FES kinase participates in KIT-ligand induced chemotaxis

FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicated in cell migration.     

Biologie des Eleonorenfalken(Falco eleonorae): 10. Der Einflu? der Horstlage auf den Bruterfolg

Zusammenfassung Der Bruterfolg des Eleonorenfalken wurde auf einer ägäischen Inselkolonie in Relation zur Horstlage untersucht. Bei Horsten mit Gratlage auf übersichtlichem Gelände war er signifikant höher (1,8 Junge/Horst) als bei den übrigen Horsten mit z. T. schlechtem Geländeüberblick (1,3 Junge/Horst). Da alle Horste gegenüber den NW-Winden geschützt lagen, waren die meisten an südlich ausgerichteten Hängen potentiell der intensiven Sonneneinstrahlung ausgesetzt. In den Horstrevieren von typisch 20 m Durchmesser gab es aber nicht immer vor Sonne geschützte Horsthöhlen, so daß bei Lufttemperaturen von 40 °C und Bodentemperaturen von bis zu 60 °C die Embryonen in den Eiern gefährdet sein können, besonders, wenn sie der direkten Sonneneinstrahlung ausgesetzt sind. Dies tritt z. B. bei einer Störung in der Brutkolonie ein. Der Bruterfolg in den sonnenexponierten Höhlen lag mit 0,8–1,3 Junge/Horst signifikant niedriger als in den sonnengeschützten Höhlen mit durchschnittlich 1,75 Junge/Horst. Neben einer normalen Infertilität von 10 % fielen weitere 8 % aller Eier in der Brutkolonie durch Sonneneinwirkung aus.

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Biology of the Eleonora's Falcon(Falco eleonorae): 10. Breeding success in relation to nest site exposition

Summary Breeding success of the Eleonora's Falcon was studied in an Aegean island colony. In nests near cliff tops with an unobstructed view of the surroundings, a significantly higher breeding success (1.8 pulli/nest) was obtained than in other nests (1.3 pulli/nest). Since all nests were chosen protected from the wind, and as the main wind comes from NW, most nests were situated on southern slopes and are thus potentially exposed to the sun. Within a falcon territory of typically 20 m diameter in size there was not always a lime stone crevice with complete shade for the eggs. At an air temperature of 40 °C and a soil temperature of up to 60 °C, excessive sunning of an unprotected egg can be lethal for the falcon embryo. Breeding success in sun exposed nests was significantly lower (0.8–1.3 pulli/nest) than in sheltered nests (1.75 pulli/nest). In addition to a normal infertility of 10 %, about 8 % of all eggs laid were lost due to sun irradiation.

     

Microcapillary-like structures prompted by phospholipase A2 activation in endothelial cells and pericytes co-cultures on a polyhydroxymethylsiloxane thin film

A thin film of poly(hydroxymethylsiloxane) (PHMS) has been deposited on glass dishes and tested as artificial support material for vascularization from mixed cultures of endothelial cells (EC) and pericytes (PC). The EC/PC co-cultures adhered massively on PHMS, with the formation of net-like microcapillary structures. Such evidence was not found on control glass substrates in the same co-culture conditions neither on PHMS for EC and PC in monocultures. The physicochemical characterization of PHMS and control glass surface by time-of-flight secondary ion mass spectrometry, X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy, pointed to the main role of the polymer hydrophobilicy to explain the observed cellular behavior. Moreover, enhanced intercellular cross-talk was evidenced by the up-regulation and activation of cytoplasmic and Ca(2+)-independent phospholipase A(2) (cPLA(2) and iPLA(2)) expression and cPLA(2) phosphorylation, leading to the cell proliferation and microcapillary formation on the PHMS surface, as evidenced by confocal microscopy analyses. Co-cultures, established with growth-arrested PCs by treatment with mitomycin C, showed an increase in EC proliferation on PHMS. AACOCF(3) or co-transfection with cPLA(2) and iPLA(2)siRNA reduced cell proliferation. The results highlight the major role played by EC/PC cross-talk as well as the hydrophobic character of the substrate surface, to promote microcapillary formation. Our findings suggest an attractive strategy for vascular tissue engineering and provide new details on the interplay of artificial substrates and capillary formation.     

Antiprotozoal activity of Brazilian plant extracts from isoquinoline alkaloid-producing families

Leishmaniasis and Chagas disease afflict the poorest countries in the world. The Brazilian flora represents a rich source for the screening of potential antiparasitic compounds. In this work, we tested the total alkaloid and ethanol extracts of nine different plants from Brazilian families which produce isoquinoline alkaloids, to determine their in vitro antiparasitic effect against L. chagasi and T. cruzi parasites. Promastigotes of L. chagasi were shown to be susceptible only to the total alkaloid extracts of A. crassiflora (EC50 value = 24.89 microg/ml), A. coriacea (EC50 value = 41.60 microg/ml), C. ovalifolia (EC50 value = 63.88 microg/ml) and G. australis (EC50 value = 37.88 microg/ml). Except for the G. australis total alkaloids, all the three extracts presented a considerable activity when tested against intracellular amastigotes. The most effective alkaloid extracts were those from A. crassiflora and C. ovalifolia, which reduced the number of infected macrophages at 25 microg/ml by 86.1% and 89.8%, respectively. Among the 18 tested extracts, 16 showed anti-Trypanosoma activity. Eight extracts (A. crassiflora, A. coriacea, C. ovalifolia, D. furfuracea, D. lanceolata, S. guianensis, X. emarginata and G. australis) were the most effective against the trypomastigotes, killing approximately 100% of the parasites at the maximal concentration of 100 microg/ml. Cytotoxicity against mammalian cells was evaluated for all extracts, but potential ones showed little or no cytotoxicity and a considerable antiparasitic effect, including D. furfuracea, D. lanceolata, G. australis, S. guianensis and X. emarginata. Plants are a rich source of natural compounds, and a powerful tool for the development of new arsenals for the therapy of protozoan diseases.     

