Comparative analysis of selenocysteine machinery and selenoproteome gene expression in mouse brain identifies neurons as key functional sites of selenium in mammals

Although dietary selenium (Se) deficiency results in phenotypes associated with selenoprotein depletion in various organs, the brain is protected from Se loss. To address the basis for the critical role of Se in brain function, we carried out comparative gene expression analyses for the complete selenoproteome and associated biosynthetic factors. Using the Allen Brain Atlas, we evaluated 159 regions of adult mouse brain and provided experimental analyses of selected selenoproteins. All 24 selenoprotein mRNAs were expressed in the mouse brain. Most strikingly, neurons in olfactory bulb, hippocampus, cerebral cortex, and cerebellar cortex were exceptionally rich in selenoprotein gene expression, in particular in GPx4, SelK, SelM, SelW, and Sep15. Over half of the selenoprotein genes were also expressed in the choroid plexus. A unique expression pattern was observed for one of the highly expressed selenoprotein genes, SelP, which we suggest to provide neurons with Se. Cluster analysis of the expression data linked certain selenoproteins and selenocysteine machinery genes and suggested functional linkages among selenoproteins, such as that between SelM and Sep15. Overall, this study suggests that the main functions of selenium in mammals are confined to certain neurons in the brain.     

The role of nonhomologous end joining and homologous recombination in the clonogenic bystander effects of mammalian cells after exposure to counted 10 MeV protons and 4.5 MeV α-particles of the PTB microbeam

We have studied the dependence of clonogenic bystander effects on defects in the pathways of DNA double-strand break (DSB) repair and on linear energy transfer (LET). The single-ion microbeam of the Physikalisch-Technische Bundesanstalt (PTB) was used to irradiate parental Chinese hamster ovary cells or derivatives deficient in nonhomologous end joining (NHEJ) or homologous recombination (HR) in the G1-phase of the cell cycle. Cell nuclei were targeted with 10 MeV protons (LET = 4.7 keV/μm) or 4.5 MeV α-particles (LET = 100 keV/μm). During exposure, the cells were confluent, allowing signal transfer through both gap junctions and diffusion. When all cell nuclei were targeted with 10 MeV protons, approximately exponential survival curves were obtained for all three cell lines. When only 10% of all cell nuclei were targeted, a significant bystander effect was observed for parental and HR-deficient cells, but not for NHEJ-deficient cells. For all three cell lines, the survival data after exposure of all cell nuclei to 4.5 MeV α-particles could be fitted by exponential curves. When only 10% of all cell nuclei were targeted, significant bystander effects were obtained for parental and HR-deficient cells, whereas for NHEJ-deficient cells a small, but significant, bystander effect was observed only at higher doses. The data suggest that bystander cell killing is a consequence of un- or misrejoined DSB which occur in bystander cells during the S-phase as a result of the processing of oxidative bistranded DNA lesions. The relative contributions of NHEJ and HR to the repairing of DSB in the late S/G2-phase may affect clonogenic bystander effects.     

Microbial community shifts as a response to efficient degradation of chlorobenzene under hypoxic conditions

Limitations in the availability of oxygen restrict aerobic biodegradation of chloroaromatic compounds in groundwater ecosystems. In this context the activity of ring-cleaving chlorocatechol dioxygenases (CC12O) is crucial for effective mineralization. Previously we demonstrated that oxygen-related enzyme characteristics of CC12O can vary widely among the Proteobacteria (Balcke et al. submitted). Here, we investigated how strains with different ability to transform intermediary 3-chlorocatechol integrate into biodegradation of chlorobenzene (CB) under low or high oxygen availability. Pseudomonas veronii UFZ B549 and Acidovorax facilis UFZ B530, which had differing oxygen affinities for CC12O, were mixed together at different proportions (20:80; 80:20), and compared for degradation of chlorobenzene under oxic (215 μM O2) and hypoxic (11 μM O2) conditions. Changes in community composition in binary mixed cultures were determined and compared with an indigenous groundwater community, cultivated under comparable conditions. Community shifts were determined by FISH (fluorescent in situ hybridization) in our model system and SSCP (single stranded conformation polymorphism) fingerprinting in the groundwater community, as well as by analysis of respiratory quinones of taxonomic value. Hypoxia led to enrichment of Acidovoracae in the groundwater and binary cultures. Under hypoxic conditions cis,cis-2-chloromuconate released to the medium by A. facilis allowed for concomitant growth of P. veronii, although its low-affinity type CC12O would not imply growth on CB. Vice versa, increasing abundance of P. veronii induced intermediary 3-chlorocatechol accumulation, which was reduced by growth of A. facilis. Thus, reduced oxygen availability caused syntrophic rather than competitive interactions.     

Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment

Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when ¹³C^β and ¹³C′ shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using ¹³C^α connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cadherins and mechanotransduction by hair cells

Mechanotransduction, the conversion of a mechanical stimulus into an electrical signal is crucial for our ability to hear and to maintain balance. Recent findings indicate that two members of the cadherin superfamily are components of the mechanotransduction machinery in sensory hair cells of the vertebrate inner ear. These studies show that cadherin 23 (CDH23) and protocadherin 15 (PCDH15) form several of the extracellular filaments that connect the stereocilia and kinocilium of a hair cell into a bundle. One of these filaments is the tip link that has been proposed to gate the mechanotransduction channel in hair cells. The extracellular domains of CDH23 and PCDH15 differ in their structure from classical cadherins and their cytoplasmic domains bind to distinct effectors, suggesting that evolutionary pressures have shaped the two cadherins for their function in mechanotransduction.     

Effects of 6-month daily supplementation with oral beta-carotene in combination or not with benzo[a]pyrene on cell-cycle markers in the lung of ferrets

Epidemiological studies have demonstrated that people who eat more fruits and vegetables (rich in carotenoids) and people who have higher serum beta-carotene (BC) levels have a lower risk of cancer, particularly lung cancer. However, the two main human intervention studies of BC supplementation (the ATBC and the CARET trials) revealed an increased risk of lung cancer among smokers and asbestos workers. Previous studies carried out in the ferret have reported that BC effects are related to dose. Here, we treated ferrets with two concentrations of oral BC (0.8 and 3.2 mg/kg body weight per day) for 6 months, using BC in a formulation also containing dl-alpha-tocopherol and ascorbyl palmitate. The effect of the smoke-derived carcinogenic agent benzo[a]pyrene (BP), with or without low-dose BC, was also analysed. We determined the protein levels and mRNA expression levels of activator protein 1 (c-Jun and c-Fos), c-Myc, cyclin D1, proliferating cellular nuclear antigen and retinoic acid receptor beta. We did not find higher levels of cell proliferation markers in the lung of ferrets treated with BC or signals of squamous metaplasia lesions either. On the other hand, although no evident signals of pulmonary carcinogenesis were observed in animals exposed to BP, BC supplementation in these animals may prevent against excess cell proliferation, since this reestablishes Jun protein and cyclin D1 mRNA levels in the lung of BP-exposed animals. In summary, these results show that the combination of BC with alpha-tocopherol and ascorbyl palmitate does not induce pro-oxidant effects in the lung of ferrets.     

Reproductive attributes of the eulophid Oomyzus sokolowskii,a biological control agent of diamondback moth,Plutella xylostella (Lepidoptera: Plutellidae)

Oomyzus sokolowskii (Kurdjumov) is a gregarious larval-pupal parasitoid of the diamondback moth, Plutella xylostella L. Under laboratory conditions, we explored the reproductive attributes including the functional response of O. sokolowskii and the impact of mutual interference among female parasitoids on progeny production and progeny sex ratio. In the functional response experiment, groups of 1–50 late instar larvae were exposed to groups of five female parasitoids for 48 h. Both Holling's disc equation (type II response) and Williams and Martinez's model (type III response) fit significantly better than the null-model assuming density-independent parasitation efficiency. Williams and Martinez's model was only marginally better than Holling's disc equation which explained 82% of the variation in the number of parasitized hosts. According to Holling's disc equation, the estimated maximum number of hosts parasitised during 48 h was 17 per group of five parasitoids, the estimated attack efficiency of a single female parasitoid (a) was 0.0024 larvae per hour, and the estimated handling time of a single female (T _h) was 14.4 h per host. In the mutual interference experiment 50 host larvae were exposed to different numbers of female parasitoids (P=5, 10, 20, 30, 40 and 50) for 48 h. With increasing numbers of conspecifics, the proportion of male progeny increased and total number of progeny per parasitoid decreased, yielding an estimated mutual interference constant (m) (±SE) of 0.95±0.14. The total number of wasps emerging from individual host pupae increased with increasing number of conspecifics. The age-specific fecundity of O. sokolowskii was investigated by providing 10 host larvae daily to individual female parasitoids. After 3 days of oviposition, 90% of the females were still alive and had achieved 76.4% of their lifetime fecundity. The proportion of female progeny decreased with female age. No relationship was found between female longevity and lifetime fecundity. The results suggest that parasitoid and host densities influence the progeny production and sex allocation strategy of O. sokolowskii. The information obtained from this study would help to develop a mass-rearing protocol for O. sokolowskii.     

Filamentous network mechanics and active contractility determine cell and tissue shape

For both cells and tissues, shape is closely correlated with function presumably via geometry-dependent distribution of tension. In this study, we identify common shape determinants spanning cell and tissue scales. For cells whose sites of adhesion are restricted to small adhesive islands on a micropatterned substrate, shape resembles a sequence of inward-curved circular arcs. The same shape is observed for fibroblast-populated collagen gels that are pinned to a flat substrate. Quantitative image analysis reveals that, in both cases, arc radii increase with the spanning distance between the pinning points. Although the Laplace law for interfaces under tension predicts circular arcs, it cannot explain the observed dependence on the spanning distance. Computer simulations and theoretical modeling demonstrate that filamentous network mechanics and contractility give rise to a modified Laplace law that quantitatively explains our experimental findings on both cell and tissue scales. Our model in conjunction with actomyosin inhibition experiments further suggests that cell shape is regulated by two different control modes related to motor contractility and structural changes in the actin cytoskeleton.