Chromosomal Manipulation by Site-Specific Recombinases and Fluorescent Protein-Based Vectors

Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e.g. embryonic stem cells). Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations

JAK2 V617F-Dependent Upregulation of PU.1 Expression in the Peripheral Blood of Myeloproliferative Neoplasm Patients

Myeloproliferative neoplasms (MPN) are multiple disease entities characterized by clonal expansion of one or more of the myeloid lineages (i.e. granulocytic, erythroid, megakaryocytic and mast cell). JAK2 mutations, such as the common V617F substitution and the less common exon 12 mutations, are frequently detected in such tumor cells and have been incorporated into the diagnostic criteria published by the World Health Organization since 2008. However, the mechanism by which these mutations contribute to MPN development is poorly understood. We examined gene expression profiles of MPN patients focusing on genes in the JAK–STAT signaling pathway using low-density real-time PCR arrays. We identified the following 2 upregulated genes in MPN patients: a known target of the JAK–STAT axis, SOCS3, and a potentially novel target, SPI1, encoding PU.1. Induction of PU.1 expression by JAK2 V617F in JAK2-wildtype K562 cells and its downregulation by JAK2 siRNA transfection in JAK2 V617F-positive HEL cells supported this possibility. We also found that the ABL1 kinase inhibitor imatinib was very effective in suppressing PU.1 expression in BCR-ABL1-positive K562 cells but not in HEL cells. This suggests that PU.1 expression is regulated by both JAK2 and ABL1. The contribution of the two kinases in driving PU.1 expression was dominant for JAK2 and ABL1 in HEL and K562 cells, respectively. Therefore, PU.1 may be a common transcription factor upregulated in MPN. PU.1 is a transcription factor required for myeloid differentiation and is implicated in erythroid leukemia. Therefore, expression of PU.1 downstream of activated JAK2 may explain why JAK2 mutations are frequently observed in MPN patients

Mixing of immiscible polymers using nanoporous coordination templates

混ざり合わないポリマーを完全に混ぜる手法を開発 -プラスチックの持つ機能を飛躍的に向上-. 京都大学プレスリリース. 2015-07-02.The establishment of methodologies for the mixing of immiscible substances is highly desirable to facilitate the development of fundamental science and materials technology. Herein we describe a new protocol for the compatibilization of immiscible polymers at the molecular level using porous coordination polymers (PCPs) as removable templates. In this process, the typical immiscible polymer pair of polystyrene (PSt) and poly(methyl methacrylate) (PMMA) was prepared via the successive homopolymerizations of their monomers in a PCP to distribute the polymers inside the PCP particles. Subsequent dissolution of the PCP frameworks in a chelator solution affords a PSt/PMMA blend that is homogeneous in the range of several nanometers. Due to the unusual compatibilization, the thermal properties of the polymer blend are remarkably improved compared with the conventional solvent-cast blend. This method is also applicable to the compatibilization of PSt and polyacrylonitrile, which have very different solubility parameters

Correction: Chronic Lung Injury by Constitutive Expression of Activation-Induced Cytidine Deaminase Leads to Focal Mucous Cell Metaplasia and Cancer.

Activation-induced cytidine deaminase (AID) is an enzyme required for antibody diversification, and it causes DNA mutations and strand breaks. Constitutive AID expression in mice invariably caused lung lesions morphologically similar to human atypical adenomatous hyperplasia (AAH), which can be a precursor of bronchioloalveolar carcinoma. Similar to AAH, mouse AAH-like lesion (MALL) exhibited signs of alveolar differentiation, judging from the expression of alveolar type II (AT2) cell marker surfactant protein C (SP-C). However, electron microscopy indicated that MALL, which possessed certain features of a mucous cell, is distinct from an AAH or AT2 cell. Although MALL developed in all individuals within 30 weeks after birth, lung tumors occurred in only 10%; this suggests that the vast majority of MALLs fail to grow into visible tumors. MALL expressed several recently described markers of lung alveolar regeneration such as p63, keratin 5, keratin 14, leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and Lgr6. Increased cell death was observed in the lungs of AID transgenic mice compared with wild-type mice. Based on these observations, we speculate that MALL is a regenerating tissue compensating for cellular loss caused by AID cytotoxicity. AID expression in such regenerating tissue should predispose cells to malignant transformation via its mutagenic activity

