講義に英語を導入することで見えてきた課題と可能性～医学類における一例の紹介～

世界トップレベルの研究を行う大学や国際化を牽引する大学を重点的に支援する文部科学省の事業として，「スーパーグローバル大学（SGU）創成支援事業」がある。金沢大学は当該事業の「グローバル化牽引型」に採択されており，SGU事業の取り組みとして，2023年度を目途に学士課程の50％の授業，大学院課程の100％の授業の英語化を目指している。本稿では，筆者が試みている医学類専門科目での授業の英語化の取り組みと，それに対する学生からの評価や声について報告し，今後の授業の英語化や課題について考察する。一連の取り組みから，「学生が講義内容を日本語で十分に理解している」という前提が，授業の英語化には肝要であることが見えてきた。　The Top Global University Project（ Super Global Universities） is a project of the Ministryof Education, Culture, Sports, Science and Technology of Japan． Kanazawa University wasselected as Type B（ Global Traction Type） university on the project． As a part of the project,Kanazawa University has been aiming that 50％ of the bachelor lectures and 100％ of thegraduate school lectures will be offered in English until 2023． In the present article, I reportmy attempt of a lecture in English in School of Medicine． Also, I introduce the evaluationand the voice from students on it． Based on the above, I discuss a future aspect of a lecturein English and issues surrounding it． These attempts revealed that “Students alreadyunderstand the contents of a lecture in Japanese very well” should be required for giving alecture in English

An Opposite Effect of the CDK Inhibitor, p18^INK4c on Embryonic Stem Cells Compared with Tumor and Adult Stem Cells

Self-renewal is a feature common to both adult and embryonic stem (ES) cells, as well as tumor stem cells (TSCs). The cyclin-dependent kinase inhibitor, p18INK4c, is a known tumor suppressor that can inhibit self-renewal of tumor cells or adult stem cells. Here, we demonstrate an opposite effect of p18 on ES cells in comparison with teratoma cells. Our results unexpectedly showed that overexpression of p18 accelerated the growth of mouse ES cells and embryonic bodies (EB); on the contrary, inhibited the growth of late stage teratoma. Up-regulation of ES cell markers (i.e., Oct4, Nanog, Sox2, and Rex1) were detected in both ES and EB cells, while concomitant down-regulation of various differentiation markers was observed in EB cells. These results demonstrate that p18 has an opposite effect on ES cells as compared with tumor cells and adult stem cells. Mechanistically, expression of CDK4 was significantly increased with overexpression of p18 in ES cells, likely leading to a release of CDK2 from the inhibition by p21 and p27. As a result, self-renewal of ES cells was enhanced. Our current study suggests that targeting p18 in different cell types may yield different outcomes, thereby having implications for therapeutic manipulations of cell cycle machinery in stem cells. © 2012 Li et al

Esrrb directly binds to Gata6 promoter and regulates its expression with Dax1 and Ncoa3

Estrogen-related receptor beta (Esrrb) is expressed in embryonic stem (ES) cells and is involved in self-renewal ability and pluripotency. Previously, we found that Dax1 is associated with Esrrb and represses its transcriptional activity. Further, the disruption of the Dax1–Esrrb interaction increases the expression of the extra-embryonic endoderm marker Gata6 in ES cells. Here, we investigated the influences of Esrrb and Dax1 on Gata6 expression. Esrrb overexpression in ES cells induced endogenous Gata6 mRNA and Gata6 promoter activity. In addition, the Gata6 promoter was found to contain the Esrrb recognition motifs ERRE1 and ERRE2, and the latter was the responsive element of Esrrb. Associations between ERRE2 and Esrrb were then confirmed by biotin DNA pulldown and chromatin immunoprecipitation assays. Subsequently, we showed that Esrrb activity at the Gata6 promoter was repressed by Dax1, and although Dax1 did not bind to ERRE2, it was associated with Esrrb, which directly binds to ERRE2. In addition, the transcriptional activity of Esrrb was enhanced by nuclear receptor co-activator 3 (Ncoa3), which has recently been shown to be a binding partner of Esrrb. Finally, we showed that Dax1 was associated with Ncoa3 and repressed its transcriptional activity. Taken together, the present study indicates that the Gata6 promoter is activated by Esrrb in association with Ncoa3, and Dax1 inhibited activities of Esrrb and Ncoa3, resulting maintenance of the undifferentiated status of ES cells. © 2016 Elsevier Inc.Embargo Period 12 month

