26 research outputs found

    Tertiary Amine-Triggered Cascade S<sub>N</sub>2/Cycloaddition: An Efficient Construction of Complex Azaheterocycles under Mild Conditions

    No full text
    In this paper, an amine-triggered cascade S<sub>N</sub>2/cycloaddtion sequence between 2-(acetoxymethyl)buta-2,3-dienoate <b>1</b> and various π-system functionalized tosylamides <b>3</b> has been reported, which provides a facile method for stereoselective construction of structurally diverse azaheterocycles

    Tertiary Amine-Triggered Cascade S<sub>N</sub>2/Cycloaddition: An Efficient Construction of Complex Azaheterocycles under Mild Conditions

    No full text
    In this paper, an amine-triggered cascade S<sub>N</sub>2/cycloaddtion sequence between 2-(acetoxymethyl)buta-2,3-dienoate <b>1</b> and various π-system functionalized tosylamides <b>3</b> has been reported, which provides a facile method for stereoselective construction of structurally diverse azaheterocycles

    HOXB7 is the substrate of PARP-1 and is poly(ADP ribosyl)ated by PARP-1.

    No full text
    <p>A. Different amounts of Flag tagged HOXB7 plasmids were transfected into SKBR3 cells, cells were harvested and permeabilized by 0.01% digitonin in PARP-1 activity reaction buffer. PARP-1 auto-modification was visualized by autoradiograph after the cell lysates were separated by SDS-PAGE (top panel). Immunoblot with anti-Flag, PARP-1 and β-actin antibodies were performed after autoradiography (bottom panels). B. GST and GST-HOXB7 fusion proteins were incubated with or without purified PARP-1 protein or <sup>32</sup>P NAD+. After 30 minutes incubation, free 32P NAD+ and unbound PARP-1 proteins were washed off with reaction buffer. Proteins on glutathione sepharose beads were then separated by SDS-PAGE and transferred to PVDF membrane and stained with Ponceau (left panel). The poly(ADP ribosyl)ated proteins were visualized by autoradiography (right panel). C. Vector control and Flag-tagged HOXB7 plasmids were transfected into SKBR3 cells. Cells were harvested and incubated with PARP-1 activity assay buffer including <sup>32</sup>P NAD+ or PARP-1 inhibitor (20 µM DPQ). Cell lysates were immunoprecipitated with anti-Flag antibody and separated by SDS-PAGE followed by autoradiography (top panel) and immunoblotting with anti-Flag antibodies (bottom panel).</p

    PARP-1 interacts and modifies other HOX proteins.

    No full text
    <p>A. SKBR3 cells were transfected with empty vector, or Flag-tagged HOXA5, B6, B7, C6 and C8, respectively. Cell lysates were immunoprecipitated with anti-Flag antibody, and western blotted with anti-PARP-1 (top panel) and Flag antibodies (bottom panel), respectively. HOXC6 transfected SKBR3 cell lysate was used as loading control. B. Vector control and Flag-tagged HOX plasmids were transfected into SKBR3 cells as indicated. Cells were harvested and incubated with PARP activity reaction buffer containing 0.01% digitonin and <sup>32</sup>P NAD+. Cells were then lysed after incubation and immunoprecipitated with anti-Flag antibody. The precipitated complexes were then separated by SDS-PAGE, transferred to the nitrocellulose membrane that was used for autoradiography (top panel) and western blot (bottom panel) using anti-PARP-1 and anti-Flag antibodies. C. Multiple sequence alignment of the C-terminal peptides of HOXA5, HOXA7, HOXB6, HOXB7, HOXC6 and HOXC8 show extent of homology. D. The ONP assay was performed with the SKBR3 cells transfected with Flag HOXA7 with or without PARP-1, and one set of the HOXA7 and PARP-1 co-transfected cells was treated with DPQ 6 hours post transfection. Cell lysates were used for ONP (top panel) and western blots (bottom panel). E. ONP assays were performed with SKBR3 cells transfected with Flag HOXA1, Flag HOXA5, Flag HOXC6 or Flag HOXB6 plasmids with or without PARP-1 plasmids, and one set of each of the HOX and PARP-1 co-transfected cells was treated with DPQ 6 hours post transfection. Cell lysates were used for ONP assays (top panel) and western blots (bottom panel). F. ONP assay was performed with the SKBR3 cells transfected with Flag-tagged HOXB6 or the Lys to Glu mutant, HOXB6K221E, with or without PARP-1. Cell lysates were used for ONP assays (top panel) and western blots (bottom panel).</p

    Defining regions of PARP-1 that interact with HOXB7.

