Ebselen inhibits NO-induced apoptosis of differentiated PC12 cells via inhibition of ASK1-p38 MAPK-p53 and JNK signaling and activation of p44/42 MAPK and Bcl-2

Ebselen, a selenium-containing heterocyclic compound, prevents ischemia-induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO-induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO-induced phosphatidyl Serine exposure, cytochrome c release, and caspase-3 activation by ebselen. Analysis of key apoptotic regulators during NO-induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling-regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal protein kinase (JNK). Moreover, ebselen inhibits NO-induced p53 phosphorylation at Ser15 and c-Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol-redox-dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl-2 in SNP-treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.     

Association of vitamin D receptor polymorphisms with osteoporosis in mexican postmenopausal women

It has been reported that Vitamin D receptor polymorphisms are associated with osteoporosis, particularly those demonstrated by the BsmI and FokI restriction enzymes. Herein we report the results of a case-control study performed in postmenopausal Mexican women. We studied 65 osteoporotic women (< or = -2.5 SD bone mineral density [BMD] of young normal females) and 57 controls (over 90% > or = -1.5 SD BMD of young normal females. Restriction enzymes BsmI and FokI were used to identify polymorphisms. Odds ratios and their 95% confidence intervals were calculated, and analysis was performed controlling for age as a covariate. The BsmI genotypes revealed a higher frequency of the bb genotype in cases than in controls, contradicting much of the literature that suggests this genotype protects females against osteoporosis. Regarding the FokI genotypes, we were unable to confirm that the FF genotype has a protective effect against osteoporosis. The inconsistencies found in the literature and the results obtained in the present work suggest to us that other genetic and nongenetic factors are involved in the occurrence of osteoporosis, confounding the results of the possible association of osteoporosis and VDR polymorphisms.     

Role for the BRCA1 C-terminal repeats (BRCT) protein 53BP1 in maintaining genomic stability

p53-binding protein-1 (53BP1) is phosphorylated in response to DNA damage and rapidly relocalizes to presumptive sites of DNA damage along with Mre11 and the phosphorylated histone 2A variant, gamma-H2AX. 53BP1 associates with the BRCA1 tumor suppressor, and knock-down experiments with small interfering RNA have revealed a role for the protein in the checkpoint response to DNA damage. By generating mice defective in m53BP1 (m53BP1(tr/tr)), we have created an animal model to further explore its biochemical and genetic roles in vivo. We find that m53BP1(tr/tr) animals are growth-retarded and show various immune deficiencies including a specific reduction in thymus size and T cell count. Consistent with a role in responding to DNA damage, we find that m53BP1(tr/tr) mice are sensitive to ionizing radiation (gamma-IR), and cells from these animals exhibit chromosomal abnormalities consistent with defects in DNA repair. Thus, 53BP1 is a critical element in the DNA damage response and plays an integral role in maintaining genomic stability.     

Prediction of the membrane-spanning beta-strands of the major outer membrane protein of Chlamydia

There is preliminary experimental evidence indicating that the major outer-membrane protein (MOMP) of Chlamydia is a porin. We tested this hypothesis for the MOMP of the mouse pneumonitis serovar of Chlamydia trachomatis using two secondary structure prediction methods. First, an algorithm that calculates the mean hydrophobicity of one side of putative beta-strands predicted the positions of 16 transmembrane segments, a structure common to known porins. Second, outer loops typical of porins were assigned using an artificial neural network trained to predict the topology of bacterial outer-membrane proteins with a predominance of beta-strands. A topology model based on these results locates the four variable domains (VDs) of the MOMP on the outer loops and the five constant domains on beta-strands and the periplasmic turns. This model is consistent with genetic analysis and immunological and biochemical data that indicate the VDs are surface exposed. Furthermore, it shows significant homology with the consensus porin model of the program FORESST, which contrasts a proposed secondary structure against a data set of 349 proteins of known structure. Analysis of the MOMP of other chlamydial species corroborated our predicted model.     

Antiviral immune responses in the absence of organized lymphoid T cell zones in plt/plt mice

The paucity of lymph node (LN) T cells (plt) mutation in mice results in strongly reduced T cell numbers in LNs and homing defects of both dendritic cells (DCs) and naive T cells. In this study, we investigated the functional significance of the plt phenotype for the generation of antiviral immune responses against cytopathic and noncytopathic viruses. We found that DC-CD8(+) T cell contacts and the initial priming of virus-specific T cells in plt/plt mice occurred mainly in the marginal zone of the spleen and in the superficial cortex of LNs. The magnitude of the initial response and the maintenance of protective memory responses in plt/plt mice was only slightly reduced compared with plt/+ controls. Furthermore, plt/plt mice mounted rapid neutralizing antiviral B cell responses and displayed normal Ig class switch. Our data indicate that the defective homing of DCs and naive T cells resulting from the plt/plt mutation results in a small, but not significant, effect on the induction of protective antiviral T and B cell immunity. Overall, we conclude that the spatial organization of secondary lymphoid T cell zones via the CCR7-CC chemokine ligand 19/CC chemokine ligand 21 pathway is not an absolute requirement for the initial priming and the maintenance of protective antiviral T and B cell responses.     

Delayed striate cortical activation during spatial attention

Recordings of event-related potentials (ERPs) and event-related magnetic fields (ERMFs) were combined with functional magnetic resonance imaging (fMRI) to study visual cortical activity in humans during spatial attention. While subjects attended selectively to stimulus arrays in one visual field, fMRI revealed stimulus-related activations in the contralateral primary visual cortex and in multiple extrastriate areas. ERP and ERMF recordings showed that attention did not affect the initial evoked response at 60-90 ms poststimulus that was localized to primary cortex, but a similarly localized late response at 140-250 ms was enhanced to attended stimuli. These findings provide evidence that the primary visual cortex participates in the selective processing of attended stimuli by means of delayed feedback from higher visual-cortical areas.     

Translocation mechanism of long sugar chains across the maltoporin membrane channel

Maltoporin allows permeation of long maltodextrin chains. It tightly binds the amphiphilic sugar, offering both hydrophobic interactions with a helical lane of aromatic residues and H bonds with ionic side chains. The minimum-energy path of maltohexaose translocation is obtained by the conjugate peak refinement method, which optimizes a continuous string of conformers without applying constraints. This reveals that the protein is passive while the sugar glides screw-like along the aromatic lane. Near instant switching of sugar hydroxyl H bond partners results in two small energy barriers (of approximately 4 kcal/mol each) during register shift by one glucosyl unit, in agreement with a kinetic analysis of experimental dissociation rates for varying sugar chain lengths. Thus, maltoporin functions like an efficient translocation "enzyme," and the slow rate of the register shift (approximately 1/ms) is due to high collisional friction.     

Parathyroid hormone effects on signaling pathways in endothelial cells vary with peptide concentration

We have previously reported that parathyroid hormone (PTH) has specific effects on a human umbilical vein endothelial cell line. Further studies were performed to characterize the signaling cascades initiated by PTH. We report that PTH induced the appearance of voltage sensitive calcium channels. Furthermore, PTH increased ceramide but not diacylglycerol content. Since elevations in [Ca(2+)](i) and phospholipid turnover are signals for the activation of protein kinase C (PKC), the cells were screened for PKC isoforms. PTH induced a redistribution of the PKCepsilon to the particulate fractions of cell homogenates. In summary, PTH induced PKC translocation through a calcium-phospholipid pathway in an endothelial cell line.     

Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells

Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-gamma secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.