Reaction of Picolinamides with Ketones Producing a New Type of Heterocyclic Salts with an Imidazolidin-4-One Ring

Reactions of picolinamides with 1,3-propanesultone in methanol followed by the treatment with ketones led to a series of previously unknown chemical transformations, yielding first pyridinium salts (2a–f), with a protonated endocyclic nitrogen atom, and then heterocyclic salts (3a–j) containing an imidazolidin-4-one ring. The structures of intermediate and final products were determined by IR and 1H, 13C NMR spectroscopy, and X-ray study. The effects of the ketone and alcohol structures on the product yield were studied by quantum-chemical calculations. The stability of salts 3a–j towards hydrolysis and alcoholysis makes them excellent candidates for the search for new types of biologically active compounds

The Immunophilin-Like Protein XAP2 Is a Negative Regulator of Estrogen Signaling through Interaction with Estrogen Receptor α

XAP2 (also known as aryl hydrocarbon receptor interacting protein, AIP) is originally identified as a negative regulator of the hepatitis B virus X-associated protein. Recent studies have expanded the range of XAP2 client proteins to include the nuclear receptor family of transcription factors. In this study, we show that XAP2 is recruited to the promoter of ERα regulated genes like the breast cancer marker gene pS2 or GREB1 and negatively regulate the expression of these genes in MCF-7 cells. Interestingly, we show that XAP2 downregulates the E2-dependent transcriptional activation in an estrogen receptor (ER) isoform-specific manner: XAP2 inhibits ERα but not ERβ-mediated transcription. Thus, knockdown of intracellular XAP2 levels leads to increased ERα activity. XAP2 proteins, carrying mutations in their primary structures, loose the ability of interacting with ERα and can no longer regulate ER target gene transcription. Taken together, this study shows that XAP2 exerts a negative effect on ERα transcriptional activity and may thus prevent ERα-dependent events

XAP2 interacts with ERα but has no effect on the intracellular level of ERα.

<p>(A) HC11 cells where transiently transfected with XAP2 siRNA (msiXAP2) (lanes 2,4) or a scrambled siRNA (Scr) (lanes 1,3). 48 h after transfection, cells were treated with DMSO (−E₂) or 10 nM E₂ (+E₂) for 1 h before harvest. Whole cell extracts were prepared and Western blot experiments were performed with indicated antibodies; β-actin was used as a loading control. (B) The ERα protein levels shown in (A) were quantified by measuring the density of specific bands and normalizing to β-actin progein levels. The ERα/β-actin ratio in Scr (−E₂) cells was arbitrarily set to 1 and data were expressed as means ± SE of three independent experiments. (C) HC11 cells were treated for 1 h with DMSO (−) or E₂. Whole cell extract (WCE) was prepared and immunoprecipitation (IP) experiments were performed using a XAP2 antibody (lanes 3–4). The presence of ERα protein was monitored by Western blot analysis. WCE (lane 2) and an IgG antibody (lane 1) show the positive and negative controls, respectively. Data shown here is representative of three independent experiments. (D) Radioactively labeled proteins XAP2 and ERα synthesized by <i>in vitro</i> translation were mixed in equal amounts. After incubation (for details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025201#s2" target="_blank">Materials and Methods</a>), XAP2 antibodies or IgG antibodies were added to each protein mixture. Precipitated complexes were analyzed by SDS-PAGE. Data shown here is representative of three independent experiments.</p

XAP2/ERα interaction is crucial for XAP2 to inhibit ERα-mediated transcription.

<p>(A) Schematic illustration of the XAP2 protein. The locations of the PPlase-like domain and the three TPRs are showed and mutation constructs used in this study are indicated. (B) HeLa cells were transiently co-transfected with expression vectors encoding indicated XAP2 mutations together with ERα and a 3×ERE-TATA-Luc reporter. 3 h after transfection, cells were treated with DMSO or 10 nM E₂ for 48 h. Whole cell extracts (WCE) were prepared and luciferase activity was measured. Reporter gene activity was determined and normalized to β-galactosidase. Results were compared to luciferase activity of E₂ dependent ERα-induced reporter activity, which were arbitrarily set to 1. (C) HeLa cells were transiently co-transfected with expression vectors encoding indicated cMyc-tagged XAP2 mutations together with ERα. 24 h after transfection, cells were treated with DMSO or 10 nM E₂ for 1 h and immunoprecipitation (IP) experiments were performed using an ERα antibody. The presence of XAP2 proteins was monitored by Western blot analysis using c-Myc antibody. WCE (input) and an IgG antibody (IgG) show the positive and negative controls, respectively. XAP2 protein levels were quantified by measuring the density of specific bands. WT XAP2 transfected (+E₂) cells was arbitrarily set to 1. Data were expressed as means ± SD of three independent experiments performed in triplicate. *, <i>P</i><0.05 (Student's <i>t</i> test).</p

XAP2 represses ERα but not ERβ mediated transcription.

