The vanilloid receptor-related TRP channels (TRPV1-6) mediate thermosensation, pain perception and epithelial Ca2+ entry. As the specificity of TRPV channel heteromerization and determinants governing the assembly of TRPV subunits were largely elusive, we investigated the TRPV homo- and heteromultimerization. To analyze the assembly of TRPV subunits in living cells, we generated fluorescent fusion proteins or FLAG-tagged TRPV channel subunits. The interaction between TRPV subunits was assessed by analysis of the subcellular colocalization, fluorescence resonance energy transfer and coimmunoprecipitation. Our results demonstrate that TRPV channel subunits do not combine arbitrarily. With the exception of TRPV5 and TRPV6, TRPV channel subunits preferentially assemble into homomeric complexes. Truncation of TRPV1, expression of cytosolic termini of TRPV1 or TRPV4 and construction of chimeric TRPV channel subunits revealed that the specificity and the affinity of the subunit interaction is synergistically provided by interaction modules located in the transmembrane domains and in the cytosolic termini. The relative contribution of intramolecularly linked interaction modules presumably controls the overall affinity and the specificity of TRPV channel assembly.

Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.

The low extracellular pH of inflamed or ischemic tissues enhances painful sensations by sensitizing and activating the vanilloid receptor 1 (TRPV1). We report here that activation of TRPV1 results in a marked intracellular acidification in nociceptive dorsal root ganglion neurons and in a heterologous expression system. A characterization of the underlying mechanisms revealed a Ca 2؉ -dependent intracellular acidification operating at neutral pH and an additional as yet unrecognized direct proton conductance through the poorly selective TRPV1 pore operating in acidic extracellular media. Large organic cations permeate through the activated TRPV1 pore even in the presence of physiological concentrations of Na ؉ , Mg 2؉ , and Ca 2؉ . The wide pore and the unexpectedly high proton permeability of TRPV1 point to a proton hopping permeation mechanism along the water-filled channel pore. In acidic media, the high relative proton permeability through TRPV1 defines a novel proton entry mechanism in nociceptive neurons.During ischemia or inflammation, pain sensation is augmented by the acidic extracellular pH (pH ext ). 1 A␦-and C-fiber neurons sense extracellular acid by means of two different classes of cation channels, namely TRPV1, the founding member of the vanilloid receptor-like transient receptor potential channel family (1, 2), and the acid-sensing ion channels (ASIC) (3, 4). Both channel types are expressed in small diameter dorsal root ganglion (DRG) neurons, and their role in mediating inflammatory hyperalgesia has been proven by gene deletion techniques (5-7). TRPV1, a poorly selective cation channel, integrates multiple pain-inducing stimuli, including noxious heat, vanilloids, and acidic extracellular pH (1, 8 -10). Inflammatory mediators such as bradykinin, serotonin, histamine, or prostaglandins further stimulate TRPV1 activity either by protein kinase C-dependent signals (11-13), by a release from a phosphatidylinositol 4,5-bisphosphate-dependent inhibition (14,15), by formation of 12-lipoxygenase products (16), or by a protein kinase A-mediated recovery from inactivation (17). Furthermore, extracellular acidification shifts the activation of TRPV1 toward lower temperature or ligand thresholds by protonation of an amino acid located in the vicinity of the pore loop (18). Although the role of the external pH ext in modulating TRPV1 activity is well established, a possible impact of TRPV1 activation on the intracellular pH has not been studied.A Ca 2ϩ influx component through activated voltage-or ligand-gated cation channels has been recognized to lower the intracellular pH (pH i ) in neurons or in neuroendocrine cells (19,20). We therefore asked the question whether activation of the Ca 2ϩ -permeable TRPV1 may also mediate intracellular acidification in native rat dorsal root ganglion neurons or in a heterologous expression system. Our results provide evidence for two independent TRPV1-mediated acidification signals, including an as yet unrecognized direct proton conductance through the activated TRPV1 p...

