Efflux of amino acids from cells treated with LTX109.

<p>Exponentially growing yeast cells were washed, resuspended in water, and challenged with 70 µg/ml LTX109 (black bars) or water (grey bars) for 16 minutes. Amino acids (one letter code) in the extracellular medium were subsequently measured by HPLC. Each data point is the average of three individual measurements ± standard deviation.</p

<i>S. cerevisiae</i> genes that confer LTX109 resistance upon deletion.

<p>n, number of mutants identified.</p

Activity of LTX109 against yeast biofilm.

<p>Confocal Laser Scanning Microscopy of <i>S. cerevisiae</i> (Σ1278<i>b</i>) biofilm. Cells were grown in Lab-Tek™ Chamber Slide™ System; Permanox - (NUNC, Denmark) in 1 ml synthetic complete medium After 12 hours, the cells were exposed to 0 µg/ml LTX109 (control) or 70 µg/ml LTX109 for another 5 hours. The biofilm cells were then stained with Syto9 (green) and propidium iodide (red) LIVE/DEAD stain before confocal laser scanning microscopy. Images are 3D reconstructions of biofilm made from 2 µm thick images in stacks of 20 individual images. CLSM was perform with a Zeiss LSM510 microscope using a 63x/0.95NA a water immersion lens. Life dead staining of biofilm treated with LTX109 was repeated in four independent experiments. White bar is 30 µm.</p

Fungicidal properties of LTX109 and amphotericin B.

<p>Time-kill kinetics of exponentially growing yeast cells exposed to water (circles) or five times the MIC of LTX109 (40 µg/ml) (squares) or amphotericin B (10 µg/ml) (triangles). Viability was examined every half hour as CFUs. Each data point is the average of three individual measurements ± standard deviation.</p

Transport of H⁺, K⁺ and a fluorescent dye by cells treated with LTX109.

<p>(A) Glucose-induced acidification of medium by yeast cells. Exponentially growing <i>S. cerevisiae</i> was washed and suspended in sterile water and exposed to 100 µg/ml LTX109 (squares) or water (circles) before glucose addition at time zero. Medium pH was measured and H⁺ concentration calculated from pH = −log [H⁺]. Each data point is the average of three individual measurements with standard deviations as error bars. (B) Potassium release from yeast cells. Exponentially growing yeast cells were washed, resuspended in water, and challenged with 100 µg/ml LTX109 (squares) or water (circles) at time zero. Potassium release was measured using flame atomic absorption spectrometry in binary increasing intervals. Each data point is the average of three individual measurements ± standard deviation. (C) Nomarski (left) and fluorescent (right) microscopy of SYTOX Green-stained yeast cells. Exponential growing cells were exposed to 100 µg/ml LTX109 and SYTOX Green uptake was monitored. Cells treated with SYTOX Green and 0 µg/ml LTX109 served as control. SYTOX green uptake upon LTX109 treatment was observed in three independent experiments.</p

Models of <i>FLO</i> gene regulation by amino-acid transporters.

<p>Extracellular amino acids induce the SPS complex at the plasma membrane (PM) and the activated complex elicits gene expression of amino acid transporters <i>DIP5</i> and <i>GNP1</i>. Dip5 and Gnp1 activate <i>FLO11</i> transcription in a manner dependent on activity of the PKA-pathway (A). Inactive SPS signaling indirectly leads to increased transcript levels of <i>GAP1</i>. Gap1 increases the amino acid pool concentration, which in turn triggers a PKA independent signal for induction of <i>FLO</i> genes (B). PM, plasma membrane. NM, nuclear membrane.</p

<i>DIP5</i> and <i>GNP1</i> are essential for biofilm formation and <i>DIP5</i> depletion abolishes pseudohyphal differentiation of Sfl1^Q320STOP.

<p>Biofilm formation on a polystyrene surface was tested in the Sfl1^Q320STOP trp1 background (RB52) of Sfl1^Q320STOP<i>dip5</i> (RB317), Sfl1^Q320STOP gnp1 (RB196) and compared to ∑1278b strain 10560-2B, ∑1278b <i>dip5 gnp1</i> (RB157) and the wildtype strain CEN.PK110-16D. After 24 hours growth, in either synthetic complete 0.1% glucose or synthetic complete 2.0% glucose, biofilms were visualized by crystal violet staining and then washed three times (A). Homozygous diploid strains in the Sfl1^Q320STOP background were incubated on low-ammonium SLAD medium for 3 days at 30°C to test for pseudohyphal growth (B).</p

Mutant alleles restore invasive growth in wildtype strain and invasiveness correlates with increased <i>FLO11</i> mRNA.

