Improvement of ligninolytic properties in the hyper lignin-degrading fungus Phanerochaete sordida YK-624 using a novel gene promoter

autho

Efficient expression of laccase gene from white-rot fungusSchizophyllum communein a transgenic tobacco plant

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Cloning and Transcriptional Analysis of the Gene Encoding 5-Aminolevulinic Acid Synthase of the White-Rot Fungus Phanerochaete sordida YK-624

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Fingolimod (FTY720) Stimulates Ca²⁺/Calcineurin Signaling in Fission Yeast

<div><p>Fingolimod hydrochloride (FTY720) is the first in class of sphingosine 1-phosphate (S1P) receptor modulator approved to treat multiple sclerosis via down-regulation of G protein-coupled S1P receptor 1 by its phosphorylated form (FTY720-P). Many studies have revealed that FTY720 exerts various biological effects, including antitumor activities, angiogenesis inhibition, Ca²⁺ mobilization and apoptosis, independently of S1P receptors. However, the exact mechanisms underlying their effects or signaling pathways mediated by FTY720 have not been completely established. To gain further insights into molecular mechanisms of FTY720 action, the effect of FTY720 on Ca²⁺ signaling in fission yeast was analyzed. The addition of Ca²⁺ enhanced the sensitivity induced by FTY720, and mutants lacking genes required for calcium homeostasis, including calcineurin and its downstream transcription factor, <u>P</u>pb1-<u>r</u>esponsive zinc finger protein (Prz1), were hypersensitive to FTY720 and CaCl₂. The effect of FTY720 on calcineurin signaling was monitored by utilizing a luciferase reporter construct fused to three tandem repeats of the calcineurin-dependent response element (CDRE), which gives an accurate measure of calcineurin activity. The addition of FTY720 increased calcineurin activity as well as Ca²⁺ influx in a concentration-dependent manner. Notably, the FTY720-mediated Ca²⁺ influx and calcineurin activation were reduced markedly by the deletion of <i>yam8</i>⁺ or <i>cch1</i>⁺ encoding putative subunits of a Ca²⁺ channel. Consistently, the deletion of Pmk1 mitogen-activated protein kinase (MAPK), which plays an important role in the activation of the Yam8/Cch1 channel, markedly decreased the intracellular Ca²⁺ levels upon FTY720 treatment. These results suggest that the FTY720-stimulated Ca²⁺/calcineurin signaling activation partly involves the Yam8/Cch1 channel in fission yeast.</p> </div

Phosphorylated FTY720 (FTY720-P) failed to stimulate

<p>Ca²⁺/calcineurin signaling. (A) Effect of FTY720 and FTY720-P on intracellular Ca²⁺ levels. The wild-type cells harboring <i>adh1</i>-GFP-19-AEQ were treated with indicated concentrations of FTY720 or FTY720-P, and experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4(A)</a>. Ethanol containing NaOH was used as vehicle (Materials and Methods). The histogram was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4 (A)</a>. Bars, SD. (B) A serial dilution assay of the wild-type (wt) cells grown in rich YPD medium containing the indicated concentrations of FTY720 or FTY720-P. (C) Left: Real-time monitoring of calcineurin activity in living cells stimulated by FTY720 or FTY720-P. Wild-type cells harboring the multicopy plasmid (wt 3xCDRE::luc(R2.2)) reporter vector, pKB5723 (wt 3xCDRE) were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g003" target="_blank">Figure 3(B)</a>. The data shown are representative of multiple experiments. Right: Graph shows the peak heights of 3xCDRE::luc(R2.2) reporter activity. The data were averaged from three independent experiments. Bars, SD. (D) The effect of FTY720-P induced rise of intracellular Ca²⁺ levels in ABC transporter knockout cells. The wt or Δ<i>bfr1</i>Δ<i>pmd1</i> cells harboring <i>adh1</i>-GFP-19-AEQ were treated with indicated concentrations of FTY720-P, and experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4(A)</a>. ** p<0.01; Significantly different from vehicle (using two-way ANOVA). ## p<0.01; Significantly different from wild-type cells (using Williams’ test).</p

Pmk1 MAP kinase activated the FTY720-induced Ca²⁺ influx.

<p>(A) Knockout of Pmk1 MAP kinase suppressed the FTY720-induced Ca²⁺ influx. WT and Δ<i>pmk1</i> cells were monitored as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4 (A)</a>. (B) Knockout of Pmk1 MAP kinase suppressed the FTY720-induced calcineurin signaling. WT and Δ<i>pmk1</i> cells were monitored as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g003" target="_blank">Figure 3 (B)</a>. </p

FTY720 stimulates Ca²⁺/calcineurin signaling via the Yam8/Cch1 channel.

