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.     

Peer drug associations and emotional restraint: Causes or consequences of adolescents' drug use?

A 3-wave longitudinal design was used to examine the relationships among emotional restraint, peer drug associations, and gateway drug use in a sample of 1,256 middle school students. Structural equation modeling was used to compare 3 models: (1) One model viewed drug use as a consequence of emotional restraint and peer variables; (2) 1 viewed drug use as a cause of restraint and peer variables; and (3) 1 included reciprocal effects. All 3 models fit the data fairly well. However, the reciprocal model fit the data significantly better than either of the others. Within this model, low emotional restraint was significantly related to subsequent increases in gateway drug use among boys. In contrast, peer drug models and peer pressure were not related to subsequent changes in gateway drug use. Changes in peer drug models were, however, predicted by previous levels of gateway drug use. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Sequential tripping strategy for a transmission network back-upprotection expert system

This paper describes a sequential tripping strategy used in a wide area back-up protection expert system (BPES) to combat situations in which protection relays have maloperated or information is missing. The BPES is an innovative back-up protection scheme designed to prevent the occurrence of widespread blackouts. The BPES evaluates the certainty that transmission lines are likely to be affected by the fault and uses a sequential tripping strategy to isolate the fault if a firm decision is not available due to maloperated relays and/or missing information. The mode of analysis and the sequential tripping strategy ensures that the BPES will clear a fault at minimum risk to the network. An example is included to demonstrate how the certainty factor based sequential tripping strategy is employed by the BPES to clear a fault which occurred on the South Western part of the UK National Grid System     

Investigation on Single-Mode–Multimode– Single-Mode Fiber Structure

This paper presents an investigation on a single-mode-multimode-single-mode fiber structure. A one-way guided-mode propagation analysis for the circular symmetry waveguide is employed to model the light propagation and the approximated formulations are derived and evaluated concerning the accuracy. Phase conjunction of the multimode interference within the fiber structure is revealed. A simple way to predict and analyze the spectral response of the structure is presented through the space to wavelength mapping with the derived approximated formulations. The prediction of spectral response is verified numerically and experimentally.     

Uniform and optimal schemes for stiff initial-value problems

We formulate a class of difference schemes for stiff initial-value problems, with a small parameter ε multiplying the first derivative. We derive necessary conditions for uniform convergence with respect to the small parameter ε, that is the solution of the difference scheme u_i^h satisfies |u_i^h−u(x_i)| Ch, where C is independent of h and ε. We also derive sufficient conditions for uniform convergence and show that a subclass of schemes is also optimal in the sense that |u_i^h−u(x_i)| C min (h, ε). Finally, we show that this class contains higher-order schemes.     

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.     

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.