In vitro assembly of gap junctions

Gap junction structures were assembled in vitro from octyl-β- -glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = B = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg²⁺ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.     

The Thermosensitive Potassium Channel TREK-1 Contributes to Coolness-Evoked Responses of Grueneberg Ganglion Neurons

Neurons of the Grueneberg ganglion (GG) residing in the vestibule of the murine nose are activated by cool ambient temperatures. Activation of thermosensory neurons is usually mediated by thermosensitive ion channels of the transient receptor potential (TRP) family. However, there is no evidence for the expression of thermo-TRPs in the GG, suggesting that GG neurons utilize distinct mechanisms for their responsiveness to cool temperatures. In search for proteins that render GG neurons responsive to coolness, we have investigated whether TREK/TRAAK channels may play a role; in heterologous expression systems, these potassium channels have been previously found to close upon exposure to coolness, leading to a membrane depolarization. The results of the present study indicate that the thermosensitive potassium channel TREK-1 is expressed in those GG neurons that are responsive to cool temperatures. Studies analyzing TREK-deficient mice revealed that coolness-evoked responses of GG neurons were clearly attenuated in these animals compared with wild-type conspecifics. These data suggest that TREK-1 channels significantly contribute to the responsiveness of GG neurons to cool temperatures, further supporting the concept that TREK channels serve as thermoreceptors in sensory cells. Moreover, the present findings provide the first evidence of how thermosensory GG neurons are activated by given temperature stimuli in the absence of thermo-TRPs.     

Clinical and immunological responses in metastatic melanoma patients vaccinated with a high-dose poly-epitope vaccine

Background

Safety and cellular immunogenicity of rising doses and varying regimens of a poly-epitope vaccine were evaluated in advanced metastatic melanoma. The vaccine comprised plasmid DNA and recombinant modified vaccinia virus Ankara (MVA) both expressing a string (Mel3) of seven HLA.A2/A1 epitopes from five melanoma antigens.

Methods

Forty-one HLA-A2 positive patients with stage III/IV melanoma were enrolled. Patient groups received one or two doses of DNA.Mel3 followed by escalating doses of MVA.Mel3. Immunisations then continued eight weekly in the absence of disease progression. Epitope-specific CD8+ T cell responses were evaluated using ex-vivo tetramer and IFN-γ ELISPOT assays. Safety and clinical responses were monitored.

Results

Prime-boost DNA/MVA induced Melan-A-specific CD8+ T cell responses in 22/31 (71%) patients detected by tetramer assay. ELISPOT detected a response to at least one epitope in 10/31 (32%) patients. T cell responder rates were <50% with low-dose DNA/MVA, or MVA alone, rising to 91% with high-dose DNA/MVA. Among eight patients showing evidence of clinical benefit—one PR (24 months+), five SD (5 months+) and two mixed responses—seven had associated immune responses. Melan-A-tetramer+ immunity was associated with a median 8-week increase in time-to-progression (P = 0.037) and 71 week increase in survival (P = 0.0002) compared to non-immunity. High-dose vaccine was well tolerated. The only significant toxicities were flu-like symptoms and injection-site reactions.

Conclusions

DNA.Mel3 and MVA.Mel3 in a prime-boost protocol generated high rates of immune response to melanoma antigen epitopes. The treatment was well tolerated and the correlation of immune responses with patient outcomes encourages further investigation.     

An 86Y-labeled mirror-image oligonucleotide: influence of Y-DOTA isomers on the biodistribution in rats

A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide (86)Y (t(1/2) = 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5'-aminohexyl UGA CUG ACU GAC-3'; MW 3975) was performed using (S)-p-SCN-Bn-DOTA. (86)Y radiolabeling of the DOTA-L-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the (86)Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)](-) was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)](-) isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)](-) and conjugated to the L-RNA. The identity of the [(86)Y-DOTA]-L-RNA species was finally established by comparison of the radiometric ((86)Y) and UV-visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [(86)Y((S)-p-NH2-Bn-DOTA)](-) complex isomers, while no significant differences were observed for the [(86)Y-DOTA]-L-RNA isomers. High renal excretions were found for the [(86)Y((S)-p-NH 2-Bn-DOTA)](-) complex isomers as well as for the L-RNA isomers.     

CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation

Mature donor T cells cause graft-versus-host disease (GVHD), but they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity of allogeneic bone marrow transplantation. Suppression of GVHD with maintenance of GVT activity is a desirable outcome for clinical transplantation. We have previously shown that donor-derived CD4+CD25+ regulatory T cells inhibit lethal GVHD after allogeneic bone marrow transplantation across major histocompatibility complex (MHC) class I and II barriers in mice. Here we demonstrate that in host mice with leukemia and lymphoma, CD4+CD25+ regulatory T cells suppress the early expansion of alloreactive donor T cells, their interleukin-2-receptor (IL-2R) alpha-chain expression and their capacity to induce GVHD without abrogating their GVT effector function, mediated primarily by the perforin lysis pathway. Thus, CD4+CD25+ T cells are potent regulatory cells that can separate GVHD from GVT activity mediated by conventional donor T cells.     

Solid-solid interface adsorption of proteins and enzymes in nanophase-separated amphiphilic conetworks

Amphiphilic polymer conetworks (APCNs) are materials with a very large interface between their hydrophilic and hydrophobic phases due to their nanophase-separated morphologies. Proteins were found to enrich in APCNs by up to 2 orders of magnitude when incubated in aqueous protein solutions, raising the question of the driving force of protein uptake into APCNs. The loading of poly(2-hydroxyethyl acrylate)-linked by-poly(dimethylsiloxane) (PHEA-l-PDMS) with heme proteins (myoglobin, horseradish peroxidase, hemoglobin) and lipases was studied under variation of parameters such as incubation time, pH, concentration of the protein solution, and conetwork composition. Adsorption of enzymes to the uncharged interface is the main reason for protein uptake, resulting in protein loading of up to 23 wt %. Experimental results were supported by computation of electrostatic potential maps of a lipase, indicating that hydrophobic patches are responsible for the adsorption to the interface. The findings underscore the potential of enzyme-loaded APCNs in biocatalysis and as sensors.     

Sulphate-mediated phosphorus mobilization in riverine sediments at increasing sulphate concentration, River Spree, NE Germany

The study focuses on the response of a sulphate rich lowland river (River Spree) to a further increase in sulphate concentration as a result of mining activities in its catchments. It was hypothesized that riverine sediments could be conservative against an increase in sulphate concentration relating to both the intensity of sulphate reduction and the accompanying P mobilization. The usually lower amount of organic matter, compared to lakes or wetlands, and the high contents of iron oxides in the Spree sediment from discharged mining waters should counteract an enhanced P mobilization. Three short-term incubation experiments were carried out to test the sensitivity of different sediment horizons (0–10, 10–20 and 20–30 cm), the influence of temperature (5 and 25 °C) and the effect of a rising sulphate concentration (2.6–7.8 mM) on P mobilization rates (PMR) and sulphate reduction rates (SRR). Contrary to our initial hypothesis sulphate played a key role for P mobilization in riverine sediments because (1) all sulphate treated horizons showed a significant increase in pore water P concentrations, (2) increasing sulphate concentrations led to rising SRR and PMR, (3) the highest response on sulphate-mediated P mobilization was observed by a temperature enhancement of 20 °C. PMR increased one order of magnitude at all tested sulphate concentrations, but these increases in PMR only slightly effected the P concentrations in the overlying water. In conclusions, an increase of internal P load is only expected in case of doubling the recent in situ sulphate concentrations, but extended warm periods as an effect of climate change or increasing temperature, respectively, could be of more importance.