<p>(A–B) HeLa cells were transiently co-transfected with 1 ng of ERα (A) or ERβ (B) expression vectors together with increasing amounts of XAP2 (1–10 ng) together with 100 ng of a 3×ERE-TATA-Luc reporter. (C–D) HeLa cells were transiently transfected with 1 ng of ERα (C) or ERβ (D) expression vectors upon increasing amounts of XAP2 (1–10 ng) together with 100 ng of a pS2 promoter luciferase reporter construct. 3 h after transfection, cells were treated with DMSO or 10 nM E₂ for 48 h. Whole cell extracts (WCE) were prepared and luciferase activity was measured. Reporter gene activity was determined and normalized to β-galactosidase. Results were compared to basic luciferase activity of the reporter constructs, which were arbitrarily set to 1. Data were expressed as means ± SD of three independent experiments performed in triplicate. *, <i>P</i><0.05 (Student's <i>t</i> test).</p

Suppressive effects of XAP2 on ERα-mediated gene expression.

<p>(A) The expression of endogenous pS2 gene in MCF-7 cells was monitored following the transfection with XAP2 siRNA (hsiXAP2) or scramble siRNA (Scr). After 48 h of transfection, cells were treated with DMSO (veh) or 10 nM E₂ (E2) for 6 h before harvest. The mRNA levels of pS2 were determined by real-time RT-PCR and activity of scramble siRNA transfected E₂ treated samples were arbitrarily set to 1. (B) The expression of endogenous GREB1 gene in MCF-7 cells was monitored following the transfection with XAP2 siRNA (hsiXAP2) or scramble siRNA (Scr). After 48 h of transfection, cells were treated with DMSO or 10 nM E₂ for 6 h before harvest. The mRNA levels of GREB1 were determined by real-time RT-PCR and activity of scramble siRNA transfected E₂ treated samples were arbitrarily set to 1. (C) ERα-independent (−ERα) or dependent (+ERα) activation of 3×ERE-TATA-Luc reporter gene transcription in HeLa cells was monitored following co-transfection with XAP2 siRNA (hsiXAP2) or scramble siRNA (Scr). After 4 h of transfection, cells were treated with DMSO or 10 nM E₂ for 48 h before reporter gene activity was determined. Activity of scramble siRNA transfected DMSO treated cell samples were arbitrarily set to 1. (D) XAP2 and β-actin protein levels in MCF-7 and HeLa cells transfected with XAP2 siRNA (hsiXAP2) or Scramble siRNA (Scr) were determined by Western blot. (E) The protein expressions on Western blot in (D) were quantified by density of specific bands and normalized to β-actin. The XAP2/β-actin ratio in Scr sequence-transfected cells was arbitrarily set to 1. Data were expressed as means ± SE of three independent experiments performed in duplicate. *, <i>P</i><0.05 (Student's <i>t</i> test).</p

Fragmentation Through Polymerization (FTP): A new method to fragment DNA for next-generation sequencing.

Fragmentation of DNA is the very important first step in preparing nucleic acids for next-generation sequencing. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is a simple, robust, and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for direct use in NGS library construction and allows the elimination of the additional step of reparation of DNA ends

An expedient synthesis of a picolinamide-based betain bearing a 3-sulfonatopropyl substituent

3-[2-(Aminocarbonyl)pyridinium-1-yl]propane-1-sulfonate, a promising medicinal betain, was prepared by a one-pot synthesis with a 89% yield via N-alkylation of ethyl picolinate with 1,3-propanesultone in MeCN followed by ammonolysis. The reaction involving picolinamide in MeOH followed by the treatment with acetone afforded a novel 3,3-dimethyl- 1-oxo-2,3-dihydro-1H-imidazo[1,5-a]pyridin-4-ium 3-methoxypropane-1-sulfonate