Rapid preparation of giant unilamellar vesicles.

We report here a rapid evaporation method that produces in high yield giant unilamellar vesicles up to 50 microns in diameter. The vesicles are obtained after only 2 min and can be prepared from different phospholipids, including L-alpha-phosphatidylcholine (lecithin), dipalmitoleoyl L-alpha-phosphatidylcholine, and beta-arachidonoyl gamma-palmitoyl L-alpha-phosphatidylcholine. Vesicles can be produced in distilled water and in Hepes, phosphate, and borate buffers in the pH range of 7.0 to 11.5 with ionic strengths up to 50 mM. The short preparation time allows encapsulation of labile molecular targets or enzymes with high catalytic activities. Cell-sized proteoliposomes have been prepared in which gamma-glutamyltransferase (EC 2.3.2.2) was functionally incorporated into the membrane wall.

Spatial localization of calcium channels in giant fiber lobe neurons of the squid (Loligo opalescens).

Whole-cell voltage clamp was used to investigate the properties and spatial distribution of fast-deactivating (FD) Ca channels in squid giant fiber lobe (GFL) neurons. Squid FD Ca channels are reversibly blocked by the spider toxin omega-Agatoxin IVA with an IC50 of 240-420 nM with no effect on the kinetics of Ca channel gating. Channels with very similar properties are expressed in both somatic and axonal domains of cultured GFL neurons, but FD Ca channel conductance density is higher in axonal bulbs than in cell bodies at all times in culture. Channels presumably synthesized during culture are preferentially expressed in the growing bulbs, but bulbar Ca conductance density remains constant while Na conductance density increases, suggesting that processes determining the densities of Ca and Na channels in this extrasomatic domain are largely independent. These observations suggest that growing axonal bulbs in cultured GFL neurons are not composed entirely of "axonal" membranes because FD Ca channels are absent from the giant axon in situ but, rather, suggest a potential role for FD Ca channels in mediating neurotransmitter release at the motor terminals of the giant axon.

Transport of cytoskeletal elements in the squid giant axon.

In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon.

Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.

Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

Chloroplast DNA variation and the recent radiation of the giant senecios (Asteraceae) on the tall mountains of eastern Africa.

Chloroplast DNA restriction-site variation was surveyed among 40 accessions representing all 11 species of giant senecios (Dendrosenecio, Asteraceae) at all but one known location, plus three outgroup species. Remarkably little variation (only 9 variable sites out of roughly 1000 sites examined) was found among the 40 giant senecio accessions, yet as a group they differ significantly (at 18 sites) from Cineraria deltoidea, the closest known relative. This pattern indicates that the giant senecios underwent a recent dramatic radiation in eastern Africa and evolved from a relatively isolated lineage within the Senecioneae. Biogeographic interpretation of the molecular phylogeny suggests that the giant senecios originated high on Mt. Kilimanjaro, with subsequent dispersion to the Aberdares, Mt. Kenya, and the Cherangani Hills, followed by dispersion westward to the Ruwenzori Mountains, and then south to the Virunga Mountains, Mt. Kahuzi, and Mt. Muhi, but with dispersion back to Mt. Elgon. Geographic radiation was an important antecedent to the diversification in eastern Africa, which primarily involved repeated altitudinal radiation, both up and down the mountains, leading to morphological parallelism in both directions. In general, the plants on a given mountain are more closely related to each other than they are to plants on other mountains, and plants on nearby mountains are more closely related to each other than they are to plants on more distant mountains. The individual steps of the geographic radiation have occurred at various altitudes, some clearly the result of intermountain dispersal. The molecular evidence suggests that two species are extant ancestors to other species on the same or nearby mountains.

Carbohydrate gluing, an architectural mechanism in the supramolecular structure of an annelid giant hemoglobin.

We report a carbohydrate-dependent supramolecular architecture in the extracellular giant hemoglobin (Hb) from the marine worm Perinereis aibuhitensis; we call this architectural mechanism carbohydrate gluing. This study is an extension of our accidental discovery of deterioration in the form of the Hb caused by a high concentration of glucose. The giant Hbs of annelids are natural supramolecules consisting of about 200 polypeptide chains that associate to form a double-layered hexagonal structure. This Hb has 0.5% (wt) carbohydrates, including mannose, xylose, fucose, galactose, glucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). Using carbohydrate-staining assays, in conjunction with two-dimensional polyacrylamide gel electrophoresis, we found that two types of linker chains (L1 and L2; the nomenclature of the Hb subunits followed that for another marine worm, Tylorrhynchus heterochaetus) contained carbohydrates with both GlcNAc and GalNAc. Furthermore, two types of globins (a and A) have only GlcNAc-containing carbohydrates, whereas the other types of globins (b and B) had no carbohydrates. Monosaccharides including mannose, fucose, glucose, galactose, GlcNAc, and GalNAc reversibly dissociated the intact form of the Hb, but the removal of carbohydrate with N-glycanase resulted in irreversible dissociation. These results show that carbohydrate acts noncovalently to glue together the components to yield the complete quaternary supramolecular structure of the giant Hb. We suggest that this carbohydrate gluing may be mediated through lectin-like carbohydrate-binding by the associated structural chains ("linkers").

