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.     

Contribution of the Internal and External Oxygen to the Oxidation of Microencapsulated Fish Oil

Microencapsulation aims to protect polyunsaturated fatty acids against oxidation by embedding oil droplets in a solid matrix. In such a system the internal (dissolved and entrapped) and external (in the environment) oxygen can be differentiated. The study aims to quantify the impact of both oxygen sources on the oxidation of microencapsulated fish oil. The impact of the solubilized oxygen in bulk fish oil is investigated by saturating the oil with nitrogen, synthetic air, and pure oxygen. Even though more dissolved oxygen results in more oxidation products, the difference between the oxidation of the nitrogen and air saturated oil is significant but low. For encapsulated fish oil powders, the internal oxygen is modified by preparing oil‐in‐water emulsions under atmospheric and inert conditions. The feed is atomized and spray dried with either nitrogen or air. Powders are stored under vacuum and in vials and the hydroperoxides and anisidine value are determined in the total‐ and encapsulated oil. The internal oxygen has a minor impact, whereas the external oxygen is the main determinant for autoxidation. Apart from oxidizing the non‐encapsulated oil, the external O₂ penetrates into the particle and reacts with the encapsulated oil. Practical Applications: Comparing the contribution of the internal and external oxygen to the oxidative stability shows that the internal O₂ plays a minor role and can be neglected. This means that the emulsion preparation as well as the spray drying process can be conducted under ambient conditions. An inert production is not extending the shelf life significantly as long as the external O₂ determines oxidation. The focus should be on optimizing the diffusion barrier properties of the wall matrix to reduce the penetration of the external oxygen into the particle system. Alternatively, packaging solution reducing the external O₂ will extend the shelf life of the microencapsulated oil.     

Validity of the PAS-ADD for detecting psychiatric symptoms in adults with learning disability (mental retardation)

The Psychiatric Assessment Schedule for Adults with Developmental Disability (PAS-ADD) is a semi-structured interview for use with respondents who have learning disability and for key informants. This report investigates the ability of the instrument to detect symptoms that had been found to exist during routine clinical assessment of the patients. Field trials involved 95 referred patients with learning disability and a key informant for each sample member. Clinical opinions of the referring psychiatrists were sought using a symptom checklist. Referrer checklist symptoms and PAS-ADD data were both factor analysed. Validity testing involved (a) computation of correlations between PAS-ADD factors and checklist data and (b) comparison of PAS-ADD and referrers' diagnoses. Results indicated good validity for the PAS-ADD in relation to psychotic symptoms and depressive symptoms. Anxiety symptom identification was not well validated, probably due to small numbers. Expansive mood identified by the referrers was not detected by the PAS-ADD because there is currently no corresponding section in the interview. Where the PAS-ADD produced a diagnosis (in 58 members of the sample), 44 were in agreement with the referrer. Probability of diagnosis by PAS-ADD increased with the number of relevant active symptoms identified by the referrer. The PAS-ADD has been shown in a previous report to have the sensitivity to detect mental disorders not known to psychiatric services. For psychotic and depressive conditions, our results showed that symptom detection was in good agreement with the information provided by the referring psychiatrists on their patients. The PAS-ADD needs a section on hypomania and further investigation of its detection of anxiety disorders.     

Cervical lymph nodes metastases from a pituitary carcinoma

Ipratropium bromide is a synthetic derivative of atropine with little absorption when used in inhalation, and therefore little secondary effects. The authors review its pharmacological properties and therapeutic efficacy in the treatment of asthma in children. Combined nebulized inhalation of ipratropium bromide and beta 2 sympathomimetic results in a more efficient and more sustained bronchodilatation than beta 2 sympathicomimetic alone in the treatment of acute asthma in children. Ipratropium bromide should be usefully introduced in the therapeutic scheme of acute asthma in children. Further studies will be necessary in order to determine its efficacy and tolerance in infants.     

Finite-difference time-domain simulation of scattering from objectsin continuous random media

A three-dimensional (3D) finite-difference time-domain (FDTD) scheme is introduced to model the scattering from objects in continuous random media. FDTD techniques have been previously applied to scattering from random rough surfaces and randomly placed objects in a homogeneous background, but little has been done to simulate continuous random media with embedded objects where volumetric scattering effects are important. In this work, Monte Carlo analysis is used in conjunction with FDTD to study the scattering from perfectly electrically conducting (PEC) objects embedded in continuous random media. The random medium models under consideration are chosen to be inhomogeneous soils with a spatially fluctuating random permittivities and prescribed correlation functions. The ability of frequency averaging techniques to discriminate objects in this scenarion is also briefly investigated. The simulation scheme described in this work can be adapted and used to help in interpreting the scattered field data from targets in random environments such as geophysical media, biological media, or atmospheric turbulence     

Ultra-strong, well-apodised Bragg gratings in chalcogenide rib waveguides

The first ultra-strong, near-perfect, raised-apodised Bragg gratings in As/sub 2/S/sub 3/ chalcogenide rib waveguides using /spl lambda/=532 nm light and a modified Sagnac holographic writing setup are demonstrated. Good agreement is achieved between the experimental results and the numerical modelling of the gratings using the transfer matrix analysis for thin film structures.     

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.