In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part I: acellular assay

Acellular assay of calf thymus DNA ± rat liver microsomal S9 fraction coupled with ³²P-postlabelling was used to study the genotoxic potential of organic compounds bound onto PM10 particles collected in three European cities—Prague (CZ), Kosice (SK) and Sofia (BG) during summer and winter periods. B[a]P alone induced DNA adduct levels ranging from 4.8 to 768 adducts/10⁸ nucleotides in the concentration dependent manner. However, a mixture of 8 c-PAHs with equimolar doses of B[a]P induced 3.7–757 adducts/10⁸ nucleotides, thus suggesting the inhibition of DNA adduct forming activity by interaction among various PAHs. Comparison of DNA adduct levels induced by various EOMs indicates higher variability among seasons than among localities. DNA adduct levels for Prague collection site varied from 19 to 166 adducts/10⁸ nucleotides, for Kosice from 22 to 85 and for Sofia from 6 to 144 adducts/10⁸ nucleotides. Bioactivation with S9 microsomal fraction caused 2- to 7-fold increase in DNA adduct levels compared to −S9 samples, suggesting a crucial role of indirectly acting genotoxic EOM components, such as PAHs. We have demonstrated for the first time a significant positive correlation between B[a]P content in EOMs and total DNA adduct levels detected in the EOM treated samples (R = 0.83; p = 0.04). These results suggest that B[a]P content in EOM is an important factor for the total genotoxic potential of EOM and/or B[a]P is a good indicator of the presence of other genotoxic compounds causing DNA adducts. Even stronger correlation between the content of genotoxic compounds in EOMs and total DNA adduct levels detected (R = 0.94; p = 0.005) was found when eight c-PAHs were taken into the consideration. Our findings support a hypothesis that a relatively limited number of EOM components is responsible for a major part of its genotoxicity detectable as DNA adducts by ³²P-postlabelling.     

Overproduction of bioactive retinoic acid in cells expressing disease-associated mutants of retinol dehydrogenase 12

Retinol dehydrogenase 12 (RDH12) is an NADP(+)-dependent oxidoreductase that in vitro catalyzes the reduction of all-trans-retinaldehyde to all-trans-retinol or the oxidation of retinol to retinaldehyde depending on substrate and cofactor availability. Recent studies have linked the mutations in RDH12 to severe early-onset autosomal recessive retinal dystrophy. The biochemical basis of photoreceptor cell death caused by mutations in RDH12 is not clear because the physiological role of RDH12 is not yet fully understood. Here we demonstrate that, although bi-directional in vitro, in living cells, RDH12 acts exclusively as a retinaldehyde reductase, shifting the retinoid homeostasis toward the increased levels of retinol and decreased levels of bioactive retinoic acid. The retinaldehyde reductase activity of RDH12 protects the cells from retinaldehyde-induced cell death, especially at high retinaldehyde concentrations, and this protective effect correlates with the lower levels of retinoic acid in RDH12-expressing cells. Disease-associated mutants of RDH12, T49M and I51N, exhibit significant residual activity in vitro, but are unable to control retinoic acid levels in the cells because of their dramatically reduced affinity for NADPH and much lower protein expression levels. These results suggest that RDH12 acts as a regulator of retinoic acid biosynthesis and protects photoreceptors against overproduction of retinoic acid from all-trans-retinaldehyde, which diffuses into the inner segments of photoreceptors from illuminated rhodopsin. These results provide a novel insight into the mechanism of retinal degeneration associated with mutations in RDH12 and are consistent with the observation that RDH12-null mice are highly susceptible to light-induced retinal apoptosis in cone and rod photoreceptors.     

Role of transamination in the mobilization of respiratory substrates in germinating seeds of castor oil plants

The metabolism of 1,4-¹⁴C-succinate and 2,3-¹⁴C-succinate and the activity of succinic semialdehyde dehydrogenase (EC 1.2.1.16) were studied in germinating seeds of castor oil plants (Ricinus communis L.). Succinate metabolism involved succinate dehydrogenase and was sensitive to metabolites of the γ-aminobutyric acid shunt. Considerable accumulation of the label in amino acids reflected the progression of transamination reactions. Succinic semialdehyde dehydrogenase was purified from the endosperm of castor oil plants. Kinetic characteristics of the enzyme were evaluated. Our study indicates that the mobilization of respiratory substrates during germination of castor oil plants is related to active transamination of ketoacids in the Krebs cycle and involves the γ-aminobutyric acid shunt.     

The genetic structure of cattle populations (Bos taurus) in northern Eurasia and the neighbouring Near Eastern regions: implications for breeding strategies and conservation

We investigated the genetic structure and variation of 21 populations of cattle (Bos taurus) in northern Eurasia and the neighbouring Near Eastern regions of the Balkan, the Caucasus and Ukraine employing 30 microsatellite markers. By analyses of population relationships, as well as by a Bayesian-based clustering approach, we identified a genetic distinctness between populations of modern commercial origin and those of native origin. Our data suggested that northern European Russia represents the most heavily colonized area by modern commercial cattle. Further genetic mixture analyses based on individual assignment tests found that native Red Steppe cattle were also employed in the historical breeding practices in Eastern Europe, most probably for incorporating their strong and extensive adaptability. In analysis of molecular variance, within-population differences accounted for approximately 90% of the genetic variation. Despite some correspondence between geographical proximity and genetic similarity, genetic differentiation was observed to be significantly associated with the difference in breeding purpose among the European populations (percentage of variance among groups and significance: 2.99%, P = 0.02). Our findings give unique genetic insight into the historical patterns of cattle breeding practices in the former Soviet Union. The results identify the neighbouring Near Eastern regions such as the Balkan, the Caucasus and Ukraine, and the isolated Far Eastern Siberia as areas of 'genetic endemism', where cattle populations should be given conservation priority. The results will also be of importance for cost-effective management of their future utilization.     