With the aim of obtaining a carrier for combined magnetic‐field‐ and ultrasound‐targeted nucleic acid delivery, acoustically active lipospheres are prepared that comprise magnetic nanoparticles and plasmid DNA or synthetic siRNA. The lipospheres, with average diameters of 5 μm and smaller, are obtained upon shaking a mixture of soybean oil, a cationic lipid, magnetic nanoparticles, a nucleic acid, and aqueous buffer in a perfluoropropane atmosphere in a sealed vial. These lipospheres create contrast in ultrasound imaging and display greatly increased magnetophoretic mobility and in consequence greatly improved magnetic retention in a flow model when compared with free magnetic nanoparticles. In cell culture, the lipospheres are sedimented within minutes to the surface of cells using a gradient magnetic field. This sedimentation results in the association of about 50% of the applied plasmid DNA with the cells and in functional DNA and siRNA delivery in vitro. Under these conditions, ultrasound does not have an enhancing effect on nucleic acid delivery. When magnetic, acoustically active lipospheres carrying 125iodine‐labeled plasmid DNA are injected into the tail veins of mice, the application of a gradient magnetic field to the chests of the mice results in a two‐ to threefold enrichment of both lung lobes with the plasmid. A similar enrichment is obtained when ultrasound alone (1 MHz, 10 min) is applied. The combined application of magnetic field and ultrasound has no synergistic effect in terms of liposphere capture in the lungs. Histological analysis reveals intact lipospheres in lung capillaries. A synergistic effect of magnetic field and ultrasound is observed in site‐specific plasmid deposition in a dorsal skinfold chamber model in mice after injection into the carotis. These conditions also result in functional plasmid delivery to the vasculature after intrajugular injection.

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p < 0.01; n = 8) in vitroas well as new vessel growth ex vivo(p < 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p < 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction (p < 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis (annexin V staining, p < 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n = 3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth.

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For a few years, mineral oils and their potential adverse health effects have been a constant issue of concern in many regulatory areas such as food, cosmetics, other consumer products, and industrial chemicals. Analytically, two fractions can be distinguished: mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). This paper aims at assessing the bioaccumulative potential and associated histopathological effects of MOSH as well as the carcinogenic potential of MOAH for consumer-relevant mineral oils. It also covers the absorption, distribution, metabolism, and excretion of MOSH and MOAH upon oral and dermal exposures. The use and occurrence of consumerrelevant, highly refined mineral oils in food, cosmetics and medicinal products are summarized, and estimates for the exposure of consumers are provided. Also addressed are the challenges in characterizing the substance identity of mineral oil products under REACH. Evidence from more recent autopsy and biopsy studies, along with information on decreasing food contamination levels, indicates a low risk for adverse hepatic lesions that may arise from the retention of MOSH in the liver. With respect to MOAH, at present there is no indication of any carcinogenic effects in animals dermally or orally exposed to highly refined mineral oils and waxes. Such products are used not only in cosmetics but also in medicinal products and as additives in food contact materials. The safety of these mineral oilcontaining products is thus indirectly documented by their prevalent and long-term use, with a simultaneous lack of clinical and epidemiological evidence for adverse health effects.

The zebrafish MAGUK protein Nagie oko is a member of the evolutionarily conserved Crumbs protein complex and functions as a scaffolding protein involved in the stabilization of multi-protein assemblies at the tight junction. During zebrafish embryogenesis, mutations in nagie oko cause defects in both epithelial polarity and cardiac morphogenesis. We used deletion constructs of Nagie oko in functional rescue experiments to define domains essential for cell polarity, maintenance of epithelial integrity and cardiac morphogenesis. Inability of Nagie oko to interact with Crumbs proteins upon deletion of the PDZ domain recreates all aspects of the nagie oko mutant phenotype. Consistent with this observation, apical localization of Nagie oko within the myocardium and neural tube is dependent on Oko meduzy/Crumbs2a. Disruption of direct interactions with Patj or Lin-7, two other members of the Crumbs protein complex, via the bipartite L27 domains produces only partial nagie oko mutant phenotypes and does not impair correct junctional localization of the truncated Nagie oko deletion protein within myocardial cells. Similarly, loss of the evolutionarily conserved region 1 domain, which mediates binding to Par6, causes only a subset of the nagie oko mutant epithelial phenotypes. Finally, deletion of the C-terminus, including the entire guanylate kinase and the SH3 domains, renders the truncated Nagie oko protein inactive and recreates all features of the nagie oko mutant phenotype when tested in functional complementation assays. Our observations reveal a previously unknown diversity of alternative multi-protein assembly compositions of the Crumbs–Nagie-oko and Par6-aPKC protein complexes that are highly dependent on the developmental context.