The genetic variation of RELN expression in schizophrenia and bipolar disorder

Reelin plays an important role in the development and function of the brain and has been linked to different neuropsychiatric diseases. To further clarify the connection between reelin and psychiatric disorders, we studied the factors that influence the expression of reelin gene (RELN) and its different isoforms. We examined the total expression of RELN, allelic expression, and two alternative RELN isoforms in postmortem brain samples from patients with schizophrenia and bipolar disorder, as well as unaffected controls. We did not find a significant reduction in the total expression of RELN in schizophrenia or bipolar disorder. However, we did find a significant reduction of the proportion of the short RELN isoform, missing the C-terminal region in bipolar disorder, and imbalance in the allelic expression of RELN in schizophrenia. In addition, we tested the association between variation in RELN expression and rs7341475, an intronic SNP that was found to be associated with schizophrenia in women. We did not find an association between rs7341474 and the total expression of RELN either in women or in the entire sample. However, we observed a nominally significant effect of genotype-by-sex interaction on the variation in microexon skipping. Women with the risk genotype of rs7341475 (GG) had a higher proportion of microexon skipping, which is the isoform predominant in tissues outside the brain, while men had the opposite trend. Finally, we tested 83 SNPs in the gene region for association with expression variation of RELN, but none were significant. Our study further supports the connection between RELN dysfunction and psychiatric disorders, and provides a possible functional role for a schizophrenia associated SNP. Nevertheless, the positive associations observed in this study needs further replication as it may have implications for understanding the biological causes of schizophrenia and bipolar disorder.     

Modest Attenuation of HIV-1 Vpu Alleles Derived from Elite Controller Plasma

In the absence of antiretroviral therapy, infection with human immunodeficiency virus type 1 (HIV-1) can typically not be controlled by the infected host and results in the development of acquired immunodeficiency. In rare cases, however, patients spontaneously control HIV-1 replication. Mechanisms by which such elite controllers (ECs) achieve control of HIV-1 replication include particularly efficient immune responses as well as reduced fitness of the specific virus strains. To address whether polymorphisms in the accessory HIV-1 protein Vpu are associated with EC status we functionally analyzed a panel of plasma-derived vpu alleles from 15 EC and 16 chronic progressor (CP) patients. Antagonism of the HIV particle release restriction by the intrinsic immunity factor CD317/tetherin was well conserved among EC and CP Vpu alleles, underscoring the selective advantage of this Vpu function in HIV-1 infected individuals. In contrast, interference with CD317/tetherin induced NF-κB activation was little conserved in both groups. EC Vpus more frequently displayed reduced ability to downregulate cell surface levels of CD4 and MHC class I (MHC-I) molecules as well as of the NK cell ligand NTB-A. Polymorphisms potentially associated with high affinity interactions of the inhibitory killer immunoglobulin-like receptor (KIR) KIR2DL2 were significantly enriched among EC Vpus but did not account for these functional differences. Together these results suggest that in a subgroup of EC patients, some Vpu functions are modestly reduced, possibly as a result of host selection.     

Virulence and in planta movement of Xanthomonas hortorum pv. pelargonii are affected by the diffusible signal factor (DSF)‐dependent quorum sensing system

Xanthomonas hortorum pv. pelargonii (Xhp), the causal agent of bacterial blight in pelargonium, is the most threatening bacterial disease of this ornamental worldwide. To gain an insight into the regulation of virulence in Xhp, we have disrupted the quorum sensing (QS) genes, which mediate the biosynthesis and sensing of the diffusible signal factor (DSF). Mutations in rpfF (encoding the DSF synthase) and rpfC (encoding the histidine sensor kinase of the two‐component system RfpC/RpfG) and overexpression of rpfF showed a significant reduction in incidence and severity of the disease on pelargonium. Confocal laser scanning microscopy images of inoculated plants with a green fluorescent protein (GFP)‐labelled wild‐type strain showed that the pathogen is homogeneously dispersed in the lumen of xylem vessels, reaching the apex and invading the intercellular spaces of the leaf mesophyll tissue within 21 days. In contrast, the rpfF and rpfC knockout mutants, as well as the rpfF‐overexpressing strain, remained confined to the vicinity of the inoculation site. The rpfF and rpfC mutants formed large incoherent aggregates in the xylem vessels that might interfere with upward movement of the bacterium within the plant. Both mutants also formed extended aggregates under in vitro conditions, whereas the wild‐type strain formed microcolonies. Expression levels of putative virulence genes in planta were substantially reduced within 48 h after inoculation with the QS mutants when compared with the wild‐type. The results presented indicate that an optimal DSF concentration is crucial for successful colonization and virulence of Xhp in pelargonium.     

