Organized and Sustainable Education Program for Drug Abuse Prevention by Yogo-teachers

　学校における喫煙・飲酒・薬物乱用防止教育の充実には，問題行動が顕在化する中学校期だけでなく小学校期 における指導の推進が重要であり，系統的な指導計画を立て，指導者や時間の確保，教材作成などに組織的に取 組み，継続可能なプログラム開発を行う必要がある。そこで，地区内12 校の養護教諭が協働して，発達段階に応 じた系統的・組織的かつ継続可能な地区共通の指導計画を開発し，各校の教育課程・年間計画に位置付けた実践 研究を行った。その結果，指導計画の実施状況は，小学校11 校中，学級活動10 校，ミニ保健指導10 校，長期 休業前指導6 校，広報活動9 校となり，特別支援学校1 校では広報活動のみを行うことができた。小学校におけ る喫煙・飲酒・薬物乱用防止教育の推進には，学校保健活動の中核的役割を担う養護教諭が専門性を活かし協働 して，系統的な指導計画を各校の教育課程に位置付け組織的で継続可能なプログラムとする取組が有効であった

Inositol Depletion Restores Vesicle Transport in Yeast Phospholipid Flippase Mutants

In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases

Identification of <i>ART5</i> as a multicopy suppressor of flippase mutations.

<p>(A) Suppression of growth defects by overexpressing <i>ART5</i> in Cdc50p-depleted mutants carrying a mutation synthetically lethal with <i>cdc50</i>Δ. Cells were grown to early log phase in SGA-Ura medium, washed, and adjusted to a concentration of 2.5 × 10⁷ cells/ml. Drops of 4 μl from 5-fold serial dilutions were spotted onto a YPDA (Cdc50p-depleted) or YPGA (Cdc50p-expressed) agar plate, followed by incubation at 30°C for 1 day. The strains used were YKT1286 (<i>P</i>_<i>GAL1</i><i>-CDC50 gcs1</i>Δ), KKT116 (<i>P</i>_<i>GAL1</i><i>-CDC50 fpk1</i>Δ), and YKT1649 (<i>P</i>_<i>GAL1</i><i>-CDC50 neo1–101</i>), all carrying YEplac195 (vector), pKT1263 (p<i>CDC50</i>), or pKT1720 (p<i>ART5</i>). (B) Suppression of growth defects in flippase mutants by overexpression of <i>ART5</i>. Cells were grown and examined as in (A), except that the <i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ mutant was incubated at 25°C for 2 days. The strains used were YKT1511 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), all carrying each of the plasmids in (A), except YKT1932 carried pKT1469 (p<i>NEO1</i>) as a positive control. (C) Suppression of the defects in membrane trafficking in flippase mutants by overexpression of <i>ART5</i>. Cells were grown in YPDA medium at 25°C for 14 h (Cdc50p-depleted <i>crf1</i>Δ) or at 30°C for 12 h (Neo1p-depleted), followed by microscopic observation of small- or middle-budded cells. The percent of cells with polarized GFP- or mRFP-Snc1p was determined (n>100) and is shown with the mean ± standard deviation of three independent experiments. Representative images are shown. The strains used were YKT1933 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ <i>GFP-SNC1</i>) and YKT1910 (<i>P</i>_<i>GAL1</i><i>-NEO1 mRFP-SNC1</i>), both carrying each of the plasmids in (A), except YKT1910 carried pKT1469 (p<i>NEO1</i>) as a positive control. Bar: 5 μm. (D) Failure of <i>ART5</i> overexpression to suppress the alkylphosphocholine resistance and phospholipid-binding peptide sensitivity in a flippase mutant. Cells were grown to early log phase in SDA-Ura medium, washed, and adjusted to a concentration of 5.0 × 10⁶ cells/ml. Drops of 10 μl and 4 μl from 5-fold serial dilutions were spotted onto SDA-Ura containing 5.0 μg/ml miltefosine and YPDA containing 0.5 μg/ml papuamide B (pap B) or 2.0 μM duramycin agar plates, respectively, followed by incubation at 30°C for 1 day. The strains used were YKT1066 (WT) and YKT715 (<i>lem3</i>Δ), both carrying YEplac195 (vector) or pKT1720 (p<i>ART5</i>).</p

Flippase mutations are not suppressed by activation of either the Ino2p-Ino4p pathway or the unfolded protein response.