<p>(A) The wild-type (wt), Δ<i>yam8</i>, Δ<i>cch1</i>, or Δ<i>yam8</i>Δ<i>cch1</i> cells harboring pKB6892 (<i>adh1</i>-GFP-19-AEQ) were treated with 10 μM FTY720 or vehicle (basal), and the experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4 (A)</a>. The histogram was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4 (A)</a>. Bars, SD. (B) Effects of GdCl₃ on the FTY720-induced increase in the cytoplasmic Ca²⁺ level. The experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4 (A)</a>, except that 1 mM or 10 mM of GdCl₃ were also added to the EMM medium. The histogram was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g004" target="_blank">Figure 4A</a>. (C) Deletion of Yam8 or Cch1 reduced markedly calcineurin activation induced by FTY720. The wild-type (wt), Δ<i>yam8</i>, Δ<i>cch1</i>, or Δ<i>yam8</i>Δ<i>cch1</i> cells harboring the reporter plasmid (wt 3×CDRE::luc(R2.2)) were monitored as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g003" target="_blank">Figure 3(B)</a>.　(D) Deletion of Yam8 or Cch1 enhanced the sensitivity to FTY720. A serial dilution assay of the wild-type (wt), Δ<i>yam8</i>, Δ<i>cch1</i>, and Δ<i>yam8</i>Δ<i>cch1</i> mutant cells grown in rich YPD medium containing the indicated concentrations of FTY720. </p

Fission yeast sensitivity to FTY720.

<p>(A) Fission yeast cells are sensitive to FTY720. A serial dilution assay of the wild-type strain grown in YPD medium or YPD medium containing the indicated concentrations of FTY720 in the absence (Left: YPD) or presence (Right: + 100 mM CaCl₂) of 100 mM CaCl₂. 　Cells were incubated for 3 days at 27°C. (B) Quantitative measurements of cell growth in the presence of FTY720. The cells were grown in liquid YES cultures to an OD₆₆₀ of 0.3 and were treated with the drugs (FTY720) at the concentrations indicated, and the quantitative measurements of cell growth rates were performed using a microplate reader (Sunrise^TM series, Tecan, Switzerland). A representative for three independent curves is presented. (C) Addition of CaCl₂ exacerbated the fission yeast sensitivity to FTY720. Wild-type cells were cultured in YES liquid medium and treated with 100 mM CaCl₂ in the absence or presence of indicated concentrations of FTY720, and the growth curve of the cells were shown by measuring OD₆₆₀ for 10 h. (D) Graph shows the OD₆₆₀ at 10 h of the cells, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g001" target="_blank">Figure 1(B) and (C)</a>. The data were averaged from three independent experiments. Bars, SD.</p

FTY720 stimulates the calcineurin/Prz1 signaling pathway.

<p>(A) Left: Translocation of GFP-Prz1 to the nucleus is induced by FTY720 addition and requires calcineurin. Wild-type (wt), or calcineurin-null cells (Δ<i>ppb1</i>) expressing GFP-Prz1 were grown in EMM medium at 27°C and analyzed by fluorescence microscopy to observe GFP-Prz1 localization (GFP-Prz1). The cells were incubated with or without 10 μM FTY720 for 10 min or in the presence of 200 mM CaCl₂ for 10 min at 27 °C.  Arrowheads indicate cells whose nuclei show intense GFP fluorescence. The bar indicates 10 μm. Right: The percentage of cells in (A) showing intense nuclear fluorescence of GFP-Prz1 was measured. At least 300 cells were counted. (B) Real-time monitoring of calcineurin activity in living cells stimulated by FTY720. Wild-type cells harboring the multicopy plasmid wt 3×CDRE::luc(R2.2) reporter vector pKB5723 (wt 3×CDRE) or the multicopy plasmid carrying the mutant version of the CDRE (mt 3×CDRE::luc(R2.2)) reporter vector pKD2767 (mt 3×CDRE) were incubated with D-luciferin and treated with various concentrations of FTY720, as indicated. Using a luminometer, light emission levels expressed as relative light units (RLU) were measured per minutes for 3 h. The data shown are representative of multiple experiments. Right: Graph shows the peak heights of 3×CDRE::luc(R2.2) (wt 3×CDRE) or the mutant 3×CDRE::luc(R2.2) (mt 3×CDRE) reporter activity. The data were averaged from three independent experiments. Bars, SD. (C) Stimulation of calcineurin signaling induced by FTY720 addition requires calcineurin/Prz1 signaling. Wild-type and <i>ppb1</i>─ and <i>prz1</i>─null cells harboring the multicopy plasmid (wt 3×CDRE::luc(R2.2)) reporter vector were treated with 10 μM FTY720 and monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g003" target="_blank">Figure 3B</a>. (D) Real-time monitoring of calcineurin activity in living cells stimulated by CaCl₂. Cells as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g003" target="_blank">Figure 3 (B)</a> were treated with various concentrations of CaCl₂, as indicated, and monitored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081907#pone-0081907-g003" target="_blank">Figure 3 (B)</a>. </p