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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.
Replication
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Thus, for the growth in the presence of the ribonuclease III, the RNA phages were obliged to use sequences and structures which avoided signals to activate such ribonuclease activities. It was believed therefore that in vivo both the plus and minus strand templates were themselves single strands with nascent molecules as single-stranded tails (Zinder 1980). The double-stranded material was found following the usual deproteinization procedures. Apparently, the strands hybridized to each other during extraction (Erikson and Franklin 1966; Robertson and Zinder 1969) and were either artefacts of the extraction generated by the collapse of the native replicative intermediates or structures no longer involved in the RNA replication (Bishop and Levintow 1971). The electron microscopic study of the non-denatured R17 replicative intermediates (Thach and Thach 1973) and the biochemical characterization of the nascent M12 and Qβ RNA in extracts from both RNase I− and RNase III−E. coli strain AB105 (Keil and Hofschneider 1973; Kindler et al. 1973) were consistent with this conclusion.
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).
Airway reconstruction using decellularized aortic xenografts in a dog model
Published in Organogenesis, 2020
Shao-Fei Cheng, Song Wu, Qian-Ping Li, Hong-Yang Sang, Zheng-Yang Fan
Vessels were decellularized using a complex process recently developed by our team involving hypotonic breakdown, multiple enzymatic digestions and detergent treatments, and mechanical means.15 Briefly, the harvested bovine carotid arteries were placed in hypotonic and hypertonic Tris (hydroxymethyl) aminomethane hydrochloride (Tris–HCl, Sigma, St. Louis, MO) with 0.02% ethylenediamine tetraacetic acid (EDTA, Gibco, Grand Island, NY) and 100kIU/ml aprotinin (Sigma, St. Louis, MO) at 4°C for 24 hours. Afterward, the carotid arteries were agitated in Tris–HCl with 1% octylphenoxypolyethanol (Triton X-100, Sigma, St. Louis, MO) and 0.02% EDTA at 4°C for 24 hours. These decellularized carotid arteries were washed with Hank’s (Gibco, Grand Island, NY) four times for periods of 15 minutes each. These procedures were followed by incubation with 2 mg/mL ribonuclease A (RNase A, Boehringer, Mannheim, Germany) and 10 mg/mL deoxyribonuclease I (DNase I, Boehringer, Mannheim, Germany) at 37°C for 1–2 hours. The carotid arteries were subsequently placed in Tris–HCl with 1% Triton X-100 at 48°C for 24 hours. Then, the carotid arteries were agitated in Tris–HCl with 1% Triton X-100 and 0.02% EDTA at 4°C for 24 hours. These decellularized carotid arteries were rinsed in Hank’s (pH7.4) at 4°C for 48 hours to remove residual substances. Finally, the decellularized bovine carotid arteries were sealed and irradiated for sterilization. The detailed protocol is presented in Table 1.
Novel RNASET2 Pathogenic Variants in an East Asian Child with Delayed Psychomotor Development
Published in Fetal and Pediatric Pathology, 2018
Yan Sun, Xuyun Hu, Jiqing Song, Yanyan Hu, Caihong Liu, Guimei Li
Ribonuclease T2 is the only member of the Rh/T2/S family of acid ribonuclease. Its function is involved in lysosomal degradation of ribosomal RNA. Its presence in locations where there is no RNA suggests that this ribonuclease may have other functions (9). Subsequent studies revealed its antiangiogenic and antitumorigenic effects independent of its ribonuclease capacity (9–12). Based on this feature, human recombination RNASET2 has been proposed as a potential anticancer agent in recent studies (12,14–16). Moreover, genome-wide association study (GWAS) results also revealed relations between Single Nucleotide Polymorphisms (SNPs) on RNASET2 and Graves' disease, Crohn's disease, rheumatoid arthritis, vitiligo and other autoimmune diseases (17–25). RNASET2 mutation was also reported in patients with CLWM and AGS. The distinctive findings of CLWM are bilateral temporal lobe cysts combined with a specific pattern of multifocal white matter lesions, normo- or microcephaly, and severe psychomotor retardation in a nonprogressive clinical course, while AGS is another inherited leukodystrophy with cerebral calcification. Nevertheless, the underlining basis between RNASET2 mutation and congenital brain infection-like phenotypes is still not clear.