cell walls. The pure system was built based on purified components from E. coli and the concentration of each component. A few examples of artificial cells involving viruses and their uses are described. A robust method (in vivo) is carried out to demonstrate the use of 60 bp synthetic recombination sequences to assemble multi-fragment expression vectors in Saccharomyces cerevisiae. This method is useful for the construction of high-throughput strains and complex pathways. The integration of the assembled constructs is facilitated by the introduction of a double strand break by meganuclease I-SceI. The use of meganuclease I-SceI increases the efficiency and integration of the same construct by 95% [58]. Wang et al. generated a synthetic 170 bp dsDNA containing numerous specific neuraminidase inhibitor (NAI) resistance mutations as a positive control in downstream assays using synthetic paired-long oligonucleotides. The diagnosis of resistance mutations, molecular diagnosis, and fast molecular testing with respect to influenza virus drug resistance are the significant applications of the aforementioned advancement [59].