A convenient method to discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in human liver microsomes

Liver microsomes are a frequently used probe to investigate the phase I metabolism of xenobiotics in vitro. Structures containing nucleophilic hetero-atoms are possible substrates for cytochrome P450 enzymes (P450) and flavin-containing monooxygenases (FMO). Both enzymes are located in the endoplasmatic reticulum of hepatocytes and both need oxygen and NADPH as cofactors. The common method to distinguish between the two enzyme systems is to use the thermal inactivation of FMO and to inhibit P450 completely with carbon monoxide, N-octylamine or N-benzylimidazole. In the literature no indication could be found that the heat inactivation of FMO does not affect any of the human P450 enzymes or that the overall P450 inhibitors inhibit the different human P450 enzymes sufficiently and do not affect the FMO. The effect of N-benzylimidazole and heat inactivation was tested on specific activities of seven P450 enzymes in human liver microsomes, 1A2, 2A6, 2C9, 2C19, 2D6, 3A4/5, and 2E1, using methoxyresorufin O-demethylation, coumarin 7-hydroxylation, (S)-warfarin 4-hydroxylation, (S)-(+)-mephenytoin 4-hydroxylation, dextrometorphan O-demethylation, oxidation of denitronifedipine, and chlorzoxazone 6-hydroxylation respectively. The sulfoxidation of methimazole (MMI) was used as a specific probe for the determination of FMO activity. Methimazole sulfoxidation was compared with the well known assay for FMO metabolism, the formation of N,N-dimethylaniline (DMA) N-oxide, to be confirmed as an exclusively FMO mediated reaction. The participation of P450 and FMO in the sulfoxidation of four sulfur containing peptides, ametryne; terbutryne, prometryne and methiocarb was investigated using human liver microsomes. All four reactions were demonstrated to be catalysed predominantly by cytochrome P450.     

Aggregation dynamics of particles in a microchannel due to an applied magnetic field

Functionalized magnetic microspheres have promising applications in different microfluidic devices including MEMS-scale biosensors. These particles exhibit magnetic field-induced aggregation, which can be harnessed to achieve several practical tasks in microfluidic devices. For this, the particle aggregation needs to be well characterized. Herein, a numerical simulation and experimental validation of particle-chaining is presented. Simulations show that the particle aggregation time scales linearly with a group parameter. The predicted growth of one- two- and three-particle chains with time shows a similar trend as that found in the experiments. The results of the study could help predicting the performance of magnetic aggregate-based lab-on-a-chip devices.     

Zur Analytik von Zuckercouleuren I. Mitteilung: Differenzierung der Couleurklassen mittels Curiepunkt-Pyrolyse-Capillargaschromatographie-Massenspektrometrie

Summary After an introduction on the production, classification, legislative regulations, toxicology, and analysis of caramel colours, a report is given on the examination of these colourings by Curie-point pyrolysis-capillary gas chromatography-mass spectrometry. This method enables the differentiation between the four classes of caramel colours on the basis of the most concentrated of more than 100 identified pyrolysis products, which requires small quantities of substance (100 g) and short periods of time (1 h).

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Zur Analytik von Zuckercouleuren I. Mitteilung: Differenzierung der Couleurklassen mittels Curiepunkt-Pyrolyse-Capillargaschromatographie-Massenspektrometrie

Zusammenfassung Nach einer Einführung zur Herstellung, Klassifizierung, rechtlichen Stellung, Toxikologie und Analytik der Zuckercouleure wird über die Untersuchung dieser Farbstoffe mittels Curiepunkt-Pyrolyse-Capillargaschromatographie-Massenspektrometrie berichtet. Anhand der konzentrationsstärksten der über 100 identifizierten Pyrolyseprodukte ist es möglich, die vier Couleurklassen bei geringem Substanz- (100 g) and Zeitbedarf (1 h) sicher zu differenzieren.

     

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.     

Unidirectional bubble growth in microchannels with asymmetric surface features

The growth of vapor bubbles is studied numerically in a microchannel with asymmetric surface features. The channel design is chosen such that evaporation results in vapor bubbles growing only along a predefined direction. The principle relies on capillary forces and the pinning/depinning of three-phase contact lines at sharp edges of the wall geometry. Analytical expressions are derived predicting the direction of bubble growth and allowing to assess the robustness of a specific channel geometry in terms of supporting unidirectional bubble growth. From these expressions design rules for microchannels incorporating geometrical parameters and the wall contact angle of the liquid phase can be derived. The numerical calculations are performed based on an extended Volume-of-Fluid method accounting for phase change. The results confirm that under specific conditions, vapor bubbles only expand in one direction, thereby corroborating the analytical model. The presented concept may find applications in designing microchannels for stabilized flow boiling or micropumps/-actuators relying on phase change.     

Ignition characteristics of gasless reactions

Initiation energy requirements for condensed phase systems were determined experimentally and analytically and found to obey a diffusion-limited reaction model. A model is described for the evaluation of the threshold input energy either by computer calculations or by approximate formulas. The use of dimensionless variables greatly reduces the necessary number of computer runs and simplifies the derivation of explicit expressions. The results are useful both for parametric study and theoretical prediction. It was found that initiation is promoted by low thermal conductance, low heat capacity, small particle sizes, and high heat of reaction. This method of analysis can also assess the effects of varying the energy deposition time. The predicted data are in reasonable agreement with experimental results which were obtained using pulsed and continuous wave lasers. The theoretical model is expected to be useful in the study of reaction phenomena associated with thermite and intermetallic pyrotechnics as well as in the study of certain metallurgical processes.     

The Foreign-Language Press in American Press History

A review of U.S. press histories reveals a common failure to consider immigration and the formation of an ethnic press as constituent elements of an American culture and addresses the need for a cultural history of the media that breaks with current practices.