<p>Mutations from invasive descendants of glutamine- and ammonium-limited populations were reconstituted in the wildtype strain (A) and tested for invasive growth (B). Distribution of strains with <i>pho^T672A</i> (RB389), <i>sfl1Δ</i> (RB595), PTR3^647::C…N (RB567), <i>gdh^D206E</i> (RB484), <i>gap1Δ</i> (RB318) (C). mRNA levels for FLO adhesion genes (italic) and amino acid permease genes (italic bold) are shown for glutamine-limited mutants Ptr3^647::C…N and Sfl1^Q320STOP after growth to mid-exponential phase in liquid YPD (D). Presented are log2-transformed ratios of transcripts. The color code bar illustrates fold-change in mRNA in mutants relative to the wildtype strain CEN.PK113-7D. For the entire clustering of genes with more than 2-fold altered mRNA levels see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041272#pone.0041272.s002" target="_blank">Fig. S2</a>.</p

Dip5 interacts with Gnp1 and Mep2.

<p>Physical interaction between Dip5 and transporters Hxt1, Mep1, Mep2, Dip5, Gnp1 was tested with the membrane protein-based split-ubiquitin system in the CEN.PK background. Ade^- His^- cells (THY.AP4) expressing the C-terminal part of ubiquitin fused to Dip5 and the transcriptional activator PLV were mated to Ade^- His^- cells (THY.AP5) expressing the N-terminal part of ubiquitin fused to either Hxt1, Mep1, Mep2, Dip5 or Gnp1. Interacting protein-pairs formed a functional ubiquitin that released PLV for induction of the HIS3 and ADE2 genes, enabling growth on synthetic complete medium without adenine and histidine.</p

Hubungan layanan bimbingan dan konseling dengan kedisiplinan peserta didik di Sekolah Menengah Pertama Muhammadiyah 13 Surabaya

Kedisiplinan merupakan bagian penting dalam pendidikan baik konteks pendidikan formal maupun informal. Kedisiplinan hendaknya dipandang sebagai kekuatan positif untuk memebtuk dan mengontrol perilaku siswa. Disiplin merupakan kesadaran akan pentingnya ketertiban dan keberaturan dalam kehidupan. Lemahnya tingkat kedisiplinan akan berdampak pada masalah disiplin dalam bentuk pelanggaran terhadap tata tertib sekolah. Bimbingan dan konseling sebagai bagian integral dari pendidikan mempunyai tanggung jawab mengatasi masalah disiplin siswa di sekolah melalui pelayanan bimbingan. Adanya program bimbingan dan konseling diharapkan memiliki solusi untuk menolong siswa mengontrol hidupnya dalam tingkah laku sesuai norma dan bertanggung jawab. Penelitian ini dilaksanakan di SMP Muhammadiyah 13 Surabaya dengan sampel sebanyak 67 yang diambil dari siswa kelas VII, VIII, IX. Independen variabel dalam penelitian ini adalah layananan bimbinangan dan konseling dengan subvariabel layanan informal, layanan penempatan, layanan pengajaran, layanan penyuluhan, layanan hubungan masyarakat, serta preventif dan kuratif. Sedangkan dependen variabelnya adalah kedisiplinan siswa dengan subvariabel peraturan, penghargaan, hukuman, dan konsistensi. Metode analisis yang digunakan dalam penelitian ini adalah pertama, desckriptive statistics untuk mengetahui distribusi frekuensi temuan data dari lapangan. Kedua, untuk mengetahui signifikansi layanan bimbingan dan konseling dengan tingkat kedisiplinan siswa menggunakan korelasi Product Moment dengan SPSS (Statistical Package For Social Scinces) sebagai alat bantu. Hasil penelitian menunjukkan bahwa terdapat hubungan signifikan antara layanan bimbingan dan konseling dengan kediplinan siswa. Nilai korelasi sebesar 0,879 berada di antara 0,80 s/d 1,00 yang artinya hubungan kedua variabel menunjukkan arah yang sama. Apabila layanan bimbingan dan konseling mengalami kenaikan, maka kedisiplinan siswa juga akan ikut naik. Begitu pula sebaliknya, jika layanan bimbingan dan konseling menglami penurunan, maka kedisiplinan siswa juga akan menurun. Hubungan antara kedua variabel termasuk hubungan yang positif sedang