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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
Importantly, as mentioned above, RNA is much less stable than DNA and therefore presents some key methodological challenges. Most importantly, RNA degrading enzymes known as ribonuclease (RNase) can break down the RNA molecule into smaller fragments that are unusable for experiments. It is therefore recommended to always ensure good laboratory practice (i.e. always wearing clean gloves, a lab coat and cleaning all surfaces and equipment with RNase-inhibiting solution) and always use RNase-free plasticware and diethylpyrocarbonate (DEPC)-treated water when making relevant buffers and reagents.
“Biologically Active” RNA and the Immune Response*
Published in Edward P. Cohen, A. Arthur Gottlieb, Immune RNA, 2020
Cohen’s extracts, as well as Fishman’s, were impure and contained, in addition to RNA, both DNA and protein. However, the likely essential participation (if not elucidated) of RNA in the response was indicated by the extraordinary sensitivity of the active extracts to ribonuclease. Enzyme:substrate ratios as high as 1:700,000 inactivated the extracts (approximately 0.001 μg ribonuclease destroyed the biological activity of 700 μg of extract dissolved in saline in less than 10 min at 20°). Extracts dissolved in medium containing divalent cations were partially resistant to ribonuclease. In later studies Cohen found18 that divalent cations aid in stabilizing the secondary structure of the RNA (Figure 1); this is indicated by the heightened hyperchromicity of RNA dissolved in buffer containing calcium and magnesium relative to that observed for aliquots of the RNA solution dissolved in buffer without calcium and magnesium. This observation deserves special emphasis. Later sections of this book will present the capacity of RNA extracts injected into immunocompetent recipients, including humans, to affect the immune status of the recipient. Ribonuclease is present in blood and intercellular fluids and would be expected to quickly degrade such RNA. RNA dissolved in solutions containing salts in physiologic concentrations resisted degradation. Persistence of its biological activity was noted after incubation with ribonuclease if the reaction was performed in medium containing calcium and magnesium, but not if the reaction was performed in medium free of divalent cations.
The Modification of Histidine Residues
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
In addition to the direct modifications of lysine, cysteine, and tyrosine by carboethoxylation which are occasionally observed as side reactions in the use of diethylpyrocarbonate for the modification of histidine residues in proteins, an additional side reaction involving lysyl residues has been observed. Sams and Matthews50 reported isopeptide bond formation between the ϵ-amino group of lysine and an adjacent carboxylic acid. This reaction has been previously observed with ribonuclease.31 A reaction mechanism has not been described but likely involves the transient diethylpyrocarbonate modification of the carboxyl group.51
Targeted Degradation of Structured RNAs via Ribonuclease-Targeting Chimeras (RiboTacs)
Published in Expert Opinion on Drug Discovery, 2023
Salma Haj-Yahia, Arijit Nandi, Raphael I. Benhamou
Recently a novel targeted degradation method was developed to induce RNA degradation. To mediate RNA decay, researchers exploit ribonucleases (RNases) that naturally regulate the RNA lifetime and recruit them to specific transcripts via a small molecule, or Ribonuclease Targeting Chimeras (RiboTaCs). These molecules play an important role in regulating gene expression and have been implicated in various diseases such as cancer and viral infections. This approach has been shown to be promising in various in-vitro and in-vivo models, but more research is needed to fully understand the potential and limitations of this technology. To date, RiboTaC has been shown to recruit only RNase L, which is a key player in the interferon-induced antiviral response. However, in order to enrich the RiboTaC toolbox, other Ribonucleases will need to be recruited. In this review, we will discuss the current state of the RiboTaC approach, including its mechanism of action, potential therapeutic applications, and future favorable prospects.
An Overview of Hepatocellular Carcinoma with Emphasis on Dietary Products and Herbal Remedies
Published in Nutrition and Cancer, 2022
Deepa S. Mandlik, Satish K. Mandlik
Momordica charantia lectin (MCL) is a type II ribosome-inactivating protein obtained from bitter gourd, a vegetable used commonly. In Vitro and In Vivo, MCL treatment significantly reduced HCC cell growth by inducing G2/M phase arrest, autophagy and apoptosis (70). Furthermore, in cultured Hep G2 cells, MAP30 (type I ribosome-inactivating protein) isolated from bitter gourd, showed both cytostatic and cytotoxic impact. The activities were due to the induction of S phase cell cycle arrest and activation of extrinsic and intrinsic caspase apoptosis.MAP30’s anti-tumor activity has also been revealed In Vivo. RNase MC2 is a ribonuclease found in M. charantia that has been shown to increase apoptosis in both in-vivo and In Vitro studies (71). The antioxidant functions of total phenolic content, chlorogenic acid and anthocyanin content and in-vitro anticancer ability of potato. The highest antioxidant activity was found in Solanum pinnatisectum, which also had the best antiproliferative effects against liver cancer cells (72). The glycoalkaloids contained in potatoes were thought to have anti-tumor properties. In the range of 0.1–10 g/mL, treatment with potato glycoalkaloids (α-chaconine), subdued HepG2 cell progress with lower cytotoxicity to normal liver cells (73).
Advances in the discovery of microRNA-based anticancer therapeutics: latest tools and developments
Published in Expert Opinion on Drug Discovery, 2020
Kenneth K.W. To, Winnie Fong, Christy W.S. Tong, Mingxia Wu, Wei Yan, William C.S. Cho
This novel design made use of the conjugation of artificial ribonucleases or catalytic peptides to mediate the degradation of the targeted miRNAs [71,72]. A peptide nucleic acid (to mediate specific miRNA binding) has been conjugated with deithylenetriamine or a three-amino acid peptide HGG with Cu2+ co-factor (to mediate the oxidative or acid-base cleavage of miR-1323) for the treatment of neuroblastoma [73]. Another more recently developed conjugate system consisted of specific oligonucleotides and the catalytic peptide [(LRLR)G]2 that cleave the oncogenic miRNAs (miR-21 and miR-17) by trans-esterification reaction [71,72]. These new artificial ribonucleases were shown to effectively downregulate the targeted miRNAs, thereby restoring key tumor-suppressor protein expressions and inhibiting cancer proliferation without appreciable adverse effects.