Identification of Stk25 as a genetic modifier of Tau phosphorylation in Dab1-mutant mice.

Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation

Oguri K. Effect of a ligand selective for peripheral benzodiazepine receptors on the expression of rat hepatic P-450 cytochromes: assessment of the effect in vivo and in a hepatocyte culture system. Drug Metab Dispos

This paper is available online at http://www.dmd.org ABSTRACT: The peripheral benzodiazepine receptor plays a role in the translocation of cholesterol into mitochondria where steroidogenesis occurs. Sterols have been suggested to be involved in the regulation of the cytochrome P-450 (CYP)2B subfamily as the endogenous suppressor of this CYP. To investigate the role of cholesterol metabolites on the expression of CYPs, the effect of PK11195, a specific ligand of the peripheral benzodiazepine receptor and a stimulator of cholesterol transportation, on CYP expression was examined in rats in vivo and in cultured hepatocytes. As judged by the change in testosterone metabolic activity catalyzed by liver microsomes, i.p. injection of PK11195 into rats increased the CYP2B subfamily significantly. A trend in the induction of the CYP2A1, 2C11, and 3A isozymes was also observed. When PK11195 was given to rats together with phenobarbital, an additive effect of these compounds on testosterone metabolic activity was observed. In cultured hepatocytes, PK11195 exhibited the same effect on CYP expression as seen in vivo, but the magnitude of the effect was much greater than that observed in vivo. The inductive effect of PK11195 toward the CYP2B and 3A subfamilies was 2.3-and 6.5-fold greater, respectively, than that with phenobarbital. The inductive effect of PK11195 was confirmed by immunoblotting with antibodies against CYP2A, 2B, 2C, and 3A proteins. These results indicate that PK11195 has an inductive effect on several subfamilies of CYPs by directly acting on liver cells and has no ability to suppress the expression of these CYPs. This observation suggests that, if certain sterols play a role in the suppressive control of the CYP2B subfamily, they are produced in organelles other than the mitochondria

Formation of False Context Fear Memory Is Regulated by Hypothalamic Corticotropin-Releasing Factor in Mice

Traumatic events frequently produce false fear memories. We investigated the effect of hypothalamic corticotropin-releasing factor (CRF) knockdown (Hy-Crf-KD) or overexpression (Hy-CRF-OE) on contextual fear memory, as fear stress-released CRF and hypothalamic–pituitary–adrenal axis activation affects the memory system. Mice were placed in a chamber with an electric footshock as a conditioning stimulus (CS) in Context A, then exposed to a novel chamber without CS, as Context B, at 3 h (B-3h) or 24 h (B-24h). The freezing response in B-3h was intensified in the experimental mice, compared to control mice not exposed to CS, indicating that a false fear memory was formed at 3 h. The within-group freezing level at B-24h was higher than that at B-3h, indicating that false context fear memory was enhanced at B-24h. The difference in freezing levels between B-3h and B-24h in Hy-Crf-KD mice was larger than that of controls. In Hy-CRF-OE mice, the freezing level at B-3h was higher than that of control and Hy-Crf-KD mice, while the freezing level in B-24h was similar to that in B-3h. Locomotor activity before CS and freezing level during CS were similar among the groups. Therefore, we hypothesized that Hy-Crf-KD potentiates the induction of false context fear memory, while Hy-CRF-OE enhances the onset of false fear memory formation

Additional file 2 of BMP and activin membrane-bound inhibitor regulate connective tissue growth factor controlling mesothelioma cell proliferation

Additional file 2: Table S3

Additional file 3 of BMP and activin membrane-bound inhibitor regulate connective tissue growth factor controlling mesothelioma cell proliferation

Additional file 3. Unprocessed western blot images for Fig. 1. Unprocessed western blot images for Fig. 2. Unprocessed western blot images for Fig. 3. Unprocessed western blot images for Figure S3. Unprocessed western blot images for Figure S4

Additional file 1 of BMP and activin membrane-bound inhibitor regulate connective tissue growth factor controlling mesothelioma cell proliferation

