Identification and function of thermosensory neurons in Drosophila larvae

Although the ability to sense temperature is critical for many organisms, the underlying mechanisms are poorly understood. Using the calcium reporter yellow cameleon 2.1 and electrophysiological recordings, we identified thermosensitive neurons and examined their physiologic response in Drosophila melanogaster larvae. In the head, terminal sensory organ neurons showed increased activity in response to cooling by < or =1 degrees C, heating reduced their basal activity, and different units showed distinct response patterns. Neither cooling nor heating affected dorsal organ neurons. Body wall neurons showed a variety of distinct response patterns to both heating and cooling; the diverse thermal responses were strikingly similar to those described in mammals. These data establish a functional map of thermoresponsive neurons in Drosophila larvae and provide a foundation for understanding mechanisms of thermoreception in both insects and mammals.     

Lipid peroxidation, DNA damage, and cellular morphology of R1 Rhabdomyosarcoma cell line irradiated in vitro by gamma-rays with different dose rates

The study examines the relationship between lipid peroxidation, DNA damage, and cell morphology after the exposure of R1 Rhabdomyosarcoma cells to two different dose-rates of gamma rays. Exponential cultures of R1 cells were irradiated with single dose of 5 Gy at high dose rate (0.833 Gy/min) and low dose rate (0.0707 Gy/min). The concentration of two aldehydes, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), were determined. DNA damage induction and repair were measured by using the alkaline version of the comet assay. Cellular alteration was also estimated microscopically as was the frequency of cells with micronuclei and proportion of apoptosis and necrosis. These parameters were evaluated immediately (time 0) and after different times up to 48 h of incubation in 37 degrees C, after irradiation. Results indicate that a low dose rate in comparison to high dose rate caused a significantly higher increase of aldehydes concentration observed at 12 h, followed by obviously higher DNA damage at 48 h and altered cellular morphology. The inverse dose-rate effect estimated for the gamma rays Co-60 source was found to be related to the measured biochemical and morphological parameters.     

Myosin heavy chain isoform composition and stretch activation kinetics in single fibres of Xenopus laevis iliofibularis muscle

Skeletal muscle is composed of specialized fibre types that enable it to fulfil complex and variable functional needs. Muscle fibres of Xenopus laevis , a frog formerly classified as a toad, were the first to be typed based on a combination of physiological, morphological, histochemical and biochemical characteristics. Currently the most widely accepted criterion for muscle fibre typing is the myosin heavy chain (MHC) isoform composition because it is assumed that variations of this protein are the most important contributors to functional diversity. Yet this criterion has not been used for classification of Xenopus fibres due to the lack of an effective protocol for MHC isoform analysis. In the present study we aimed to resolve and visualize electrophoretically the MHC isoforms expressed in the iliofibularis muscle of Xenopus laevis, to define their functional identity and to classify the fibres based on their MHC isoform composition. Using a SDS-PAGE protocol that proved successful with mammalian muscle MHC isoforms, we were able to detect five MHC isoforms in Xenopus iliofibularis muscle. The kinetics of stretch-induced force transients (stretch activation) produced by a fibre was strongly correlated with its MHC isoform content indicating that the five MHC isoforms confer different kinetics characteristics. Hybrid fibre types containing two MHC isoforms exhibited stretch activation kinetics parameters that were intermediate between those of the corresponding pure fibre types. These results clearly show that the MHC isoforms expressed in Xenopus muscle are functionally different thereby validating the idea that MHC isoform composition is the most reliable criterion for vertebrate skeletal muscle fibre type classification. Thus, our results lay the foundation for the unequivocal classification of the muscle fibres in the Xenopus iliofibularis muscle and for gaining further insights into skeletal muscle fibre diversity.     

Inhibitory effect of ascorbic acid post-treatment on radiation-induced chromosomal damage in human lymphocytes in vitro

In the present study, the effect of exposure to ascorbic acid (vitamin C) after gamma-ray-induced chromosomal damage in cultured human lymphocytes was examined to explore the mechanism by which this antioxidant vitamin protects irradiated cells Non-irradiated lymphocytes were exposed to increasing concentrations of ascorbic acid (1-100 micro g/ml) and DNA damage was estimated using chromosomal aberration analysis and the comet assay. The results showed that ascorbic acid did not influence the frequency of chromosomal aberrations in non-irradiated cells, except at the highest concentration (20 micro g/ml), which induced breakage-type chromosomal aberrations. Vitamin C at the concentration of 50 micro g/ml caused DNA damage detected by the comet assay. A significant (34%) decrease in the frequency of chromosomal aberrations was observed in lymphocytes exposed to gamma-radiation and then cultured in the presence of ascorbic acid (1 micro g/ml). The removal of DNA breaks in cells exposed to 2 Gy of gamma-radiation was accelerated in the presence of ascorbic acid as determined by the comet assay, suggesting that it may stimulate DNA repair processes.     