The nature of LINER galaxies: Ubiquitous hot old stars and rare accreting black holes

Context. Galaxies, which often contain ionised gas, sometimes also exhibit a so-called low-ionisation nuclear emission line region (LINER). For 30 years, this was attributed to a central mass-accreting supermassive black hole (more commonly known as active galactic nucleus, AGN) of low luminosity, making LINER galaxies the largest AGN sub-population, which dominate in numbers over higher luminosity Seyfert galaxies and quasars. This, however, poses a serious problem. While the inferred energy balance is plausible, many LINERs clearly do not contain any other independent signatures of an AGN. Aims. Using integral field spectroscopic data from the CALIFA survey, we compare the observed radial surface brightness profiles with what is expected from illumination by an AGN. Methods. Essential for this analysis is a proper extraction of emission lines, especially weak lines, such as Balmer H beta lines, which are superposed on an absorption trough. To accomplish this, we use the GANDALF code, which simultaneously fits the underlying stellar continuum and emission lines. Results. For 48 galaxies with LINER-like emission, we show that the radial emission-line surface brightness profiles are inconsistent with ionisation by a central point-source and hence cannot be due to an AGN alone. Conclusions. The most probable explanation for the excess LINER-like emission is ionisation by evolved stars during the short but very hot and energetic phase known as post-AGB. This leads us to an entirely new interpretation. Post-AGB stars are ubiquitous and their ionising effect should be potentially observable in every galaxy with the gas present and with stars older than ~1 Gyr unless a stronger radiation field from young hot stars or an AGN outshines them. This means that galaxies with LINER-like emission are not a class defined by a property but rather by the absence of a property. It also explains why LINER emission is observed mostly in massive galaxies with old stars and little star formation.

DUst around NEarby Stars. The survey observational results

Context. Debris discs are a consequence of the planet formation process and constitute the fingerprints of planetesimal systems. Their solar system counterparts are the asteroid and Edgeworth-Kuiper belts. Aims. The DUNES survey aims at detecting extra-solar analogues to the Edgeworth-Kuiper belt around solar-type stars, putting in this way the solar system into context. The survey allows us to address some questions related to the prevalence and properties of planetesimal systems. Methods. We used Herschel/PACS to observe a sample of nearby FGK stars. Data at 100 and 160 μm were obtained, complemented in some cases with observations at 70 μm, and at 250, 350 and 500 μm using SPIRE. The observing strategy was to integrate as deep as possible at 100 μm to detect the stellar photosphere. Results. Debris discs have been detected at a fractional luminosity level down to several times that of the Edgeworth-Kuiper belt. The incidence rate of discs around the DUNES stars is increased from a rate of ~12.1% ± 5% before Herschel to ~20.2% ± 2%. A significant fraction (~52%) of the discs are resolved, which represents an enormous step ahead from the previously known resolved discs. Some stars are associated with faint far-IR excesses attributed to a new class of cold discs. Although it cannot be excluded that these excesses are produced by coincidental alignment of background galaxies, statistical arguments suggest that at least some of them are true debris discs. Some discs display peculiar SEDs with spectral indexes in the 70–160 μm range steeper than the Rayleigh-Jeans one. An analysis of the debris disc parameters suggests that a decrease might exist of the mean black body radius from the F-type to the K-type stars. In addition, a weak trend is suggested for a correlation of disc sizes and an anticorrelation of disc temperatures with the stellar age.

