C–H bond activation of benzene and thiophene by photochemically generated rhenocene cation

A cationic rhenocene-acetonitrile adduct [Cp2Re(NCMe)](BF4)(1) reacted with an excess of benzene, thiophene, 2-methylthiophene, and pyrrole under UV irradiation to afford the C–H bond activation products [Cp2Re(H)R]BF4 (R = phenyl, 2-thienyl, 2-(5-methylthienyl), 2-pyrrolyl) in high yields. In cases of thiophene derivatives and pyrrole, α-C–H bonds are selectively activated. A plausible mechanism involves the photodissociation of acetonitrile from 1 to generate a coordinatively unsaturated rhenocene cation [Cp2Re]+. When 2,5-dimethylthiophene and dibenzothiophene, having no α-C–H bonds, were used as substrates, products of the activation of other C–H bonds were formed first, but they isomerized to thermodynamically more stable η11-S-coordinated complexes in refluxing acetone. On the other hand, irradiation of the η1-S-coordinated complexes reproduced the original C–H bond activation products. Because of the cationic character, [Cp2Re(H)R]BF4 were readily deprotonated by triethylamine to give neutral rhenocene derivatives Cp2ReR. When R is thienyl or 2-(5-methylthienyl), treatment of Cp2ReR with HBF4⋅Et2O and MeI resulted in protonation and methylation to give [Cp2Re(H)R]BF4 (R = and [Cp2Re(Me)R]I. Thermolysis of [Cp2Re(Me)R]I in the presence of PPh3 unexpectedly resulted in migration of R to the Cp ring to give [(2 thienyl C5H4)CpRe(PPh3)]I

Deletion of Long Isoform of Eukaryotic Elongation Factor 1Bδ Leads to Audiogenic Seizures and Aversive Stimulus-Induced Long-Lasting Activity Suppression in Mice

Alternative splicing enables a gene to give rise to diverse protein products. The Eef1d gene produces two isoforms: a short isoform that encodes translation elongation factor 1Bδ (eEF1Bδ1), and a long isoform that encodes the heat shock-responsive transcription factor eEF1BδL. Previously, we found that eEF1BδL was a splice variant that was specific to the brain and testis, and the protein encoded is thought to have a function in the central nervous system. In this study, we generated knockout (KO) mice of C57BL/6J background that selectively lacked a specific exon in Eef1d for the long isoform. These KO mice lacked eEF1BδL, but not eEF1Bδ1, in the brain. Although the KO mice showed normal anxiety-related and learning behavior in behavioral tests, some showed severe seizures in response to loud sounds (90 dBA), an audiogenic seizures (AGS) response. Furthermore, after the KO mice had been subjected to the fear conditioning test, they showed remarkably decreased locomotor activity in their home cage and in the open-field and elevated plus-maze tests. After the fear conditioning test, a significant decrease in brain weight, atrophy of the hippocampus and midbrain, and reduced cortical layer thickness were observed in the KO mice. We also found a compensatory increase in the eEF1Bδ1 level and elevated protein synthesis with the induction of endoplasmic reticulum stress markers in these mice. Our results suggest that eEF1BδL has an important role in normal brain function especially when exposed to external stimuli

Integrated stress response regulates GDF15 secretion from adipocytes, preferentially suppresses appetite for a high-fat diet and improves obesity

The eIF2α phosphorylation-dependent integrated stress response (ISR) is a signaling pathway that maintains homeostasis in mammalian cells exposed to various stresses. Here, ISR activation in adipocytes improves obesity and diabetes by regulating appetite in a non-cell-autonomous manner. Adipocyte-specific ISR activation using transgenic mice decreases body weight and improves glucose tolerance and obesity induced by a high-fat diet (HFD) via preferential inhibition of HFD intake. The transcriptome analysis of ISR-activated adipose tissue reveals that growth differentiation factor 15 (GDF15) expression is induced by the ISR through the direct regulation of the transcription factors ATF4 and DDIT3. Deficiency in the GDF15 receptor GFRAL abolishes the adipocyte ISR-dependent preferential inhibition of HFD intake and the anti-obesity effects. Pharmacologically, 10(E), 12(Z)-octadecadienoic acid induces ISR-dependent GDF15 expression in adipocytes and decreases the intake of the HFD. Based on our findings the specific activation of the ISR in adipocytes controls the non-cell-autonomous regulation of appetite

A cell-autonomous role for JSAP1 and JLP in mouse cerebellar Purkinje cell survival

We previously reported that the structurally related JNK scaffolding proteins JSAP1 and JLP play functionally redundant and key roles in maintaining mouse cere-bellar Purkinje cell (PC) survival, which may be related to their regulation of axonal transport. The JSAP1 and JLP proteins are also widely expressed in other cell types throughout the brain. Notably, in the cerebellum, JSAP1 is abundantly expressed in the pinceau, a cluster of basket cell (BC) axons and termini that surround the PC axon initial segment. Thus, it is possible that BC-expressed JSAP1 plays a role in PC survival. To investigate this possibility, we generated and analyzed mice containing a PC/BC-specific double knockout (DKO) of Jsap1 and Jlp (Jsap1:Jlp cDKO mice). These mice exhibited PC axonal dystrophy, followed by progressive PC loss, consistent with the phenotypes previously reported for PC-specific Jsap1 and Jlp DKO mice. Furthermore, we found that the phenotypes of Jsap1:Jlp cDKO PCs were rescued by PC-specific transgenic expression of JSAP1. Taken together, these results indicate that BC-expressed JSAP1 plays little or no role in PC survival, and that PC-expressed JSAP1 and JLP play cell-autonomous roles in preventing PC degeneration

A novel mouse model of muscle wasting

Background Formation of 43S and 48S preinitiation complexes plays an important role in muscle protein synthesis. There is no muscle-wasting mouse model caused by a repressed 43S preinitiation complex assembly. Objective The aim of the present study was to develop a convenient mouse model of skeletal muscle wasting with repressed 43S preinitiation complex assembly. Material and methods A ligand-activatable PERK derivative Fv2E-PERK causes the phosphorylation of eukaryotic initiation factor 2α (eIF2α), which inhibits 43S preinitiation complex assembly. Thus, muscle atrophic phenotypes, intracellular signaling pathways, and intracellular free amino acid profiles were investigated in human skeletal muscle α-actin (HSA) promoter-driven Fv2E-PERK transgenic (Tg) mice. Results HSA-Fv2E-PERK Tg mice treated with the artificial dimerizer AP20187 phosphorylates eIF2α in skeletal muscles and leads to severe muscle atrophy within a few days of ligand injection. Muscle atrophy was accompanied by a counter regulatory activation of mTORC1 signaling. Moreover, intracellular free amino acid levels were distinctively altered in the skeletal muscles of HSA-Fv2E-PERK Tg mice. Conclusions As a novel model of muscle wasting, HSA-Fv2E-PERK Tg mice provide a convenient tool for studying the pathogenesis of muscle loss and for assessing putative therapeutics

Quantitative Dynamics of Chromatin Remodeling during Germ Cell Specification from Mouse Embryonic Stem Cells

SummaryGerm cell specification is accompanied by epigenetic remodeling, the scale and specificity of which are unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome

A role for mDia, a Rho-regulated actin nucleator, in tangential migration of interneuron precursors.

神経細胞の配置メカニズムを解明－抑制性神経前駆細胞に特有の移動の機構が明らかに. 京都大学プレスリリース. 2012-1-16.In brain development, distinct types of migration, radial migration and tangential migration, are shown by excitatory and inhibitory neurons, respectively. Whether these two types of migration operate by similar cellular mechanisms remains unclear. We examined neuronal migration in mice deficient in mDia1 (also known as Diap1) and mDia3 (also known as Diap2), which encode the Rho-regulated actin nucleators mammalian diaphanous homolog 1 (mDia1) and mDia3. mDia deficiency impaired tangential migration of cortical and olfactory inhibitory interneurons, whereas radial migration and consequent layer formation of cortical excitatory neurons were unaffected. mDia-deficient neuroblasts exhibited reduced separation of the centrosome from the nucleus and retarded nuclear translocation. Concomitantly, anterograde F-actin movement and F-actin condensation at the rear, which occur during centrosomal and nuclear movement of wild-type cells, respectively, were impaired in mDia-deficient neuroblasts. Blockade of Rho-associated protein kinase (ROCK), which regulates myosin II, also impaired nuclear translocation. These results suggest that Rho signaling via mDia and ROCK critically regulates nuclear translocation through F-actin dynamics in tangential migration, whereas this mechanism is dispensable in radial migration

