Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADH:decyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.     

Ion-imprinted beads for molecular recognition based mercury removal from human serum

The aim of this study is to prepare ion-imprinted polymers which can be used for the selective removal of mercury ions [Hg(2+)] from human serum. N-Methacryloyl-(L)-cysteine (MAC) was chosen as the complexing monomer. In the first step, Hg(2+) was complexed with MAC and the Hg(2+)-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-cysteine) (MIP) beads were synthesized by suspension polymerization. After that, the template ions (i.e., Hg(2+)) were removed using thiourea (0.5%, v/v) in 0.05 M HCl. The specific surface area of the MIP beads was found to be 59.04 m(2)/g with a size range of 63-140 micro m in diameter and the swelling ratio was 91.5%. According to the elemental analysis results, the MIP beads contained 87.0 micro mol MAC/g polymer. The maximum adsorption capacity was 0.45 mg Hg(2+)/g beads. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. The relative selectivity coefficients of MIP beads for Hg(2+)/Cd(2+), Hg(2+)/Zn(2+) were 14.7 and 21.5 times greater than the non-imprinted (NIP) matrix, respectively. The MIP beads could be used many times without decreasing in their adsorption capacities significantly.     

Description and mapping of the resistance of DBA/2 mice to TNF-induced lethal shock

In our search for genes that inhibit the inflammatory effects of TNF without diminishing its antitumor capacities we found that, compared with C57BL/6 mice, DBA/2 mice exhibit a dominant resistance to TNF-induced lethality. Tumor-bearing (C57BL/6 x DBA/2)(BXD)F(1) mice completely survived an otherwise lethal TNF/IFN-gamma-antitumor therapy with complete regression of the tumor. This was not the case for C57BL/6 mice. Genetic linkage analysis revealed that TNF resistance is linked to a major locus on distal chromosome 6 and a minor locus on chromosome 17. Compared with littermate controls, chromosome substitution mice carrying a DBA/2 chromosome 6 in a C57BL/6 background were significantly protected against TNF and TNF/IFN-gamma, albeit less so than DBA/2 mice. Definition of a critical region of 13 Mb on chromosome 6 was the highest mapping resolution obtained. Further analysis of candidate genes may provide a powerful tool to control TNF-induced pathologies in humans.     

Synthesis of DNA-oligonucleotides damaged by arylamine-modified 2'-deoxyguanosine

C8-Arylamine-dG adducts bearing a labile N-formamidine group at the exocyclic amino function were converted into their corresponding 5'-O-DMTr-3'-O-phosphoramidite-C8-arylamine-dG derivatives. These compounds were used for the automated synthesis of site-specifically modified oligonucleotides. These oligonucleotides were characterized by ESI-MS and enzymatic digestion and studied for their CD properties and Tm values.     

A new method to prevent carry-over contaminations in two-step PCR NGS library preparations

Two-step PCR procedures are an efficient and well established way to generate amplicon libraries for NGS sequencing. However, there is a high risk of cross-contamination by carry-over of amplicons from first to second amplification rounds, potentially leading to severe misinterpretation of results. Here we describe a new method able to prevent and/or to identify carry-over contaminations by introducing the K-box, a series of three synergistically acting short sequence elements. Our K-boxes are composed of (i) K1 sequences for suppression of contaminations, (ii) K2 sequences for detection of possible residual contaminations and (iii) S sequences acting as separators to avoid amplification bias. In order to demonstrate the effectiveness of our method we analyzed two-step PCR NGS libraries derived from a multiplex PCR system for detection of T-cell receptor beta gene rearrangements. We used this system since it is of high clinical relevance and may be affected by very low amounts of contaminations. Spike-in contaminations are effectively blocked by the K-box even at high rates as demonstrated by ultra-deep sequencing of the amplicons. Thus, we recommend implementation of the K-box in two-step PCR-based NGS systems for research and diagnostic applications demanding high sensitivity and accuracy.     

Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25

The cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibody ipilimumab induces immune-mediated long-term control of metastatic melanoma in a fraction of patients. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, thus far clinically useful, immunologically relevant biomarkers that predict treatment efficiency have been elusive. Here, we show that neutralization of IL-2 or blocking the α and β subunits of the IL-2 receptor (CD25 and CD122, respectively) abolished the antitumor effects and the accompanying improvement of the ratio of intratumoral T effector versus regulatory cells (Tregs), which were otherwise induced by CTLA-4 blockade in preclinical mouse models. CTLA-4 blockade led to the reduction of a suppressive CD4⁺ T cell subset expressing Lag3, ICOS, IL-10 and Egr2 with a concomitant rise in IL-2-producing effector cells that lost FoxP3 expression and accumulated in regressing tumors. While recombinant IL-2 improved the therapeutic efficacy of CTLA-4 blockade, the decoy IL-2 receptor α (IL-2Rα, sCD25) inhibited the anticancer effects of CTLA-4 blockade. In 262 metastatic melanoma patients receiving ipilimumab, baseline serum concentrations of sCD25 represented an independent indicator of overall survival, with high levels predicting resistance to therapy. Altogether, these results unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides the first immunologically relevant biomarker, namely elevated serum sCD25, that predicts resistance to CTLA-4 blockade in patients with melanoma.     

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.