Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

Deliberately Losing Control of C−H Activation Processes in the Design of Small-Molecule-Fragment Arrays Targeting Peroxisomal Metabolism

Combined photochemical arylation, “nuisance effect” (S_NAr) reaction sequences have been employed in the design of small arrays for immediate deployment in medium-throughput X-ray protein–ligand structure determination. Reactions were deliberately allowed to run “out of control” in terms of selectivity; for example the ortho-arylation of 2-phenylpyridine gave five products resulting from mono- and bisarylations combined with S_NAr processes. As a result, a number of crystallographic hits against NUDT7, a key peroxisomal CoA ester hydrolase, have been identified.     

Anesthetic considerations in the child with Gaucher disease

Alpidem, an imidazopyridine that acts at the gamma-aminobutyric acid/benzodiazepine receptor complex, has been reported to be an effective anxiolytic with a more favorable side effect profile than benzodiazepines. The effect of alpidem was investigated in an 8-week, open, clinical trial in 13 patients with panic disorder, with or without agoraphobia. Three patients were responders (much improved or very much improved), five patients were nonresponders, and five patients dropped out after less than 6 weeks of treatment. Significant improvement was seen in the sample as a whole for spontaneous panic attacks, phobic avoidance, and anticipatory anxiety. Most improvement occurred during the first 4 weeks of treatment, and responders had milder panic disorder at baseline. Adverse effects were generally mild. After 8 weeks of treatment, taper of medication over 2 weeks occurred without significant worsening of panic disorder symptoms. The efficacy of alpidem in the treatment of panic disorder remains uncertain and requires assessment in a controlled trial.     

Purification and characterization of an endopeptidase from Lactobacillus delbrueckii subsp. bulgaricus B14

An intracellular endopeptidase was purified from cell-free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 by anion exchange chromatography on DEAE-Sepharose, hydroxyapatite chromatography, second anion exchange chromatography on Mono-Q, and metal-chelating affinity chromatography. The endopeptidase was a monomer with a molecular mass of approximately 70 kDa determined by SDS-PAGE and gel filtration. Various oligopeptides (e.g. Met-enkephalin, bradykinin) were hydrolysed by the endopeptidase. Exopeptidase activity and cleavage of dipeptides or tripeptides was not observed. The K_M value for the cleavage of Metenkephalin was 1.2 mM. Temperature and pH optima were 47 °C and pH 7.7, respectively. The endopeptidase was inhibited by the classical agents for metal-dependent (EDTA) and serine (DFP) enzymes. Activity was increased by Co²⁺ and Mg²⁺, no effect was observed with Ca²⁺. After inhibition with EDTA, enzyme activity could be restored fully by Co²⁺. Activity was inhibited by Zn²⁺, Mn²⁺, Fe²⁺, Cu²⁺, Cd²⁺ and Hg²⁺. The N-terminal sequence of the endopeptidase was determined as: H₂N-Val-Arg-Gly-Gly-Ser-Gly-Asp-Thr-Thr-Val-0H.     

Performance-Analyse und -Optimierung in der Softwareentwicklung

Nicht-funktionale Anforderungen an ein Softwaresystem beschreiben Aspekte, die nicht direkt die Funktionalit?t, wie sie der Benutzer sieht, betreffen.     

Simultaneous design of power stage and controller for switchingpower supplies

This paper develops a method for simultaneously designing the power stage and controller for a switching power supply. The method utilizes a numerical optimization procedure, which facilitates computer-aided design. It is found that better performance can be achieved than with a traditional two-step design process, where the power stage and controller are designed sequentially. Optimization and simulation results for a buck power converter are presented to illustrate the design process and benefits     

