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.     

Fixed mesh front‐tracking methodology for finite element simulations

A direct front‐tracking method using an Eulerian–Lagrangian formulation is developed in two space dimensions. The front‐tracking method is general in that it can track any type of interface once its local velocity is specified or has been determined by calculation. The method uses marker points to describe the interface position and tracks the interface evolution on a fixed finite‐element mesh, including growth, contraction, splitting and merging. Interfacial conditions are applied directly at the interface position. The method is applied to three scenarios that involve different interface conditions and are based on energy and mass diffusion. The three calculations are for the dendritic solidification of a pure substance, the cellular growth of an alloy, and the Ostwald ripening of silica particles in silicon. Numerical results show that very complicated interface morphologies and topological changes can be simulated properly and efficiently. Copyright © 2004 John Wiley & Sons, Ltd.     

Balloon dilatation in acute myocardial infarct in routine clinical practice: results of the register of the Working Society of Leading Cardiologic Hospital Physicians in 4,625 patients

Balloon angioplasty as the treatment of first choice in the setting of an acute myocardial infarction (AMI) is gaining widespread acceptance because of favourable results from specialised centres concerning high patency rates and low mortality. This study reports the results of angioplasty for AMI at large community hospitals during 1992-1995. 4625 procedures were performed at 68 centres of the Arbeitsgemeinschaft Leitender Kardiologischer Krankenhaus?rzte (ALKK). The age of the patients was 60.8 +/- 11.3 years, with 75.1% men. The infarct related artery was the left anterior descendent in 43%, the right coronary artery in 37%, the circumflex artery in 16%, a bypass graft in 2.3% and the left main stem in 1.4% of patients. The success rate (residual stenosis < 50%) of the intervention was 86%. There was a wide range of procedures per centre, with a median of 40 AMI angioplasties per year and centre. The amount of angioplasties for AMI in relation to all angioplasties performed during this period rose from 5.2% in 1992 to 5.9% in 1995 (p = 0.01). Local complications at the puncture site occurred in 3.2%, with the need for a surgical intervention in 1.1% of patients. In 273 (5.9%) of the patients a second angioplasty was performed during the hospital stay. Aortocoronary bypass surgery was performed in 3% of the patients. Hospital mortality was 9.5% (438/4625 patients). The mortality rate remained constant during the years investigated (1992: 10.6%; 1993: 8.6%; 1994: 9.7%; 1995: 9.8%; p = ns). Higher mortality was observed in older patients, patients with multiple vessel disease, the left anterior descending artery or a bypass graft as infarct related artery as well as in patients with failed reperfusion (residual stenoses > 50%). Hospitals with a case load of more than 40 angioplasties for AMI per year showed a lower mortality as compared to the others. In clinical practice at large community hospitals results of angioplasty for AMI concerning mortality, complications and technical success rate are comparable to those of highly specialised centres. The absolute numbers of angioplasties for AMI increased constantly over the years.     

Kinetocardiotocography--follow-up of diazepam poisoning

Report about continuous fetal monitoring of heart rate and movements 9-30 hours after diazepam-intoxication. in an early stage after intoxication fetal movements are associated with FHF-decelerations, later on with accelerations. A sinusoidal like pattern may be caused by fetal suckling movements.     

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.