Chemically tagging the Hyades Supercluster. A homogeneous sample of F6-K4 kinematically selected northern stars

Stellar kinematic groups are kinematical coherent groups of stars that might have a common origin. These groups are dispersed throughout the Galaxy over time by the tidal effects of both Galactic rotation and disc heating, although their chemical content remains unchanged. The aim of chemical tagging is to establish that the abundances of every element in the analysis are homogeneus among the members. We study the case of the Hyades Supercluster to compile a reliable list of members (FGK stars) based on our chemical tagging analysis. For a total of 61 stars from the Hyades Supercluster, stellar atmospheric parameters (T_eff, log g, ξ, and [Fe/H]) are determined using our code called StePar, which is based on the sensitivity to the stellar atmospheric parameters of the iron EWs measured in the spectra. We derive the chemical abundances of 20 elements and find that their [X/Fe] ratios are consistent with Galactic abundance trends reported in previous studies. The chemical tagging method is applied with a carefully developed differential abundance analysis of each candidate member of the Hyades Supercluster, using a well-known member of the Hyades cluster as a reference (vB 153). We find that only 28 stars (26 dwarfs and 2 giants) are members, i.e. that 46% of our candidates are members based on the differential abundance analysis. This result confirms that the Hyades Supercluster cannot originate solely from the Hyades cluster.

Magnetic activity and differential rotation in the young Sun-like stars KIC 7985370 and KIC 7765135

Aims. We present a detailed study of the two Sun-like stars KIC 7985370 and KIC 7765135, to determine their activity level, spot distribution, and differential rotation. Both stars were previously discovered by us to be young stars and were observed by the NASA Kepler mission. Methods. The fundamental stellar parameters (vsini, spectral type, T_eff, log g, and [Fe/H]) were derived from optical spectroscopy by comparison with both standard-star and synthetic spectra. The spectra of the targets allowed us to study the chromospheric activity based on the emission in the core of hydrogen Hα and Ca ii infrared triplet (IRT) lines, which was revealed by the subtraction of inactive templates. The high-precision Kepler photometric data spanning over 229 days were then fitted with a robust spot model. Model selection and parameter estimation were performed in a Bayesian manner, using a Markov chain Monte Carlo method. Results. We find that both stars are Sun-like (of G1.5 V spectral type) and have an age of about 100–200 Myr, based on their lithium content and kinematics. Their youth is confirmed by their high level of chromospheric activity, which is comparable to that displayed by the early G-type stars in the Pleiades cluster. The Balmer decrement and flux ratio of their Ca ii-IRT lines suggest that the formation of the core of these lines occurs mainly in optically thick regions that are analogous to solar plages. The spot model applied to the Kepler photometry requires at least seven persistent spots in the case of KIC 7985370 and nine spots in the case of KIC 7765135 to provide a satisfactory fit to the data. The assumption of the longevity of the star spots, whose area is allowed to evolve with time, is at the heart of our spot-modelling approach. On both stars, the surface differential rotation is Sun-like, with the high-latitude spots rotating slower than the low-latitude ones. We found, for both stars, a rather high value of the equator-to-pole differential rotation (dΩ ≈ 0.18 rad d^-1), which disagrees with the predictions of some mean-field models of differential rotation for rapidly rotating stars. Our results agree instead with previous works on solar-type stars and other models that predict a higher latitudinal shear, increasing with equatorial angular velocity, that can vary during the magnetic cycle.

No surviving evolved companions of the progenitor of SN 1006

Type Ia supernovae are thought to occur when a white dwarf made of carbon and oxygen accretes sufficient mass to trigger a thermonuclear explosion(1). The accretion could be slow, from an unevolved (main-sequence) or evolved (subgiant or giant) star(2,3) (the single-degenerate channel), or rapid, as the primary star breaks up a smaller orbiting white dwarf(3,4) (the double-degenerate channel). A companion star will survive the explosion only in the single-degenerate channel(5). Both channels might contribute to the production of type Ia supernovae(6,7), but the relative proportions of their contributions remain a fundamental puzzle in astronomy. Previous searches for remnant companions have revealed one possible case for SN 1572 (refs 8, 9), although that has been questioned(10). More recently, observations have restricted surviving companions to be small, main-sequence stars(11-13), ruling out giant companions but still allowing the single-degenerate channel. Here we report the results of a search for surviving companions of the progenitor of SN 1006 (ref. 14). None of the stars within 4 arc minutes of the apparent site of the explosion is associated with the supernova remnant, and we can firmly exclude all giant and subgiant stars from being companions of the progenitor. In combination with previous results, our findings indicate that fewer than 20 per cent of type Ia supernovae occur through the single-degenerate channel.

