Protrusive growth from giant liposomes driven by actin polymerization

Development of protrusions in the cell is indispensable in the process of cell motility. Membrane protrusion has long been suggested to occur as a result of actin polymerization immediately beneath the cell membrane at the leading edge, but elucidation of the mechanism is insufficient because of the complexity of the cell. To study the mechanism, we prepared giant liposomes containing monomeric actin (100 or 200 μM) and introduced KCl into individual liposomes by an electroporation technique. On the electroporation, the giant liposomes deformed. Most importantly, protrusive structure grew from the liposomes containing 200 μM actin at rates (ranging from 0.3 to 0.7 μm/s) similar to those obtained in the cell. The deformation occurred in a time range (30 ∼ 100 s) similar to that of actin polymerization monitored in a cuvette (ca. 50 s). Concomitant with deformation, Brownian motion of micron-sized particles entrapped in the liposomes almost ceased. From these observations, we conclude that actin polymerization in the liposomes caused the protrusive formation.

CtBP-dependent activities of the short-range Giant repressor in the Drosophila embryo

There are at least three short-range gap repressors in the precellular Drosophila embryo: Krüppel, Knirps, and Giant. Krüppel and Knirps contain related repression motifs, PxDLSxH and PxDLSxK, respectively, which mediate interactions with the dCtBP corepressor protein. Here, we present evidence that Giant might also interact with dCtBP. The misexpression of Giant in ventral regions of transgenic embryos results in the selective repression of eve stripe 5. A stripe5-lacZ transgene exhibits an abnormal staining pattern in dCtBP mutants that is consistent with attenuated repression by Giant. The analysis of Gal4-Giant fusion proteins identified a minimal repression domain that contains a sequence motif, VLDLS, which is conserved in at least two other sequence-specific repressors. Removal of this sequence from the native Giant protein does not impair its repression activity in transgenic embryos. We propose that Giant-dCtBP interactions might be indirect and mediated by an unknown bZIP subunit that forms a heteromeric complex with Giant. We also suggest that the VLDLS motif recruits an as yet unidentified corepressor protein.

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").

Library of medium-resolution Fiber Optic Echelle spectra of F, G, K, and M field dwarfs to giant stars

We present a library of Penn State Fiber Optic Echelle (FOE) observations of a sample of field stars with spectral types F to M and luminosity classes V to I. The spectral coverage is from 3800 to 10000 Å with a nominal resolving power of 12,000. These spectra include many of the spectral lines most widely used as optical and near-infrared indicators of chromospheric activity such as the Balmer lines (Hα to H epsilon), Ca II H & K, the Mg I b triplet, Na I D_1, D_2, He I D_3, and Ca II IRT lines. There are also a large number of photospheric lines, which can also be affected by chromospheric activity, and temperature-sensitive photospheric features such as TiO bands. The spectra have been compiled with the goal of providing a set of standards observed at medium resolution. We have extensively used such data for the study of active chromosphere stars by applying a spectral subtraction technique. However, the data set presented here can also be utilized in a wide variety of ways ranging from radial velocity templates to study of variable stars and stellar population synthesis. This library can also be used for spectral classification purposes and determination of atmospheric parameters (T_eff, log g, [Fe/H]). A digital version of all the fully reduced spectra is available via ftp and the World Wide Web (WWW) in FITS format.

Giant magnetoresistance in ultrasmall graphene based devices

By computing spin-polarized electronic transport across a finite zigzag graphene ribbon bridging two metallic graphene electrodes, we demonstrate, as a proof of principle, that devices featuring 100% magnetoresistance can be built entirely out of carbon. In the ground state a short zigzag ribbon is an antiferromagnetic insulator which, when connecting two metallic electrodes, acts as a tunnel barrier that suppresses the conductance. The application of a magnetic field makes the ribbon ferromagnetic and conductive, increasing dramatically the current between electrodes. We predict large magnetoresistance in this system at liquid nitrogen temperature and 10 T or at liquid helium temperature and 300 G.

Una mostra de traducció de literatura juvenil: James and the Giant Peach

En aquest Treball Fi de Grau (TFG) farem una anàlisi comparativa de James and the Giant Peach i les seues traduccions al català i al castellà. Pararem especial atenció al fet que es tracta d’un exemple de literatura juvenil, a l’hora d’analitzar els elements típics d’aquest gènere, com ara noms propis, elements culturals, expressions pragmàtiques, etc. El nostre objectiu principal serà extraure unes conclusions ben fonamentades sobre les tendències pel que fa a la traducció de literatura juvenil.