4a).

TPA/NaB, tetra-decanoyl phorbol-13-acetate–sodium butyrate reactivation control lysate loaded at a 1:10 dilution. S1 in the supplemental material) (23). Mapping of LMP1 signaling motifs and cellular pathways. Les collections du Musée national de l'Éducation The HK1 NPC cell line infected with the EBV recombinant Akata strain (a generous gift from George Tsao, Hong Kong University, Hong Kong) were maintained with 0.8 mg/ml neomycin selection in RPMI 1640 medium supplemented with 10% fetal bovine serum as previously described (28, 34).

On a HFD, however, the levels of these intermediates were significantly elevated in ABCG1 BAC Tg liver (Figure 5C). To determine if the CTAR1 and CTAR2 domains contribute to EBV genome loss, 293 cells infected with CTAR1 or CTAR2 motif mutants were analyzed. P values below 0.0001 were considered significant. Notably, Out et al observed that TC levels correlate with the impact of ABCG1 deficiency on lesion area.24 In our study, TC levels were approximately 700 mg/dL, perhaps a range where ABCG1 has little effect on atherosclerosis. HPLC analysis of (C) cholesterol ester and (D) triglyceride levels from wild-type (WT, n=4), ABCG1 BAC Tg (Tg, n=5), ABCG1−/− (KO, n=5), and rescued (Resc, n=4) mice after 8 weeks on a high-fat high-cholesterol diet. Compared to wild-type controls, selective overexpression of ABCG1 did not alter the levels of any lipids, unlike the results reported by Kennedy et al20 who found that excess ABCG1 protected from diet-induced tissue lipid accumulation. $28.95. Differentiation by lifting epithelial monolayers to an air-liquid interface is a physiological stimulus for inducing lytic reactivation (11, 20, 21). A recent study of organotypic raft culture of keratinocytes demonstrated that the differentiation-dependent induction of the immediately early proteins BZLF1 (Z) and BRLF1 (R) is dependent on the expression of LMP1 (11). This site uses cookies.

LMP1 contributes to permissive infection in differentiating epithelia. It is plausible that cyclin D1 may facilitate the outgrowth of EBV-infected cells by promoting the retention of EBV genomes. Only 3 left. DNA extraction and quantitative PCR.Frozen cell pellets were extracted for total DNA with the GeneJET genomic DNA purification kit with RNase treatment (Thermo Scientific).

Fold changes were calculated relative to HK1 EBV (shSC) cells, averaged from three replica titrations, and compared in five independent experiments. A, Species-specific QRT-PCR analysis of ABCG1 mRNA levels in macrophages treated with ethanol (vehicle), 10 μmol/L of 22-R-OH-cholesterol and 9-cis-RA (LXR/RXR), 1 μmol/L TO901317, or 50 μg/mL Ac-LDL for 24 hours, analyzed by 1-way ANOVA with a Newman-Keuels post test. View cart for details.

A prior study in AdAH cells supports the possibility that epithelial cells that retain EBV infection are not integrated, as the ability to retain EBV genomes was not a heritable trait (6). Download, Latent Membrane Protein 1 Is a Novel Determinant of Epstein-Barr Virus Genome Persistence and Reactivation, Sign In to Email Alerts with your Email Address. Something went wrong. ABCG1 is appropriately overexpressed in ABCG1 BAC Tg mice. Data were analyzed by 1-way ANOVA with a Newman-Keuls posttest; *P<0.05, ***P<0.001 relative to no acceptor. large éventail de possibilité. Three independent congenic C57Bl/6 lines were developed, and the line with highest human ABCG1 expression, estimated at approximately 9 BAC copies, was used in this study. Conclusions— Transgenic human ABCG1 does not influence atherosclerosis in apoE−/− mice but may participate in the regulation of tissue cholesterol biosynthesis.
For detailed methodology, please see the supplemental materials (available online at http://atvb.ahaournals.org). Data were analyzed by 1-way ANOVA with a Newman-Keuls posttest; *P<0.05, ***P<0.001 relative to no acceptor. Deletion of the PXQXT TRAF-binding motif in CTAR1 (ΔPXQXT), but not mutation of the CTAR2 residue (Y384G) critical for NF-κB activation, resulted in a more progressive loss of EBV genomes, stabilizing at a 90% loss by 46 days (Fig.

Although there is no question that ABCG1 deficiency compromises cholesterol efflux to HDL, whether excess ABCG1 facilitates efflux is more challenging. Transgenic ABCG1 alters cholesterol biosynethetic intermediate levels in liver. Recently, Basso et al developed a BAC Tg model of murine ABCG1 overexpression and observed a 39% and 52% increase in lesion area in en face and cross sectional analyses, respectively, when crossed to LDLR−/− mice and fed a HFD of 21.2% fat and 0.2% cholesterol for 12 weeks.22 Increased lesions were associated with increased levels of the proinflammatory markers tumor necrosis factor (TNF)-α and MCP-1, arguing for a proatherogenic and proinflammatory role of ABCG1, which presumably overrides the enhanced efflux to HDL also observed in macrophages from this model. Compared to controls, cholesterol levels were unchanged in ABCG1 BAC Tg liver on chow, but significantly increased after 14 weeks on a HFD (Figure 5A). As an alternative, cells were stained by EBER-ISH to compare the numbers of infected cells lost.

Download figureDownload PowerPointFigure 5. These increases in ABCG1 expression were maintained in apoE−/− mice (not shown).

Although LMP1 is a latent transcript expressed in type II and III latencies, it is also expressed in the lytic cycle (11). To confirm functionality of the human ABCG1 transgene, we bred ABCG1 BAC Tg mice onto an ABCG1−/− backgound (Figure 3A). QRT-PCR analysis confirmed expression of human ABCG1 mRNA across multiple tissues (supplemental Figure I).
Compared to wild-type controls, ABCG1−/− mice exhibited significantly elevated CE (Figure 3C) and significantly decreased TG levels (Figure 3D), and both of these phenotypes were completely rescued by expression of human ABCG1 in lieu of endogenous murine ABCG1. Isotopic 3H-cholesterol efflux assays were performed on thioglycollate-elicited primary peritoneal macrophages. Download, Quantitation of EBV genomes by averaged copy numbers per cell. 3a). Techniques administratives. 1b) (4, 6). At 3 months (92 days) of serial passage, HK1 EBV-Akata cells stably expressing cyclin D1 resulted in a 21% reduction in EBER positivity (76.3% EBER-ISH+) compared to that of cells grown in the presence of neomycin (97.3% EBER-ISH+) (Fig. 3b). Because the ABCG1 antibody used in these experiments may not recognize murine and human ABCG1 equally well, it is not possible to determine the contribution of human ABCG1 to total ABCG1 protein levels in our mice. Sterols analyzed include cholesterol (ChGC), lathosterol (Lath), lanosterol (Lano), desmosterol (Desmo), 7α-OH-cholesterol (7aOH), 24-OH-cholesterol (24OH), 27-OH-cholesterol (27-OH), and cholestanol (CholGC). Because plasma apoE levels have been reported to be increased in ABCG1−/− mice,21 we tested the converse possibility that overexpression of ABCG1 may lead to decreased apoE levels. A major challenge in understanding the role of EBV infection in epithelial cancer is the difficulty in establishing EBV-infected cell lines, which stands in contrast to the EBV-immortalizing properties of B lymphocytes. Stable expression of cyclin D1 was detected by immunoblotting for the HA tag in transduced 293 and HK1 EBV-infected cell lines, and although the level of LMP1 was increased by cyclin D1 expression in 293 cells, the same did not occur in HK1 EBV-infected cells (Fig. Understanding whether cholesterol intermediate levels are altered in ABCG1-deficicient mice may shed further light on the potential role for ABCG1 in sterol biosynthesis. Food Microbiology, 1986, 3, 157-159 Use of a small scale insulated fermentation to simulate the scotch malt whisky fermentation M. Lavery, H. Chamberlain and D. R. Berry Department of Bioscience and B iotechnology, University of Strathclyde, 204 George Street, Glasgow G1 1XW, Scotland Received 1 7 February 1986 A small scale malt whisky fermentation has been carried out in an … . 4a). 1d).