    No full text
    <p>A. The molecular structure of PARP-1 and the deletion constructs used in the present study are shown in the panel. B, C. Full-length PARP-1 and different truncations of PARP-1 proteins as indicated were <i>in vitro</i> transcribed and translated (TNT) in presence of the <sup>35</sup>S-methionine and subjected to GST-HOXB7 or GST pull-down assay (left panels Fig. 2B and 2C). The right panels of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040644#pone-0040644-g002" target="_blank">Figure 2B and 2C</a> show the input (20%) of the PARP-1 full-length and truncation proteins from TNT products. D. Plasmids coding Flag-tagged HOXB7 and Myc-tagged wild type PARP1 or first zinc-finger deleted PARP1 were cotransfected into MCF-7 cells. Cell lysates were immunoprecipitated with anti-Flag antibody. PARP-1 and HOXB7 protein were detected with anti-Myc or anti-Flag antibodies.</p

    Syntheses of Thiophene–Thiophene-Linked Corrorin Dimers with Tunable Near-Infrared Absorption and Distinctive Reactivity

    No full text
    Thiahexaphyrinone 1 and thia–dipyrrin-appended corrorin 2 have been synthesized. Surprisingly, further oxidation of compound 2 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in dichloromethane afforded dimer 3 with two molecules of compound 2 linked at the α-carbon atoms of the thienyl units. Treatment of compound 3 with DDQ in MeOH and SnCl2 in tetrahydrofuran/H2O afforded the dimethoxy-attached dimer 4 and hydrogenated dihydroxy-attached dimer 5, respectively. These results provide the first examples for synthesizing thiophene-linked porphyrinoid dimers with tunable near-infrared absorption and chirality

    Activating Mutations in <i>PIK3CA</i> Lead to Widespread Modulation of the Tyrosine Phosphoproteome

    No full text
    The human oncogene <i>PIK3CA</i> is frequently mutated in human cancers. Two hotspot mutations in <i>PIK3CA</i>, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis

    BGLF4-induced mitotic phosphorylation network.

    No full text
    <p>Linkage of BGLF4 up-regulated phosphoproteins to mitosis was obtained using the DAVID bioinformatics resource and manual literature curation. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005346#ppat.1005346.s009" target="_blank">S4 Table</a>.</p

    Regulation of known APC/C substrates by BGLF4.

    No full text
    <p>(A) Fold change in protein level of APC/C substrates as measured by MS. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005346#ppat.1005346.s007" target="_blank">S2 Table</a>. (B) Western blot validation of BGLF4 mediated up-regulation of individual APC/C substrates in Dox inducible Akata (EBV+) cells expressing wild-type BGLF4, but not SUMO binding-deficient and kinase-dead mutants (BGLF4<sup>mSIM-N</sup> and BGLF4<sup>KD</sup>). Immunoblot analysis of the same cell lysates from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005346#ppat.1005346.g002" target="_blank">Fig 2C</a> using antibodies as indicated. (C) EBV replication induces the accumulation of TOP2A and Aurora B. Western blot analysis of cell extracts from Akata-BX1 (EBV+) treated as indicated using anti-Aurora B, anti-TOP2A and anti-β-actin antibodies as indicated. (D) and (E) SAC inhibition suppresses production of extracellular EBV virus. Supernatant virion DNA (D) and cell associated viral DNA (E) from Akata (EBV+) cells treated as indicated was determined by PCR. The experiments were carried out in three biological replicates with similar results and the representative results are presented. Relative PCR product intensity was quantified by ImageJ software.</p
    corecore