Multidrug Sensitive Yeast Strains, Useful Tools for Chemical Genetics

The budding yeast Saccharomyces cerevisiae is a useful eukaryote model organism for application to chemical biology studies, for example, drug screening, drug evaluation, and target identification. To use yeast for chemical biology research, however, it has been necessary to construct yeast strains suitable for various compounds because of their high drug resistance. Hence, the deletion of all multidrug resistance genes except for those that are important for viability and for genetic experiments/manipulation could increase the drug sensitivity without influencing the transformation, mating, or sporulation efficiency. There are two major factors conferring multidrug resistance in S. cerevisiae: one is the drug efflux system and the other is the permeability barrier. We therefore constructed a strain which shows high sensitivity to multiple drugs by disrupting the drug efflux system using ATP-binding cassette transporters and suppressing the membrane barrier system by introducing an ERG6-inducible system. In this review, we discuss the construction of our multidrug-sensitive yeast strains and their application in chemical biology

Amphidinolide H, a Potent Cytotoxic Macrolide, Covalently Binds on Actin Subdomain 4 and Stabilizes Actin Filament

AbstractThe actin-targeting toxins have not only proven to be invaluable tools in studies of actin cytoskeleton structure and function but they also served as a foundation for a new class of anticancer drugs. Here, we describe that amphidinolide H (AmpH) targets actin cytoskeleton. AmpH induced multinucleated cells by disrupting actin organization in the cells, and the hyperpolymerization of purified actin into filaments of apparently normal morphology in vitro. AmpH covalently binds on actin, and the AmpH binding site is determined as Tyr200 of actin subdomain 4 by mass spectrometry and halo assay using the yeast harboring site-directed mutagenized actins. Time-lapse analyses showed that AmpH stimulated the formation of small actin-patches, followed by F-actin rearrangement into aggregates via the retraction of actin fibers. These results indicate that AmpH is a novel actin inhibitor that covalently binds on actin

Stimulation of microtubule-based transport by nucleation of microtubules on pigment granules

Microtubule (MT)-based transport can be regulated through changes in organization of MT transport tracks, but the mechanisms that regulate these changes are poorly understood. In Xenopus melanophores, aggregation of pigment granules in the cell center involves their capture by the tips of MTs growing toward the cell periphery, and granule aggregation signals facilitate capture by increasing the number of growing MT tips. This increase could be explained by stimulation of MT nucleation either on the centrosome or on the aggregate of pigment granules that gradually forms in the cell center. We blocked movement of pigment granules to the cell center and compared the MT-nucleation activity of the centrosome in the same cells in two signaling states. We found that granule aggregation signals did not stimulate MT nucleation on the centrosome but did increase MT nucleation activity of pigment granules. Elevation of MT-nucleation activity correlated with the recruitment to pigment granules of a major component of MT-nucleation templates, γ-tubulin, and was suppressed by γ-tubulin inhibitors. We conclude that generation of new MT transport tracks by concentration of the leading pigment granules provides a positive feedback loop that enhances delivery of trailing granules to the cell center

慢性肉芽腫症患者好中球よりのリソゾーム酵素放出について

Lysosomal enzyme release from PMN exposed to STZ was examined using PMN from normal and CGD donors. Normal PMN showed an increase of extracellular lysosomal enzyme activity and a marked reduction of totol (extra-plus intra-cellular) enzyme activity after phagocytosis. On the other hand, in PMN from CGD patients, such a reduction of total enzyme activity was not observed and much more enzyme than normal was released extracellularly. When the supernatant from PMN from CGD patients after phagocytosis of STZ was incubated with an artificial H2O2 generating system, glucose plus glucose oxidase, the enzyme activities were greatly suppressed as the amount of H2O2 rose. A similar result was obtained with the addition of glucose oxidase during phagocytosis in PMN from CGD patients. These findings suggest that the presence of H2O2 might suppress lysosomal enzyme activities and result in inhibition of lysosomal enzyme release.This work was supported in parts by Grants-in-Aid for Scientific Research (Project Nos. 437010 and 544050) from the Ministry of Education, Science, and Culture of Japan

ヒトナチュラル・キラー（NK）細胞の電顕的考察

An electron microscopic study of human natural killer (NK) cells showed abundant cytoplasm and relatively large mitochondria. They also had various kinds of granules in their cytoplasm. The mode of binding to the tumor cell (K 562) was tight, and villi protruded from NK cells to the target cells. Some target cells were already lysed after incubation for 5 minutes at 4°C.This work was supported in parts by Grants-in-Aid for Scientific Research (Project Nos. 544050 and 56770468) from the Ministry of Education, Science and Culture of Japan

白血病細胞の α-マンノシダーゼと β-ガラクトシダーゼのアイソザイムパターン

The isoenzyme profiles of α-mannosidase and β-galactosidase in leukemic cells were examined with DEAE-Sephadex chromatography. A clear difference of these constituent isoenzymes was found between normal lymphocytes and granulocytes. In leukemic cells, the components of a-mannosidase eluting early (fraction I to III) from the chromatography were specific for myelogenous leukemia. No case of acute non-T/ non-B lymphocytic leukemia (ALL) or T-cell leukemia demonstrated these components. Leukemic cells except for T-cell leukemia also demonstrated significant increase of an α-mannosidase component which eluted with 0.2 M of KCl (fraction VI). There was no type specific alteration of β-galactosidase isoenzymes among examined leukemias. ALL and T-cell leukemia had isoenzymes of β-galactosidase similar to those of normal lymphocytes. However, an increase of an β-galactosidase component eluted with 0.3M of KCl (fraction VII) was found in acute myelocytic leukemia and chronic myelo (mono) cytic leukemia. These enzyme analysis may be useful for investigating intrinsic abnormalities in leukemic cells.This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan