Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells

Klimaschutz im Luftverkehr: vom EU-Emissionshandel zu CORSIA

Als ein Baustein zur Erreichung des Ziels eines CO2-freien Wachstums des internationalen Luftverkehrs hat die Generalversammlung der Internationalen Zivilluftfahrtorganisation die Einführung der marktbasierten Klimaschutzmaßnahme CORSIA ab 2020 beschlossen. CORSIA wird auch im Vergleich zum bislang in Europa auf den Luftverkehr angewendeten Emissionshandelssystem EU-ETS vorgestellt. Das neue umweltpolitische Instrument wird anschließend sowohl aus umwelt- als auch aus transaktionskosten-ökonomischer und wettbewerbspolitischer Sicht für den Passagierluftverkehr kritisch gewürdigt

P3-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP for the Rapid Activation of the Na+,K+-ATPase

This is the published version, also available here: http://dx.doi.org/10.1016/S0006-3495(00)76387-9.P3-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP (pHP-caged ATP) has been investigated for its application as a phototrigger for the rapid activation of electrogenic ion pumps. The yield of ATP after irradiation with a XeCl excimer laser (λ = 308 nm) was determined at pH 6.0–7.5. For comparison, the photolytic yields of P3-[1-(2-nitrophenyl)]ethyl ATP (NPE-caged ATP) and P3-[1,2-diphenyl-2-oxo]ethyl ATP (desyl-caged ATP) were also measured. It was shown that at λ = 308 nm pHP-caged ATP is superior to the other caged ATP derivatives investigated in terms of yield of ATP after irradiation. Using time-resolved single-wavelength IR spectroscopy, we determined a lower limit of 106 s−1 for the rate constant of release of ATP from pHP-caged ATP at pH 7.0. Like NPE-caged ATP, pHP-caged ATP and desyl-caged ATP bind to the Na+,K+-ATPase and act as competitive inhibitors of ATPase function. Using pHP-caged ATP, we investigated the charge translocation kinetics of the Na+,K+-ATPase at pH 6.2–7.4. The kinetic parameters obtained from the electrical measurements are compared to those obtained with a technique that does not require caged ATP, namely parallel stopped-flow experiments using the voltage-sensitive dye RH421. It is shown that the two techniques yield identical results, provided the inhibitory properties of the caged compound are taken into account. Our results demonstrate that under physiological (pH 7.0) and slightly basic (pH 7.5) or acidic (pH 6.0) conditions, pHP-caged ATP is a rapid, effective, and biocompatible phototrigger for ATP-driven biological systems

Effect of connective tissue grafting on buccal bone changes based on cone beam computed tomography scans in the aesthetic zone of single immediate implants:A 1-year randomized controlled trial

BACKGROUND Connective tissue grafting has a beneficial effect on the peri-implant mucosa, but the effect of grafting the buccal mucosa on buccal bone thickness (BBT) has not been investigated, although BBT is proposed to be a key factor for the soft-tissue contour. The aim of this trial was to assess the outcome of a connective tissue graft (CTG) in the aesthetic zone of single immediate implants on the change of BBT according to cone beam computed tomography (CBCT) scan analysis. METHODS In a 1-year randomized controlled trial, 60 patients received an immediately placed implant and provisionalization, either combined with CTG (test group) or without CTG (control group). CBCTs were taken pre-operatively (T$_{pre}$ ) and 1 year after definitive restoration (T$_{2}$ ). Any change in BBT was assessed at different implant levels. Additionally, the change in mid-buccal mucosal level (MBML) and approximal marginal bone level were assessed. RESULTS Fifty-five patients were available for statistical analysis (test group, n = 28; control group, n = 27). At T$_{2}$ , the average change in BBT was significantly larger in the test group (-0.84 ± 0.61 mm) than in the control group (-0.46 ± 0.54 mm, P = 0.02). A MBML gain of 0.07 ± 0.85 mm in the test and a MBML loss -0.52 ± 1.16 mm in the control group was observed at T$_{2}$ . Average loss of marginal bone was 0.05 ± 0.33 mm and 0.01 ± 0.38 mm, respectively. CONCLUSIONS The application of CTG in the aesthetic zone of immediately placed and provisionalized implants is accompanied with more loss of BBT, but at the same time better maintains the mid-buccal mucosal level

Experimental infections of different carp strains with the carp edema virus (CEV) give insights into the infection biology of the virus and indicate possible solutions to problems caused by koi sleepy disease (KSD) in carp aquaculture

Outbreaks of koi sleepy disease (KSD) caused by carp edema virus (CEV) may seriously affect populations of farmed common carp, one of the most important fish species for global food production. The present study shows further evidence for the involvement of CEV in outbreaks of KSD among carp and koi populations: in a series of infection experiments, CEV from two different genogroups could be transmitted to several strains of naïve common carp via cohabitation with fish infected with CEV. In recipient fish, clinical signs of KSD were induced. The virus load and viral gene expression results confirm gills as the target organ for CEV replication. Gill explants also allowed for a limited virus replication in vitro. The in vivo infection experiments revealed differences in the virulence of the two CEV genogroups which were associated with infections in koi or in common carp, with higher virulence towards the same fish variety as the donor fish. When the susceptibility of different carp strains to a CEV infection and the development of KSD were experimentally investigated, Amur wild carp showed to be relatively more resistant to the infection and did not develop clinical signs for KSD. However, the resistance could not be related to a higher magnitude of type I IFN responses of affected tissues. Despite not having a mechanistic explanation for the resistance of Amur wild carp to KSD, we recommend using this carp strain in breeding programs to limit potential losses caused by CEV in aquaculture