Interaction Between the a3 Region of Factor VIII and the TIL'E’ Domains of the von Willebrand Factor

The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL’E’ domains of VWF. Binding between the FVIII a3 region and VWF TIL’E’ was here observed using NMR spectroscopy, where chemical shift changes were localized to two β-sheet regions on the edge of TIL’E’ upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL’E’ as well as mutants of TIL’E’, which further highlights the importance of the β-sheet region of TIL’E’ for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the β-sheet region of TIL’E’ is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex

Retinal glycoprotein enrichment by concanavalin a enabled identification of novel membrane autoantigen synaptotagmin-1 in equine recurrent uveitis.

Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology

Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide

We investigated toxicity of 2-3 layered >1 μm sized graphene oxide (GO) and reduced graphene oxide (rGO) in mice following single intratracheal exposure with respect to pulmonary inflammation, acute phase response (biomarker for risk of cardiovascular disease) and genotoxicity. In addition, we assessed exposure levels of particulate matter emitted during production of graphene in a clean room and in a normal industrial environment using chemical vapour deposition. Toxicity was evaluated at day 1, 3, 28 and 90 days (18, 54 and 162 μg/mouse), except for GO exposed mice at day 28 and 90 where only the lowest dose was evaluated. GO induced a strong acute inflammatory response together with a pulmonary (Serum-Amyloid A, Saa3) and hepatic (Saa1) acute phase response. rGO induced less acute, but a constant and prolonged inflammation up to day 90. Lung histopathology showed particle agglomerates at day 90 without signs of fibrosis. In addition, DNA damage in BAL cells was observed across time points and doses for both GO and rGO. In conclusion, pulmonary exposure to GO and rGO induced inflammation, acute phase response and genotoxicity but no fibrosis

Zap-Pano: a photocaged prodrug of the KDAC inhibitor panobinostat

We report the synthesis and biological evaluation of a light-activated (caged) prodrug of the KDAC inhibitor panobinostat (Zap-Pano). We demonstrate that addition of the 4,5-dimethoxy-2-nitrobenzyl group to the hydroxamic acid oxygen results in an inactive prodrug. In two cancer cell lines we show that photolysis of this compound releases panobinostat and an unexpected carboxamide analogue of panobinostat. Photolysis of Zap-Pano causes an increase in H3K9Ac and H3K18Ac, consistent with KDAC inhibition, in an oesophageal cancer cell line (OE21). Irradiation of OE21 cells in the presence of Zap-Pano results in apoptotic cell death. This compound is a useful research tool, allowing spatial and temporal control over release of panobinostat

Ethnic differences in cardiovascular risk in rheumatic disease: focus on Asians

Rheumatic diseases are associated with high cardiovascular morbidity and mortality. Considerable differences exist in the frequency of cardiovascular disease (CVD) risk factors and events among people of different ethnic origins, but little is known of the ethnic variations in the relative distribution of CVD risk factors and the degree of atherosclerosis in patients with rheumatic diseases. Understanding this variation will provide insight into the underlying pathogenesis of CVD in patients with rheumatic diseases, and aid in future studies of the detection and management of this complication. In general, although Asian patients seem to have fewer background CVD risk factors and are less affected by metabolic syndrome (MetS) than their non-Asian counterparts, those with rheumatic disease are equally as susceptible to CVD. Furthermore, it seems that systemic inflammation and mechanisms that do not involve conventional CVD risk factors and MetS have an important role in the development of atherosclerosis in patients with rheumatic diseases. Here we examine the frequency of conventional CVD risk factors and the prevalence of MetS in both Asian and non-Asian patients with selected rheumatic diseases. We also discuss the burden of CVD, as evaluated using various surrogate markers in these patients, and their overall CVD mortality rate. © 2011 Macmillan Publishers Limited. All rights reserved.postprin