80 research outputs found

    Inducible Ablation of <i>COUP-TFII</i> at Pre-puberty Stage Leads to Infertility and Hypogonadism.

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    <p>A) Scheme of inducible ablation of <i>COUP-TFII</i> at Pre-puberty stage. Tamoxifen or oil was intraperitonealy injected into P14 animals to induce the deletion of <i>COUP-TFII</i> gene, and mice were sacrificed at P90. B) Immunoblotting of COUP-TFII was performed to examine the deletion efficiency in mutant mice, and tissues were collected from the comparison littermates. F/F, Tam: <i>COUP-TF<sup>flox/flox</sup></i> treated with tamoxifen: Cre/+ F/F, Oil: <i>Cre-ER<sup>TM (+/βˆ’)</sup> COUP-TFII<sup>flox/flox</sup></i> treated with oil and Cre/+ F/F, Tam: <i>Cre-ER<sup>TM (+/βˆ’)</sup> COUP-TFII<sup>flox/flox</sup></i> treated with tamoxifen. C) The photograph depicts the appearance of male reproduction organs from 3-month-old littermate. D) Relative weight of reproduction organs normalized with body weight. Results are expressed as the mean (Β±SD) of the ratios for each genotype. Statistical comparison was done with a student test. * P<0.05, ** P<0.01</p

    COUP-TFII Plays Roles in Testis Organogenesis and Progenitor Leydig cell Formation.

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    <p>A) Immunohistochemistry for COUP-TFII at embryonic 18.5 (E18.5) and P7. Tamoxifen was injected to pregnant mothers at E18.5. B) Immunohistochemistry for Leydig cell markers, 3Ξ²-HSD at E18.5, P14 and P21. Arrow indicated progenitor Leydig cells, and arrowhead was fetal Leydig cells. C) Quantitative results of progenitor Leydig cell number peri-seminiferous tubule. Data in (C) indicate meanΒ±SD. * P<0.05; ** P<0.01. D) H& E staining of the testes and epididymis from P60 littermate of control and mutant mice. Spermatazoa was indicated by arrow. Immunohistochemistry result for Leydig cell markers, 3Ξ²-HSD and EST. E) The photograph depicts the appearance of testes from control and mutant mice at E18.5 and P14.</p

    Arrest of Spermatogenesis at the Round Spermatid Stage in the Null Mice.

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    <p>H&E staining of paraffin-embedded testes (A, A') and epididymis (B). A' is the large magnification of the box area in the A. (C) Quantitative realtime RT-PCR analysis of the germ cell differentiation markers. RNA was isolated from 3-months-old littermates. Expression levels of each gene were normalized to the levels of the 18sRNA (nβ€Š=β€Š6). Data in (C) indicate the meanΒ±SD. * P<0.05; ** P<0.01</p

    Arylations of Substituted Enamides by Aryl Iodides: Regio- and Stereoselective Synthesis of (<i>Z</i>)‑β-Amido-Ξ²-Arylacrylates

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    Arylations of substituted enamides by aryl iodides were achieved for the first time via an unusual PdCl<sub>2</sub>(COD)/Ag<sub>3</sub>PO<sub>4</sub> catalytic system. A broad range of (<i>Z</i>)-Ξ²-amido-Ξ²-arylacrylates were prepared regio- and stereoselectively in a highly efficient manner

    Inactivation of the Host Lipin Gene Accelerates RNA Virus Replication through Viral Exploitation of the Expanded Endoplasmic Reticulum Membrane

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    <div><p>RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of <i>PAH1</i> (<u>p</u>hosphatidic <u>a</u>cid phospho<u>h</u>ydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in <i>pah1Ξ”</i> yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in <i>pah1</i>Ξ” yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the <i>Arabidopsis</i> Pah2p in <i>Nicotiana benthamiana</i> decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.</p></div

    Deletion of the single yeast lipin gene (<i>PAH1</i>) enhances TBSV repRNA accumulation.

