Protein microarray assay for the screening of SH3 domain interactions

Analysis of cellular signal transduction processes increasingly focuses on the systematic characterization of complete protein interaction networks. Understanding the interplay of signaling components enables insight into the molecular basis of diverse diseases such as cancer. This paves the way for the rational design of specific therapeutics. Protein interactions are often mediated by conserved modular domains, e.g., SH3-domains, which recognize proline-rich sequences in their cognate ligands. In the course of this study, different microarray formats (reactive silane monolayers and nitrocellulose on glass slides) and assay work flows were evaluated to develop a microarray based screening assay that permits the reliable identification of interactions between certain target proteins with a set of SH3 domains. Nine representative SH3 domains which were produced and purified as GST-fusion proteins were spotted on the microarray substrates and probed with two well-characterized ligands, the Nef protein from HIV-1 and the human protein Sam68. The best results from these low-density model arrays were obtained with nitrocellulose slides. We show that a straightforward and highly robust detection of ligand binding is achieved by staining with a fluorescently labeled antibody directed against the N-terminal His-tag attached to these proteins. The optimized assay protocol reported here allows for the identification of SH3-interactions with high reproducibility and adequate signal-to-background and signal-to-noise ratios, as well as the quantitative determination of relative binding affinities.     

Analysis of special surfactants by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry

Multidimensional gas-chromatographic analyses of olesochemically based nonionic, anionic and several cationic surfactants in industrial cleaners are demonstrated. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry allows the simultaneous determination of fatty alcohols, fatty alcohol sulphates and alkyl polyglucosides. In addition, the determination of fatty alcohol ethoxylates up to C₁₀EO₈ (highest degree of ethoxylation) and C₁₈EO₅ (longest C-chain at an ethoxylation degree of five) and the analysis of fatty alcohol alkoxylates that contain ethoxy (EO) and propoxy (PO) groups could be realized. Because of decomposition in the injector and a weak EI-fragmentation, cationic surfactants such as alkyl benzyl dimethyl ammonium chloride could also be identified by their characteristic fragments. Thermogravimetric analyses confirmed that the temperature in a normal GC injector is not high enough to cause thermal decomposition of esterquats. However, we could demonstrate that a modified silylation procedure forms decomposition products of esterquats in the GC injector which are detectable by GC × GC–(TOF)MS and allows the identification of such GC-atypical analytes.     

Asymptotic Palm likelihood theory for stationary point processes

In the present paper, we propose a Palm likelihood approach as a general estimating principle for stationary point processes in $\mathbf{R}^d$ for which the density of the second-order factorial moment measure is available in closed form or in an integral representation. Examples of such point processes include the Neyman–Scott processes and the log Gaussian Cox processes. The computations involved in determining the Palm likelihood estimator are simple. Conditions are provided under which the Palm likelihood estimator is strongly consistent and asymptotically normally distributed.     

Highly efficient synthesis of enantiopure diacetylated C(2)-symmetric diols by ruthenium- and enzyme-catalyzed dynamic kinetic asymmetric transformation (DYKAT)

Highly efficient synthesis of enantiopure diacetates of 2,4-pentanediol and 2,5-hexanediol starting from commercially available mixtures of the diols (dl/meso approximately 1:1) has been realized by combining a fast ruthenium-catalyzed epimerization with an enzymatic transesterification. The in situ coupling of these two processes produces the diacetates in high yield in >99 % enantiomeric excess.     

Effects of lactate and acetate on the determination of serum ethyl glucuronide by CZE

The analysis of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in serum with an optimized CZE assay is reported. The method uses a 0.1-mm id fused-silica capillary of 50 cm effective length that is coated with linear polyacrylamide, a pH 4.4 nicotinic acid/epsilon-aminocaproic acid (EACA) BGE, reversed polarity and indirect analyte detection. The assay is based on a 1:1 dilution of serum with deionized water and has LODs for EtG, lactate and acetate of 3.8 x 10(-7) M, 2.60 x 10(-6 )M and 2.18 x 10(-6 )M, respectively. Separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) and its quantification are shown to be possible for a wide range of lactate (stacker) and acetate (destacker) concentrations, macrocomponents that have an impact on the CZE behavior of EtG and that change after intake of ethanol. The assay has been successfully applied to the analysis of EtG, lactate and acetate in (i) sera of volunteers that ingested known amounts of alcohol and (ii) samples of patients that were classified (teetotalers and social drinkers vs. alcohol abusers) via analysis of carbohydrate-deficient transferrin.     

