HPLC determination of arginases inhibitor N-(ω)-hydroxy-nor-L-arginine using core-shell particle column and LC-MS/MS identification of principal metabolite in rat plasma

For the purpose of in vivo pharmacokinetic studies, an HPLC method was developed and validated for the quantification of N-(ω)-hydroxy-nor-L-arginine, L-arginine and N-(ω)-ethyl-L-arginine (internal standard) in rat plasma. Sample processing involved a solid-phase extraction on the Waters MCX cartridges and on-line pre-column derivatization of the analytes with o-phthaldialdehyde and 3-mercaptopropionic acid. Separation of the derivatives was carried out on a core-shell Kinetex C18 column in a gradient elution mode with a mobile phase consisting of methanol and water (pH=3.00 adjusted with formic acid). Fluorimetric detection with the excitation/emission wavelengths of 235/450 nm was used. The method was validated according to the FDA guidelines and applied to pilot pharmacokinetic experiments. An unknown metabolite was extracted from the plasma of Wistar rats after a single bolus of N-(ω)-hydroxy-nor-L-arginine (i.v. 10 mg kg(-1)). The metabolite was identified as nor-L-arginine using mass spectrometry. Validated method was successfully used for pilot pharmacokinetic experiment on rats.     

New monodisperse magnetic polymer microspheres biofunctionalized for enzyme catalysis and bioaffinity separations

Magnetic macroporous PGMA and PHEMA microspheres containing carboxyl groups are synthesized by multi-step swelling and polymerization followed by precipitation of iron oxide inside the pores. The microspheres are characterized by SEM, IR spectroscopy, AAS, and zeta-potential measurements. Their functional groups enable bioactive ligands of various sizes and chemical structures to couple covalently. The applicability of these monodisperse magnetic microspheres in biospecific catalysis and bioaffinity separation is confirmed by coupling with the enzyme trypsin and huIgG. Trypsin-modified magnetic PGMA-COOH and PHEMA-COOH microspheres are investigated in terms of their enzyme activity, operational and storage stability. The presence of IgG molecules on microspheres is confirmed.     

Tumor necrosis factor-alpha potentiates genotoxic effects of benzo[a]pyrene in rat liver epithelial cells through upregulation of cytochrome P450 1B1 expression

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant, which may contribute to the development of human cancer. The ultimate carcinogenic BaP metabolite produced by cytochrome P450 enzymes (CYP), such as CYP1A1 and CYP1B1, anti-BaP-7,8-diol-9,10-epoxide, binds covalently to DNA and causes mutations. The levels of various CYP isoforms can be significantly modulated under inflammatory conditions. As the chronic inflammation is known to contribute to carcinogenesis, we investigated interactions of a major proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), and BaP in regulation of the expression of CYP1A1/1B1 and induction of DNA damage in rat liver epithelial WB-F344 cells. TNF-alpha enhanced induction of CYP1B1, while it simultaneously suppressed the BaP-induced CYP1A1 expression. The observed deregulation of CYP1 induction was found to be associated with a significantly enhanced formation of DNA adducts. The elevated DNA damage corresponded with increased phosphorylation of p53 tumor suppressor at Ser-15 residue, enhanced accumulation of cells in the S-phase of cell cycle and potentiation of BaP-induced apoptosis. Inhibition of CYP1B1 by fluoranthene significantly decreased both the formation of DNA adducts and the induction of apoptosis in WB-F344 cells treated with BaP and TNF-alpha, thus suggesting that this isoform might be responsible for genotoxic effects of BaP in nonparenchymal liver cells. Our results seem to indicate that inflammatory conditions might enhance genotoxic effects of carcinogenic polycyclic aromatic hydrocarbons through upregulation of CYP1B1 expression.     

