Деякі проблеми використання тимчасово зайнятих земель

<div><p>Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype <em>Yersinia enterocolitica</em> which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different <em>Yersinia</em> mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, <em>Clostridium difficile</em> toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal <em>GILZ</em> promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the <em>GILZ</em> promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of <em>GILZ</em> promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.</p> </div

Expression and purification of recombinant GST-C2IN-p53.

<p><i>A</i>. Scheme of GST-C2IN-p53 fusion construct. <i>B</i>. Time course of GST-C2IN-p53 expression in <i>E. coli</i> Rosetta. Samples were harvested after time points indicated (0, 1, 3, 6 and 20 h) and subjected to SDS-PAGE followed by Coomassie staining (top) or Western blot analysis using a p53 antibody (bottom). GST-C2IN-p53 is denoted by an arrow. <i>C</i>. Purification of GST-C2IN-p53 fusion protein by heparin affinity chromatography. GST-C2IN-p53 was isolated from whole cell extracts by heparin-based chromatography and eluted by a linear salt gradient as monitored by UV absorbance. Subsequently, fractions collected during affinity chromatography were characterized by immunoblot analysis with p53 antibody.</p

Characterization of GST-C2IN-p53 <i>in vitro</i>.

<p><i>A.</i> Detection of different domains of the fusion protein by Western blotting. Increasing amounts of GST-C2IN-p53 (50, 100 and 200 ng) were separated by electrophoresis and subsequently analyzed by Western blotting with different antibodies (anti-C2IN, anti-p53, anti-GST). <i>B</i>. DNA-binding of purified GST-C2IN-p53. Increasing amounts of GST-C2IN-p53 were incubated with a biotin-labeled oligonucleotide duplex containing a p53-responsive element (p53-RE). Complex formation was then monitored by native PAGE followed by detection with streptavidin-POD (Stv-POD). C2IN served as negative control and was visualized by a C2IN antibody. The star denotes a biotin moiety, while the circle represents the GST-tag of the fusion protein.</p

The ß-cyclodextrin derivative AMBnTßCD but not methyl-ß-cyclodextrin protects Vero cells from intoxication with C2 toxin when administered at 10 µM final concentration.

<p>Vero cells grown in 24-well plates were treated for 1 h at 37°C with either AMBnTßCD (10 µM) or methyl-ß-cyclodextrin (MßCD, 10 µM) and subsequently cells were challenged with C2 toxin (200 ng/ml C2IIa+100 ng/ml C2I). After 1.5 and 2.5 h of incubation at 37°C, pictures were taken to determine the percentages of round cells. Values are given as mean ± S.D. (n = 3) and significance was tested for each time point between toxin-treated samples and samples treated with either AMBnTßCD plus C2 toxin or MßCD plus C2 toxin by using the student's t-test (***p<0.0005; n. s. = not significant).</p

AMBnTßCD inhibits the pH-dependent membrane translocation of C2 toxin across cytoplasmic membranes of intact cells.

<p>Vero cells were incubated for 30 min at 37°C with 100 nM Baf A1 and subsequently for 30 min at 4°C in serum-free medium with C2 toxin (400 ng/mL C2IIa+200 ng/ml C2I) or without toxin for control. Then, the medium was removed and cells were exposed to a short acidic shift with warm medium (5 min, pH 4.5, 37°C, Baf A1) to trigger pore formation by C2IIa and membrane translocation of C2I. In parallel, cells were exposed for 5 min to neutral medium (37°C, pH 7.5, Baf A1) as a control. In some samples, AMBnTßCD (10 µM) was present during acidic shift. Subsequently, the medium was changed and cells were further incubated at 37°C in neutral medium, still in the presence of Baf A1 to prevent the normal uptake of C2 toxin via acidified endosomes. Pictures were taken after 30 and 90 min of incubation. The percentages of round (i.e. intoxicated) cells were determined from the pictures, values are given as mean ± S.D. (n = 3). Significance was tested for each time point between samples, treated with C2 under acidic conditions in the absence or presence of AMBnTßCD by using the student's t-test (***p<0.0005).</p

AMBnTßCD blocks the single pores formed by C2IIa and Ib in planar lipid membranes.

<p><i>A</i>. Currents through single C2IIa (left) and Ib (right) channels at 50 mV applied voltage. The two topmost tracks represent the AMBnTßCD-free control experiments. The fast flickering between open and closed states (1/f noise) was mostly but not completely removed here by averaging over 100-ms time interval. In the presence of increasing AMBnTßCD, the channels were spontaneously blocked (three lower current tracks). The dashed lines represent zero current levels. <i>B</i>. Typical time histograms of AMBnTßCD-induced C2IIa (top) and Ib (bottom) current fluctuations. Original current recordings collected with 15 kHz filter and 50 kHz sampling were additionally filtered with a 300 Hz filter to exclude most of the high-frequency 1/f events. T_off represents the time channels spent in the blocked state and T_on is the time between successful blockages. Data were fitted by direct single-exponential (i.e. log probability) fitting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023927#pone.0023927-Sigworth1" target="_blank">[79]</a>. The fits were obtained using variable metrics as a search method and maximum likelihood as a minimization method. Measurements were performed in 1 M KCl solutions at pH 6 and 50 mV applied voltage. 0.4 µM and 2.3 µM AMBnTßCD concentrations were used for the C2IIa and Ib time-histograms, respectively, as statistically more represented.</p

The time point of AMBnTßCD application determines the protective effect of this compound against intoxication of Vero cells with C2 toxin.