A spectroscopy study of nearby late-type stars, possible members of stellar kinematic groups

Context. Nearby late-type stars are excellent targets for seeking young objects in stellar associations and moving groups. The origin of these structures is still misunderstood, and lists of moving group members often change with time and also from author to author. Most members of these groups have been identified by means of kinematic criteria, leading to an important contamination of previous lists by old field stars. Aims. We attempt to identify unambiguous moving group members among a sample of nearby-late type stars by studying their kinematics, lithium abundance, chromospheric activity, and other age-related properties. Methods. High-resolution echelle spectra (R ~ 57 000) of a sample of nearby late-type stars are used to derive accurate radial velocities that are combined with the precise Hipparcos parallaxes and proper motions to compute galactic-spatial velocity components. Stars are classified as possible members of the classical moving groups according to their kinematics. The spectra are also used to study several age-related properties for young late-type stars, i.e., the equivalent width of the lithium Li i 6707.8 Å line or the R'_HK index. Additional information like X-ray fluxes from the ROSAT All-Sky Survey or the presence of debris discs is also taken into account. The different age estimators are compared and the moving group membership of the kinematically selected candidates are discussed. Results. From a total list of 405 nearby stars, 102 have been classified as moving group candidates according to their kinematics. i.e., only ~25.2% of the sample. The number reduces when age estimates are considered, and only 26 moving group candidates (25.5% of the 102 candidates) have ages in agreement with the star having the same age as an MG member.

Discovery of "isolated" co-moving T Tauri stars in Cepheus

Context. During the course of a large spectroscopic survey of X-ray active late-type stars in the solar neighbourhood, we discovered four lithium-rich stars packed within just a few degrees on the sky. Although located in a sky area rich in CO molecular regions and dark clouds, the Cepheus-Cassiopeia complex, these very young stars are projected several degrees away from clouds in front of an area void of interstellar matter. As such, they are very good "isolated" T Tauri star candidates. Aims. We present optical observations of these stars conducted with 1-2 m class telescopes. We acquired high-resolution optical spectra as well as photometric data allowing us to investigate in detail their nature and physical parameters with the aim of testing the "runaway" and "in-situ" formation scenarios. Their kinematical properties are also analyzed to investigate their possible connection to already known stellar kinematic groups. Methods. We use the cross-correlation technique and other tools developed by us to derive accurate radial and rotational velocities and perform an automatic spectral classification. The spectral subtraction technique is used to infer chromospheric activity level in the Hα line core and clean the spectra of photospheric lines before measuring the equivalent width of the lithium absorption line. Results. Both physical (lithium content, chromospheric, and coronal activities) and kinematical indicators show that all stars are very young, with ages probably in the range 10-30 Myr. In particular, the spectral energy distribution of TYC4496-780-1 displays a strong near-and far-infrared excess, typical of T Tauri stars still surrounded by an accretion disc. They also share the same Galactic motion, proving that they form a homogeneous moving group of stars with the same origin. Conclusions. The most plausible explanation of how these "isolated" T Tauri stars formed is the "in-situ" model, although accurate distances are needed to clarify their connection with the Cepheus-Cassiopeia complex. The discovery of this loose association of "isolated" T Tauri stars can help to shed light on atypical formation processes of stars and planets in low-mass clouds.

Chromospheric activity and rotation of FGK stars in the solar vicinity. An estimation of the radial velocity jitter

Context. Chromospheric activity produces both photometric and spectroscopic variations that can be mistaken as planets. Large spots crossing the stellar disc can produce planet-like periodic variations in the light curve of a star. These spots clearly affect the spectral line profiles, and their perturbations alter the line centroids creating a radial velocity jitter that might “contaminate” the variations induced by a planet. Precise chromospheric activity measurements are needed to estimate the activity-induced noise that should be expected for a given star. Aims. We obtain precise chromospheric activity measurements and projected rotational velocities for nearby (d ≤ 25 pc) cool (spectral types F to K) stars, to estimate their expected activity-related jitter. As a complementary objective, we attempt to obtain relationships between fluxes in different activity indicator lines, that permit a transformation of traditional activity indicators, i.e., Ca II H & K lines, to others that hold noteworthy advantages. Methods. We used high resolution (~50 000) echelle optical spectra. Standard data reduction was performed using the IRAF ECHELLE package. To determine the chromospheric emission of the stars in the sample, we used the spectral subtraction technique. We measured the equivalent widths of the chromospheric emission lines in the subtracted spectrum and transformed them into fluxes by applying empirical equivalent width and flux relationships. Rotational velocities were determined using the cross-correlation technique. To infer activity-related radial velocity (RV) jitter, we used empirical relationships between this jitter and the R’_HK index. Results. We measured chromospheric activity, as given by different indicators throughout the optical spectra, and projected rotational velocities for 371 nearby cool stars. We have built empirical relationships among the most important chromospheric emission lines. Finally, we used the measured chromospheric activity to estimate the expected RV jitter for the active stars in the sample.