RAL-1 controls multivesicular body biogenesis and exosome secretion

Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plasma membrane. Despite their importance in various processes, the molecular mechanisms controlling their formation and release remain unclear. Using nematodes and mammary tumor cells, we show that Ral GTPases are involved in exosome biogenesis. In Caenorhabditis elegans, RAL-1 localizes at the surface of secretory MVBs. A quantitative electron microscopy analysis of RAL-1–deficient animals revealed that RAL-1 is involved in both MVB formation and their fusion with the plasma membrane. These functions do not involve the exocyst complex, a common Ral guanosine triphosphatase (GTPase) effector. Furthermore, we show that the target membrane SNARE protein SYX-5 colocalizes with a constitutively active form of RAL-1 at the plasma membrane, and MVBs accumulate under the plasma membrane when SYX-5 is absent. In mammals, RalA and RalB are both required for the secretion of exosome-like vesicles in cultured cells. Therefore, Ral GTPases represent new regulators of MVB formation and exosome release.     

Comparative multilocus phylogeography of two Palaearctic spruce bark beetles: influence of contrasting ecological strategies on genetic variation

While phylogeographic patterns of organisms are often interpreted through past environmental disturbances, mediated by climate changes, and geographic barriers, they may also be strongly influenced by species‐specific traits. To investigate the impact of such traits, we focused on two Eurasian spruce bark beetles that share a similar geographic distribution, but differ in their ecology and reproduction. Ips typographus is an aggressive tree‐killing species characterized by strong dispersal, whereas Dendroctonus micans is a discrete inbreeding species (sib mating is the rule), parasite of living trees and a poor disperser. We compared genetic variation between the two species over both beetles’ entire range in Eurasia with five independent gene fragments, to evaluate whether their intrinsic differences could have an influence over their phylogeographic patterns. We highlighted widely divergent patterns of genetic variation for the two species and argue that the difference is indeed largely compatible with their contrasting dispersal strategies and modes of reproduction. In addition, genetic structure in I. typographus divides European populations in a northern and a southern group, as was previously observed for its host plant, and suggests past allopatric divergence. A long divergence time was estimated between East Asian and other populations of both species, indicating their long‐standing presence in Eurasia, prior to the last glacial maximum. Finally, the strong population structure observed in D. micans for the mitochondrial locus provides insights into the recent colonization history of this species, from its native European range to regions where it was recently introduced.     

hNaf1 is required for accumulation of human box H/ACA snoRNPs, scaRNPs, and telomerase

The human telomerase ribonucleoprotein particle (RNP) shares with box H/ACA small Cajal body (sca)RNPs and small nucleolar (sno)RNPs the proteins dyskerin, hGar1, hNhp2, and hNop10. How dyskerin, hGar1, hNhp2, and hNop10 assemble with box H/ACA scaRNAs, snoRNAs, and the RNA component of telomerase (hTR) in vivo remains unknown. In yeast, Naf1p interacts with H/ACA snoRNP proteins and may promote assembly of Cbf5p (the yeast ortholog of dyskerin) with nascent pre-snoRNAs. Here we show that the human HsQ96HR8 protein, thereafter termed hNaf1, can functionally replace endogenous Naf1p in yeast. HeLa hNaf1 associates with dyskerin and hNop10 as well as box H/ACA scaRNAs, snoRNAs, and hTR. Reduction of hNaf1 steady-state levels by RNAi significantly lowers accumulation of these components of box H/ACA scaRNP, snoRNP, and telomerase. hNaf1 is found predominantly in numerous discrete foci in the nucleoplasm and fails to accumulate within Cajal bodies or nucleoli. Altogether, these results suggest that hNaf1 intervenes in early assembly steps of human box H/ACA RNPs, including telomerase.     

Endophytic fungi from flowers, capsules and seeds of Eucalyptus globulus

The main goal of this work was to detect whether Cytospora chrysosperma and Fusicoccum eucalypti are present as endophytes of symptomless hypocotyls, cotyledons, flowers, capsules, peduncles of flowers in order to interpret an earlier finding of their presence in seeds, seedling stems and twigs of E. globulus. Segments from these organs as well as from bark and the xylem from flower peduncles were surface-sterilized and plated on 2% malt-agar. All plates were incubated at 24 degrees C for six weeks or more depending on the growth rate of fungi. C. chrysosperma was asymptomatically present in flowers, capsules, hypocotyls, cotyledons and peduncles. F. eucalypti was isolated from asymptomatic flowers and capsules. It is probable that C. chrysosperma spreads during seed germination colonizing seedling stems through hypocotyl and cotyledon.     

Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.