36 research outputs found

    Western blot of tight junction proteins ZO-1 and claudin-3 in IPEC-J2 cells treated with deoxynivalenol (DON).

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    <p>Cells were grown on inserts and incubated for 24, 48 or 74 hours with DON (0 or 2000 ng/mL) applied from apical or basolateral side in complete medium. ZO-1 (225 kDa) and claudin-3 (22 kDa) expression was analysed by immunoblotting. The housekeeping protein GAPDH (37 kDa) was used as loading control.</p

    Cellular distribution of the tight junction protein claudin-3 (CLDN-3) in IPEC-J2 monolayers treated with deoxynivalenol (DON).

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    <p>Cells were grown on inserts and incubated for 24, 48 or 74 hours either without DON (upper panel) or with 2000 ng/mL DON applied from apical (middle panel) or basolateral side (lower panel) in complete medium. Monolayers were stained for the tight junction associated protein claudin-3 and nuclei stained with DAPI, then detected by immunofluorescence microscopy. All micrographs were taken under identical exposure time and in the centre of each membrane. Micrographs are representative for 3 separate experiments with similar results. Scale bar  =  50 µm.</p

    Influence of deletions in <i>KlCAT8</i> and <i>KlSIP4</i> on carbon utilization.

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    <p><i>K</i>. <i>lactis</i> wild-type (JA6), <i>Klcat8</i>Δ (yIG8), <i>Klsip4</i>Δ (JA6/DS4) and <i>Klcat8</i>Δ<i>Klsip4</i>Δ (yIG8/DS4) strains were pregrown in YP medium with 2% glucose (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139464#sec013" target="_blank">Material and Methods</a>) and spotted in serial 10-fold dilutions on minimal plates with 2% of the indicated carbon sources. Plates were incubated at 30°C for 4 days.</p

    Effect of deoxynivalenol (DON) on total cell count of IPEC-J2.

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    <p>Cells were grown on inserts and incubated for 24, 48 or 74 hours with DON (0–4000 ng/mL) applied from apical or basolateral side in complete medium. Data are given as means (±SEM) in triplicates from three separate experiments. ***p≤0.001 vs. DON0.</p

    Sip4 is involved in expression of carnitine shuttle genes in <i>K</i>. <i>lactis</i>.

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    <p>(A) Schematic overview of metabolic pathways and key genes essential for the utilization of non-fermentable carbon source ethanol in yeast as found in the yeast genome database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139464#pone.0139464.ref033" target="_blank">33</a>]. (B) RNA levels of genes related to the carnitine shuttle in <i>sip4</i>Δ, <i>cat8</i>Δ and <i>sip4</i>Δ<i>cat8</i>Δ mutants relative to congenic wild-type strains were determined by qRT-PCR in <i>S</i>. <i>cerevisiae</i> (left panel) and <i>K</i>. <i>lactis</i> (right panel). Cultures were shifted from 2% glucose to 3% ethanol medium for 2 hours at an OD<sub>600</sub> of 0.8 to 1.0. Gene expression levels were normalized to the reference gene <i>HEM2</i> and quantified relative to wild-type levels (set to 1.0; dashed line) Data points and error bars represent mean values ± standard deviations obtained with three independent biological samples each measured in technical triplicates. Asterisks indicate statistically significant differences compared to wild-type (<i>t</i>-test; *<i>P</i> < 0.001; ns, not significant). (C) Protein levels of Yat2 in wild-type and mutant strains. In <i>S</i>. <i>cerevisiae</i> (left) the chromosomal <i>YAT2</i> gene was tagged with a (HA)<sub>6</sub>-epitope in wild-type and <i>cat8</i>Δ and <i>sip4</i>Δ mutant backgrounds (strains CMY196, CMY198 and CMY197), respectively. In <i>K</i>. <i>lactis</i> the strains JA6 (WT), yIG8 (<i>cat8</i>Δ) and JA6/DS4 (<i>sip4</i>Δ) were transformed with centromeric plasmid pCM68 (<i>KlYAT2-6HA</i>::pKATUC4) or empty vector (pKATUC4). Cultures were grown in glucose or ethanol medium and the indicated amounts of protein extracts were probed by Western blotting with anti-HA antibody (top panel). Nop1, detected with an anti-Nop1 antibody served as loading control (bottom panel). The position of KlYat2-(HA)<sub>6</sub> (104.6 kDa) and ScYat2-(HA)<sub>6</sub> (110.5 kDa) are indicated by arrows. Numbers refer to molecular markers in kDa.</p

    Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts

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    <div><p>Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK) functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of <i>Saccharomyces cerevisiae</i> to that of <i>Kluyveromyces lactis</i>. In high glucose, <i>S</i>. <i>cerevisiae</i> displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive) while <i>K</i>. <i>lactis</i> has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative), which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the <i>Saccharomyces</i> lineag<i>e</i>. We find that in <i>K</i>. <i>lactis</i>, but not in <i>S</i>. <i>cerevisiae</i>, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of <i>KlSIP4</i> gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in <i>K</i>. <i>lactis</i>. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and KlCat8, to selected CSREs and provide evidence that KlSip4 counteracts KlCat8-mediated transcription activation by competing for binding to some but not all CSREs. The finding that the hierarchical relationship of these transcription factors differs between <i>K</i>. <i>lactis</i> and <i>S</i>. <i>cerevisiae</i> and that the sets of target genes have diverged contributes to explaining the phenotypic differences in metabolic life-style.</p></div

    Cellular distribution of the tight junction protein ZO-1 in IPEC-J2 monolayers treated with deoxynivalenol (DON).

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    <p>Cells were grown on inserts and incubated for 24, 48 or 74 hours either without DON (upper panel) or with 2000 ng/mL DON applied from apical (middle panel) or basolateral side (lower panel) in complete medium. Monolayers were stained for the tight junction associated protein ZO-1 and nuclei stained with DAPI, then detected by immunofluorescence microscopy. All micrographs were taken under identical exposure time and in the centre of each membrane. Micrographs are representative for 3 separate experiments with similar results. Scale bar  =  50 µm.</p

    Deoxynivalenol (DON) enlarged nucleic area (µm<sup>2</sup>) of IPEC-J2 cells.

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    <p>xCells were incubated for 24, 48 or 74 hours with DON (0–4000 ng/mL) in complete medium applied from apical (ap) or basolateral (bl) side. Data are given as means (±SEM) from three separate experiments.</p><p>***p≤0.001 vs control.</p

    Impact of deoxynivalenol (DON) on transepithelial electrical resistance (TEER) in polarised IPEC-J2 layers.

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    <p>Cells were grown on inserts and incubated for 24, 48 or 72 hours with DON (0–4000 ng/mL) applied from apical or basolateral side in complete medium. TEER values are expressed in kOhms per insert (0.3 cm<sup>2</sup>) with 1 kOhm being the level of confluence. Data are given as means (±SEM) from at least 14 separate experiments. ***p≤0.001 vs. DON0.</p

    Cell cycle analysis of IPEC-J2 cells treated with deoxynivalenol (DON).

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    <p>Cells were grown on inserts and synchronised for 24 hours in serum free medium and then incubated for 24, 48 or 74 hours with DON (0 or 2000 ng/mL) applied from apical or basolateral side in complete medium. After staining with propidium iodide DNA content was analysed by FACS. Data given are means (±SEM) from five separate experiments. **p≤0.01 vs. DON0, ***p≤0.001 vs. DON0.</p
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