¹¹³Cd Nuclear Magnetic Resonance as a Probe of Structural Dynamics in a Flexible Porous Framework Showing Selective O₂/N₂ and CO₂/N₂ Adsorption

Two new isomorphous three-dimensional porous coordination polymers, {[Cd­(bpe)_0.5(bdc)­(H₂O)]·EtOH}_<i>n</i> (<b>1</b>) and {[Cd­(bpe)_0.5(bdc)­(H₂O)]·2H₂O}_<i>n</i> (<b>2</b>) [bpe = 1,2-bis­(4-pyridyl)­ethane, and H₂bdc = 1,4-benzenedicarboxylic acid], have been synthesized by altering the solvent media. Both structures contain one-dimensional channels filled with metal-bound water and guest solvent molecules, and desolvated frameworks show significant changes in structure. However, exposure to the solvent vapors (water and methanol) reverts the structure back to the as-synthesized structure, and thus, the reversible flexible nature of the structure was elucidated. The flexibility and permanent porosity were further reinforced from the CO₂ adsorption profiles (195 and 273 K) that show stepwise uptake. Moreover, a high selectivity for O₂ over N₂ at 77 K was realized. The framework exhibits interesting solvent vapor adsorption behavior with dynamic structural transformation depending upon the size, polarity, and coordination ability of the solvent molecules. Further investigation was conducted by solid state ¹¹³Cd nuclear magnetic resonance (NMR) spectroscopy that unambiguously advocates the reversible transformation “pentagonal-bipyramidal CdO₆N → octahedral CdO₅N” geometry in the desolvated state. For the first time, ¹¹³Cd NMR has been used as a probe of structural flexibility in a porous coordination polymer system

Evidences suggesting the link between AID and lung cancer.

<p>A, Immunohistochemistry for AID in MALLs. MALL is indicated by an arrowhead. B, HE staining of the lung of AID Tg mice with lung adenoma. An adenoma is demarcated by a dotted line. Region indicated by a rectangle is enlarged in C. C, Arrowheads indicate MALLs within lung adenoma. Immunohistochemistry for D, ΔNp63: E, keratin 5; F, keratin 14; G, Lgr 5; and H, Lgr 6 in lung adenoma.</p

Characteristics of MALL cells as immature mucous cells.

<p>Electron microscopic findings of MALL: A, Overview of MALL cells. B, Magnified view of the apical part of MALL cells. Arrows indicate immature secretory vesicles in the cytoplasm of MALL cells. An asterisk indicates a basal cell adjacent to MALL cells. C, PAS staining demonstrates that positively stained materials are in the apex of MALL cells (arrowheads). D, Electron micrograph showing a phagocytic epithelial cell within MALL (asterisk). Inset, higher power view of the phagocytic epithelial cell. Arrows indicate the basement membrane. Arrowheads indicate residual bodies within the cell. AS, alveolar space; PAS, periodic-acid Schiff.</p

Time course of MALL count and size in the right lung of AIDon mice and wild-type mice.

<p>A, In the left panel, mean density of MALLs in sections per 1 cm² is plotted against mice age in weeks. A solid line with closed symbol and a gray line with open symbol represent AIDon and wild-type mice, respectively. Error bars represent standard errors for data from three mice, except for a wild-type mouse and a 75-week-old AIDon mouse each of which was analyzed singly. Area of a dotted rectangle was enlarged in the right panel. B, Cross-sectional area with individual MALLs plotted. Mean values are indicated by red lines. P values calculated by t-test between groups are indicated by brackets.</p