Screening library evolution through automation of solution preparation

The quality of the compound library is a critical success factor in every high-throughput screening campaign. Screening solutions have to be prepared with a high level of process control to ensure the correct identity and initial concentration of each compound. However, even under optimized storage conditions, a certain level of degradation in solution cannot be avoided. Therefore, regular quality control and eventual removal of solutions from the screening deck is necessary. Because solution preparation, especially the weighing of compounds, is a tedious and often manual task, a regular resolubilization of compounds is difficult to achieve. By complete automation of the solution preparation, the authors have laid the foundation for a life cycle management of screening solutions. They demonstrate how a combination of quality and process control leads to a continuous improvement of the screening library. In presenting an automation concept, they show how a series of innovative process optimizations led to a high-performance system that achieves full industrialization of solution preparation.     

Chloroplast ultrastructure in leaves of tobacco plants with the introduced gene for the acyl-lipid Δ9-desaturase from <Emphasis Type="Italic">Synechococcus vulcanus</Emphasis> at normal and low temperature

Ultrastructural changes in chloroplasts of tobacco plants (Nicotiana tabacum L.) with the introduced desC gene for the acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were investigated during plant acclimation to cold. Control plants were transformed with an empty pGA482 binary vector. At optimum growth temperature, a decreased number of grana and thylakoids and an increased number of plastoglobules and their larger area were observed in transgenic plants when compared to control ones. In control plants, acclimation to cold (6 days at 10°C) resulted in the larger areas of chloroplasts and grana. These changes indicated starting cold-induced injuries manifested in swelling of the stroma and a slight decrease in the total number of thylakoids in the chloroplast. In contrast, transgenic plants responded to cold by reducing the chloroplast, granal, and plastoglobule areas. Meantime, the number of thylakoids per granum increased noticeably. The total number of thylakoids in the chloroplast increased from 123 to 203. It was concluded that expression of the acyl-lipid Δ9-desaturase gene in tobacco plants provided for the formation of the cell ultrastructure similar to one characteristic of cold-tolerant plants.     

Cryopreservation of seeds and protocorms of rare temperate orchids

The efficiency of cryopreservation of seeds of five rare and endangered species of temperate orchids belonging to Platanthera and Dactylorhiza genera followed by their asymbiotic culture in vitro, as well as of in vitro cultured D. fuchsii protocorms (specific stage of orchid embryo development after release from the seed coat) was investigated. Germination rates of seeds after their exposure to liquid nitrogen were species-depended and could be either higher or lower than in the unfrozen control. There was no significant difference between growth rates of protocorms of the same species obtained from seeds collected in various Russia regions and cultured for 5 months. After vitrification, 9% of D. fuchsii protocorms with a larger diameter of 1200 μm survived cryopreservation; however, their growth was retarded for three months when compared to control protocorms.     

Microtubule-binding proteins CLASP1 and CLASP2 interact with actin filaments

Cell morphogenesis requires dynamic communication between actin filaments and microtubules which is mediated, at least in part, by direct structural links between the two cytoskeletal systems. Here, we examined interaction between the CLIP-associated proteins (CLASP) CLASP1 and CLASP2, and actin filaments. We demonstrate that, in addition to a well-established association with the distal ends of microtubules, CLASP2alpha co-localizes with stress fibers, and that both CLASP1alpha and CLASP2alpha co-immunoprecipitate with actin. GFP-CLASP2alpha exhibits retrograde flow in the lamellipodia of Xenopus primary fibroblasts and in the filopodia of Xenopus spinal cord neurons. A deletion mapping analysis reveals that both the microtubule-binding domain of CLASP2 (which is homologous between all CLASPs) and the N-terminal dis1/TOG domain of CLASP2alpha (which is homologous between alpha isoforms) possess actin-binding activity. Fluorescence resonance energy transfer experiments demonstrate significant energy transfer between YFP-CLASP2alpha and CFP-actin. Our results indicate that CLASPs function as actin/microtubule crosslinkers in interphase cells. We propose that CLASPs facilitate recognition of actin filaments by the plus ends of growing microtubules at the initial stages of actin-microtubule interaction. Cell Motil.     

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue. Our results reveal large domains of constant phase together with regions of quasi-random phase, which are correlated to respectively high- and low-intensity regions in the standard SHG images. A comparison with polarization-resolved SHG highlights the crucial role of relative fibril polarity in determining the SHG signal intensity. Indeed, it appears that even a well-organized noncentrosymmetric structure emits low SHG signal intensity if it has no predominant local polarity. This work illustrates how the complex architecture of noncentrosymmetric scatterers at the nanoscale governs the coherent building of SHG signal within the focal volume and is a key advance toward a complete understanding of the structural origin of SHG signals from tissues.