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

Constitutive Expression of AID Leads to Tumorigenesis

Genome stability is regulated by the balance between efficiencies of the repair machinery and genetic alterations such as mutations and chromosomal rearrangements. It has been postulated that deregulation of class switch recombination (CSR) and somatic hypermutation (SHM), which modify the immunoglobulin (Ig) genes in activated B cells, may be responsible for aberrant chromosomal translocations and mutations of non-Ig genes that lead to lymphocyte malignancy. However, the molecular basis for these genetic instabilities is not clearly understood. Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce both CSR and SHM in artificial substrates in fibroblasts as well as B cells. Here we show that constitutive and ubiquitous expression of AID in transgenic mice caused both T cell lymphomas and dysgenetic lesions of epithelium of respiratory bronchioles (micro-adenomas) in all individual mice. Point mutations, but not translocations, were massively introduced in expressed T cell receptor (TCR) and c-myc genes in T lymphoma cells. The results indicate that AID can mutate non-Ig genes including oncogenes, implying that aberrant AID expression could be a cause of human malignancy

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

精神科救急医療体制の現状と課題：日本公衆衛生学会モニタリング・レポート委員会精神保健福祉分野活動総括

目的　精神科救急医療体制の構築と関連する法律の整備に関して，現代の日本における課題を明らかにし，解決策を探ること。　方法　日本公衆衛生学会モニタリング・レポート委員会精神保健福祉分野のグループ活動として，2014年度から2017年度にかけて精神科救急および措置入院に関する情報収集を行った。各年次総会に提出した報告書を基に，必要に応じて文献を追加した。　結果　地域における精神科医療資源の偏在や，歴史的な精神疾患に関する認識の問題なども絡んでいるため，全国均一的な救急医療システムの構築のためには越えなければならないハードルは高い。また，強制入院の中で最も法的な強制力が強い措置入院制度に関しては，その実際的な運用を巡って全国でも地域差が大きいために，精神保健福祉法に，より具体的な記載が盛り込まれるとともに，厚生労働省から一定のガイドラインが提示されている。とくに近年は凶悪犯罪事件との関連を巡って，社会的にも関心が高まっており，一部では措置入院の保安処分化を懸念する声が上がっている。精神疾患は今や五大疾病の一つに位置づけられているが，その性質上，生活習慣病などに比べて，疫学的エビデンスが圧倒的に不足しており，これが臨床や行政の現場での対応に足並みが揃わない主要因であると考えられる。　結論　日本公衆衛生学会は，医療・福祉・行政などに携わる多職種から構成される学際的な組織である強みを活かして，多施設共同の疫学研究を主導し，措置入院解除および退院後の予後に関する，すべての関係自治体が共有しうるデータベースとしての疫学的エビデンスの構築を推進する役割を担っている

Rad54 is dispensable for the ALT pathway

10.1111/j.1365-2443.2006.01020.xGenes to Cells11111305-1315GECE