These data indicate that EBV genome loss is a common property conferred by both the B958 and China1 strains and suggest that it may localize to a conserved LMP1 signaling domain. Although the detection of a prevalent LMP1 China1 strain in NPC might be due to enhanced immune evasion properties, it is also possible that LMP1 strains may be distinguished by their role in EBV pathogenesis (18). These data confirm that the human ABCG1 transgene functionally compensates for murine ABCG1 in vivo. It is also possible that effects of ABCG1 on lesion area may require functional apoE. 1986 Maskette Handicap (G1) race results, race date, entries, field, video, contenders, probables, news, notes, & comments. We thank Anja Kerksiek and Silvia Friedrichs for technical assistance in GC and GC-MS analysis. B, Representative image of ORO-stained aortic roots from apoE−/− and ABCG1/apoE−/− mice. Download figureDownload PowerPointFigure 3. Transgenic ABCG1 does not affect atherosclerotic lesion area or macrophage cholesterol efflux. It is possible that the robust pathogenesis in the apoE−/− model may overwhelm the impact of selective ABCG1 overexpression on atherosclerosis. EBV genome copy numbers per cell at the beginning and end of each assay as defined by comparison to Namalwa cells are denoted in italics. Data represent mean and standard error of n=9 mice for WT and G1 cohorts and n=10 mice for apoE−/− and G1/apoE−/− cohorts, each assayed in triplicate. The resulting unstable infection has hampered efforts to study the determinants of EBV persistence and latency in epithelial oncogenesis. To assess atherosclerosis, ABCG1 BAC Tg mice were bred to apoE−/− animals and fed a HFD of 21.2% fat, and 0.2% cholesterol for 14 weeks, followed by aortic root lesion quantification using 4 sections per mouse. 1c). C, Isotopic 3H-cholesterol efflux assays from elicited peritoneal macrophages derived from wild-type (WT), ABCG1 BAC Tg (G1), apoE−/−, and ABCG1 BAC/apoE−/− (G1/apoE−/−) mice. ABCG1 is appropriately overexpressed in ABCG1 BAC Tg mice. On the HFD, we observed a nonsignificant trend toward reduced apoE levels in ABCG1 BAC Tg mice (supplemental Figure IV). Organisation, études de cas. Skills & Endorsements Knockdown of LMP1 in EBV Akata-infected HK1 cells cultured in the air-liquid interface suppressed lytic reactivation, as demonstrated by decreased induction of lytic proteins and infectious virus production. The central hypothesis for LMP1 as an oncogenic signaling protein is that it is consistently expressed and detected in NPC tumors (10, 13). In comparison to other human tumor viruses, such as human papillomavirus and Merkel cell polyomavirus, which are characterized by integrated viral genomes in tumor cells, EBV-associated cancers, including NPC, maintain EBV genomes as extrachromosomal episomes.

(d) Immunoblot analysis for LMP1 expression in EBV-infected and trans-complemented 293 cell lines. 2b). ABCG1 BAC Tg mice exhibited a nonsignificant trend toward decreased levels of cholesterol biosynthetic intermediates in the livers of chow-fed animals, and a significant increase in cholesterol, lathosterol, lanosterol, and desmosterol in fat-fed liver when quantified by mass spectrometry as a function of tissue dry weight. Thus, the switch to a persistent and latent infection in preneoplastic cells is likely a common driver event for cancers associated with oncogenic gammaherpesviruses. In comparison with monolayer culture, the induction of the differentiation marker involucrin indicated differentiation of HK1 cells in the 3-week ALI culture period beginning at week 1 (Fig. B, Representative image of ORO-stained aortic roots from apoE−/− and ABCG1/apoE−/− mice.

These values are summarized in Fig.

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