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.     

Role of Perceived Importance in Intergroup Contact.

Furthering G. W. Allport's (1954) contentions for optimal contact, the authors introduce a new construct: the perceived importance of contact. They propose that perceived importance is the best proximal predictor of contact's reduction of prejudice. If individuals have opportunities for contact at work or in the neighborhood, their chances to have intergroup acquaintances and friends increase. Intergroup contact among acquaintances and friends can be perceived as more or less important, which in turn determines intergroup evaluations. A 1st study shows that the new measure of perceived importance is indeed distinct from established quantity and quality indicators. The results are cross-validated in a 2nd study that also sheds light on the meaning of importance. In 3rd and 4th studies, structural equation analyses and a meta-analysis support the hypotheses. (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 inositol-1,4,5-trisphosphate system is involved in rapid effects of aldosterone in human mononuclear leukocytes

There is increasing evidence for rapid steroid action on electrolyte transport in human mononuclear leukocytes (HML). In HML, aldosterone stimulates the Na+/H+ antiporter within a few minutes. Because a variety of hormones and growth factors activate the Na+/H+ antiporter via protein kinase C and inositol phospholipids, a possible involvement of inositol-1,4,5-trisphosphate (IP3) in the rapid effects of aldosterone in HML was investigated. The stimulation of IP3 generation was started by the addition of aldosterone, concanavalin A, or other steroids. A significant increase in IP3 levels by aldosterone (1 nmol/L, P < 0.05) was found after 1 min, similar to that found after concanavalin A (25 micrograms/mL). Aldosterone caused a concentration-dependent elevation of IP3 levels, with an apparent EC50 of approximately 0.1 nmol/L. Fludrocortisone stimulated IP3 generation at similar concentrations, whereas a weaker IP3 stimulation by glucocorticoids (hydrocortisone, dexamethasone) occurred at micromolar concentrations only. Canrenone, a potent inhibitor of classical aldosterone action, was not effective up to a concentration of 100 nmol/L. These findings show kinetic and pharmacological similarities with both the functional data on Na+/H+ antiport stimulation by aldosterone and the studies of 125I-aldosterone binding to plasma membranes of HML. Thus, these data are the first to indicate an involvement of the phosphoinositide pathway in the rapid membrane effects of aldosterone.     

Thermomechanical fatigue—damage mechanisms and mechanism-based life prediction methods

An existing extensive database on the isothermal and thermomechanical fatigue behaviour of high-temperature titanium alloy EVII 834 and dispersoid-strengthened aluminum alloy X8019 in SiC particle-reinforced as well as unreinv conditions was used to evaluate both the adaptability of fracture mechanics approaches to TMF and the resulting predictive capabilities of determining material life by crack propagation consideration. Selection of the correct microstructural concepts was emphasised and these concepts were, then adjusted by using data from independent experiments in order to avoid any sort of fitting. It is shown that the cyclic /-integral (δJ_eff concept) is suitable to predict the cyclic lifetime for conditions where the total crack propagation rate is approximately identical to pure fatigue crack growth velocity. In the case that crack propagation is strongly affected by creep, the creep-fatigue damage parameter δ_CF introduced by Riedel can be successfully applied. If environmental effects are very pronounced, the accelerating influence of corrosion on fatigue crack propagation can no longer implicitly be taken into account in the fatigue crack growth law. Instead, a linear combination of the crack growth rate contributions from plain fatigue (determined in vacuum) and from environmental attack is assumed and found to yield a satisfactory prediction, if the relevant corrosion process is taken into account.     

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.