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.     

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.     

Inhalation toxicity of 1,6-hexanediamine dihydrochloride in F344/N rats and B6C3F1 mice

1,6-Hexanediamine (HDA) is a high production volume chemical which is used as an intermediate in the synthesis of paints, resins, inks, and textiles and as a corrosion inhibitor in lubricants. Two- and 13-week studies of the toxicity of the dihydrochloride salt of HDA (HDDC) were conducted in male and female Fischer 344/N rats and B6C3F1 mice using whole-body inhalation exposure. Both species were evaluated for histopathologic and reproductive effects, and rats were examined for clinical chemistry and hematologic changes. In the 2-week inhalation studies, animals were exposed to 10-800 mg HDDC/m3, 6 hr per day. All rats, all female mice, and two of five male mice in the high-exposure group died before the end of the study. Surviving mice in this group had a dose-dependent depression in body weight gain. Clinical signs were primarily related to upper respiratory tract irritation and included dyspnea and nasal discharge in both species. Treatment-related histopathologic lesions included inflammation and necrosis of the laryngeal epithelium of both species and the tracheal epithelium of mice, as well as focal inflammation and ulceration of the respiratory and olfactory nasal mucosa. In the 13-week inhalation studies, animals were exposed to HDDC at concentrations of 1.6-160 mg/m3 for 6 hr per day, 5 days per week. In addition to the base study groups, a supplemental group of rats at each exposure level was included to assess the effect of HDDC on reproduction. No treatment-related changes in organ weights or organ-to-body-weight ratios occurred in rats, and no treatment-related clinical signs or gross lesions were seen in either species. Chemical-related microscopic lesions were limited to the upper respiratory tract (larynx and nasal passages) in the two highest exposure groups and were similar in both species. These lesions included minimal to mild focal erosion, ulceration, inflammation, and hyperplasia of the laryngeal epithelium, in addition to degeneration of the olfactory and respiratory nasal epithelium. HDDC caused no significant changes in sperm morphology or vaginal cytology and no significant adverse effects on reproduction in rats or mice. Hematologic and clinical chemistry changes in rats were minor and sporadic and were not accompanied by related histologic findings. HDDC did not increase the frequency of micronucleated erythrocytes in mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Isolation and Spectroscopic Characterization of Eu@C72

A new Eu-endohedral fullerene cage, namely, Eu@C₇₂, was synthesized, separated, and a single isomer, Eu@C₇₂-I, has been isolated by multi-step HPLC for the first time and characterized by LDI-TOF mass spectrometry, UV-Vis and EPR spectroscopy. Additionally, a second isomer, Eu@C₇₂-II, has been separated by HPLC and identified by LDI-TOF mass spectrometry.     

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.