Mitochondrial ribosomal proteins (mrps) of the budding yeast, Saccharomyces cerevisiae, have been extensively characterized genetically and biochemically. However, the list of the genes encoding individual mrps is still not complete and quite a few of the mrps are only predicted from their similarity to bacterial ribosomal proteins. We have constructed a yeast strain in which one of the small subunit proteins, termed Mrp4, was tagged with S-peptide and used for affinity purification of mitochondrial ribosome. Mass spectrometric analysis of the isolated proteins detected most of the small subunit mrps which were previously identified or predicted and about half of the large subunit mrps. In addition, several proteins of unknown function were identified. To confirm their identity further, we added tags to these proteins and analyzed their localization in subcellular fractions. Thus, we have newly established Ymr158w (MrpS8), Ypl013c (MrpS16), Ymr188c (MrpS17) and Ygr165w (MrpS35) as small subunit mrps and Img1, Img2, Ydr116c (MrpL1), Ynl177c (MrpL22), Ynr022c (MrpL50) and Ypr100w (MrpL51) as large subunit mrps.
Hepatitis B virus (HBV) infection is a major risk factor for the development of chronic liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). A growing body of evidence suggests that HBV X protein (HBx) plays a crucial role in viral replication and HCC development. Here, we identified peroxiredoxin 1 (Prdx1), a cellular hydrogen peroxide scavenger, as a novel HBx-interacting protein. Coimmunoprecipitation analysis coupled with site-directed mutagenesis revealed that the region from amino acids 17 to 20 of the HBx, particularly HBx Cys17, is responsible for the interaction with Prdx1. Knockdown of Prdx1 by siRNA significantly increased the levels of intracellular HBV RNA, HBV antigens, and extracellular HBV DNA, whereas knockdown of Prdx1 did not increase the activities of HBV core, enhancer I (Enh1)/X, preS1, and preS2/S promoters. Kinetic analysis of HBV RNA showed that knockdown of Prdx1 inhibited HBV RNA decay, suggesting that Prdx1 reduces HBV RNA levels posttranscriptionally. The RNA coimmunoprecipitation assay revealed that Prdx1 interacted with HBV RNA. The exosome component 5 (Exosc5), a member of the RNA exosome complexes, was coimmunoprecipitated with Prdx1, suggesting its role in regulation of HBV RNA stability. Taken together, these results suggest that Prdx1 and Exosc5 play crucial roles in host defense mechanisms against HBV infection. IMPORTANCE Hepatitis B virus (HBV) infection is a major global health problem. HBx plays important roles in HBV replication and viral carcinogenesis through its interaction with host factors. In this study, we identified Prdx1 as a novel HBx-binding protein. We provide evidence suggesting that Prdx1 promotes HBV RNA decay through interaction with HBV RNA and Exosc5, leading to downregulation of HBV RNA. These results suggest that Prdx1 negatively regulates HBV propagation. Our findings may shed new light on the roles of Prdx1 and Exosc5 in host defense mechanisms in HBV infection.
Granulosa cells (GCs) are essential components of follicles and are involved in regulating the process of follicles development. However, comparative studies on GCs isolated from different staged follicles have not been conducted in goose. The aim of the present study was to identify the growth characteristics of goose GCs from pre-hierarchical (6–10 mm) and hierarchical (F4–F2, F1) follicles. Our results showed that the three cohorts of cells had different tolerance to collagenase and had noticeable morphological differences. The F1 granulosa layers were fully digested by 0.1% collagenase, while higher concentration (0.3%) was used for both F4–F2 and pre-hierarchical granulosa layers. In the state of suspension, the diameter of F1 individual cell was larger than the other two cohorts. However, after adhering to the culture plate, cells of F1 just had changes in the diameter accompanied by small bright spots, while both pre-hierarchical and F4–F2 GCs proliferated rapidly with spreading and irregularly shaped voids. Furthermore, all attached cells could be stained by the follicle-stimulating hormone receptor antibody. Analyses of both growth curve and the mRNA expression profiles of genes related to cellular proliferation, apoptosis, and steroidogenesis suggested that three cohorts of in vitro cultured GCs had different physiological viability and functions. Taken together, the present study not only revealed differences of the growth characteristics among three cohorts of goose GCs from pre-hierarchical, F4–F2 and F1 follicles, but also optimized the in vitro culture system of geese different staged GCs.
Theca cells, including theca interna cells and theca externa cells, are vital components of ovarian follicles. The aim of the present study is to identify a reliable method for the in vitro culture of theca cells from duck ovarian hierarchical (F4-F2) follicles. We improved the method for cell separation by using trypsin to further remove granular cells, and we increased the concentration of fetal bovine serum used in in vitro culture to improve cytoactivity. Cell antibody immunofluorescence (IF) showed that all inoculated cells could be stained by the CYP17A1/19A1 antibody but not by the FSHR antibody, which could stain granulosa cells. Furthermore, morphological differences were observed between the outlines of theca interna and externa cells and in their nuclei. Growth curve and CYP17A1/19A1 mRNA relative expression analyses suggested that the growth profile of theca interna cells may have been significantly different from that of theca externa cells in vitro. Theca interna cells experienced the logarithmic phase on d1–d2, the plateau phase on d2–d3, and the senescence phase after d3, while theca externa cells experienced the logarithmic phase on d1–d3, the plateau phase on d3–d5, and the senescence phase after d5. Taken together, these results suggested that we have successfully established a reliable theca cell culture model and further defined theca cell characteristics in vitro.
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