A peptide corresponding to the C-terminal region of pleiotrophin inhibits angiogenesis in vivo and in vitro

Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both α(ν) β(3) integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN(112-136) ) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN(112-136) inhibited binding of PTN to α(ν) β(3) integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β(3) . Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing α(ν) β(3) -selective antibody. PTN(112-136) did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β(3) Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPβ/ζ by siRNA or by c-src inhibition, suggesting that PTN(112-136) may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN(112-136) was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123-132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN(112-136) induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to α(ν) β(3) integrin.     

Dimensionality reduction of free energy profiles of benzene in silicalite-1: calculation of diffusion coefficients using transition state theory

This study describes the development and application of a new computational methodology for calculating the self-diffusivity of sorbate molecules strongly confined within shape-selective nanoporous materials. An umbrella sampling strategy, employing repulsive walls to confine the sorbate within specific regions of the pore space, is invoked to extract free energy profiles with respect to the sorbate degrees of freedom. Based on these profiles, it is shown how the multidimensional problem of translational diffusion of benzene in flexible silicalite-1 can be reduced first to a six-dimensional problem, then to a three-dimensional (3D) problem and finally, to a 1D problem. A 3D free energy distribution is accumulated as a function of the benzene centre of mass position and ultimately reduced to a set of 1D profiles for the benzene centre of mass along the pore axes. From these profiles, the rate constants for jumps executed by benzene between sorption sites are calculated using transition state theory; from the latter rate constants, the low-occupancy self-diffusivity is obtained using the MESoRReD method [Kolokathis PD, Theodorou DN. On solving the master equation in spatially periodic systems. J Chem Phys. 2012;137:034112]. The activation energy for diffusion and preferred orientations in the various sorption states in silicalite are in very favourable agreement with available experimental measurements.     

MKK7 and ARF

Sensing, integrating, and processing of stressogenic signals must be followed by accurate differential response(s) for a cell to survive and avoid malignant transformation. The DNA damage response (DDR) pathway is vital in this process, as it deals with genotoxic/oncogenic insults, having p53 as a nodal effector that performs most of the above tasks. Accumulating data reveal that other pathways are also involved in the same or similar processes, conveying also to p53. Emerging questions are if, how, and when these additional pathways communicate with the DDR axis. Two such stress response pathways, involving the MKK7 stress-activated protein kinase (SAPK) and ARF, have been shown to be interlocked with the ATM/ATR-regulated DDR axis in a highly ordered manner. This creates a new landscape in the DDR orchestrated response to genotoxic/oncogenic insults that is currently discussed.     

Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin''s action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions.     

The influence of storage on the “chemical age” of red wines

Storage conditions and duration have a considerable influence on wine quality. Optimum temperature and humidity conditions may improve wine quality through ageing, while incorrect or excessively long storage leads to negative results. In order to evaluate the global effects of storage on red wine composition, 20 Sangiovese wines were stored in two different conditions (cellar or house) for a period of 2 years and analysed every 6 months. Untargeted LC–MS analysis showed various putative markers for the type and length of conservation (i.e. pigments, flavanols, pantothenic acid etc.), while targeted LC–MS confirmed and expanded these results within specific metabolic groups. The results of multivariate analysis showed that wines stored in the cellar changed little even after 2 years of storage, while wines stored in typical domestic conditions (house) developed approximately four times faster, reaching a composition similar to wines stored in the cellar for 2 years after just 6 months. The formation of several monosulfonated flavanols during domestic ageing provided the first evidence in wine of a reaction between wine tannins—both catechins and proanthocyanidins—and the exogenous antioxidant bisulfite. Moreover, ageing in domestic conditions appeared to induce an accelerated decrease in wine pigments, while specifically promoting the formation of pinotin A-like pigments and the hydrolysis of flavonol glycosides.     

Disruption of Clathrin‐Mediated Trafficking Causes Centrosome Overduplication and Senescence

The Hsc70 cochaperone, G cyclin‐associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin‐dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin‐mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin‐dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication.