ATP13A1 is the functional homologue of Spf1.

<p>(<b>A</b>) Immunostaining of HeLa cells with α-ATP13A1 and DAPI (nucleus staining) indicates perinuclear localization for ATP13A1. Scale bar: 10µm. (<b>B</b>) Amount of spliced XBP1 (a result of ER stress) was assayed by qPCR analysis. Silencing of ATP13A1 in HeLa cells causes an increase in the spliced form. (<b>C</b>) Western blot of BiP protein reveals that its amount is increased upon silencing of ATP13A1 in HeLa cells. Blot was also probed with an antibody against GAPDH as a loading control. (<b>D</b>) Mass Spectrometry analysis reveals a specific increase in GlcCer levels in cells silenced for ATP13A1 suggesting enhanced activity of GlcCer synthase. (<b>E</b>) Fluorescent cholera toxin subunit B (CT-B) was used to stain the plasma membrane ganglioside GM1. Cells treated with an siRNA against ATP13A1 display accumulation of GM1. Scale bar: 10µm.</p

Spf1 affects protein mannosylation and its ATPase activity is required for its molecular function.

<div><p>(A) Western blot of three GPI anchored proteins that undergo known mannosylation (Gas1, Ccw14 and Yps1) reveals that this modification is reduced in Δ<i>spf1</i> compared to WT. </p> <p>(B) Western blot of Gas1 in WT and Δ<i>spf1</i> cells without any plasmid, with a plasmid containing full-length SPF1 gene, or with plasmid containing the full-length gene carrying a mutation rendering the protein ATPase-dead. Lack of rescue in the ATPase dead mutant confirms that the reason for the defect in Gas1 maturation is the absence of the functional ATPase activity of Spf1. </p></div

Biosynthesis of sphingolipids is interrupted due to deletion of SPF1.

<p>(<b>A</b>) WT and Δ<i>spf1</i> cells were plated in serial dilution on YPD plates without and with Aureobasidin A, an IPC synthase inhibitor. Loss of SPF1 increases the sensitivity to this inhibitor. (<b>B</b>) The levels of different types of IPC, were measured by mass spectrometry and are mostly lower in Δ<i>spf1</i> cells compared to WT. (<b>C</b>) The levels of different types of MIPC and M(IP)2C, were measured by mass spectrometry and are mostly lower in Δ<i>spf1</i> cells compared to WT. * P value<0.05, ** P value<0.01, *** P value<0.001. </p

Δ<i>spf1</i> cells are more sensitive to the ergosterol biosynthesis inhibitor Terbinafine and display change in ergosterol distribution compared to WT.

<p>(<b>A</b>) WT and Δ<i>spf1</i> cells were plated in serial dilution on YPD plates without and with Terbinafine, an ergosterol biosynthesis inhibitor. Loss of SPF1 causes growth inhibition in the presence of this inhibitor. (<b>B</b>) WT and Δ<i>spf1</i> cells were stained for sterol distribution by fillipin staining. While most of the Δ<i>spf1</i> cells exhibit homogeneous sterol distribution, the WT shows a non-homogenous sterol staining pattern. Scale bar: 5µm.</p

Spf1 affects the exit of GPI anchor proteins from the ER.

<p>WT and ∆<i>spf1</i> cells expressing tagged GPI anchored proteins (YFP-Ccw14 and RFP-Gas1) were imaged. While these proteins are normally localized to the cell periphery and the vacuole, a deletion of SPF1 enhances ER retention. Scale bar: 5µm. </p

World of Children as Media Users

This dissertation consists of two parts - a theoretical and an analytical, empirical part. In the theoretical segment, there is a summary of theoretical knowledge about celebrities and their relationship to society and the public, a comparison between celebrities and real authorities, an overview of mass-communication and its influence on the socialisation of children and a series of advantages and disadvantages of the impact of mass-communication on children. The final stage of the theoretical segment characterises, from the point of view of developmental psychology, the two age groups that will be compared in the second segment. The empirical segment answers such questions as how much do children use media in their leisure time, what types of media are most popular amongst them, which celebrities do the children admire and why are these celebrities important for them in particular. The empirical segment is based on analysis of data taken from questionnaires created purposely for qualitative-quantitative research. These questionnaires were given to students in the 4th and 6th grades of an elementary school in Polepy in the Czech Republic (ages 10/11 and 12/13 respectively). The goal of this dissertation is to show the trending habits of children as mass- media users

GM1 ganglioside-independent intoxication by Cholera toxin

<div><p>Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (Le^X) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the Le^X glycan <i>in vitro</i> when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to Le^X. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.</p></div

Effect of UDCA bile acid supplementation.

<p>(<b>A</b>) and (<b>B</b>) effects of UDCA bile acid supplementation on the activity of CYP450 catalysed reactions in the <i>Npc1</i> mouse model of plus or minus 0.5% ursodeoxycholic acid (UDCA, w/w) measured at (<b>A</b>) 6 weeks and (<b>B</b>) 9 weeks of age, shown as a function of percent of age-matched control littermates. Methoxyresorufin-O-dealkylation (MROD); ethoxyresorufin-O-dealkylation (EROD); pentoxyresorufin-O-dealkylation (PROD); benzoxyresorufin-O-dealkylation (BROD). Data are presented as mean ± SEM, n = 6 (3 males and 3 females), * <i>p—</i>value < 0.05, ** <i>p—</i>value < 0.01, *** <i>p—</i>value < 0.001, **** <i>p—</i>value < 0.0001 calculated using an unpaired nonparametric test Mann-Whitney test. (<b>C</b>) Relative protein quantitation of Cyp3a isolated from liver of 6-week old male mice found a significant decrease in <i>Npc1</i>^<i>+/-</i> and <i>Npc1</i>^<i>-/-</i> untreated mice compared <i>Npc1</i>^<i>+/+</i> untreated mice. UDCA treatment restored <i>Npc1</i>^<i>-/-</i> Cyp3a levels to that of <i>Npc1</i>^<i>+/+</i> mice while have no effect on the <i>Npc1</i>^<i>+/-</i> or <i>Npc1</i>^<i>+/+</i> Cyp3a values. (<b>D</b>) Relative protein quantitation of Cyp1a2 isolated from liver of 6-week old male mice found a significant decrease in <i>Npc1</i>^<i>+/-</i> and <i>Npc1</i>^<i>-/-</i> untreated mice compared <i>Npc1</i>^<i>+/+</i> untreated mice. UDCA treatment restored <i>Npc1</i>^<i>-/-</i> Cyp1a2 levels to that of <i>Npc1</i>^<i>+/+</i> mice while decreasing the levels for <i>Npc1</i>^<i>+/-</i> and <i>Npc1</i>^<i>+/+</i> Cyp1a2 values. Data are presented as mean ± SD, n = 3. * <i>p—</i>value < 0.05, ** <i>p—</i>value < 0.01, *** <i>p—</i>value < 0.001 calculated using two-tailed, unpaired t test. (<b>F</b>) Representative western blot analysis of Cyp3a and Cyp1a2 in the mouse model of <i>Npc1</i> plus or minus UDCA treatment.</p

Enzyme activity rates, and relevant statistics of the P450 activity assays after treatment with UDCA intergenotypic comparisons.

<p>Enzyme activity rates, and relevant statistics of the P450 activity assays after treatment with UDCA intergenotypic comparisons.</p

Enzyme activity rates, and relevant statistics of the P450 activity assays.

<p>Enzyme activity rates, and relevant statistics of the P450 activity assays.</p