<p>Vero cells were grown in 24-well plates and AMBnTßCD (10 µM) was applied to the cell medium either 30, 15 or 5 min before C2 toxin (200 ng/ml C2IIa+100 ng/ml C2I) was added to the cells or AMBnTßCD was added together with the toxin into the medium. In parallel, AMBnTßCD was added to the cells 5, 15, 30 or 60 min after the toxin. For a control, cells were treated with medium alone or with C2 toxin in the absence of AMBnTßCD. The cells were incubated for 3 h at 37°C and pictures were taken to determine the percentages of round cells. Values are given as mean ± S.D. (n = 3) and significance was tested for each sample treated with C2 toxin and AMBnTßCD against cells treated with C2 toxin only using the student's t-test (***p<0.0005; **p<0.005; *p<0.05; n. s. = not significant).</p

AMBnTßCD protects Vero cells from intoxication with iota toxin from <i>C. perfringens</i> and inhibits the pH-dependent membrane translocation of the toxin.

<p><i>A</i>. Time- and concentration-dependent inhibition of the intoxication of Vero cells with iota toxin. Vero cells grown in 24-well dishes to subconfluency were treated for 30 min at 37°C with 2, 5, 10 and 20 µM final concentrations of AMBnTßCD or without AMBnTßCD for control. Iota toxin (200 ng/ml Ib+100 ng/ml Ia) was added and cells were further incubated at 37°C with the toxin in the absence or presence of AMBnTßCD. Pictures were taken after the indicated incubation periods, the number of total cells and round cells were counted and the percentages of round cells calculated. Values are given as mean ± S.D. (n = 3) and significance was tested for each time point between iota toxin-treated samples without and with the respective concentration of AMBnTßCD by using the student's t-test (***p<0.0005; ***<0.005; *<0.05). <i>B</i>. The time point of AMBnTßCD application determines the protective effect against iota toxin. AMBnTßCD (10 µM) was applied to Vero cells at 30, 15 or 5 min before iota toxin (200 ng/ml Ib+100 ng/ml Ia), together with iota toxin or 5, 15 or 30 min after the toxin. As a control, cells were treated with medium or with iota toxin alone. The cells were incubated for 3 h at 37°C and pictures were taken to determine the percentages of round cells. Values are given as mean ± S.D. (n = 3) and significance was tested between cells treated with iota toxin alone and cells treated with toxin and AMBnTßCD by using the student's t-test (***p<0.0005; **p<0.005). <i>C</i>. AMBnTßCD inhibits the pH-dependent membrane translocation of iota toxin across the cytoplasmic membranes of intact Vero cells. Cells were incubated for 30 min at 37°C with 100 nM Baf A1 and subsequently for 30 min at 4°C in serum-free medium with iota toxin (1000 ng/mL Ib+500 ng/ml Ia) or without toxin for control. Then, 10 µM of AMBnTßCD were added (for control no AMBnTßCD) and the pH of the medium was adjusted to 4.5 with HCl (for control pH 7.5) and cells were exposed for 15 min to 37°C to trigger pore formation by Ib membrane translocation of Ia. Subsequently, cells were further incubated at 37°C in neutral medium containing Baf A1 and pictures were taken after 45 min and 3 h of incubation. The percentages of round (i. e. intoxicated) cells were determined, values are given as mean ± S.D. (n = 3). Significance was tested for each time point between samples treated with iota toxin under acidic condition in the absence or presence of AMBnTßCD by using the student's t-test (***p<0.0005).</p

AMBnTßCD blocks the transmembrane pores formed by C2IIa and Ib in planar lipid membranes at the multichannel level.

<p><i>A</i>. Multichannel C2II (top) and Ib (bottom)-induced conductance changed by AMBnTßCD addition. The current recordings were additionally filtered over 500-ms time interval. 0.1 M KCl solutions at pH 6 were buffered by MES. Recordings were taken at 20 mV applied voltage. <i>B</i>. Typical multichannel titration curves for C2IIa (left) and Ib (right)-modified membranes show about 15 times lower IC₅₀ values for C2II channels. All multichannel measurements were taken at 20 mV applied voltage.</p

Effect of AMBnTßCD on receptor binding of C2 toxin.

<p>Vero cells were incubated for 30 min at 4°C with C2 toxin (200 ng/ml C2IIa+100 ng/ml C2I) to enable toxin binding to the receptor on the cell surface. Then, the medium was removed and cells were washed to remove any unbound toxin. Fresh medium containing 10 µM of AMBnTßCD was added and cells were further incubated at 37°C to trigger internalization of the cell-bound C2 toxin. As a control, cells were incubated with fresh medium without AMBnTßCD or left untreated. After 3 h, pictures were taken to determine the percentages of round cells (scale bar = 100 µm) (<i>A</i>). Values are given as mean ± S.D. (n = 3) and significance was tested between toxin-treated samples with or without AMBnTßCD by using the student's t-test (***p<0.0005). <i>B</i>. Western blot detection of cell-associated C2I protein. Equal amounts of cell lysate proteins were subjected to SDS-PAGE, blotted and C2I was visualized in a Western blot with a specific antibody against the N-terminal domain of C2I. Purified C2I protein was run as a control in the same gel (not shown).</p