<p>(A) Activation of the Ino2p-Ino4p pathway by the <i>opi1</i>Δ mutation does not suppress the growth defects of flippase mutants. Cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>, except the cells were incubated at 30°C or 25°C for 1 day or 2 days, respectively. The strains used were YKT38 (wild type, WT), YKT1938 (<i>opi1</i>Δ), YKT1510 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ), YKT1939 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ <i>opi1</i>Δ), YKT1120 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), YKT1940 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>opi1</i>Δ), YKT1660 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), and YKT1941 (<i>P</i>_<i>GAL1</i><i>-NEO1 opi1</i>Δ). (B, C) Activation of the unfolded protein response pathway (UPR) does not suppress the growth defects of flippase mutants. (B) Expression of the UPR-activating <i>HAC1</i> (238 type S238A) mutant. Cells were grown in SG-Leu medium, washed, and adjusted to a concentration of 1.0 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto an SG-Leu, SD-Leu, or SD-Leu without inositol agar plate, followed by incubation at 30°C for 1.5 days. The strains used were KKT466 (<i>hac1</i>Δ), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), all carrying either YCplac111 (Vector) or pKT2139 [p<i>HAC1</i> (238 type S238A)]. (C) Treatment with TM or DTT. Cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>. Cells were spotted onto a YPGA, YPDA containing 0.5 μg/ml TM, or SD containing 6.5 mM DTT agar plate, followed by incubation at 30°C for 1 day. The strains used were YKT1066 (wild type, WT), KKT466 (<i>hac1</i>Δ), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>).</p

Suppression of the growth defects in flippase mutants by decreased synthesis of PI.

<p>(A) Effects of inositol depletion on steady state phospholipid composition in the flippase mutants. Cells were grown in SD medium with or without inositol at 30°C for 12 h or 16 h (Neo1p-depleted). Phospholipids were extracted and quantified by liquid chromatography mass spectrometry as described in the Materials and Methods. The data represent mole percentage (mol%) of total phospholipids, with mean values ± standard deviation (n = 3). Asterisks indicate a significant difference in the Student’s <i>t</i> test (*: <i>P</i> < 0.05; **: <i>P</i> < 0.005). The strains used were YKT1066 (wild type, WT), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>). (B) Suppression of the cold-sensitive growth defects in the <i>cdc50</i>Δ mutant by partial depletion of Pis1p. Cells were pregrown to early log phase in SD medium containing 2% raffinose and 2% or 0.005% galactose for 2 days, washed, and adjusted to a concentration of 1.0 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto the same medium and YPDA, followed by incubation at the indicated temperature for the indicated time. The strains used were YKT1066 (wild type, WT), YKT1697 (<i>cdc50</i>Δ), YKT1942 (<i>P</i>_<i>GAL1</i><i>-PIS1</i>), and YKT1943 (<i>P</i>_<i>GAL1</i><i>-PIS1 cdc50</i>Δ). (C) Effects of Pis1p depletion on steady state phospholipid composition in the <i>cdc50</i>Δ mutant. Cells were pregrown and grown in SD medium containing 2% raffinose and 0.005% galactose for 4 days, in total, at 30°C. Phospholipids were extracted and quantified as in (A). The strains used were those in (B). (D) Aureobasidin A (AbA) treatment does not suppress the growth defects of flippase mutants. Cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>. Cells were spotted onto a YPGA, YPDA, or YPDA containing 75 ng/ml AbA agar plate, followed by incubation at 30°C for 1 day. The strains used were YKT1066 (wild type, WT), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>). (E) <i>ART5</i> overexpression suppressed the growth defects of flippase mutants more efficiently than the <i>CHO1</i> overexpression. Cells were pregrown to early log phase in SG-Leu medium, washed, and adjusted to a concentration of 2.5 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto an SDA agar plate with or without inositol, followed by incubation at 30°C for 1 day or 1.5 days (the <i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ mutant). The strains used were YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), carrying YEplac181 (vector), pKT1259 (p<i>CDC50</i>), pKT1788 (p<i>NEO1</i>), pKT1719 (p<i>ART5</i>), or pKT1754 (p<i>CHO1</i>).</p

Suppression of the defects in flippase mutants by depletion of inositol from culture medium.

<p>(A) Suppression of the growth defects. Cells were pregrown to early log phase in YPGA medium, washed, and adjusted to a concentration of 1.0 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto an SD agar plate with or without inositol, followed by incubation at 30°C for 1.5 days. The strains used were YKT1066 (wild type, WT), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), YKT1909 (<i>P</i>_<i>GAL1</i><i>-NEO1 P</i>_<i>GAL1</i><i>-CDC50</i>), and YKT1887 (<i>P</i>_<i>GAL1</i><i>-NEO1 P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ). (B) Suppression of growth defects in the flippase null mutants. Cells were pregrown to early log phase in SD medium without inositol, and cell growth was examined as in (A), except that they were incubated at 30°C, 25°C, or 18°C for 1.5, 2, or 5 days, respectively. The strains used were YKT1066 (wild type, WT), YKT1944 (<i>cdc50</i>Δ), and YKT1945 (<i>cdc50</i>Δ <i>dnf1</i>Δ). YKT1945 was constructed by tetrad dissection of spores from a <i>cdc50</i>Δ/<i>CDC50 DNF1</i>/<i>dnf1</i>Δ heterozygous diploid on an inositol-depleted SD agar plate. (C) Suppression of the defects in endocytic recycling of Snc1p. Cells were grown in SD medium with or without inositol at 30°C for 12 h or 16 h (Neo1p-depleted), followed by microscopic observation of small- or middle-budded cells. The percent of cells with mRFP-Snc1p in polarized sites was determined (n>100) and is shown with the mean ± standard deviation of three independent experiments. Representative images are shown. The strains used were YKT1912 (<i>mRFP-SNC1</i>, WT), YKT1936 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ <i>mRFP-SNC1</i>), YKT1937 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>mRFP-SNC1</i>), and YKT1910 (<i>P</i>_<i>GAL1</i><i>-NEO1 mRFP-SNC1</i>). Bar: 5 μm. (D) Restoration of the plasma membrane location of GFP-Snc1p-pm. Cells were grown in SD-Ura or SD-Leu medium with or without inositol at 30°C for 16 h. More than 100 cells were microscopically observed, and the percent of cells with internally accumulated GFP-Snc1p-pm was determined. The strains used were YKT1066 (wild type, WT), YKT1120 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1660 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), carrying pKT1444 (pRS416-GFP-SNC1-pm) or pKT1491 (pRS315-GFP-SNC1-pm). Bar: 5 μm. (E) Formation of secretory vesicles. Cells were grown in SD with or without inositol at 30°C for 12 h, followed by a shift to 37°C for 2 h. Secretory vesicles were fractionated by the Nycodenz density gradient, and each fraction was measured for mRFP fluorescence intensity and the total amount of phospholipid phosphates. Strains used were YKT1844 (<i>sec6–4 mRFP1-Lact-C2</i>) and YKT1854 (<i>sec6–4 P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>mRFP1-Lact-C2</i>).</p