Linking hematopoiesis to endochondral skeletogenesis through analysis of mice transgenic for collagen X

Each skeletal element where marrow develops is first defined by a hypertrophic cartilage blueprint. Through programmed tissue substitution, the cartilaginous skeletal model is replaced by trabecular bone and marrow, with accompanying longitudinal tissue growth. During this process of endochondral ossification, hypertrophic cartilage expresses a unique matrix molecule, collagen X. Previously we reported that transgenic mice with dominant interference collagen X mutations develop variable skeleto-hematopoietic abnormalities, manifested as growth plate compressions, diminished trabecular bone, and reduced lymphatic organs (Nature 1993, 365:56). Here, histology and flow cytometry reveal marrow hypoplasia and impaired hematopoiesis in all collagen X transgenic mice. A subset of mice with perinatal lethality manifested the most severe skeletal defects and a reduction of marrow hematopoiesis, highlighted by a lymphocyte decrease. Thymic reduction is accompanied by a paucity of cortical immature T cells, consistent with the marrow's inability to replenish maturing cortical lymphocytes. Diminished spleens exhibit indistinct lymphatic nodules and red pulp depletion; the latter correlates with erythrocyte-filled vascular sinusoids in marrows. All mice display reduced B cells in marrows and spleens, and elevated splenic T cells. These hematopoietic defects underscore an unforeseen link between hypertrophic cartilage, endochondral ossification, and establishment of the marrow microenvironment required for blood cell differentiation.     

The FK506-binding protein of the malaria parasite, Plasmodium falciparum, is a FK506-sensitive chaperone with FK506-independent calcineurin-inhibitory activity

We have identified an immunophilin of the FKBP family in Plasmodium falciparum that contains a conserved peptidyl prolyl isomerase (PPIase) and tetratricopeptide repeat (TPR) domains. The 35 kDa protein was named FKBP35 and expressed in bacteria. Recombinant FKBP35 exhibited potent PPIase and protein folding activities against defined substrates in vitro, suggesting that it is a parasitic chaperone. Both activities were inhibited by macrolide immunosuppressant drugs, ascomycin (a FK506 derivative) and rapamycin, but not by cyclosporin A, providing biochemical evidence of its inclusion in the FKBP family. Interestingly, FKBP35 inhibited purified plasmodial calcineurin (protein phosphatase 2B) in the absence of any drug. In the parasite's cell, FKBP35 exhibited a stage-specific nucleocytoplasmic shuttling and did not co-localize with calcineurin. FKBP35 associated with plasmodial heat shock protein 90 (Hsp90), another member of the chaperone superfamily, via the TPR domain. Geldanamycin, a Hsp90 inhibitor, and ascomycin inhibited P. falciparum growth in a synergistic fashion. Extensive search of the P. falciparum genome revealed no other FKBP sequence, implicating PfFKBP35 as a highly significant antimalarial drug target. Thus, the single FKBP of Plasmodium is an essential parasitic chaperone with a novel drug-independent calcineurin-inhibitory activity.     

Modeling the Filler Phase Interaction with Solidification Front in Al(TiC) Composite Produced by the In Situ Method

This paper presents simulation results of the interaction of TiC nanoparticle in liquid aluminum. The behavior of the TiC particle in the frontal interaction region stems from the operation of a system of such forces as gravity, viscous flow drag force, and Saffman force. The difference in density between the TiC and the aluminum matrix makes the particle fall, regardless of the radius dimension; while the Saffman force—which accounts for the local velocity gradient of the liquid aluminum—causes that particles with the smallest radii considered in the calculations 6.4 × 10⁻⁸ m; 7 × 10⁻⁸ m; 7.75 × 10⁻⁸ m; 9.85 × 10⁻⁸ m are repelled from the solidification front and the particles with 15.03 × 10⁻⁸ m are attracted to it. The viscosity growth in the course of casting caused by the lowering temperature reduces this effect, though the trend is maintained. The degree to which the particle is attracted to the front clearly depends on the velocity gradient of the liquid phase. For a very small gradient of 0.00001 m/s, the particle is at its closest position relative to the front.     

Serial passage in an insect host indicates genetic stability of the human probiotic Escherichia coli Nissle 1917

Background and objectivesThe probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to effectively prevent and alleviate intestinal diseases. Despite the widespread medical application of EcN, we still lack basic knowledge about persistence and evolution of EcN outside the human body. Such knowledge is important also for public health aspects, as in contrast to abiotic therapeutics, probiotics are living organisms that have the potential to evolve. This study made use of experimental evolution of EcN in an insect host, the red flour beetle Tribolium castaneum, and its flour environment.MethodologyUsing a serial passage approach, we orally introduced EcN to larvae of T.castaneum as a new host, and also propagated it in the flour environment. After eight propagation cycles, we analyzed phenotypic attributes of the passaged replicate EcN lines, their effects on the host in the context of immunity and infection with the entomopathogen Bacillus thuringiensis, and potential genomic changes using WGS of three of the evolved lines.ResultsWe observed weak phenotypic differences between the ancestral EcN and both, beetle and flour passaged EcN lines, in motility and growth at 30°C, but neither any genetic changes, nor the expected increased persistence of the beetle-passaged lines. One of these lines displayed distinct morphological and physiological characteristics.Conclusions and implicationsOur findings suggest that EcN remains rather stable during serial passage in an insect. Weak phenotypic changes in growth and motility combined with a lack of genetic changes indicate a certain degree of phenotypic plasticity of EcN.Lay SummaryFor studying adaptation of the human probiotic Escherichia coli strain Nissle 1917, we introduced it to a novel insect host system and its environment using a serial passage approach. After passage, we observed weak phenotypic changes in growth and motility but no mutations or changes in persistence inside the host.