Differences in mRNA expression,protein content,and enzyme activity of superoxide dismutases in type II pneumocytes of acute and chronic lung injury

The lung is protected against oxidative stress by a variety of antioxidants and type II pneumocytes seem to play an important role in antioxidant defense. Previous studies have shown that inhalation of NO₂ results in acute and chronic lung injury. How the expression and enzyme activity of antioxidant enzymes are influenced in type II cells of different inflammatory stages has yet not been studied. To elucidate this question, we exposed rats to 10 ppm NO₂ for 3 or 20 days to induce acute or chronic lung injury. From these and air-breathing rats, type II pneumocytes were isolated. The mRNA expression and protein content of CuZnSOD and MnSOD as well as total SOD-specific enzyme activity were determined. For the acute lung injury (3 d NO₂), the expression of CuZnSOD mRNA was significantly increased, while MnSOD expression was significantly reduced after 3 days of NO 2 exposure. For the chronic lung injury (20 d NO₂), CuZnSOD expression was still enhanced, while MnSOD expression was comparable to control. In parallel to CuZnSOD mRNA expression, the protein amount was significantly increased in acute and chronic lung injury however MnSOD protein content exhibited no intergroup differences. Total SOD enzyme activity showed a significant decrease after 3 days of NO₂ exposure and was similar to control after 20 days. We conclude that during acute and chronic lung injury in type II pneumocytes expression and protein synthesis of CuZnSOD and MnSOD are regulated differently.     

Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients

Fragile X‐associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55–200 CGG repeats in the 5′‐UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA‐binding proteins by the expanded CGG repeats. Sam68 is an RNA‐binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA‐binding proteins sequentially, first Sam68, then hnRNP‐G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing‐regulatory function. Consequently, Sam68‐responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain‐of‐function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.     

Unikaryon phyllotretae sp. n. (Protista,Microspora), a new microsporidian pathogen of Phyllotreta undulata (Coleoptera; Chrysomelidae)

The microsporidium Unikaryon phyllotretae sp. n., a new pathogen of Phyllotreta undulata, is described based on light microscopic and ultrastructural characteristics. Microscopic examination of parasitized individuals revealed two types of spores. The majority of the spores were of the first type, which are oval and measured 2.74±0.17×1.93±0.17 μm when fresh. Fresh spores of the second type (very rare) are elongated and measured 4.39±0.18×1.61±0.20 μm. All life stages have single nuclei. Sporogony ends with uninucleate single sporoblasts and spores. The spores were only observed in Malpighian tubules. The isofilar polar filament of the parasite has six to eight coils, and a well-developed polaroplast was of the lamellated type, with closely packed anterior lamellae and loosely packed posterior lamellae.     

Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species

Background

The Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring.

Methodology/Principal Findings

Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster.

Conclusion

This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity.     

Der Bau der Canini bei den Paulchoffatiidae (Multituberculata; Ober-Jura)

Canines are preserved among Multituberculata only in the upper jaw of the Paulchoffatiidae, the Pinheirodontidae and the North American genusGliodon Engelmann &; Callison, 1999. They resemble the anterior premolars (p^1–3) in the morphology of their crown, but they differ from them by the presence of only one root. In the present paper, 126 isolated canines of the Paulchoffatiidae from the Guimarota coal-pit in Portugal are treated. They show a wide morphological variation, from bicuspid to pentacuspid expression, and they can be grouped into 18 morphological units (Tab. 1). The bicuspid canines (11 specimens) show one buccal and one lingual cusp, arranged side by side, the latter normally being larger than the former. In the tricuspid canines (32 specimens) the cusps (one buccal and two lingual) are arranged in a triangle, with a great variation in the position of the cusps to each other. Tetracuspid canines (69 specimens) are the dominating group. Two buccal and two lingual cusps are present, which differ markedly in their largeness and their position to each other. The teeth differ also much in the shape of their crown. The pentacuspid canines (14 specimens) show two buccal and three lingual cusps. The variation in the arrangement of the cusps and in the shape of the crown is similar as in the tetracuspid specimens. One or two small cuspules can be present at the anterior border of the crown additionally to the main cusps in the canines. Tricuspid and tetracuspid canines are present also in the skulls of the Paulchoffatiidae, whereas bicuspid and pentacuspid canines are known only as isolated specimens. In the Pinheirodontidae the canines are tricuspid or tetracuspid, inGlirodon they are unicuspid. The Paulchoffatiidae and Pinheirodontidae — Paulchoffatiid line sensuKielan-Jaworowska &; Hurum, 2001 — are characterized by increasing premolarization of the upper canines. With that they differ markedly from all other multituberculates where the canini become reduced.     

Generation of carbon monoxide and iron from hemeproteins in the presence of 7,8-dihydroneopterin

7,8-Dihydroneopterin and neopterin are secreted by human and primate macrophages after activation by interferon-gamma in a ratio of 2:1. 7,8-Dihydroneopterin is known to suppress radical-mediated processes, but it is also able in the presence of iron ions to generate superoxide radical anion and hydroxyl radicals from molecular oxygen. Effects of 7,8-dihydroneopterin were investigated on (met)myoglobin and (met)hemoglobin. Addition of 7,8-dihydroneopterin to heme proteins in air-saturated solution resulted in dose-dependent cleavage of the porphyrin moiety. The liberation of non-heme iron and carbon monoxide originating from the cleaved porphyrin was quantified. Both were generated at equimolar concentrations with a linear correlation coefficient of 0.9. Addition of ferrous iron significantly accelerated the pteridine-mediated cleaving of the porphyrin. However, the total yield of porphyrin cleaved was controlled by the pterin rather than by the ferrous ion concentration. 7,8-Dihydroneopterin is assumed to reduce the heme iron in intact protein molecules, thereby preparing the conditions for binding of oxygen and carbon monoxide as ligands. Beyond that, it is concluded that hydroxyl radicals might be generated via reduction of molecular oxygen to superoxide anion in the autoxidation process and dismutation to hydrogen peroxide and subsequent Fenton reaction.     

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.     

Pre-experience of social exclusion suppresses cortisol response to psychosocial stress in women but not in men

Lack of social support and social exclusion is associated with adverse effects for mental and physical health. Additionally, women appear to be more vulnerable to social triggers of health disturbances. The hypothalamus–pituitary–adrenocortical-axis (HPA axis) might play a key role in this context as it has been shown both to relate to psychosocial conditions and health outcomes and to respond differentially depending on gender. In a previous experiment we found no effects of exclusion alone (operationalized via Cyberball) on cortisol secretion. Here we examine the effects of a social exclusion pre-experience on psychological and cortisol responses to a public speaking stressor. Subjects (33 m, 34 f) were randomly assigned to social exclusion (SE) or one of two control conditions (exclusion attributed to technical default (TD) and social inclusion (SI)). Afterwards salivary cortisol and psychological responses to a public speaking paradigm were assessed. Exclusion pre-treatment does not affect psychological responses to public speaking stress though with respect to cortisol significant. Cyberball by gender and Cyberball by gender by time interactions are found. SE-women show a blunted cortisol stress response to public speaking while cortisol responses of SE-men fall between SI-men and TD-men. Pre-experience of social exclusion leads to a blunted cortisol response to stress in women but not in men. This factor might contribute to the higher vulnerability to social triggers of health disturbances observed in women.     

The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

Summary The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.