Redox-active silica nanoparticles. Part 1. Electrochemistry and catalytic activity of spherical, nonporous silica particles with nanometric diameters and covalently bound redox-active modifications

Nonporous spherical silica nanoparticles resulting from a controlled St?ber process are covalently surface modified with redox-active molecules. Ferrocene, a ruthenium(II) complex with an N2P2Cl2 ligand set, and a sterically hindered biphenylamine are used as modifiers. The resulting materials are characterized by physical, spectroscopic, electrochemical, and chemical methods. The cyclic voltammetric behavior is studied in detail and reveals effects of charge transport by electron hopping along the surface of particles adsorbed on a Pt electrode. The ruthenium(II) complex remains catalytically active with respect to hydrogenation upon immobilization on the particles. Thus, the respective material provides a heterogenized homogeneous hydrogenation catalyst on a solid support.     

A new approach to determine method detection limits for compound-specific isotope analysis of volatile organic compounds

Compound-specific isotope analysis (CSIA) has been established as a useful tool in the field of environmental science, in particular in the assessment of contaminated sites. What limits the use of gas chromatography/isotope ratio mass spectrometry (GC/IRMS) is the low sensitivity of the method compared with GC/MS analysis; however, the development of suitable extraction and enrichment techniques for important groundwater contaminants will extend the fields of application for GC/IRMS. So far, purge and trap (P&T) is the most effective, known preconcentration technique for on-line CSIA with the lowest reported method detection limits (MDLs in the low microg/L range). With the goal of improving the sensitivity of a fully automated GC/IRMS analysis method, a commercially available P&T system was modified. The method was evaluated for ten monoaromatic compounds (benzene, toluene, para-xylene, ethylbenzene, propylbenzene, isopropylbenzene, 1,2,3-trimethylbenzene, 1,2,4-trimethylbenzene, 1,3,5-trimethylbenzene, fluorobenzene) and ten halogenated volatile organic compounds (VOCs) (dichloromethane, cis-1,2-dichloroethene, trans-1,2-dichloroethene, carbon tetrachloride, chloroform, 1,2-dichloroethane, trichloroethene, tetrachlorethene, 1,2-dibromoethane, bromoform). The influence of method parameters, including purge gas flow rates and purge times, on delta13C values of target compounds was evaluated. The P&T method showed good reproducibility, high linearity and small isotopic fractionation. MDLs were determined by consecutive calculation of the delta13C mean values. The last concentration for which the delta13C value was within this iterative interval and for which the standard deviation was lower than +/-0.5 per thousand for triplicate measurements was defined as the MDL. MDLs for monoaromatic compounds between 0.07 and 0.35 microg/L are the lowest values reported so far for continuous-flow isotope ratio measurements using an automated system. MDLs for halogenated hydrocarbons were between 0.76 and 27 microg/L. The environmental applicability of the P&T-GC/IRMS method in the low-microg/L range was demonstrated in a case study on groundwater samples from a former military air field contaminated with VOCs.     

Direct simultaneous determination of eight sweeteners in foods by capillary isotachophoresis

A method for isotachophoretic determination of sweeteners of different character in candies and chewing gums was developed. A capillary of 0.8 mm ID and 90 mm effective length made of fluorinated ethylene-propylene copolymer is filled with an electrolyte system consisting of 10 mM HCl + 14 mM Tris, pH 7.7 (leading electrolyte) and 5 mM L-histidine + 5 mM Tris, pH 8.3 (terminating electrolyte). The analysis is performed at a driving current of 200 microA and for detection current is decreased to 100 microA. Boric acid is added to the aqueous sample solution to form borate complexes with substances of polyhydroxyl nature and make them migrate isotachophoretically. Using conductivity detection, the calibration curves in the tested concentration range up to 2.5 mM were linear for all components of interest: acesulfame K, saccharine, aspartame, cyclamate, sorbitol, mannitol, lactitol, and xylitol. The concentration detection limits ranged between 0.024 and 0.081 mM. Good precision of the ITP method is evidenced by favorable RSD values ranging from 0.8 to 2.8% obtained at the analyte concentration of 1.0 mM (n = 6). The analysis time was about 20 min. Simplicity, accuracy, and low cost of analyses make ITP an alternative procedure to methods used so far for the determination of ionizable sweeteners.     

Structure of cyclic nucleoside phosphonate ester prodrugs: an inquiry

The configuration at phosphorus in cyclic (S)-HPMPC (1, cidofovir) and (S)-HPMPA (2) phenyl ester (5 and 6, respectively) diastereomers ((R(p))-5, (R(p))-6, (S(p))-6) was determined by X-ray crystallography and correlated to their (1)H and (31)P NMR spectra in solution. (R(p))-5 and (R(p))-6 have chair conformations with the nucleobase substituent equatorial and the P-OPh axial. Perhaps surprisingly, (S(p))-6 is (a, a) in the crystal and exists largely as an equilibrium of (a, a)/(e, e) conformers in chloroform or acetonitrile.