Molecular basis for Jagged-1/Serrate ligand recognition by the Notch receptor.

We have mapped a Jagged/Serrate-binding site to specific residues within the 12th EGF domain of human and Drosophila Notch. Two critical residues, involved in a hydrophobic interaction, provide a ligand-binding platform and are adjacent to a Fringe-sensitive residue that modulates Notch activity. Our data suggest that small variations within the binding site fine-tune ligand specificity, which may explain the observed sequence heterogeneity in mammalian Notch paralogues, and should allow the development of paralogue-specific ligand-blocking antibodies. As a proof of principle, we have generated a Notch-1-specific monoclonal antibody that blocks binding, thus paving the way for antibody tools for research and therapeutic applications

Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia.The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity.S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or T-ALL-associated mutations lead to conformational changes of the NRR that permit metalloprotease cleavage

Systems biology reveals key tissue-specific metabolic and transcriptional signatures involved in the response of Medicago truncatula plant genotypes to salt stress

Salt stress is an important factor limiting plant productivity by affecting plant physiology and metabolism. To explore salt tolerance adaptive mechanisms in the model legume Medicago truncatula, we used three genotypes with differential salt-sensitivity: TN6.18 (highly sensitive), Jemalong A17 (moderately sensitive), and TN1.11 (tolerant). Cellular damage was monitored in roots and leaves 48 h after 200 mM NaCl treatment by measuring lipid peroxidation, nitric oxide, and hydrogen peroxide contents, further supported by leaf stomatal conductance and chlorophyll readings. The salt-tolerant genotype TN1.11 displayed the lowest level of oxidative damage, in contrast to the salt sensitive TN6.18, which showed the highest responses. Metabolite profiling was employed to explore the differential genotype-related responses to stress at the molecular level. The metabolic data in the salt tolerant TN1.11 roots revealed an accumulation of metabolites related to the raffinose pathway. To further investigate the sensitivity to salinity, global transcriptomic profiling using microarray analysis was carried out on the salt-stressed sensitive genotypes. In TN6.18, the transcriptomic analysis identified a lower expression of many genes related to stress signalling, not previously linked to salinity, and corresponding to the TIR-NBS-LRR gene class. Overall, this global approach contributes to gaining significant new insights into the complexity of stress adaptive mechanisms and to the identification of potential targets for crop improvement

Cytokine-induced killer cells express CD39, CD38, CD203a, CD73 ectoenzymes and P1 adenosinergic receptors

Cytokine-induced killer (CIK) cells, a heterogeneous T cell population obtained by in vitro differentiation of peripheral blood mononuclear cells (PBMC), represent a promising immunological approach in cancer. Numerous studies have explored the role of CD38, CD39, CD203a/PC-1, and CD73 in generating extracellular adenosine (ADO) and thus in shaping the tumor niche in favor of proliferation. The findings shown here reveal that CIK cells are able to produce extracellular ADO via traditional (CD39/CD73) and/or alternative (CD38/CD203a/CD73 or CD203a/CD73) pathways. Transcriptome analysis showed the mRNA expression of these molecules and their modulation during PBMC to CIK differentiation. When PBMC from normal subjects or cancer bearing patients were differentiated into CIK cells under normoxic conditions, CD38 and CD39 were greatly up-regulated while the number of CD203a, and CD73 positive cells underwent minor changes. Since hypoxic conditions are often found in tumors, we asked whether CD39, CD38, CD203a, and CD73 expressed by CIK cells were modulated by hypoxia. PBMC isolated from cancer patients and differentiated into CIK cells in hypoxic conditions did not show relevant changes in CD38, CD39, CD73, CD203a, and CD26. CIK cells also expressed A1, A2A, and A2B ADO receptors and they only underwent minor changes as a consequence of hypoxia. The present study sheds light on a previously unknown functional aspect of CIK cells, opening the possibility of pharmacologically modulated ADO-generating ectoezymes to improve CIK cells performance

Inflammation and tissue repair markers distinguish the nodular sclerosis and mixed cellularity subtypes of classical Hodgkin's lymphoma

