Electronic (e-)cigarettes have emerged in recent years as putative alternative to conventional tobacco cigarettes. These products do not contain typical carcinogens that are present in tobacco smoke, due to the lack of combustion. However, besides nicotine, hazards can also arise from other constituents of liquids, such as solvents, flavors, additives and contaminants. In this study, we have analyzed 28 liquids of seven manufacturers purchased in Germany. We confirm the presence of a wide range of flavors to enhance palatability. Although glycerol and propylene glycol were detected in all samples, these solvents had been replaced by ethylene glycol as dominant compound in five products. Ethylene glycol is associated with markedly enhanced toxicological hazards when compared to conventionally used glycerol and propylene glycol. Additional additives, such as coumarin and acetamide, that raise concerns for human health were detected in certain samples. Ten out of 28 products had been declared "free-of-nicotine" by the manufacturer. Among these ten, seven liquids were identified containing nicotine in the range of 0.1-15 µg/ml. This suggests that "carry over" of ingredients may occur during the production of cartridges. We have further analyzed the formation of carbonylic compounds in one widely distributed nicotine-free brand. Significant amounts of formaldehyde, acetaldehyde and propionaldehyde were only found at 150 °C by headspace GC-MS analysis. In addition, an enhanced formation of aldehydes was found in defined puff fractions, using an adopted machine smoking protocol. However, this effect was delayed and only observed during the last third of the smoking procedure. In the emissions of these fractions, which represent up to 40 % of total vapor volume, similar levels of formaldehyde were detected when compared to conventional tobacco cigarettes. By contrast, carbonylic compounds were hardly detectable in earlier collected fractions. Our data demonstrate the necessity of standardized machine smoking protocols to reliably address putative risks of e-cigarettes for consumers.

The hepatitis B virus (HBV) X protein (HBx) was originally suggested to be a viral transcriptional activator, but its functional mechanisms are still unclear. In this study we have analysed the intracellular localization of HBx in transfected cells and demonstrate that its compartmentalization is dependent on overall expression levels. HBx was exclusively or predominantly localized in the nuclei in weakly expressing cells. However, elevated cellular levels correlated with its accumulation in the cytoplasm, suggesting that the capacity of HBx for nuclear compartmentalization might be limited. Cytoplasmic HBx was detected either as punctate granular staining or in dispersed, finely granular patterns. We have further analysed the detailed cytoplasmic compartmentalization, using confocal microscopy, and show no association with the endoplasmic reticulum, plasma membrane or lysosomes, but a substantial association of HBx with mitochondria. However, a major fraction of cytoplasmic HBx did not localize in mitochondria, indicating the presence of two distinctly compartmentalized cytoplasmic populations. Furthermore, high levels of HBx expression led to an abnormal mitochondrial distribution, involving clumping and organelle aggregation, which was not observed at lower expression levels. The data presented here provide novel insights into the compartmentalization of HBx and may prove important for future evaluations of its functions, both in the viral life-cycle and in the pathology of HBV-related liver disease.

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To investigate CD40 signaling complex formation in living cells, we used green fluorescent protein (GFP)-tagged CD40 signaling intermediates and confocal life imaging. The majority of cytoplasmic TRAF2-GFP and, to a lesser extent, TRAF3-GFP, but not TRAF1-GFP or TRAF4-GFP, translocated into CD40 signaling complexes within a few minutes after CD40 triggering with the CD40 ligand. The inhibitor of apoptosis proteins cIAP1 and cIAP2 were also recruited by TRAF2 to sites of CD40 signaling. An excess of TRAF2 allowed recruitment of TRAF1-GFP to sites of CD40 signaling, whereas an excess of TRAF1 abrogated the interaction of TRAF2 and CD40. Overexpression of TRAF1, however, had no effect on the interaction of TRADD and TRAF2, known to be important for tumor necrosis factor receptor 1 (TNF-R1)-mediated NF-B activation. Accordingly, TRAF1 inhibited CD40-dependent but not TNF-R1-dependent NF-B activation. Moreover, down-regulation of TRAF1 with small interfering RNAs enhanced CD40/ CD40 ligand-induced NF-B activation but showed no effect on TNF signaling. Because of the trimeric organization of TRAF proteins, we propose that the stoichiometry of TRAF1-TRAF2 heteromeric complexes ((TRAF2) 2 -TRAF1 versus TRAF2-(TRAF1) 2 ) determines their capability to mediate CD40 signaling but has no major effect on TNF signaling. CD40 and its ligand CD40L1 /CD154 are members of the tumor necrosis factor (TNF) receptor and TNF ligand family and represent major regulators of lymphocyte function (1). Aside from T-and B-cells, CD40 and CD40L are expressed in a variety of non-lymphocytic cell types including monocytes, dendritic cells, fibroblasts, smooth muscle, and endothelial cells (1). The CD40/CD40L system plays a critical role in the regulation of thymus-dependent humoral immune responses but also contributes to chronic inflammatory processes in autoimmune diseases, neurodegenerative disorders, graft-versus-host disease, cancer, and atherosclerosis (1).Engagement of CD40 results in the recruitment of members of the TNF receptor-associated factor (TRAF) adaptor protein family (1, 2). In addition, triggering of CD40 leads to Janus family kinase 3 (Jak3)-dependent activation of signal transducers and activators of transcription (STAT) proteins and to activation of the Src-related tyrosine kinase Lyn (3-6). TRAF proteins couple TNF receptors and Toll/interleukin-1 receptor family members to pathways leading to the activation of the inhibitor of I-B kinases and kinases of the mitogen-activated protein kinase (MAPK) family (2). All members of the TRAF family share a conserved C-terminal homology domain of ϳ180 amino acids (TRAF domain), which mediates interactions with the above mentioned receptors and the majority of cytosolic factors known for their TRAF binding capacity, including kinases, inhibitor of apoptosis proteins, and death domain adaptor proteins (2). With the exception of TRAF1, the N-terminal domain of all six known mammalian TRAFs comprise a single RING finger followed by several zinc finger motifs (2) that are important for ...

Material Supplementary 5.DC1http://www.jimmunol.org/content/suppl/2010/07/06/jimmunol.090355