Niche differentiation between diploid and hexaploid <Emphasis Type="Italic">Aster amellus</Emphasis>

The maintenance of separated diploid and polyploid populations within a contact zone is possible due to both prezygotic and postzygotic isolation mechanisms. Niche differentiation between two cytotypes may be an important prezygotic isolating mechanism and can be studied using reciprocal transplant experiments. We investigated niche differentiation between diploid and hexaploid Aster amellus in their contact zone in the Czech Republic. Diploid populations are confined to habitats with low productivity, whereas hexaploid populations occur in habitats with both low and high productivity. Thus, we chose three diploid populations and six hexaploid populations, three in each of the two different habitat types. We analyzed habitat characteristics and carried out reciprocal transplant experiments in the field using both seeds and adult plants. Sites of diploid and hexaploid populations differed significantly in vegetation and soil properties. The mean number of juveniles was higher at sites of home ploidy level than at sites of foreign ploidy level, suggesting niche differentiation between the two cytotypes. On the other hand, transplanted adult plants survived at all sites and juvenile plants were able to establish at some sites of the foreign cytotype. Furthermore, the mean number of juveniles, survival, and flowering percentages were higher at home sites than at foreign sites, indicating local adaptation. We conclude that niche differentiation between the two cytotypes and local adaptation within each cytotype may contribute to the maintenance of diploid and hexaploid populations of A. amellus in their contact zone. Moreover, further factors, such as differences in flowering phenology and exclusion of minority cytotypes, should also be considered.     

Differences in AM fungal root colonization between populations of perennial Aster species have genetic reasons

We tested the hypothesis whether differences between plant populations in root colonization by arbuscular mycorrhizal (AM) fungi could be caused by genetic differentiation between populations. In addition, we investigated whether the response to AM fungi differs between plants from different populations and if it is affected by the soil in which the plants are cultivated. We used Aster amellus, which occurs in fragmented dry grasslands, as a model species and we studied six different populations from two regions, which varied in soil nutrient concentration.We found significant differences in the degree of mycorrhizal colonization of plant roots between regions in the field. To test if these differences were due to phenotypic plasticity or had a genetic basis, we performed a greenhouse experiment. The results suggested that Aster amellus is an obligate mycotrophic plant species with a high dependency upon mycorrhiza. Plant biomass was affected only by soil, and not by population or the interaction between the population and the soil. Mycorrhizal colonization was significantly affected by all three factors (soil, population, interaction of soil and population). Plants from the population originating from the soil with lower nutrient availability developed more mycorrhiza even when grown in soil with higher nutrient availability. The correspondence between mycorrhizal colonization of plants in the field and in both soils in the pot experiment suggests that the observed differences in root colonization have a genetic basis.     

Development and application of a miniaturized gel electrophoresis device for protein analysis

The present article describes a miniaturized polyacrylamide slab gel electrophoresis-chip (PASGE-Chip) that can rapidly separate a set of predefined samples as well as cell lysate samples for clinical diagnosis. The chip consists of a polymethyl methacrylate (PMMA) upper unit (25 x 30 x 10 mm, width x length x depth) with integrated buffer chambers, running electrodes and loading wells and a bottom unit comprising a silicon dioxide-coated silicon plate with embossed gel chamber (11 x 15 x 0.37 mm). This miniaturized device was designed to be fast, easy to use and cheap to produce. The polyacrylamide slab gel electrophoresis can be performed in less than 10 min with low voltage. The gel-to-gel repeatability is around 3.8%. The limit of detection is approx. 10 ng as determined by Coomassie staining of selected standard proteins, and corresponds to a 10-fold increase in sensitivity as compared with a common size PAGE analysis device (e.g. 10 x 7 cm). The device was successfully applied to peptide mass fingerprint analysis, protein sequencing and ultra-sensitive immunodetection, and the performance was compared to a commonly used regular PAGE device.     

Occurrence of bifidobacteria in faeces of calves fed milk or a combined diet

The development of faecal bacteria composition in calves fed milk or a combined diet was investigated from 4 to 21 days of age. On day 7, bifidobacteria in faeces of milk-fed calves already increased from about 7.6 to 9.2 log CFU/g and did not change until the end of the study, whereas in calves fed the combined diet bifidobacteria only moderately increased to 7.9 log CFU/g and decreased slowly until day 21. The counts of bifidobacteria in calves on a combined diet were significantly (p < 0.01) lower compared to those in milk-fed calves. Bifidobacterial counts determined by cultivation or by fluorescence in situ hybridisation (FISH) did not differ significantly. Our results showed that the occurrence of bifidobacteria in calf faeces is highly dependent on the diet composition. Faecal bacteria flora of calves fed exclusively by milk is rich in bifidobacteria, but in calves on a combined diet coliforms dominated.