Background: Classical Hodgkin's lymphoma (cHL), although a malignant disease, has many features in common with an inflammatory condition. The aim of this study was to establish the molecular characteristics of the two most common cHL subtypes, nodular sclerosis (NS) and mixed cellularity (MC), based on molecular profiling and immunohistochemistry, with special reference to the inflammatory microenvironment. Methods: We analysed 44 gene expression profiles of cHL whole tumour tissues, 25 cases of NS and 19 cases of MC, using Affymetrix chip technology and immunohistochemistry. Results: In the NS subtype, 152 genes showed a significantly higher expression, including genes involved in extracellular matrix (ECM) remodelling and ECM deposition similar to wound healing. Among these were SPARC, CTSK and COLI. Immunohistochemistry revealed that the NS-related genes were mainly expressed by macrophages and fibroblasts. Fifty-three genes had a higher expression in the MC subtype, including several inflammation-related genes, such as C1Qα, C1Qβ and CXCL9. In MC tissues, the C1Q subunits were mainly expressed by infiltrating macrophages. Conclusions and interpretations: We suggest that the identified subtype-specific genes could reflect different phases of wound healing. Our study underlines the potential function of infiltrating macrophages in shaping the cHL tumour microenvironment

Genome wide in silico SNP-tumor association analysis

BACKGROUND: Carcinogenesis occurs, at least in part, due to the accumulation of mutations in critical genes that control the mechanisms of cell proliferation, differentiation and death. Publicly accessible databases contain millions of expressed sequence tag (EST) and single nucleotide polymorphism (SNP) records, which have the potential to assist in the identification of SNPs overrepresented in tumor tissue. METHODS: An in silico SNP-tumor association study was performed utilizing tissue library and SNP information available in NCBI's dbEST (release 092002) and dbSNP (build 106). RESULTS: A total of 4865 SNPs were identified which were present at higher allele frequencies in tumor compared to normal tissues. A subset of 327 (6.7%) SNPs induce amino acid changes to the protein coding sequences. This approach identified several SNPs which have been previously associated with carcinogenesis, as well as a number of SNPs that now warrant further investigation CONCLUSIONS: This novel in silico approach can assist in prioritization of genes and SNPs in the effort to elucidate the genetic mechanisms underlying the development of cancer

Influence of Genetic Background and Tissue Types on Global DNA Methylation Patterns

Recent studies have shown a genetic influence on gene expression variation, chromatin, and DNA methylation. However, the effects of genetic background and tissue types on DNA methylation at the genome-wide level have not been characterized extensively. To study the effect of genetic background and tissue types on global DNA methylation, we performed DNA methylation analysis using the Affymetrix 500K SNP array on tumor, adjacent normal tissue, and blood DNA from 30 patients with esophageal squamous cell carcinoma (ESCC). The use of multiple tissues from 30 individuals allowed us to evaluate variation of DNA methylation states across tissues and individuals. Our results demonstrate that blood and esophageal tissues shared similar DNA methylation patterns within the same individual, suggesting an influence of genetic background on DNA methylation. Furthermore, we showed that tissue types are important contributors of DNA methylation states

Correction: S1PR1 drives a feedforward signalling loop to regulate BATF3 and the transcriptional programme of Hodgkin lymphoma cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype

Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling

Suppression of the Nrf2-Dependent Antioxidant Response by Glucocorticoids and 11β-HSD1-Mediated Glucocorticoid Activation in Hepatic Cells

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key transcription factor regulating a plethora of detoxifying enzymes and antioxidant genes involved in drug metabolism and defence against oxidative stress. The glucocorticoid receptor (GR) is a ligand-induced transcription factor involved in the regulation of energy supply for metabolic needs to cope with various stressors. GR activity is controlled by glucocorticoids, which are synthesized in the adrenal glands and regenerated mainly in the liver from inactive cortisone by 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1).; Using transfected HEK-293 cells and hepatic H4IIE cells we show that glucocorticoids, activated by 11β-HSD1 and acting through GR, suppress the Nrf2-dependent antioxidant response. The expression of the marker genes NQO1, HMOX1 and GST2A was suppressed upon treatment of 11β-HSD1 expressing cells with cortisone, an effect that was reversed by 11β-HSD1 inhibitors. Furthermore, our results demonstrate that elevated glucocorticoids lowered the ability of cells to detoxify H(2)O(2). Moreover, a comparison of gene expression in male and female rats revealed an opposite sexual dimorphism with an inverse relationship between 11β-HSD1 and Nrf2 target gene expression.; The results demonstrate a suppression of the cellular antioxidant defence capacity by glucocorticoids and suggest that elevated 11β-HSD1 activity may lead to impaired Nrf2-dependent antioxidant response. The gender-specific differences in hepatic expression levels of 11β-HSD1 and Nrf2 target genes and the impact of pharmacological inhibition of 11β-HSD1 on improving cellular capacity to cope with oxidative stress warrants further studies in vivo