Middle East Respiratory Syndrome Coronavirus Accessory Protein 4a Is a Type I Interferon Antagonist

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory infection with as yet unclear epidemiology. We previously showed that MERS-CoV counteracts parts of the innate immune response in human bronchiolar cells. Here we analyzed accessory proteins 3, 4a, 4b, and 5 for their abilities to inhibit the type I interferon response. Accessory protein 4a was found to block interferon induction at the level of melanoma differentiation-associated protein 5 (MDA5) activation presumably by direct interaction with double-stranded RNA.     

Superparamagnetic iron oxide nanoparticles as radiosensitizer via enhanced reactive oxygen species formation

Internalization of citrate-coated and uncoated superparamagnetic iron oxide nanoparticles by human breast cancer (MCF-7) cells was verified by transmission electron microscopy imaging. Cytotoxicity studies employing metabolic and trypan blue assays manifested their excellent biocompatibility. The production of reactive oxygen species in iron oxide nanoparticle loaded MCF-7 cells was explained to originate from both, the release of iron ions and their catalytically active surfaces. Both initiate the Fenton and Haber-Weiss reaction. Additional oxidative stress caused by X-ray irradiation of MCF-7 cells was attributed to the increase of catalytically active iron oxide nanoparticle surfaces.     

A field study on the dependence of the fecundity of Daphnia spec. on food concentration

Summary The dependency of the fecundity of Daphnia spec. on the food concentration was studied in Lake Constance. To eliminate the influence of temperature the number of eggs per adult female was plotted against the concentration of particulate carbon (<50 m) at the estimated time of the production of the eggs. The curve obtained is similar to the results of laboratory investigations. The minimum food concentration for egg production is slightly above 0.2 mg C/1; at 0.7 mg C/1 the curve reaches a plateau. The absolute values of the threshold concentration and the incipient limiting level are compared to laboratory results. It appears that the shift of the field curve to higher concentrations is caused by the imcomplete availability of the 50 m particulate carbon fraction to the daphnids. Daphnia in Lake Constance seem to be food limited during most of the year.     

Structural and functional fingerprint of the mitochondrial ATP-binding cassette transporter Mdl1 from Saccharomyces cerevisiae

The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 microm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation.     

Quantitative detection of siRNA and single-stranded oligonucleotides: relationship between uptake and biological activity of siRNA

The quantitative detection of oligomeric nucleic acids including short double-stranded RNA in cells and tissues becomes increasingly important. Here, we describe a method for the detection of siRNA in extracts prepared from mammalian cells, which is based on liquid hybridization with a ³²P-labelled probe followed by a nuclease protection step. The limit of detection of absolute amounts of siRNA is in the order of 10–100 amol. This methodology is suited to quantitatively follow the spontaneous uptake of siRNA by mammalian cells, i.e. without the use of carrier substances. This protocol may also be used to detect extremely low amounts of other kinds of short nucleic acids, including antisense oligonucleotides.     

Delivery of nucleic acid into mammalian cells by anthrax toxin

Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.     

Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment

We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-alpha-D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose. A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-alpha-D-mannosyl-D-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-O-alpha-D-mannosyl-D-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-alpha-D-mannosyl-D-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.