Abstract-Vascular endothelial (VE)-cadherin is the major adhesion molecule of endothelial adherens junctions. It plays an essential role in controlling endothelial permeability, vascular integrity, leukocyte transmigration, and angiogenesis. Elevated levels of soluble VE-cadherin are associated with diseases like coronary atherosclerosis. Previous data showed that the extracellular domain of VE-cadherin is released by an unknown metalloprotease activity during apoptosis. In this study, we used gain-of-function analyses, inhibitor studies, and RNA interference experiments to analyze the proteolytic release of VE-cadherin in human umbilical vein endothelial cells (HUVECs). We found that VE-cadherin is specifically cleaved by the disintegrin and metalloprotease ADAM10 in its ectodomain, releasing a soluble fragment and generating a carboxyl-terminal membrane-bound stub, which is a substrate for a subsequent ␥-secretase cleavage. This ADAM10-mediated proteolysis could be induced by Ca 2ϩ influx and staurosporine treatment, indicating that ADAM10-mediated VE-cadherin cleavage contributes to the dissolution of adherens junctions during endothelial cell activation and apoptosis, respectively. In contrast, protein kinase C activation or inhibition did not modulate VE-cadherin processing. Increased ADAM10 expression was functionally associated with an increase in endothelial permeability. Remarkably, our data indicate that ADAM10 activity also contributes to the thrombin-induced decrease of endothelial cell-cell adhesion. Moreover, knockdown of ADAM10 in HUVECs as well as in T cells by small interfering RNA impaired T-cell transmigration. Taken together, our data identify ADAM10 as a novel regulator of vascular permeability and demonstrate a hitherto unknown function of ADAM10 in the regulation of VE-cadherindependent endothelial cell functions and leukocyte transendothelial migration. Key Words: endothelium Ⅲ metalloprotease Ⅲ endothelial permeability Ⅲ VE-cadherin T he endothelium represents the major physical barrier in the extravasation of blood components and leukocytes to the surrounding tissue. Impairment of this barrier by inflammatory mediators leads to disintegration of endothelial junctions and an increase in permeability and formation of edema. However, the molecular mechanisms regulating the cohesion of endothelial junctions are still poorly understood. Vascular endothelial (VE)-cadherin, which represents the major component of endothelial adherens junctions, is a crucial determinant of vascular integrity. This 130-kDa cell surface glycoprotein that mediates homotypic Ca 2ϩ -dependent cell adhesion also plays a central role in vasculogenesis, angiogenesis, and the regulation of macromolecular permeability. 1 Several inflammatory mediators and vasoactive factors, including thrombin, 2 have the potential to disrupt the VEcadherin complex and to increase vascular permeability providing the basis of edematous tissue injury in many diseases states including sepsis, ischemia/reperfusion, and acute respiratory dist...

CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.

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An ideal system to investigate individual determinants of the replication process of (+)-strand RNA viruses is a cell-free extract that supports viral protein and RNA synthesis in a synchronized manner. Here, we applied a translation/replication system based on cytoplasmic extracts of Nicotiana tabacum cells to Tomato bushy stunt virus (TBSV) RNA. In vitro translated TBSV proteins p33 and p92 form viral replicase, which, in the same reaction, accomplishes the entire replication cycle on exogenous TBSV DI or full-length RNA. Tests of mutant TBSV RNAs confirmed the template specificity of the in vitro replication reaction. Complementation experiments ascertained the significance of an earlier identified TBSV host factor. Interestingly, formation of the viral replicase occurs also in the absence of concurrent protein synthesis demonstrating that translation and RNA replication are not functionally linked in this system. Our studies with cell-free extracts of a plant host thus confirmed earlier findings and enabled novel insights into the TBSV RNA replication process.

<p>The economic and technical efficiency of geothermal plants is often impaired by corrosion, scaling and biological fouling. In Germany, the highly saline fluid of the North German Basin is known to cause severe corrosion. Meanwhile geothermal plants in the southern Molasse Basin, one of the most extensively exploited geothermal regions in Germany, are troubled by carbonate scaling. One possible solution is the employment of a scale inhibitor. A novel scaling inhibitor is evaluated in field- and laboratory tests. This inhibitor consists of a polysaccharide backbone structure and branches of polyacrylic- and maleic acid copolymer.</p><p>The laboratory tests with different scaling inhibitor concentrations were designed to observe the biodegradation of the scaling inhibitor in an anaerobic environment similar to the conditions found in heat exchangers of geothermal plants. The concentration of inhibitor was quantified by UV/VIS and liquid chromatography (LC). Molecular biological techniques (PCR, DGGE, Microbiome analysis) were used to characterize the biocenosis on metal surfaces and in fluids of the experiments.</p><p>During the experiment the concentration of inhibitor decreased up to  3 % of the initial concentration. The formation of methane and acetate was observed which indicates a biological degradation by acetoclastic methanogenesis. Hydrogen formation was observed in setups containing steel coupons. This implies that hydrogen is primarily formed by corrosion processes and in tests with active microorganisms hydrogen was consumed completely. Various fermentative bacteria classified as Clostridia and Firmicutes as well as methanogenic archaea were identified. In some experiments sulfate reducing bacteria were found. Those are well known to catalyze corrosion processes.</p><p>Results of field experiments in a bypass system as well as microbiological monitoring of the inhibitor application in geothermal plant located in the molasse basin will be presented.</p>