A Tripartite Synapse Model in Drosophila

Tripartite (three-part) synapses are defined by physical and functional interactions of glia with pre- and post-synaptic elements. Although tripartite synapses are thought to be of widespread importance in neurological health and disease, we are only beginning to develop an understanding of glial contributions to synaptic function. In contrast to studies of neuronal mechanisms, a significant limitation has been the lack of an invertebrate genetic model system in which conserved mechanisms of tripartite synapse function may be examined through large-scale application of forward genetics and genome-wide genetic tools. Here we report a Drosophila tripartite synapse model which exhibits morphological and functional properties similar to those of mammalian synapses, including glial regulation of extracellular glutamate, synaptically-induced glial calcium transients and glial coupling of synapses with tracheal structures mediating gas exchange. In combination with classical and cell-type specific genetic approaches in Drosophila, this model is expected to provide new insights into the molecular and cellular mechanisms of tripartite synapse function

Selective Chemical Labeling of Natural T Modifications in DNA.

We present a chemical method to selectively tag and enrich thymine modifications, 5-formyluracil (5-fU) and 5-hydroxymethyluracil (5-hmU), found naturally in DNA. Inherent reactivity differences have enabled us to tag 5-fU chemoselectively over its C modification counterpart, 5-formylcytosine (5-fC). We rationalized the enhanced reactivity of 5-fU compared to 5-fC via ab initio quantum mechanical calculations. We exploited this chemical tagging reaction to provide proof of concept for the enrichment of 5-fU containing DNA from a pool that contains 5-fC or no modification. We further demonstrate that 5-hmU can be chemically oxidized to 5-fU, providing a strategy for the enrichment of 5-hmU. These methods will enable the mapping of 5-fU and 5-hmU in genomic DNA, to provide insights into their functional role and dynamics in biology.R.E.H. is supported by The University of Cambridge, F.K. is supported by the Wellcome Trust, and A.B.S. is supported by the Herchel Smith Fund. The Balasubramanian group is core- funded by a Wellcome Trust Senior Investigator Award and by Cancer Research UK. Departmental NMR facilities are supported by EPSRC grant EP/K039520/1.This is the final version. It was first published by ACS at http://pubs.acs.org/doi/abs/10.1021/jacs.5b03730

Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania.

BACKGROUND: 5-Hydroxymethyluracil (5hmU) is a thymine base modification found in the genomes of a diverse range of organisms. To explore the functional importance of 5hmU, we develop a method for the genome-wide mapping of 5hmU-modified loci based on a chemical tagging strategy for the hydroxymethyl group. RESULTS: We apply the method to generate genome-wide maps of 5hmU in the parasitic protozoan Leishmania sp. In this genus, another thymine modification, 5-(β-glucopyranosyl) hydroxymethyluracil (base J), plays a key role during transcription. To elucidate the relationship between 5hmU and base J, we also map base J loci by introducing a chemical tagging strategy for the glucopyranoside residue. Observed 5hmU peaks are highly consistent among technical replicates, confirming the robustness of the method. 5hmU is enriched in strand switch regions, telomeric regions, and intergenic regions. Over 90% of 5hmU-enriched loci overlapped with base J-enriched loci, which occurs mostly within strand switch regions. We also identify loci comprising 5hmU but not base J, which are enriched with motifs consisting of a stretch of thymine bases. CONCLUSIONS: By chemically detecting 5hmU we present a method to provide a genome-wide map of this modification, which will help address the emerging interest in the role of 5hmU. This method will also be applicable to other organisms bearing 5hmU.FK is supported by the Wellcome Trust, DB is supported by the Herchel Smith Fund, REH is supported by the University of Cambridge and the Herchel Smith Fund, GRM was supported by Trinity College and the Herchel Smith fund, PVD is supported by Marie Curie fellowship and the Wellcome Trust. The Balasubramanian group is core-funded by a Wellcome Trust Senior Investigator Award (099232/Z/12/Z) and Cancer Research UK(C14303/A17197)

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Delta orf1 and Delta orf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Delta orf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Delta orf1, Delta orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule

トウヨウ エイワ ジョガクイン ダイガク ソツギョウセイ ノ キャリア ケイセイ ノ ジッタイ ト イシキ チョウサ ホウコク

The main object of this paper is to investigate the work careers and life careersof Toyo Eiwa University alumnae, and to examine what influenced their decision makingand their feelings about their careers.The authors prepared an original sociological and opinion survey on all 2930alumnae, grouped according to their graduate years. The survey was conducted onan unregistered basis in July 2007. The questionnaires were mailed to the alumnaewhose graduate year is even. Seven hundred sixty two (25% ) of the alumnae respondedto the survey. The response rate was not high enough to come to any definite conclusionon the behavior of and thought concerning the career building of Toyo EiwaUniversity alumunae. However, the authors believe that it is very important to investigatethe results and report on findings that may be useful in reviewing and improvingcolledge education.The alumnae association of Toyo Eiwa University kindly allowed use of its listof members, and Hajime Sasaki, the head officer of the career section of Toyo EiwaUniversity, helped us to elaborate the questionnaires and to contact the alumnae.The paper consists of five sections: 1) the purpose of the study; 2) the report onthe entire survey; 3) the relation between the first job decision making and its results;4) the timing of retirement from the first job and life course; 5) the free opinion of thealumnae and some findings. Sections 1 and 2 were written by Hayashi; Section 3 waswritten by Hasegawa; Section 4 was written by Arita; Section 5 was written by Kawasaki

C/EBPβ Promotes Transition from Proliferation to Hypertrophic Differentiation of Chondrocytes through Transactivation of p57Kip2

BACKGROUND: Although transition from proliferation to hypertrophic differentiation of chondrocytes is a crucial step for endochondral ossification in physiological skeletal growth and pathological disorders like osteoarthritis, the underlying mechanism remains an enigma. This study investigated the role of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) in chondrocytes during endochondral ossification. METHODOLOGY/PRINCIPAL FINDINGS: Mouse embryos with homozygous deficiency in C/EBPbeta (C/EBPbeta-/-) exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBPbeta-/- chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBPbeta in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBPbeta. In fact, a DNA cell cycle histogram revealed that the C/EBPbeta overexpression caused accumulation of cells in the G0/G1 fraction. Among cell cycle factors, microarray and real-time RT-PCR analyses have identified the cyclin-dependent kinase inhibitor p57(Kip2) as the transcriptional target of C/EBPbeta. p57(Kip2) was co-localized with C/EBPbeta in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/EBPbeta deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/EBPbeta in the p57(Kip2) promoter between -150 and -130 bp region containing a putative C/EBP motif. The knockdown of p57(Kip2) by the siRNA inhibited the C/EBPbeta-induced chondrocyte hypertrophy. Finally, when we created the experimental osteoarthritis model by inducing instability in the knee joints of adult mice of wild-type and C/EBPbeta+/- littermates, the C/EBPbeta insufficiency caused resistance to joint cartilage destruction. CONCLUSIONS/SIGNIFICANCE: C/EBPbeta transactivates p57(Kip2) to promote transition from proliferation to hypertrophic differentiation of chondrocytes during endochondral ossification, suggesting that the C/EBPbeta-p57(Kip2) signal would be a therapeutic target of skeletal disorders like growth retardation and osteoarthritis