Durability of polymeric glazing materials for solar applications

The economic viability of solar collector systems for domestic hot water (DHW) generation is strongly linked to the cost of such systems. Installation and hardware costs must be reduced by 50% to allow significant market penetration. An attractive approach to cost reduction is to replace glass and metal parts with less expensive, lighter weight polymeric components. Weight reduction decreases the cost of shipping, handling, and installation. The use of polymeric materials also allows the benefits and cost savings associated with well established manufacturing processes, along with savings associated with improved fastening, reduced part count, and overall assembly refinements. A key challenge is to maintain adequate system performance and assure requisite durability for extended lifetimes. Results of preliminary and ongoing screening tests for a large number of candidate polymeric glazing materials are presented. Based on these results, two specific glazings with moderate and poor weathering stability are selected to demonstrate how a service lifetime methodology can be applied to accurately predict the optical performance of these materials during in-service use. A summary is given for data obtained by outdoor exposure and indoor testing of polyvinyl chloride (PVC) and high temperature modified polycarbonate copolymer (coPC) materials, and an initial risk analysis is given for the two materials. Screening tests and analyses for service lifetime prediction are discussed. A methodology that provides a way to derive correlations between degradation experienced by materials exposed to controlled accelerated laboratory exposure conditions and materials exposed to in-service conditions is given, and a validation is presented for the methodology based upon durability test results for PVC and coPC.     

The high-resolution time-of-flight spectrometer TOFTOF

The TOFTOF spectrometer is a multi-disc chopper time-of-flight spectrometer for cold neutrons at the research neutron source Heinz Maier-Leibnitz (FRM II). After five reactor cycles of routine operation the characteristics of the instrument are reported in this article. The spectrometer features an excellent signal to background ratio due to its remote position in the neutron guide hall, an elaborated shielding concept and an s-shaped curved primary neutron guide which acts i.a. as a neutron velocity filter. The spectrometer is fed with neutrons from the undermoderated cold neutron source of the FRM II leading to a total neutron flux of 10¹⁰n/cm²/s in the continuous white beam at the sample position distributed over a continuous and particularly broad wavelength spectrum. A high energy resolution is achieved by the use of high speed chopper discs made of carbon-fiber-reinforced plastic. In the combination of intensity, resolution and signal to background ratio the spectrometer offers new scientific prospects in the fields of inelastic and quasielastic neutron scattering.     

RAP46 is a negative regulator of glucocorticoid receptor action and hormone-induced apoptosis

RAP46 was first identified by its ability to bind the glucocorticoid receptor. It has since been reported to bind several cellular proteins, including the anti-apoptotic protein Bcl-2, but the biological significance of these interactions is unknown. Here we show that RAP46 binds the hinge region of the glucocorticoid receptor and inhibits DNA binding and transactivation by the receptor. We further show that overexpression of RAP46 in mouse thymoma S49.1 cells inhibits glucocorticoid-induced apoptosis. Conversely, glucocorticoid-induced apoptosis and transactivation were enhanced after treating S49.1 cells with the immunosuppressant rapamycin, which down-regulates cellular levels of BAG-1, the mouse homolog of RAP46. The effect of rapamycin can, however, be overcome by overexpression of RAP46. These results together identify RAP46 as a protein that controls glucocorticoid-induced apoptosis through its negative regulatory action on the transactivation property of the glucocorticoid receptor.     

Location of the sacral pedicle, foramina, and ala on the lateral aspect of the sacrum: a radiographic study

Twenty-one adult dry-bone sacral specimens were used to quantitatively determine the location of the sacral pedicle, foramina, and ala on the lateral radiographic view of the sacrum. The anterior and posterior sacral foramina from S1 to S3, the midlines of the anterior sacrum and cephalad border of the S1 vertebral body, and the lateral limit of the lateral sacral mass were outlined with wires. A lateral radiograph was taken, and measurements were made directly from the radiograph. The average sacral pedicle height for both male and female specimens was approximately 20 mm for S1, 12 mm for S2, and 7 mm for S3. The sacral foramina height averaged approximately 13 mm for S1 and S2, and 10 mm for S3. The average ala and S1 body-ala angles were 88 degrees and 35 degrees. The distance from the ala tip to the anterior aspect of the sacrum averaged 12 mm, and the average anterior height of the S1 vertebral body above the ala was 11 mm. These measurements, in conjunction with inlet and outlet radiographs, may aid in the recognition of the vital structures of the sacrum on the lateral radiographic view and enhance the safety of dorsal sacral screw placement.