Association of Rex-1 to target genes supports its interaction with Polycomb function

Rex-1/Zfp42 displays a remarkably restricted pattern of expression in preimplantation embryos, primary spermatocytes, and undifferentiated mouse embryonic stem (ES) cells and is frequently used as a marker gene for pluripotent stem cells. To understand the role of Rex-1 in selfrenewal and pluripotency, we used Rex-1 association as a measure to identify potential target genes, and carried out chromatin-immunoprecipitation assays in combination with gene specific primers to identify genomic targets Rex-1 associates with. We find association of Rex-1 to several genes described previously as bivalently marked regulators of differentiation and development, whose repression in mouse embryonic stem (ES) cells is Polycomb Group-mediated, and controlled directly by Ring1A/B. To substantiate the hypothesis that Rex-1 contributes to gene regulation by PcG, we demonstrate interactions of Rex-1 and YY2 (a close relative of YY1) with Ring1 proteins and the PcG-associated proteins RYBP and YAF2, in line with interactions reported previously for YY1. We also demonstrate the presence of Rex-1 protein in both trophectoderm and Inner Cell Mass of the mouse blastocyst and in both ES and in trophectoderm stem (TS) cells. In TS cells, we were unable to demonstrate association of Rex-1 to the genes it associates with in ES cells, suggesting that association may be cell-type specific. Rex-1 might fine-tune pluripotency in ES cells by modulating Polycomb-mediated gene regulation.     

The Mutational Landscape of Acute Promyelocytic Leukemia Reveals an Interacting Network of Co-Occurrences and Recurrent Mutations

Preliminary Acute Promyelocytic Leukemia (APL) whole exome sequencing (WES) studies have identified a huge number of somatic mutations affecting more than a hundred different genes mainly in a non-recurrent manner, suggesting that APL is a heterogeneous disease with secondary relevant changes not yet defined. To extend our knowledge of subtle genetic alterations involved in APL that might cooperate with PML/RARA in the leukemogenic process, we performed a comprehensive analysis of somatic mutations in APL combining WES with sequencing of a custom panel of targeted genes by next-generation sequencing. To select a reduced subset of high confidence candidate driver genes, further in silico analysis were carried out. After prioritization and network analysis we found recurrent deleterious mutations in 8 individual genes (STAG2, U2AF1, SMC1A, USP9X, IKZF1, LYN, MYCBP2 and PTPN11) with a strong potential of being involved in APL pathogenesis. Our network analysis of multiple mutations provides a reliable approach to prioritize genes for additional analysis, improving our knowledge of the leukemogenesis interactome. Additionally, we have defined a functional module in the interactome of APL. The hypothesis is that the number, or the specific combinations, of mutations harbored in each patient might not be as important as the disturbance caused in biological key functions, triggered by several not necessarily recurrent mutations.     

Isolation and characterization of 10 polymorphic dinucleotide microsatellite markers for llama and guanaco

We isolated and characterized five polymorphic dinucleotide microsatellite markers from llama (Lama glama) and five from guanaco (Lama guanicoe). All loci were assayed on wild llamas and guanacos from Argentina, as all of the primers were able to amplify in both species.     

Interaction of rat liver 3-D-(-)-hydroxybutyrate aopdehydrogenase with phospholipids

The interaction of phospholipids with pure, catalytically inactive rat liver 3-d-(—)-CoA hydroxybutyrate apodehydrogenase (apoHBD) was examined, (a) A relationship could be established between density of packing of phospholipid molecules at the interface and apoHBD activation, namely, the larger the area per polar head, the higher the lipid molar efficiency. In this context, codispersion of lecithins with phospholipids that were inactive or scarcely active per se, such as phosphatidylethanolamine and lysophosphatidylcholine (miristoyl; Iysod₁₄) increased the activating efficiency of lecithins, (b) ApoHBD formed tightly bound, catalytically active complexes with lecithin liposomes and micelles (diC₁₀ + lysoC₁₄; cetylphosphorylcholine), but a phospholipid-water interface was not essential for HBD activity since a molecular dispersion of diheptanoyl lecithin (diC₇) activated apoHBD to a limited extent. ApoHBD formed loosely bound, catalytically inactive complexes with multilayer vesicles, but HBD activity could be restored by sonication or by adding liposome to those complexes. Unlike liposomes and micelles, apoHBD interaction with multilayer vesicles did not involve a hydrophobic contribution, which was apparently necessary for apoHBD activation, (c) LysoC₁₄, did₁₀ + lysoC₁₄, and cetylphosphorylcholine micelles activated apoHBD but diC₇ micelles inhibited the HBD activity of the apoHBD-diC₇ (monomer) complex. The inhibition decreased when the medium ionic strength was increased. Liposomes and diCi₁₀ + lysoC₁₄ micelles activated and stabilized apoHBD much more efficiently than pure lysoC₁₄ or cetylphosphorylcholine micelles, (d) The mode of aggregation of the activating phospholipid strongly affected the kinetics of the HBD reaction. With liposomes the reaction showed an initial lag (or induction) period whose duration varied over a range of 3 to 15 min, depending on the activating phospholipid; with diC₇ monomers and micelles the kinetics was linear throughout, while with multilayer vesicles the lag was virtually infinite since HBD activity was insignificant, (e) Energies of activation for apoHBD-diC₁₄ complexes, either below or above the lecithin gel-to-liquid crystalline transition temperature were not significantly different, in accordance with apoHBD interaction with the proximal end of the hydrocarbon chains, that is, the less subject to phase transitions. With a diC₁₄-substituted mitochondrial preparation, however, no HBD activity was detected below 24 °C (near the gel-to-liquid crystalline transition temperature of diC₁₄), thus indicating that, in the inner membrane, apoHBD interacts with the whole length of the fatty acyl chain and, consequently, is sensitive to phase transition.     

Growth and differentiation of Vero cells cultivated in three-dimensional type 1 collagen

Vero cells grown in a three-dimensional arrangement of type 1 collagen were phenotypically altered. Such alterations consisted of variations in cellular form and reactivity to toluidine blue. After 22 days of incubation the Vero cells formed three-dimensional arrangements in both tubular form and cellular mass. A reduction in the collagenous substrate was observed in the culture and the alteration noted in the birefringence of the extracellular matrix was attributed to enzymatic action. This model demonstrates a good in vitro system for the study of collagenase activation and for in vitro tests with collagen.     

Anion channel activity is necessary to induce ethylene synthesis and programmed cell death in response to oxalic acid

Oxalic acid is thought to be a key factor of the early pathogenicity stage in a wide range of necrotrophic fungi. Studies were conducted to determine whether oxalate could induce programmed cell death (PCD) in Arabidopsis thaliana suspension cells and to detail the transduction of the signalling pathway induced by oxalate. Arabidopsis thaliana cells were treated with millimolar concentrations of oxalate. Cell death was quantified and ion flux variations were analysed from electrophysiological measurements. Involvement of the anion channel and ethylene in the signal transduction leading to PCD was determined by using specific inhibitors. Oxalic acid induced a PCD displaying cell shrinkage and fragmentation of DNA into internucleosomal fragments with a requirement for active gene expression and de novo protein synthesis, characteristic hallmarks of PCD. Other responses generally associated with plant cell death, such as anion effluxes leading to plasma membrane depolarization, mitochondrial depolarization, and ethylene synthesis, were also observed following addition of oxalate. The results show that oxalic acid activates an early anionic efflux which is a necessary prerequisite for the synthesis of ethylene and for the PCD in A. thaliana cells.