The Anticancer Drug Ellipticine Activated with Cytochrome P450 Mediates DNA Damage Determining Its Pharmacological Efficiencies: Studies with Rats,Hepatic Cytochrome P450 Reductase Null (HRN?) Mice and Pure Enzymes

Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b₅ alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b₅, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b₅, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.     

Simulierte Gegenstromchromatographie

Simulated Countercurrent Chromatography. This paper reviews simulated countercurrent chromatography, a separation process that has received only little attention. In contrast to standard batchwise operated chromatography, a periodical mode of operation is used. The feed and collection lines are shifted along the column, thus simulating countercurrent operation of the liquid phase and the stationary solid phase. As a result, higher product yield and higher product purity are obtained. At the same time the amount of solid phase and solvent phase required in comparison to discontinuous chromatography is reduced. Industrial applications have been developed since 1964 by Universal Oil Products (UOP). Large scale separation processes in the petrochemical and the sugar industries are already in operation. On the bench scale this process is used to separate biotechnological products and pharmaceuticals. The need to solve difficult separation problems (isomeric or racemic mixtures, biomolecules, polymers) will encourage further developments. Together with improved process-simulation tools and process control, simulated countercurrent chromatography will be established as a powerful separation technique with widespread industrial applicability.     

Management of hypoparathyroidism during pregnancy--report of twelve cases

Rats exposed to Pneumocystis carinii mount antibody responses to a broad band migrating on western blot with an apparent molecular weight of 45-55 kDa. One antigen within this band, designated p55, is uniformly recognized by P. carinii exposed rats. Although the gene encoding the p55 antigen had been previously cloned, the location of this antigen within the organism was unknown. Prior attempts to localize the protein were unsuccessful. A monospecific polyclonal antiserum raised against a carboxyl-terminal 15-oligomer peptide yielded specific reactivity with a single 55 kDa band on a western blot of P. carinii. Using this antiserum, little to no reactivity could be detected with P. carinii organisms by immunofluorescence assay (IFA). However, zymolyase treatment of P. carinii dramatically increased the intensity and proportion of organisms reactive by IFA. Zymolyase, an enzyme with beta-1,3 glucanase activity, has previously been shown to remove the electron dense outer layer of the P. carinii cell wall, exposing an electron lucent layer. Immunoelectron microscopy performed on zymolyase treated organisms showed the majority of labeling occurs within the cell wall.     

In-plane neutron radiography for studying the influence of surface treatment and design of cathode flow fields in direct methanol fuel cells

The influence of surface treatment and design of cathode flow fields in direct methanol fuel cells was investigated by in-plane neutron radiography and measurements of cell performance and pressure drop along the cathode channels. A specially designed test cell and neutron radiography set-up allows for studying the water distribution in an in-plane viewing direction. A temporal resolution of down to 10 s was used while an image resolution of approximately 80 μm could be obtained. The cathode flow fields were either impregnated by a hydrophobizing or hydrophilizing agent or left untreated. It turned out that hydrophobic channel walls lead to the formation of large water droplets, which partially block the air flow in the cathode channels. Their periodical growth and discontinuous removal leads to an unstable and fluctuating operation. Hydrophilized cathode flow fields, on the other hand, ensure a stable operation due to removal of excess water by a continuous water film. Two different cell designs including untreated cathode flow fields with either dual-channel or grid design were compared. The grid flow field was superior with regard to the stability of cell performance and less prone to the formation and removal of water droplets.     

Water Evolution in Direct Methanol Fuel Cell Cathodes Studied by Synchrotron X‐Ray Radiography

Water evolution, distribution, and removal in the cathodes of a running direct methanol fuel cell were investigated by means of synchrotron X‐ray radiography. Radiographs with a spatial resolution of around 5 μm were taken every 5 s. Special cell designs allowing for through‐plane and in‐plane viewing were developed, featuring two mirror‐symmetrical flow field structures consisting of one channel with the through‐plane design. Evolution and discharge of water droplets and the occurrence of water accumulations in selected regions of the channels were investigated. These measurements revealed a nonuniform distribution of water in the channels. Both irregular and periodic formation of water droplets were observed. In‐plane measurements revealed, that the droplets evolve between adjacent carbon fiber bundles of the gas diffusion layer. The water distribution within the channel cross‐section fits very well to the pressure difference between cathode channel inlet and outlet. The quick discharge of water droplets causes sudden decreases of the pressure difference up to 4.5 mbar.     

Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1

Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H:quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using ³²P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast, the formation of AAI-DNA adducts was catalyzed by CYP1B1 with the A133S mutation. Our experimental model confirms the importance of the hydroxyl group possessing amino acids in the active center of CYP1A1 and 1A2 for AAI nitroreduction.     

Evidence-based medicine: quality and comparability of clinical trials investigating the efficacy of prostaglandin F(2α) for the treatment of bovine endometritis

The objective of this study was to evaluate the quality and comparability of published literature, and to summarize the effect of prostaglandin F(2α) (PGF(2α)) for the treatment of endometritis. It has been postulated that there is a dearth of high-level evidence-based research results in veterinary medicine. Also, there is a marked variation in the quality of studies in veterinary and animal science. Post-partum uterine infections occur commonly in dairy cattle and are reported to have a negative impact on reproductive performance. A comprehensive literature search was conducted utilizing online databases revealing a total of 2723 references. After applying specific exclusion criteria, a total of 68 trials were eligible for further analysis. These articles were evaluated utilizing specific parameters listed in an evaluation form such as randomization and the involvement of control groups. The analysis revealed that more than half of the trials (51·5%) were at least 20 years old. Furthermore, we found that about one third (36·8%) of all trials were controlled and randomized, while 3 of those (4·4%) were also blinded. Of those trials which calculated a calving-to-conception interval (n=30), 50% of the authors claimed an improvement, which was statistically significant in 23·3% of the cases. We conclude that there is a wide discrepancy between research results investigating the efficacy of PGF(2α).