<i>S</i>. <i>cerevisiae</i> strains used in this study.

<p>YKT and KKT strains are isogenic derivatives of YEF473 and BY4743, respectively.</p><p>Only relevant genotypes are described.</p><p><i>S</i>. <i>cerevisiae</i> strains used in this study.</p

The growth defects of flippase mutants were suppressed by mutations in inositol transporters.

<p>(A) Suppression of the flippase mutations by overexpression was specific to <i>ART5</i>. Cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>, except that the <i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ mutant was incubated at 25°C for 2 days. The strains used were YKT1511 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ) and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), both carrying YEplac195 (vector), pKT1720 (p<i>ART5</i>), pKT2135 (p<i>RIM8</i>), pKT2136 (p<i>ROD1</i>), pKT2137 (p<i>ROG3</i>), pKT1881 (p<i>ALY1</i>), pKT1882 (p<i>ALY2</i>), pKT2088 (p<i>ART5</i>-<i>PYm</i>), or pKT2138 (p<i>ART5</i>-<i>AMm</i>). YKT1511 and YKT1932 also carried pKT1263 (p<i>CDC50</i>) and pKT1469 (p<i>NEO1</i>), respectively, as positive controls. (B) Suppression of the growth defects of flippase mutants by mutations in inositol transporters. Cells were pregrown to early log phase in SGA-Ura or YPGA medium, and cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>. The strains used were YKT1066 (wild type, WT), YKT1866 (<i>itr1</i>Δ <i>itr2</i>Δ), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1934 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ <i>itr1</i>Δ), YKT1935 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ <i>itr1</i>Δ <i>itr2</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), YKT1915 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>itr1</i>Δ), YKT1914 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>itr1</i>Δ <i>itr2</i>Δ), YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), YKT1877 (<i>P</i>_<i>GAL1</i><i>-NEO1 itr1</i>Δ), and YKT1881 (<i>P</i>_<i>GAL1</i><i>-NEO1 itr1</i>Δ <i>itr2</i>Δ). YKT1529, YKT1513, and YKT1932 also carried YEplac195 (vector) or pKT1720 (p<i>ART5</i>).</p

Methylation Analysis of DNA Mismatch Repair Genes Using DNA Derived from the Peripheral Blood of Patients with Endometrial Cancer: Epimutation in Endometrial Carcinogenesis

Germline mutation of DNA mismatch repair (MMR) genes is a cause of Lynch syndrome. Methylation of MutL homolog 1 (MLH1) and MutS homolog 2 (MSH2) has been detected in peripheral blood cells of patients with colorectal cancer. This methylation is referred to as epimutation. Methylation of these genes has not been studied in an unselected series of endometrial cancer cases. Therefore, we examined methylation of MLH1, MSH2, and MSH6 promoter regions of peripheral blood cells in 206 patients with endometrial cancer using a methylation-specific polymerase chain reaction (MSP). Germline mutation of MMR genes, microsatellite instability (MSI), and immunohistochemistry (IHC) were also analyzed in each case with epimutation. MLH1 epimutation was detected in a single patient out of a total of 206 (0.49%)—1 out of 58 (1.72%) with an onset age of less than 50 years. The patient with MLH1 epimutation showed high level MSI (MSI-H), loss of MLH1 expression and had developed endometrial cancer at 46 years old, complicated with colorectal cancer. No case had epimutation of MSH2 or MSH6. The MLH1 epimutation detected in a patient with endometrial cancer may be a cause of endometrial carcinogenesis. This result indicates that it is important to check epimutation in patients with endometrial cancer without a germline mutation of MMR genes