Analysis of the Wnt/B-catenin/TCF4 pathway using SAGE,genome-wide microarray and promoter analysis: Identification of BRI3 and HSF2 as novel targets

The Wnt signaling pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. β-catenin, a cytoplasmic component, plays a major role in the transduction of canonical Wnt signaling. The aim of this study was to identify novel genes that are regulated by active β-catenin/TCF signaling in hepatocellular carcinoma-derived Huh7 cells with high (transfected) and low β-catenin/TCF activities. High TCF activity Huh7 cells led to earlier and larger tumor formation when xenografted into nude mice. SAGE (Serial Analysis of Gene Expression), genome-wide microarray and in silico promoter analysis were performed in parallel, to compare gene expression between low and high β-catenin/TCF activity clones, and also those that had been rescued from the xenograft tumors. SAGE and genome-wide microarray data were compared and contrasted. BRI3 and HSF2 were identified as novel targets of Wnt/β-catenin signaling after combined analysis and confirming experiments including qRT-PCR, ChIP, luciferase assay and lithium treatment.     

Structural characterization of an ATPase active F1-/V1 -ATPase (alpha3beta3EG) hybrid complex

Co-reconstitution of subunits E and G of the yeast V-ATPase and the alpha and beta subunits of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) resulted in an alpha(3)beta(3)EG hybrid complex showing 53% of the ATPase activity of TF(1). The alpha(3)beta(3)EG oligomer was characterized by electron microscopy. By processing 40,000 single particle projections, averaged two-dimensional projections at 1.2-2.4-nm resolution were obtained showing the hybrid complex in various positions. Difference mapping of top and side views of this complex with projections of the atomic model of the alpha(3)beta(3) subcomplex from TF(1) (Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., Sekimoto, Y., Kambara, M., Saika, K., Kagawa, Y., and Yoshida, M. (1997) Structure 5, 825-836) demonstrates that a seventh mass is located inside the shaft of the alpha(3)beta(3) barrel and extends out from the hexamer. Furthermore, difference mapping of the alpha(3)beta(3)EG oligomer with projections of the A(3)B(3)E and A(3)B(3)EC subcomplexes of the V(1) from Caloramator fervidus (Chaban, Y., Ubbink-Kok, T., Keegstra, W., Lolkema, J. S., and Boekema, E. J. (2002) EMBO Rep. 3, 982-987) shows that the mass inside the shaft is made up of subunit E, whereby subunit G was assigned to belong at least in part to the density of the protruding stalk. The formation of an active alpha(3)beta(3)EG hybrid complex indicates that the coupling subunit gamma inside the alpha(3)beta(3) oligomer of F(1) can be effectively replaced by subunit E of the V-ATPase. Our results have also demonstrated that the E and gamma subunits are structurally similar, despite the fact that their genes do not show significant homology.     

Zinc and copper status in acute pancreatitis: an experimental study

Metal ions are required as active components of several proteins, including pancreatic enzymes, and they can play important roles in the etiopathogenesis of acute pancreatitis. In the present study, we measured the concentrations of zinc (Zn) and copper (Cu) in both serum and pancreatic tissue, as markers of trace element status in an experimental acute pancreatitis model. Twenty-four male Wistar rats were divided into two groups: the experimental group (N=24) and the control group (N=10). Acute pancreatitis was induced by injection of 48% ethyl alcohol into the common biliary duct. The animals were sacrificed 24 h later to detect the concentrations of Zn and Cu. There was no significant difference in tissue Zn and Cu concentrations between control and experimental groups (p<0.05). However, in the acute pancreatitis group, serum Zn and Culevels were very significantly lower (p<0.001 and p<0.0001, respectively). In conclusuion, these findings suggested that altered mineral metabolism in serum and pancreatic tissue may have contributed to the pathophysiology of acute pancreatitis.     

The effect of various liposome formulations on insulin penetration across Caco-2 cell monolayer

The aim of the study was to determine the penetration properties of various insulin containing liposome formulations through Caco-2 cell monolayer and to compare the in vitro test results with in vivo tests. The effect of sodium taurocholate as a penetration enhancer when it was added to the liposome formulation was also investigated. In vitro permeation experiments were performed in diffusion cells with the Caco-2 cell monolayer used as the membrane. Permeability values of various insulin containing liposome formulations through Caco-2 cells were determined (log k(insulin-solution) = -2.217 +/- 0.0723 cm.h(-1), log k(insulin-liposome) = -2.141 +/- 0.0625 cm.h(-1), log k(insulin-sodium tauroholate liposome)= -1.952 +/- 0.0623 cm.h(-1)). In vivo tests were performed in mice. Formulations were administered orally and blood glucose levels were determined and penetrations were compared with the Caco-2 cell experiment results. In conclusion, the permeability of insulin was increased across Caco-2 cell monolayer when the liposome sodium taurocholate (NaTC) formulation was used. The oral administration of insulin and NaTC incorporated liposomes significantly decreased blood glucose levels. Furthermore, it was shown that a high in vitro/in vivo correlation was observed using the Caco-2 cell monolayer model.     