The Hippo Signaling Pathway Components Lats and Yap Pattern Tead4 Activity to Distinguish Mouse Trophectoderm from Inner Cell Mass

Outside cells of the preimplantation mouse embryo form the trophectoderm (TE), a process requiring the transcription factor Tead4. Here, we show that transcriptionally active Tead4 can induce Cdx2 and other trophoblast genes in parallel in embryonic stem cells. In embryos, the Tead4 coactivator protein Yap localizes to nuclei of outside cells, and modulation of Tead4 or Yap activity leads to changes in Cdx2 expression. In inside cells, Yap is phosphorylated and cytoplasmic, and this involves the Hippo signaling pathway component Lats. We propose that active Tead4 promotes TE development in outside cells, whereas Tead4 activity is suppressed in inside cells by cell contact- and Lats-mediated inhibition of nuclear Yap localization. Thus, differential signaling between inside and outside cell populations leads to changes in cell fate specification during TE formation

Induced 2C Expression and Implantation-Competent Blastocyst-like Cysts from Primed Pluripotent Stem Cells

Soon after fertilization, the few totipotent cells of mammalian embryos diverge to form a structure called the blastocyst (BC). Although numerous cell types, including germ cells and extended-pluripotency stem cells, have been developed from pluripotent stem cells (PSCs) in vitro, generating functional BCs only from PSCs remains elusive. Here, we describe induced self-organizing 3D BC-like cysts (iBLCs) generated from mouse PSC culture. Resembling natural BCs, iBLCs have a blastocoel-like cavity and were formed with outer cells expressing trophectoderm lineage markers and with inner cells expressing pluripotency markers. iBLCs transplanted to pseudopregnant mice uteruses implanted, induced decidualization, and exhibited growth and development before resorption, demonstrating that iBLCs are implantation competent. iBLC precursor intermediates required the transcription factor Prdm14 and concomitantly activated the totipotency-related cleavage-stage MERVL reporter and 2C genes. Thus, our system may contribute to the understanding of molecular mechanisms underpinning totipotency, embryogenesis, and implantation

Trk-fused gene (TFG) regulates pancreatic beta cell mass and insulin secretory activity

The Trk-fused gene (TFG) is reportedly involved in the process of COPII-mediated vesicle transport and missense mutations in TFG cause several neurodegenerative diseases including hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P). The high coincidence ratio between HMSN-P and diabetes mellitus suggests TFG to have an important role(s) in glucose homeostasis. To examine this possibility, β-cell specific TFG knockout mice (βTFG KO) were generated. Interestingly, βTFG KO displayed marked glucose intolerance with reduced insulin secretion. Immunohistochemical analysis revealed smaller β-cell masses in βTFG KO than in controls, likely attributable to diminished β-cell proliferation. Consistently, β-cell expansion in response to a high-fat, high-sucrose (HFHS) diet was significantly impaired in βTFG KO. Furthermore, glucose-induced insulin secretion was also markedly impaired in islets isolated from βTFG KO. Electron microscopic observation revealed endoplasmic reticulum (ER) dilatation, suggestive of ER stress, and smaller insulin crystal diameters in β-cells of βTFG KO. Microarray gene expression analysis indicated downregulation of NF-E2 related factor 2 (Nrf2) and its downstream genes in TFG depleted islets. Collectively, TFG in pancreatic β-cells plays a vital role in maintaining both the mass and function of β-cells, and its dysfunction increases the tendency to develop glucose intolerance.This study was partly supported by a Grant-in-Aid for Research Activity Start-up (JSPS KAKENHI Grant Number JP15H06427) (to T.Y.) from the Ministry of Education, Science, Sports and Culture, Japan, and grants from Mitsubishi Tanabe Pharma (to T.Y.), Novartis Pharma (to T.Y.), Takeda Science Foundation (to Y.N.), Asahi Life Foundation (to Y.N.) and The Uehara Memorial Foundation (to Y.N.)