dictyNews Electronic Edition Volume 40, number 7 February 28, 2014 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Properties of a non-bioactive fluorescent derivative of differentiation- inducing factor-3, an anti-tumor agent found in Dictyostelium discoideum Yuzuru Kubohara 1, Haruhisa Kikuchi 2, Yusuke Matsuo 2, Yoshiteru Oshima 2 and Yoshimi Homma 3 1 Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan 2 Laboratory of Natural Product Chemistry, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578, Japan 3 Department of Biomolecular Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan Biology Open, in press Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives such as butoxy-DIF-3 (Bu-DIF-3) are potent anti-tumor agents. To investigate the activity of DIF- like molecules in tumor cells, we recently synthesized a green fluorescent DIF-3 derivative, BODIPY-DIF-3G, and analyzed its bioactivity and cellular localization. In this study, we synthesized a red (orange) fluorescent DIF-3 derivative, BODIPY-DIF-3R, and compared the cellular localization and bioactivities of the two BODIPY-DIF-3s in HeLa human cervical cancer cells. Both fluorescent compounds penetrated the extracellular membrane within 0.5 h and localized mainly to the mitochondria. In formalin-fixed cells, the two BODIPY-DIF-3s also localized to the mitochondria, indicating that the BODIPY-DIF-3s were incorporated into mitochondria independently of the mitochondrial membrane potential. After treatment for 3 days, BODIPY- DIF-3G, but not BODIPY-DIF-3R, induced mitochondrial swelling and suppressed cell proliferation. Interestingly, the swollen mitochondria were stainable with BODIPY-DIF-3G but not with BODIPY-DIF-3R. When added to isolated mitochondria in vitro, BOIDPY-DIF-3G dose-dependently increased the rate of O2 consumption, but BODIPY-DIF-3R did not. These results suggest that the bioactive BODIPY-DIF-3G suppresses cell proliferation at least in part by altering mitochondrial activity, whereas the non-bioactive BODIPY-DIF-3R localizes to the mitochondria but does not affect mitochondrial activity or cell proliferation. Submitted by Yuzuru Kubohara [kubohara@gunma-u.ac.jp] --------------------------------------------------------------------------- N-glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by 'off-line' liquid chromatography and mass spectrometry. Hykollari A, Dragosits M, Rendic D, Wilson IBH, Paschinger K. Electrophoresis. 2014 Feb 26. doi: 10.1002/elps.201300612. [Epub ahead of print] In this study, we have performed the first mass spectrometric analysis of N-glycans of the M31 mutant strain of the cellular slime mould Dictyostelium discoideum, previously shown to have a defect in glucosidase II. Together with glucosidase I, this enzyme mediates part of the initial processing of N-glycans; defects in either glucosidase are associated with human diseases and result in an accumulation of incorrectly-processed oligosaccharides which are not, or only poor, substrates for a range of downstream enzymes. To examine the effect of the glucosidase II mutation in Dictyostelium, we employed off-line LC-MALDI-TOF-MS in combination with chemical and enzymatic treatments and MS/MS to analyse the neutral and anionic N-glycans of the mutant as compared to the wild-type. The major neutral species were, as expected, of the composition Hex10-11 HexNAc2-3 with one or two terminal glucose residues. Consistent with the block in processing of neutral N-glycans caused by the absence of glucosidase II, fucose was apparently absent from the N-glycans and bisecting N-acetylglucosamine was rare. The major anionic oligosaccharides were sulphated and/or methylphosphorylated forms of Hex8-11 HexNAc2-3 , many of which surprisingly lacked glucose residues entirely. As anionic N-glycans are considered to be mostly associated with lysosomal enzymes in Dictyostelium, we hypothesise that glycosidases present in the acidic compartments may act on the oligosaccharides attached to such slime mould proteins. Furthermore, our chosen analytical approach enabled us, via observation of diagnostic negative-mode MS/MS fragments, to determine the fine structure of the methylphosphorylated and sulphated N-glycans of the M31 glucosidase mutant in their native state. This article is protected by copyright. All rights reserved. Submitted by Iain Wilson [iain.wilson@boku.ac.at] ============================================================== [End dictyNews, volume 40, number 7]