Blockade of vascular endothelial growth factor receptor I (VEGF-RI), but not VEGF-RII, suppresses joint destruction in the K/BxN model of rheumatoid arthritis

It was recently shown that vascular endothelial growth factor (VEGF), a growth factor for endothelial cells, plays a pivotal role in rheumatoid arthritis. VEGF binds to specific receptors, known as VEGF-RI and VEGF-RII. We assessed the physical and histological effects of selective blockade of VEGF and its receptors in transgenic K/BxN mice, a model of rheumatoid arthritis very close to the human disease. Mice were treated with anti-mouse VEGF Ab, anti-mouse VEGF-RI and -RII Abs, and an inhibitor of VEGF-RI tyrosine kinase. Disease activity was monitored using clinical indexes and by histological examination. We found that synovial cells from arthritic joints express VEGF, VEGF-RI, and VEGF-RII. Treatment with anti-VEGF-RI strongly attenuated the disease throughout the study period, while anti-VEGF only transiently delayed disease onset. Treatment with anti-VEGF-RII had no effect. Anti-VEGF-RI reduced the intensity of clinical manifestations and, based on qualitative and semiquantitative histological analyses, prevented joint damage. Treatment with a VEGF-RI tyrosine kinase inhibitor almost abolished the disease. These results show that VEGF is a key factor in pannus development, acting through the VEGF-RI pathway. The observation that in vivo administration of specific inhibitors targeting the VEGF-RI pathway suppressed arthritis and prevented bone destruction opens up new possibilities for the treatment of rheumatoid arthritis.     

Expanding the scorpion toxin alpha-KTX 15 family with AmmTX3 from Androctonus mauretanicus.

A novel toxin, AmmTX3 (3823.5 Da), was isolated from the venom of the scorpion Androctonus mauretanicus. It showed 94% sequence homology with Aa1 from Androctonus australis and 91% with BmTX3 from Buthus martensi which, respectively, block A-type K+ current in cerebellum granular cells and striatum cultured neurons. Binding and displacement experiments using rat brain synaptosomes showed that AmmTX3 and Aa1 competed effectively with 125I-labelled sBmTX3 binding. They fully inhibited the 125I-labelled sBmTX3 binding (Ki values of 19.5 pm and 44.2 pm, respectively), demonstrating unambiguously that the three molecules shared the same target in rat brain. The specific binding parameters of 125I-labelled AmmTX3 for its site were determined at equilibrium (Kd = 66 pm, Bmax = 22 fmol per mg of protein). Finally, patch-clamp experiments on striatal neurons in culture demonstrated that AmmTX3 was able to inhibit the A-type K+ current (Ki = 131 nm).     

Identification of the Chi site of Haemophilus influenzae as several sequences related to the Escherichia coli Chi site

The Escherichia coli Chi site 5'-GCTGGTGG-3' modulates the activity of the powerful dsDNA exonuclease and helicase RecBCD. Genome sequence analyses revealed that Chi is frequent on the chromosome and oriented with respect to replication on the E . coli genome. Chi is also present much more frequently than predicted statistically for a random 8-mer sequence. Although it is assumed that Chi is ubiquitous, there is virtually no proof that its features are conserved in other microorganisms. We therefore identified and analysed the Chi sequence of an organism for which the full genome sequence was available, Haemophilus influenzae . The biological test we used is based on our finding that rolling circle plasmids provide a specific substrate for RecBCD analogues in different microorganisms. Unexpectedly, several related sequences, corresponding to 5'-GNTGGTGG-3' and 5'-G(G/C)TGGAGG-3', showed Chi activity. As in E . coli , the H . influenzae Chi sites are frequent on the genome, which is in keeping with the need for frequent Chi sites for dsDNA break repair of chromosomal DNA. Although statistically over-represented, this feature is less marked than that of the E . coli Chi site. In contrast to E . coli , the H . influenzae Chi motifs are only slightly oriented with respect to the replication strand. Thus, although Chi appears to have a highly conserved biological role in attenuating exonuclease activity, its sequence characteristics and statistical representation on the genome may differ according to the particular features of the host.     

Early water-deficit effects on seminal roots morphology in barley

In order to study seminal roots morphology in barley grown under different water treatments, experiments were carried out under glasshouse-controlled conditions. Eight genotypes were cultivated under four water treatments (100, 75, 50 and 25% of field capacity). Seminal root length and root-to-shoot dry matters' ratio were measured. Root volume was assessed at three soil depths. Results showed broad genotypic differences for all traits. The effect of low and moderate water deficit was slight. In contrast, the impact of severe water treatment was strongly marked on all traits. The impact of water deficit intensity on root traits at different soil depths is discussed.     

A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps

Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [μ-C4(cpdppz)₂(phen)₄Ru₂]⁴⁺, which consists of two Ru(phen)₂dppz²⁺ moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (K_d ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins.     

miR-126-3p is essential for CXCL12-induced angiogenesis

Atherosclerosis, in the ultimate stage of cardiovascular diseases, causes an obstruction of vessels leading to ischemia and finally to necrosis. To restore vascularization and tissue regeneration, stimulation of angiogenesis is necessary. Chemokines and microRNAs (miR) were studied as pro-angiogenic agents. We analysed the miR-126/CXCL12 axis and compared impacts of both miR-126-3p and miR-126-5p strands effects in CXCL12-induced angiogenesis. Indeed, the two strands of miR-126 were previously shown to be active but were never compared together in the same experimental conditions regarding their differential functions in angiogenesis. In this study, we analysed the 2D-angiogenesis and the migration assays in HUVEC in vitro and in rat's aortic rings ex vivo, both transfected with premiR-126-3p/-5p or antimiR-126-3p/-5p strands and stimulated with CXCL12. First, we showed that CXCL12 had pro-angiogenic effects in vitro and ex vivo associated with overexpression of miR-126-3p in HUVEC and rat's aortas. Second, we showed that 2D-angiogenesis and migration induced by CXCL12 was abolished in vitro and ex vivo after miR-126-3p inhibition. Finally, we observed that SPRED-1 (one of miR-126-3p targets) was inhibited after CXCL12 treatment in HUVEC leading to improvement of CXCL12 pro-angiogenic potential in vitro. Our results proved for the first time: 1-the role of CXCL12 in modulation of miR-126 expression; 2-the involvement of miR-126 in CXCL12 pro-angiogenic effects; 3-the involvement of SPRED-1 in angiogenesis induced by miR-126/CXCL12 axis.     

EMT impairs breast carcinoma cell susceptibility to CTL-mediated lysis through autophagy induction

Epithelial to mesenchymal transition (EMT) has become one of the most exciting fields in cancer biology. While its role in cancer cell invasion, metastasis and drug resistance is well established, the molecular basis of EMT-induced immune escape remains unknown. We recently reported that EMT coordinately regulates target cell recognition and sensitivity to specific lysis. In addition to the well-characterized role for EMT in tumor phenotypic change including a tumor-initiating cell phenotype, we provided evidence indicating that EMT-induced tumor cell resistance to cytotoxic T-lymphocytes (CTLs) also correlates with autophagy induction. Silencing of BECN1 in target cells that have gone through the EMT restored CTL susceptibility to CTL-induced lysis. Although EMT may represent a critical target for the development of novel immunotherapy approaches, a more detailed understanding of the inter-relationship between EMT and autophagy and their reciprocal regulation will be a key determinant in the rational approach to future tumor immunotherapy design.