Cell Surface-Associated Anti-MUC1-Derived Signal Peptide Antibodies: Implications for Cancer Diagnostics and Therapy

The MUC1 tumor associated antigen is highly expressed on a range of tumors. Its broad distribution on primary tumors and metastases renders it an attractive target for immunotherapy. After synthesis MUC1 is cleaved, yielding a large soluble extracellular alpha subunit containing the tandem repeats array (TRA) domain specifically bound, via non-covalent interaction, to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Thus far, inconclusive efficacy has been reported for anti-MUC1 antibodies directed against the soluble alpha subunit. Targeting the cell bound beta subunit, may bypass limitations posed by circulating TRA domains. MUC1’s signal peptide (SP) domain promiscuously binds multiple MHC class II and Class I alleles, which upon vaccination, generated robust T-cell immunity against MUC1-positive tumors. This is a first demonstration of non-MHC associated, MUC1 specific, cell surfaces presence for MUC1 SP domain. Polyclonal and monoclonal antibodies generated against MUC1 SP domain specifically bind a large variety of MUC1-positive human solid and haematological tumor cell lines; MUC1-positive bone marrow derived plasma cells obtained from multiple myeloma (MM)-patients, but not MUC1 negative tumors cells, and normal naive primary blood and epithelial cells. Membranal MUC1 SP appears mainly as an independent entity but also co-localized with the full MUC1 molecule. MUC1-SP specific binding in BM-derived plasma cells can assist in selecting patients to be treated with anti-MUC1 SP therapeutic vaccine, ImMucin. A therapeutic potential of the anti-MUC1 SP antibodies was suggested by their ability to support of complement-mediated lysis of MUC1-positive tumor cells but not MUC1 negative tumor cells and normal naive primary epithelial cells. These findings suggest a novel cell surface presence of MUC1 SP domain, a potential therapeutic benefit for anti-MUC1 SP antibodies in MUC1-positive tumors and a selection tool for MM patients to be treated with the anti-MUC1 SP vaccine, ImMucin.     

Growth enhancement of transgenic poplar plants by overexpression of Arabidopsis thaliana endo-1,4–β-glucanase (cel1)

Poplar (Populus tremula) plants which had been transformed with Arabidopsis thaliana cel1 cDNA and successfully over-expressed the gene, exhibited significant phenotypic alterations which included taller plants, larger leaves, increased stem diameter, wood volume index, dry weight and a higher percentage of cellulose and hemicellulose, compared to the wild-type plants. Transgenic A. thaliana plants over-expressing A. thaliana cel1 exhibited similar levels of cel1 mRNA in the elongation zone of the flowering stem and higher levels in mature leaves when compared with wild-type plants. CEL1 protein levels in the elongation zone of the flowering stem of transgenic plants were similar or slightly higher compared to that of the wild-type plants, whereas mature leaves of transgenic plants contained a higher level of CEL1. These data indicate that in elongating zone of Arabidopsis, CEL1 level is tightly regulated. In contrast to transgenic poplar over-expressing the A. thaliana cel1, no phenotypic difference was found between A. thaliana transgenic and wild-type plants.     

AUG sequences are required to sustain nonsense-codon-mediated suppression of splicing

More than 90% of human genes are rich in intronic latent 5′ splice sites whose utilization in pre-mRNA splicing would introduce in-frame stop codons into the resultant mRNAs. We have therefore hypothesized that suppression of splicing (SOS) at latent 5′ splice sites regulates alternative 5′ splice site selection in a way that prevents the production of toxic nonsense mRNAs and verified this idea by showing that the removal of such in-frame stop codons is sufficient to activate latent splicing. Splicing control by SOS requires recognition of the mRNA reading frame, presumably recognizing the start codon sequence. Here we show that AUG sequences are indeed essential for SOS. Although protein translation does not seem to be required for SOS, the first AUG is shown here to be necessary but not sufficient. We further show that latent splicing can be elicited upon treatment with pactamycin—a drug known to block translation by its ability to recognize an RNA fold—but not by treatment with other drugs that inhibit translation through other mechanisms. The effect of pactamycin on SOS is dependent neither on steady-state translation nor on the pioneer round of translation. This effect is found for both transfected and endogenous genes, indicating that SOS is a natural mechanism.     

Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2.     

Trogocytosis is a gateway to characterize functional diversity in melanoma-specific CD8+ T cell clones

Trogocytosis, the transfer of membrane patches from target to immune effector cells, is a signature of tumor-T cell interaction. In this study, we used the trogocytosis phenomenon to study functional diversity within tumor-specific T cell clones with identical TCR specificity. MART-1(26-35)-specific CD8 T cell clones, which differed in their trogocytosis capacity (low [2D11], intermediate [2G1], high [2E2]), were generated from melanoma patients. Functional evaluation of the clones showed that the percentage of trogocytosis-capable T cells closely paralleled each clone's IFN-γ and TNF-α production, lysosome degranulation, and lysis of peptide-pulsed targets and unmodified melanoma. The highly cytotoxic 2E2 clone displayed the highest TCR peptide binding affinity, whereas the low-activity 2D11 clone showed TCR binding to peptide-MHC in a CD8-dependent manner. TCR analysis revealed Vβ16 for clones 2E2 and 2G1 and Vβ14 for 2D11. When peptide-affinity differences were bypassed by nonspecific TCR stimulation, clones 2E2 and 2D11 still manifested distinctive signaling patterns. The high-activity 2E2 clone displayed prolonged phosphorylation of ribosomal protein S6, an integrator of MAPK and AKT activation, whereas the low-activity 2D11 clone generated shorter and weaker phosphorylation. Screening the two clones with identical TCR Vβ by immunoreceptor array showed higher phosphorylation of NK, T, and B cell Ag (NTB-A), a SLAM family homophilic receptor, in clone 2E2 compared with 2G1. Specific blocking of NTB-A on APCs markedly reduced cytokine production by CD8 lymphocytes, pointing to a possible contribution of NTB-A costimulation to T cell functional diversity. This finding identifies NTB-A as a potential target for improving anti-cancer immunotherapy.