Additional file 1: Figure S1. Relative CTGF and BAMBI mRNA levels in MM cells knocked down by transfection of CTGF-targeted siRNA. Relative mRNA expression levels of BAMBI in MSTO-211H, NCI-H28, Y-MESO-8D, NCI-H2052 and NCI-H2452 cells transfected with a CTGF-targeted siRNA (20nM) are shown. BAMBI mRNA expression level was downregulated by transfection of CTGF-targeted siRNA in MSTO-211H, NCI-H28, Y-MESO-8D and NCI-H2052 cells, but not in NCI-H2452 cells. Figure S2. Relative BAMBI mRNA levels and cell proliferation rates of cells knocked down by Santa Cruz's BAMBI siRNA pool. A, Relative mRNA expression levels of BAMBI in Y-MESO-14 and Y-MESO-27 cells transfected with a Santa Cruz's BAMBI siRNA pool (20nM). BAMBI mRNA expression level was downregulated by transfection of siRNA in Y-MESO-14 and Y-MESO-27 cells. B, Graphs of proliferation rates as measured by viable cell counting assay. Figure S3. Knockdown of BAMBI does not affect the TGF-β/Smad activation in Y-MESO-27 cells. Western blotting showed p-Smad2 and p-Smad3 levels in Y-MESO-27 cells. Cells were transfected with a BAMBI-targeted siRNA (20 nM) and compared with nontransfected cells and cells transfected with control siRNA. Cells were stimulated by adding exogenous TGF-β1 and compared with cells without exogenous TGF-β1. Figure S4. Expression and subcellular localization of BAMBI protein in mesothelial and MM cell lines. A, Western blots showing BAMBI levels in seven MM cell lines and the mesothelial MeT-5A cell line. BAMBI protein was ubiquitously expressed by all mesothelioma cell lines but only weakly or below detection limits by MeT-5A cells. B, Immunofluorescence staining of Y-MESO-27 and MeT-5A cells showing that BAMBI localizes to the plasma membrane and cytosol. The nuclei were counterstained with DAPI (blue). The white arrows demarcate BAMBI expression in the cell membrane. Scale bars: 10 μm. Table S1. the oligonucleotide sequences of the siRNAs used in the study. Table S2. the oligonucleotide sequences and accession number of primers used in the study

Stk25 expression is not correlated with augmented Tau phosphorylation in a Tauopathy model or in transfected HeLa cells.

<p><b>A</b> Lysates from brains of 9.5 month old wild-type or Tg4510 mutant animals were immunostained for Stk25 and β-actin (upper panel). The average Stk25 signal normalized to β-actin from 3 separate samples from each genotype showed no statistically significant difference (lower panel). <b>B</b> Overexpression of Stk25-GFP did not lead to increased phosphorylation of co-transfected Tau in HeLa cells. Antibodies used for Westerns indicated at left: AT8 (phospho Ser202/Thr205 Tau) 5E2 total Tau. Sizes of expected Stk25-GFP and endogenous Stk25 are indicated at right.</p

Sequence comparison of exon I of the <i>APP</i> gene from C57BL/6 and BALB/c mice.

<p>The GCG repeated sequence (bold) begins 73 bp upstream of the initiator Met. There are 7 repeats in this region of the <i>APP</i> gene in BALB/c mice as compared to 4 in C57BL/6.</p

The Golgi extension and neuronal polarization phenotypes in <i>dab1</i> mutant neurons is dependent upon mouse strain background.

<p>The appearance of the Golgi apparatus was examined in 100 µm thick sections of the hippocampus at birth by immunostaining for GRASP65 and Ctip2 to identify pyramidal neurons. <b>A</b> In wild-type animals the Golgi apparatus extends several microns into the presumptive apical dendrite in the hippocampi, but in C57BL/6 <i>dab1−/−</i> mutants it is convoluted near the nucleus. The Golgi appears more elongated in the Balb/c <i>dab1−/−</i> mutant mice. Nucleus to Golgi distances were measured on isolated cells (insets). Arrowheads represent points used for measurements. <b>B</b> The nucleus to Golgi tip distances are greater for wild-type than <i>dab1−/−</i> mutants, and greater for BALB/c versus C57BL/6 <i>dab1−/−</i> mutants (*,** p<0.0001, Student's t test). <b>C</b> The number of multiple axon bearing neurons in <i>dab1−/−</i> mutant neurons is reduced on the BALB/c background as compared to the C57BL/6 background. In both cases, knocking down Stk25 leads to a further reduction in neurons with multiple axons and the development of neurons with no axons (* p<0.001 compared to the respective EV-control samples, ** p = 0.01 compared between EV-control samples, Student's t test) (Bar = 20 µm) Values for C57BL/6 samples have been published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031152#pone.0031152-Matsuki2" target="_blank">[34]</a> and are shown here for comparison to BALB/c samples.</p

Stk25 mRNA and protein levels are strain-dependent.

<p><b>A</b> Stk25 mRNA expression was 1.8-fold higher in C57BL/6 versus BALB/c strain in <i>dab1</i> −/− and wild-type mice by real-time PCR. <b>B</b> Western blot analysis of Stk25 protein levels showed higher levels of expression in <i>dab1</i> −/− and wild-type mutant animals on the C57BL/6 than the BALB/c strain. <b>C</b> Representative anti-Stk25 (upper panel) and anti-actin (lower panel) Western blots of hippocampal lysates.</p