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Nucleic Acids as Therapeutic Targets and Agents
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
One of the best-known DNA-repair enzymes is the zinc finger DNA-binding enzyme PARP-1 which becomes activated by sensing and binding to breaks mainly in single-stranded DNA, although it can also detect breaks in double-stranded DNA. For example, it can detect damage caused by exposure to topoisomerase inhibitors such as camptothecin and its derivatives, alkylating agents and cross-linking agents such as the platinum-based agents, or ionizing radiation. It then initiates DNA repair by recruiting and then ADP-ribosylating nuclear proteins such as the scaffold protein XRCC1 which directs POLB (DNA polymerase B) to replace a damaged nucleotide. Nuclear proteins associated with apoptosis may also be recruited. Therefore, the PARP-1 mediated poly(ADP-ribosyl)ation of nuclear proteins transforms DNA damage into signals that lead either to activation of repair processes such as the base-excision repair pathway or to cell death via apoptosis. There are over 16 variants of the PARP family of repair enzymes, although PARP-1 and PARP-2 are the most abundant and best studied.
Molecular Organization of Entamoeba Histolytica
Published in Roberto R. Kretschmer, Amebiasis: Infection and Disease by Entamoeba histolytica, 2020
Isaura Meza, Haydee K. Torres-Guerrero, Marco A. Meraz
Acid extracts of Entamoeba nuclei did not reveal typical histone-patterns. The nuclear proteins show a complex pattern with a broad range in molecular weights, some of which could be contaminants from the cytoplasm and the nuclear membrane. We have found enrichment of certain basic proteins in these nuclear extracts as compared with total cell acid extracts. The enriched proteins correspond to molecular weights of 19, 25, 29, and 32 kDa which are in the range of those reported for histones. It is also clear that some of the basic proteins, such as those with molecular weights of 19, 25, and 32 kDa specifically bind to Entamoeba histolytica purified DNA labeled with 32P-deoxycytidine (Figure 5C).87 Two other basic, DNA-binding proteins of 38 and 53 kDa, were not found particularly enriched in the nuclear extracts. The DNA bound to Entamoeba nuclear proteins is released with 300 mM NaCl buffer, an ionic strength that did not release the DNA from chicken erythrocyte histones. These results suggest a difference in the affinity for DNA between the basic proteins in Entamoeba and in chicken erythrocytes.
Colon Carcinogenesis: Biochemical Changes
Published in Herman Autrup, Gary M. Williams, Experimental Colon Carcinogenesis, 2019
Young S. Kim, Laurence J. Mcintyre
Since carcinogenesis involves alterations in the control of cellular growth, one area of possible biochemical changes is nuclear proteins, as these are likely to be involved in genetic control mechanisms. Boffa and Allfrey37 studied the nuclear nonhistone proteins of rat colonic epithelial cells during DMH-induced carcinogenesis. They found that the nuclei from cancer cells contained two groups of tumor-specific proteins, TNP1 and TNP2. The TNP1 protein fraction was shown to be associated with actively transcribing portions of the genome. When human colon adenocarcinoma was compared with normal colonic mucosa two similar groups of proteins were seen in the tumor cell nuclei.37
BRD7 restrains TNF-α-induced proliferation and migration of airway smooth muscle cells by inhibiting notch signaling
Published in Experimental Lung Research, 2022
Hong Li, Tian Yang, Tianjun Chen, Ya Liu, Yamei Pang, Lan Yang
Western blotting was performed according to a previously described protocol.30 Total protein was extracted using radioimmunoprecipitation assay lysis buffer containing a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Nuclear proteins were extracted using a ProteinExt Mammalian Nuclear and Cytoplasmic Protein Extraction Kit (TransGen Biotech). Protein concentrations were determined using the BCA protein assay kit (Beyotime, Shanghai, China). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after which the proteins were transferred onto polyvinylidene fluoride membranes. Membranes were blocked, probed with primary antibodies, and incubated with secondary HRP-conjugated IgG. The membranes were developed using an enhanced chemiluminescence reagent (TransGen Biotech) and images were captured using a gel imager system (Bio-Rad, Hercules, CA, USA). Primary antibodies against BRD7 (Sanying Biotech, Wuhan, China), β-actin (Sanying Biotech), Notch1 (Cell Signaling Technology, Danvers, MA, USA), anti-NICD1 (Cell Signaling Technology), Hey1 (Sanying Biotech), and Hes1 (Cell Signaling Technology) were used.
5-Aza-2’-deoxycytidine may enhance the frequency of T regulatory cells from CD4+ naïve T cells isolated from the peripheral blood of patients with chronic HBV infection
Published in Expert Review of Clinical Immunology, 2021
Yu Fang, Xiao-Dong Yuan, Hui-Hui Liu, Lin Xiang, La-Mei Chen, Yu-Chen Fan, Shuai Gao, Kai Wang
Nuclear proteins generated from cultured cells were used to determine relative levels of DNMT activity using DNMT Activity Quantification Kit. Nuclear proteins were prepared using a Nuclear Extraction Kit. In this assay, microplate wells were stably coated with a universal DNMT substrate. During the reaction, the DNMT enzyme, which transfers a methyl group from Adomet (the methyl donor molecule) to methylate DNA substrate, in the nuclear protein sample was obtained. An anti-5-methylcytosine antibody is then used to identify the methylated DNA. Enzyme activity is proportional to the amount of methylated DNA, which can be assessed through an ELISA-like reaction by measuring absorbance with a microplate reader at a wavelength of 450 nm with an optional reference wavelength of 655 nm.
Guizhi-Shaoyao-Zhimu decoction attenuates bone erosion in rats that have collagen-induced arthritis via modulating NF‐κB signalling to suppress osteoclastogenesis
Published in Pharmaceutical Biology, 2021
Shu-jun Wei, Qing Zhang, Yong-jing Xiang, Lan-yu Peng, Wei Peng, Qiang Ren, Yong-xiang Gao
RAW264.7 cells were added at 2 × 106/well in plates for 24 h. Cells were pre-treated for 3 h with GSZD (0, 200, 400 and 800 μg/mL) and then induced for 30 min by RANKL (80 ng/mL) with equivalent GSZD concentrations. Ice-cold RIPA buffer was used for total protein extraction. Nuclear (N) or cytoplasmic (C) proteins were isolated with the cytoplasmic and nuclear proteins extraction kits, and protein concentrations were determined via BCA assay. Equal amounts of proteins were separated via SDS-PAGE prior to PVDF membrane transfer. Blots were blocked for 1 h with TBST containing 5% BSA, followed by overnight incubation at 4 °C with primary antibodies. Next, membranes were washed and probed using species-matched HRP-conjugated secondary antibody for 1 h. An ECL imaging system was used to visualize protein, with Image J (v1.51, National Institutes of Health, Bethesda, MD) being used to visualize protein bands.