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.     

Exceptions that prove the rule--Using a theory of motivated social cognition to account for ideological incongruities and political anomalies: Reply to Greenberg and Jonas (2003).

A meta-analysis by J. T. Jost, J. Glaser, A. W. Kruglanski, and F. J. Sulloway (2003) concluded that political conservatism is partially motivated by the management of uncertainty and threat. In this reply to J. Greenberg and E. Jonas (2003), conceptual issues are clarified, numerous political anomalies are explained, and alleged counterexamples are incorporated with a dynamic model that takes into account differences between "young" and "old" movements. Studies directly pitting the rigidity-of-the-right hypothesis against the ideological extremity hypothesis demonstrate strong support for the former. Medium to large effect sizes describe relations between political conservatism and dogmatism and intolerance of ambiguity; lack of openness to experience; uncertainty avoidance; personal needs for order, structure, and closure; fear of death; and system threat. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

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.     

Holistic nursing--definition and realization

Single-channel properties of a delayed rectifier voltage-gated K+ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between -60 and -40 mV with a potential of half-maximal activation, E50, at -47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between -60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at -40 mV. The time constant of deactivation was 126 msec at -80 mV and 16.9 msec at -110 mV. In symmetrical 105 mM K+, the single-channel conductance (gamma) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13-15 degrees C. In Na+ -rich solution with 2.5 mM extracellular K+ gamma was 7 pS and the reversal potential was negative to -80 mV, indicating a high selectivity for K+ over Na+. gamma depended on extracellular K+ concentration (KD = 19.6 mM) and temperature (Q10 = 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel amplitude at all potentials tested with a half-maximal inhibiting concentration (IC50) of 0.6 mM. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC50 = 6.8 nM). mast cell degranulating peptide (MCDP, IC50 = 41.9 microM). In Ringer solution the membrane potential of macroscopic I-channel patches was about -65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential.