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.     

Evaluation of Atmospheric and Vacuum Residues Using Molecular Distillation and Optimization

The term atmospheric residue describes the material at the bottom of the atmospheric distillation tower having a lower boiling point limit of about 340°C; the term vacuum residue (heavy petroleum fractions) refers to the bottom of the vacuum distillation, which has an atmospheric equivalent boiling point (AEBP) above 540°C. In this work, the objective is to evaluate the behavior of different kinds of Brazilian atmospheric and vacuum residues using molecular distillation. The Falling Film Molecular Distillator was used. For the results obtained through this process, a significant range of temperature can be explored avoiding the thermal decomposition of the material. So these results are very important to the refinery decisions and improvements. The Experimental Factorial Design results showed that the temperature has more influence on the process than the feed flow rate, when a higher percentage of distillate is required.     

COPD: using nutrition to prevent respiratory function decline

Close to three-fourths of patients with chronic obstructive pulmonary disease (COPD) suffer from weight loss. Identifying a single cause for this is difficult, as several factors-including chronic mouth breathing, dyspnea, aerophagia, certain medications, and depression-often act in concert. Malnutrition can exacerbate symptoms of COPD by decreasing ventilatory muscle strength, exercise tolerance, and immunocompetence, and by increasing the risk of depression and anxiety. Goals of nutrition intervention are to prevent or reverse malnutrition without worsening the disease process and to improve respiratory function, thereby reducing morbidity and delaying mortality. Recommendations for intake of fats, carbohydrates, protein, and water must be individualized.     

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.     

Stabilisation of protein and oil in fish silage for use as a ruminant feed supplement

Fish silage was prepared from 96.5% offal obtained from Atlantic cod fillet processing and 3.5% (by wt) formic acid. The fish silage became an homogeneous liquid of low viscosity in 36–58 h at 20°C; however, the oil and protein components continued to be hydrolysed for several months. Addition of formaldehyde to fish silage (0.25 or 0.39% by wt) after liquefaction was complete, served to prevent continued protein hydrolysis and oxidative rancidity of the oil. It also decreased the development of ‘off-odours’, and the formation of total volatile bases. Fish silage was readily absorbed by hay at a ratio of 1.5 parts silage to 1.0 parts hay by weight; the product is referred to as ‘haylage’. Voluntary intake by wethers of ‘haylage’ prepared from de-oiled, formaldehyde-treated fish silage was better than for ‘haylage’ prepared from untreated fish silage. The crude protein content of ‘haylage’ rations was more than satisfactory for fattening lambs, although the energy content of ‘haylage’ rations may be a limiting factor. Retention of fish oil in the silage to increase the energy content of rations was unsuccessful because voluntary feed intake was depressed. The addition of formaldehyde to fish silage is advantageous when the product is to be fed to ruminant livestock since protein and lipid degradation in the feed, and also possibly in the rumen, are minimised and voluntary intake of ‘haylage’ by sheep is increased.     

Phosphorus Inputs to Lake Simcoe from 1990 to 2003: Declines in Tributary Loads and Observations on Lake Water Quality

Total phosphorus (TP) inputs to Lake Simcoe have led to hypolimnetic dissolved oxygen (DO) depletion and loss of cold water fish habitat. Since 1990, efforts have been made to reduce the total TP input to the lake below a defined target of 75 t/year, which was predicted to lead to reductions in spring TP concentration and improvements in end-of-summer hypolimnetic DO concentrations. The total TP load to the lake during the most recent period of record (1998/99-2003/04) ranged from 53 to 76 t/yr and averaged 67 t/yr, compared to an average of 114 t/yr estimated between 1990/91 and 1997/98 (range 85-157 t/yr). Reductions in TP loads from the catchment via tributary discharge (∼26 t) accounted for the majority of the decrease in total load between the two time periods. Total P concentrations decreased significantly in four out of six long-term monitored tributaries; however, concentrations in all six tributaries remain above the level recommended to avoid nuisance plant growth (30 μg/L). Although TP loads to the lake are currently below the target 75 t/yr, excessive growths of filamentous algae and macrophytes continue to be a problem in the nearshore zone. End-of-summer minimum hypolimnetic DO concentrations (average 4.3 mg/L, 1998/99-2003/04) remain substantially below the level (7 mg/L) that is considered protective of lake trout. Efforts to reduce TP loads to the lake therefore need to continue.