GABPα regulates Oct-3/4 expression in mouse embryonic stem cells

金沢大学大学院医学系研究科機能再生学Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts, and transcription factors Oct-3/4, Nanog, Sox2, and STAT3, are essential for their self-renewal. In this study, we searched for molecules downstream of STAT3 in ES cells. Using DNA chip analysis, we obtained GA-repeat binding protein (GABP) α. Expression of GABPα was restricted to undifferentiated ES cells and controlled by STAT3. We found that the expression level of Oct-3/4 is reduced by knockdown of GABPα. On the other hand, GABPα-overexpressing ES cells maintained the expression level of Oct-3/4 even in the absence of LIF. Moreover, the induction of Oct-3/4 repressors Cdx-2, Coup-tf1, and GCNF was stimulated by GABPα knockdown. These data suggest that GABPα upregulates the expression of Oct-3/4 via downregulation of Oct-3/4 repressors. © 2006 Elsevier Inc. All rights reserved

ETS-related transcription factors Etv4 and Etv5 are involved in proliferation and induction of differentiationassociated genes in embryonic stem (ES) cells

The pluripotency and self-renewal capacity of embryonic stem (ES) cells is regulated by several transcription factors. Here, we show that the ETS-related transcription factors Etv4 and Etv5 (Etv4/5) are specifically expressed in undifferentiated ES cells, and suppression of Oct3/4 results in down-regulation of Etv4/5. Simultaneous deletion of Etv4 and Etv5 (Etv4/5 double knock-out(dKO)) in ES cells resulted in a flat, epithelial cell-like appearance, whereas the morphology changed into compact colonies in a 2i medium (containing two inhibitors for GSK3 and MEK/ERK). Expression levels of self-renewal marker genes, including Oct3/4 and Nanog, were similar between wild-type and dKO ES cells, whereas proliferation of Etv4/5 dKO ES cells was decreased with overexpression of cyclin-dependent kinase inhibitors (p16/p19, p15, and p57). A differentiation assay revealed that the embryoid bodies derived from Etv4/5 dKO ES cells were smaller than the control, and expression of ectoderm marker genes, including Fgf5, Sox1, and Pax3, was not induced in dKO-derived embryoid bodies. Microarray analysis demonstrated that stem cell-related genes, including Tcf15, Gbx2, Lrh1, Zic3, and Baf60c, were significantly repressed in Etv4/5dKOEScells.Theartificial expression of Etv4 and/or Etv5 in Etv4/5 dKO ES cells induced re-expression of Tcf15 and Gbx2. These results indicate that Etv4 and Etv5, potentially through regulation of Gbx2 and Tcf15, are involved in the ES cell proliferation and induction of differentiation-associated genes in ES cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A

A novel in-frame deletion in the leucine zipper domain of C/EBPε leads to neutrophil-specific granule deficiency

Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by neutrophil dysfunction, bilobed neutrophil nuclei and lack of neutrophil-specific granules. Defects in a myeloid-specific transcription factor, CCAAT/enhancer binding protein-ε (C/EBPε), have been identified in two cases in which homozygous frameshift mutations led to loss of the leucine zipper domain. In this study, we report a 55-y-old woman affected with SGD caused by a novel homozygous 2-aa deletion (ΔRS) in the leucine zipper domain of the C/EBPε gene. The patient showed characteristic neutrophil abnormalities and recurrent skin infections; however, there was no history of deep organ infections. Biochemical analysis revealed that, in contrast to the two frameshift mutations, the ΔRS mutant maintained normal cellular localization, DNA-binding activity, and dimerization, and all three mutants exhibited marked reduction in transcriptional activity. The ΔRS mutant was defective in its association with Gata1 and PU.1, as well as aberrant cooperative transcriptional activation of eosinophil major basic protein. Thus, the ΔRS likely impairs protein-protein interaction with other transcription factors, resulting in a loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function, and indicate that multiple molecular mechanisms lead to SGD. © 2015 by The American Association of Immunologists, Inc

In Vivo Deficiency of Both C/EBPβ and C/EBPε Results in Highly Defective Myeloid Differentiation and Lack of Cytokine Response