Protein Interaction Analysis of Senataxin and the ALS4 L389S Mutant Yields Insights into Senataxin Post-Translational Modification and Uncovers Mutant-Specific Binding with a Brain Cytoplasmic RNA-Encoded Peptide

Senataxin is a large 303 kDa protein linked to neuron survival, as recessive mutations cause Ataxia with Oculomotor Apraxia type 2 (AOA2), and dominant mutations cause amyotrophic lateral sclerosis type 4 (ALS4). Senataxin contains an amino-terminal protein-interaction domain and a carboxy-terminal DNA/RNA helicase domain. In this study, we focused upon the common ALS4 mutation, L389S, by performing yeast two-hybrid screens of a human brain expression library with control senataxin or L389S senataxin as bait. Interacting clones identified from the two screens were collated, and redundant hits and false positives subtracted to yield a set of 13 protein interactors. Among these hits, we discovered a highly specific and reproducible interaction of L389S senataxin with a peptide encoded by the antisense sequence of a brain-specific non-coding RNA, known as BCYRN1. We further found that L389S senataxin interacts with other proteins containing regions of conserved homology with the BCYRN1 reverse complement-encoded peptide, suggesting that such aberrant protein interactions may contribute to L389S ALS4 disease pathogenesis. As the yeast two-hybrid screen also demonstrated senataxin self-association, we confirmed senataxin dimerization via its amino-terminal binding domain and determined that the L389S mutation does not abrogate senataxin self-association. Finally, based upon detection of interactions between senataxin and ubiquitin–SUMO pathway modification enzymes, we examined senataxin for the presence of ubiquitin and SUMO monomers, and observed this post-translational modification. Our senataxin protein interaction study reveals a number of features of senataxin biology that shed light on senataxin normal function and likely on senataxin molecular pathology in ALS4.     

The effects of chronic aerobic and anaerobic exercises n lymphocyte subgroups

Exercise is the strongest stress to which the body is ever exposed. The body responds to this stress through a set of physiological changes in its metabolic, hormonal and immunological systems. In this study, responses of the immune system to the long-term aerobic and anaerobic exercises have been investigated. Twenty-four sedentary male university students and officers participated in this study. Subjects were divided into two groups, each consisting of twelve people. Group-1 (age: 25.67 +/- 3.79 years, height: 174.83 +/- 5.15 cm, body mass: 72.17 +/- 8.05 kg) and Group-2 (age: 24.83 +/- 2.89 years, height: 175.3 +/- 6.68 cm, body mass: 70.67 +/- 6.15 kg). After physical examinations of the two groups, resting ECG, respiratory function tests and metabolic tests with the use of the breath by breath method were completed, and anerobic heart rates at the threshold level were determined. The first group was subjected to exercise using Monark ergometry cycles at a heart rate 10% below the threshold level for 8 weeks, 3 days a week, 30 min a day. The second group exercised at a heart rate 10% above the threshold level for 8 weeks, 3 days a week, 20 min a day. Heart rates were checked with the Polar Test during exercises. Pre-exercise (Ep) venous blood samples were taken from each group before their 1st and 24th exercises. Hb (gr), Hct (%), erythrocyte (x10(6)/microl), leukocyte (x10(6)/microl), leukocyte subpopulations (neutrophil, lymphocyte, monocyte, eosinophil, basophil %) and thrombocyte (x10(6)/microl) values were determined. CD3, CD4, CD8, CD19 and CD56 values were determined by Flow Cytometry method using monoclonal antibodies. The chronic effects of exercise were examined through a comparison of Ep blood samples at the 1st exercise with Ep blood samples at the 24th exercise. While the increase in the total leukocyte number was significant (p<0.05) in the first group, increase in the second group was found to be non-significant. When percentiles of leukocyte subpopulations were taken into consideration, changes in the first and second group were found to be non-significant. When lymphocyte subgroups were examined; in the first group a decrease in CD3 and CD4 percentiles to 7% and 12%, respectively (p<0.05) and a 65% increase (p<0.01) in the CD56 value were observed. In the second group a decrease in CD3 and CD4 percentiles to 13% and 17%, respectively (p<0.05) and a 73% increase (p<0.01) in the CD56 value were observed. The Sample-t Test and The Wilcoxon Test were used for statistical analysis.