The reaction between N(alpha)-acetyllysine methyl ester (Lys) and 2'-deoxyguanosine (dGuo) was used to study structural aspects of DNA-protein cross-link (DPC) formation. The precise structure of DPCs depended on the nature of the oxidant and cross-linking reactions in which a series of different oxidation conditions generated a distribution of adducts, principally spirodiiminodihydantoins with lysine appended at the purine position of C5 (5-Lys-Sp), C8 (8-Lys-Sp), or both C5 and C8 (5,8-diLys-Sp). Singlet oxygen oxidation of dGuo produced 5-Lys-Sp exclusively when Rose Bengal or methylene blue was used to photochemically generate (1)O2 in the presence of Lys, whereas riboflavin or benzophenone-mediated photochemistry generated lysine radicals and led to C8 adduct formation, yielding 8-Lys-Sp and 5,8-diLys-Sp. Notably, the yield of dGuo modifications from riboflavin photooxidation increased dramatically in the presence of lysine. Oxidation of deoxyguanosine/lysine mixtures with Na2IrCl6 or sulfate radicals produced both 5-Lys-Sp and 8-Lys-Sp. The same adducts were formed in single and double-stranded oligodeoxynucleotides, and these could be analyzed after nuclease digestion. Adduct formation in duplex DNA was somewhat dependent on the accessibility of lysine to C5 vs C8 of the purine. No adduct formation was detected between lysine and the other nucleobases T, C, or A. Overall, the precise location of adduct formation at C5 vs C8 of guanine appears to be diagnostic of the oxidation pathway.
Formononetin (FN), a bioactive component extracted from the red clover (Trifolium pratense L.), has been long used for treating carcinomas in China. In the present study, we aim to investigate the potential therapeutical effects of FN on cell line of prostatic adenocarcinoma (PC-3) and human prostate epithelial cells (RWPE1). These findings indicated that FN significantly inhibited the cell growth of PC-3 in a dose-dependent manner, but no such effect was observed in RWPE1 cells. The apoptotic counts were effectively increased following the treatments as shown in flow cytometry. The results from Western blotting assay suggested that FN treatment contributed to the reduced Bcl-2 protein level and the elevated Bax expression in PC-3 cells, thereby resulting in the increasing Bax/Bcl-2 ratios. Furthermore, the phosphorylated level of p38 in PC-3 cells was activated through the FN treatment, whereas the endogenous Akt phosphorylation was blocked. Collectively, our findings demonstrate that FN exerts the anticarcinogenic effect on prostate cancer in vitro, in which the underlying mechanisms are associated with enhancing the Bax/Bcl-2 ratios and regulating the p38/Akt pathway, thus triggering apoptosis in tumor cells.
The infection of host cells by porcine circovirus type 2 (PCV2) leads to extensive modulation of the gene expression levels of target cells. To uncover the pathogenesis and virus-host interactions of PCV2, a quantitative proteomic study using the stable isotope labeling with amino acids in cell culture (SILAC), coupled with mass spectrometry, was performed on PCV2-infected PK-15 cells. The SILAC-based approach identified 1341 proteins, 163 of which showed significant change in level at 72 h after infection (79 up-regulated and 84 down-regulated). The modulated proteins included a number of proteins involved in substrate transport, cytoskeletal changes, and the stress response. Changes in the expression levels of selected proteins were verified by Western blot analysis. Ingenuity Pathway Analysis was used to reveal protein and interactive pathway regulation in response to PCV2 infection. Functional network and pathway analyses could provide insights into the complexity and dynamics of virus-host cell interactions and may accelerate our understanding of the mechanisms of PCV2 infection.
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