金沢大学医薬保健研究域医学系The CCAAT/enhancer binding proteins (C/EBPs) are transcription factors involved in hematopoietic cell development and induction of several inflammatory mediators. Here, we generated C/EBPβ and C/EBPε double-knockout (bbee) mice and compared their phenotypes to those of single deficient (bbEE and BBee) and wild-type (BBEE) mice. The bbee mice were highly susceptible to fatal infections and died within 2–3 months. Morphologically, their neutrophils were blocked at the myelocytes/metamyelocytes stage, and clonogenic assays of bone marrow cells indicated a significant decrease in the number of myeloid colonies of the bbee mice. In addition, the proportion of hematopoietic progenitor cells [Lin(−)Sca1(+)c-Kit(+)] in the bone marrow of the bbee mice was significantly increased, reflecting the defective differentiation of the myeloid compartment. Furthermore, microarray expression analysis of LPS- and IFNγ-activated bone marrow-derived macrophages from bbee compared to single knockout mice revealed decreased expression of essential immune response-related genes and networks, including some direct C/EBP-targets such as Marco and Clec4e. Overall, the phenotype of the bbee mice is distinct from either the bbEE or BBee mice, demonstrating that both transcription factors are crucial for the maturation of neutrophils and macrophages, as well as the innate immune system, and can at least in part compensate for each other in the single knockout mice

Baf53a is involved in survival of mouse ES cells, which can be compensated by Baf53b

金沢大学医薬保健研究域医学系The human Baf (Brg1/Brm associated factor) complex, also known as the mammalian SWI/SNF chromatin-remodeling complex, is involved in a variety of cellular processes. The pluripotency and self-renewal abilities are major characteristics of embryonic stem (ES) cells and are regulated by the ES cell-specific BAF (esBAF) complex. Baf53a is one of the subunits of the esBAF complex. Here, we found that Baf53a was expressed in undifferentiated ES cells and that it interacted with Oct3/4. Analyses of tetracycline-inducible Baf53a conditional knockout ES cells revealed that the undifferentiated markers, including Nanog and Oct3/4, were expressed in Baf53a-deficient ES cells; however, growth of the cells was repressed, and expression of p53, p21, and cleaved Caspase 3 was increased. Cell death of Baf53a-deficient ES cells was rescued by overexpression of Baf53a, but not by the Baf53a M3 mutant (E388A/R389A/R390A). Interestingly, Baf53b, a homologue of Baf53a, rescued cell death of Baf53a-deficient ES cells. Baf53a-deficient ES cells overexpressing exogenous Baf53a or Baf53b remained in the undifferentiated state, proliferated, and repressed expression of p21. In summary, our findings suggest that Baf53a is involved in the survival of ES cells by regulating p53 and Caspase3, and that Baf53b is able to compensate for this functional aspect of Baf53a. © 2017 The Author(s)

Deficiency of CCAAT/Enhancer Binding Protein-Epsilon Reduces Atherosclerotic Lesions in LDLR-/- Mice

The CCAAT/enhancer binding proteins (C/EBPs) are transcription factors involved in hematopoietic cell development and induction of several inflammatory mediators. C/EBPε is expressed only in myeloid cells including monocytes/macrophages. Atherosclerosis is an inflammatory disorder of the vascular wall and circulating immune cells such as monocytes/macrophages. Mice deficient in the low density lipoprotein (LDL) receptor (Ldlr-/-) fed on a high cholesterol diet (HCD) show elevated blood cholesterol levels and are widely used as models to study human atherosclerosis. In this study, we generated Ldlr and Cebpe double-knockout (llee) mice and compared their atherogenic phenotypes to Ldlr single deficient (llEE) mice after HCD. Macrophages from llee mice have reduced lipid uptake by foam cells and impaired phagokinetic motility in vitro compared to macrophages from llEE mice. Also, compared to llEE mice, llee mice have alterations of lipid metabolism, and reduced atheroma and obesity, particularly the males. Peritoneal macrophages of llee male mice have reduced mRNA expression of FABP4, a fatty acid binding protein implicated in atherosclerosis. Overall, our study suggests that the myeloid specific factor C/EBPε is involved in systemic lipid metabolism and that silencing of C/EBPε could decrease the development of atherosclerosis