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    <p>(A) The role of Pah1p phosphatidate phosphatase and Dgk1p diacylglycerol kinase in lipid synthesis in yeast. To convert phosphatidic acid (PA) to diacylglycerol (DAG), Pah1p is dephosphorylated (activated) by the ER-localized Nem1p/Spo7p complex. (B) Top panel: Replication of the TBSV repRNA in wt and <i>pah1Ξ”</i> yeast was measured by Northern blotting 24 h after initiation of TBSV replication. Yeast co-expressed the TBSV (lanes 1–6) and the CNV (lanes 7–12) p33 and p92 replication proteins. The accumulation level of repRNA was normalized based on the ribosomal (r)RNA. Each sample is obtained from different yeast colonies. Middle and bottom panels: The accumulation levels of FLAG-p92 and 6Γ—His-p33 were tested by Western blotting. Each experiment was repeated. (C–D) Expression of wt Pah1p and a phosphorylation deficient, constitutively active Pah1p, called Pah1-7A, which contains alanine substitutions for all seven phosphorylation sites, reduces TBSV replication in <i>pah1Ξ”</i> and wt yeasts. Northern blotting was done as in panel B. (E) Stimulatory effect of deletion of <i>NEM1</i> and <i>SPO7</i>, which form the dephosphorylation complex in the ER membrane, on TBSV repRNA accumulation is shown by Northern blotting. Note that Nem1p and Spo7p are required to dephosphorylate Pah1p, leading to the activation and relocalization of Pah1p from the cytosol to the ER membrane.</p

    Only a small portion of the tombusvirus p33 replication protein co-localize with peroxisomes in <i>pah1Ξ” nem1Ξ”</i> yeast.

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    <p>(A) YFP-tagged p33 and CFP-Pex13 (a peroxisomal membrane marker) were co-expressed in WT (RS453) or (B) <i>pah1Ξ” nem1Ξ”</i> yeasts. The confocal images were taken 24 hours after the induction of YFP-p33 expression at 23Β°C. See further details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003944#ppat-1003944-g005" target="_blank">Fig. 5</a>.</p

    Inhibition of tombusvirus and RCNMV RNA accumulation in plants by over-expression of AtPah2p in <i>N. benthamiana</i>.

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    <p>(A) Expression of the yeast <i>PAH1</i> homolog AtPah2p (lanes 5–8) was done in <i>N. benthamiana</i> leaves by agroinfiltration. Two days later, the same leaves were infiltrated with <i>Agrobacterium</i> carrying a plasmid to launch CNV replication from the 35S promoter. The control samples were obtained from leaves expressing no proteins (lanes 1–4). Total RNA was extracted from leaves 5 days after agroinfiltration that launched CNV replication. The accumulation of CNV gRNA and subgenomic (sg)RNAs in <i>N. benthamiana</i> leaves was measured by Northern blotting (Top panel). The ribosomal RNA (rRNA) was used as a loading control and shown in agarose gel stained with ethidium-bromide (Second panel). (B) Over-expression of AtPah2p in <i>N. benthamiana</i> protects the plant from rapid necrosis caused by systemic CNV infection. The pictures were taken 12 days after agroinfiltration. (C) Inhibition of the peroxisomal TBSV replication by over-expression of AtPah2p in <i>N. benthamiana</i>. The agro-infiltrated leaves were inoculated with TBSV two days later, followed by sampling of the same leaves after 3 day of incubation. The accumulation of TBSV gRNA and subgenomic (sg)RNAs in <i>N. benthamiana</i> leaves was measured by Northern blotting. See additional details in panel A. (D) The lack of inhibition of the mitochondrial CIRV tombusvirus by over-expression of AtPah2p in <i>N. benthamiana</i>. See additional details in panel A. (E) Inhibition of the distantly related RCNMV (which uses ER membranes for replication) by over-expression of AtPah2p in <i>N. benthamiana</i>. The agro-infiltrated leaves were inoculated with RCNMV two days later, followed by sampling of the same leaves after 3 day of incubation. The accumulation of RCNMV RNA1 in <i>N. benthamiana</i> leaves was measured by Northern blotting. See additional details in panel A.</p
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