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Sample records for vesicles membrane depolarization

  1. Neuronal Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via VGLUT.

    Science.gov (United States)

    Aguilar, Jenny I; Dunn, Matthew; Mingote, Susana; Karam, Caline S; Farino, Zachary J; Sonders, Mark S; Choi, Se Joon; Grygoruk, Anna; Zhang, Yuchao; Cela, Carolina; Choi, Ben Jiwon; Flores, Jorge; Freyberg, Robin J; McCabe, Brian D; Mosharov, Eugene V; Krantz, David E; Javitch, Jonathan A; Sulzer, David; Sames, Dalibor; Rayport, Stephen; Freyberg, Zachary

    2017-08-30

    The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Membrane Trafficking and Vesicle Fusion

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 19; Issue 5. Membrane Trafficking and Vesicle Fusion: Post-Palade Era Researchers Win the Nobel Prize. Riddhi Atul Jani Subba Rao Gangi Setty. General Article Volume 19 Issue 5 May 2014 pp 421-445 ...

  3. Mechanism of pyrithione-induced membrane depolarization in Neurospora crassa.

    Science.gov (United States)

    Ermolayeva, E; Sanders, D

    1995-01-01

    Pyrithione is a general inhibitor of membrane transport in fungi and is widely used in antidandruff shampoos as an antifungal agent. An electrophysiological approach has been used to determine the mode of action of pyrithione on the plasma membrane of the model ascomycete, Neurospora crassa. At pH 5.8, pyrithione induces a dramatic dose-dependent electrical depolarization of the membrane which is complete within 4 min, amounts to 110 mV at saturating pyrithione concentrations, and is half maximal between 0.6 and 0.8 mM pyrithione. Zinc pyrithione induces a similar response but exerts a half-maximal effect at around 0.3 mM. The depolarization is strongly dependent on external pH, being almost absent at pH 8.2, at which the concentration of the uncharged form of pyrithione--which might be expected to permeate the membrane freely--is markedly lowered. However, quantitative considerations based on cytosolic buffer capacity, the pKa of pyrithione, and the submillimolar concentration at which it is active appear to preclude significant cytosolic acidification on dissociation of the thiol proton from the uncharged form of pyrithione. Current-voltage analysis demonstrates that the depolarization is accompanied by a decrease in membrane electrical conductance in a manner consistent with inhibition of the primary proton pump and inconsistent with a mode of action of pyrithione on plasma membrane ion channels. We conclude that pyrithione inhibits membrane transport via a direct or indirect effect on the primary proton pump which energizes transport and that the site of action of pyrithione is likely to be intra- rather than extracellular. PMID:7574648

  4. Role of Outer Membrane Vesicles of Bacteria

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 20; Issue 8. Role of Outer Membrance Vesicles of Bacteria. M V Jagannadham M K Chattopadhyay. General Article Volume 20 Issue 8 ... Keywords. Outer membrane ves ic les (OMVs); secretion; communication; virulence; antibiotic resistance; vaccines.

  5. Membrane Trafficking and Vesicle Fusion

    Indian Academy of Sciences (India)

    IAS Admin

    hemophagocytic syndrome) and metabolic (diabe- tes) disorders [2, 23, 33]. Mutations in the genes of the basic secretory protein machinery lead to a number of membrane trafficking diseases such as Charcot–Marie–Tooth disease, Cohen.

  6. Giant plasma membrane vesicles: models for understanding membrane organization.

    Science.gov (United States)

    Levental, Kandice R; Levental, Ilya

    2015-01-01

    The organization of eukaryotic membranes into functional domains continues to fascinate and puzzle cell biologists and biophysicists. The lipid raft hypothesis proposes that collective lipid interactions compartmentalize the membrane into coexisting liquid domains that are central to membrane physiology. This hypothesis has proven controversial because such structures cannot be directly visualized in live cells by light microscopy. The recent observations of liquid-liquid phase separation in biological membranes are an important validation of the raft hypothesis and enable application of the experimental toolbox of membrane physics to a biologically complex phase-separated membrane. This review addresses the role of giant plasma membrane vesicles (GPMVs) in refining the raft hypothesis and expands on the application of GPMVs as an experimental model to answer some of key outstanding problems in membrane biology. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Chlorovirus-Mediated Membrane Depolarization of Chlorella Alters Secondary Active Transport of Solutes▿

    Science.gov (United States)

    Agarkova, Irina; Dunigan, David; Gurnon, James; Greiner, Timo; Barres, Julia; Thiel, Gerhard; Van Etten, James L.

    2008-01-01

    Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding genes; 224 are secondary active transporters. Solute uptake experiments using seven radioactive compounds revealed that virus infection alters the transport of all the solutes. However, the degree of inhibition varied depending on the solute. Experiments with nystatin, a drug known to depolarize cell membranes, produced changes in solute uptake that are similar but not identical to those that occurred during virus infection. Therefore, these studies indicate that chlorovirus infection causes a rapid and sustained depolarization of the host plasma membrane and that this depolarization leads to the inhibition of secondary active transporters that changes solute uptake. PMID:18842725

  8. Normal chemotaxis in Dictyostelium discoideum cells with a depolarized plasma membrane potential

    NARCIS (Netherlands)

    Duijn, Bert van; Vogelzang, Sake A.; Ypey, Dirk L.; Molen, Loek G. van der; Haastert, Peter J.M. van

    1990-01-01

    We examined a possible role for the plasma membrane potential in signal transduction during cyclic AMP-induced chemotaxis in the cellular slime mold Dictyostelium discoideum. Chemotaxis, cyclic GMP and cyclic AMP responses in cells with a depolarized membrane potential were measured. Cells can be

  9. Large Deformation Mechanics of Plasma Membrane Chained Vesicles in Cells

    Science.gov (United States)

    Kosawada, Tadashi; Sanada, Kouichi; Takano, Tetsuo

    The clathrin-coated pits, vesicles and chained vesicles on the inner surface of the plasma membrane facilitate the cell to transport specific extracellular macromolecules. This cellular process is strongly involved with large mechanical deformations of the plasma membrane accompanied by changes in membrane curvature. The assembly of the clathrin coat is thought to provide curvature into the membrane. Hence, effects of in-plane shear elasticity due to these coat structure may be significant on the vesicular mechanics. In this study, large deformation mechanics of plasma membrane chained vesicles in cells have been formulated based on minimization of bending and in-plane shear strain energy of the membrane. Effects of outer surrounding cytoplasmic flat membrane upon mechanically stable shapes of the vesicles were revealed, while effects of in-plane shear elasticity were partly discussed.

  10. Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls

    Science.gov (United States)

    Spalding, E. P.; Cosgrove, D. J.

    1992-01-01

    A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H(+)-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca(2+)-channel blockers (verapamil, La3+) or by reducing extracellular free Ca2+ by treatment with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca2+ is involved. No effects of the K(+)-channel blocker tetraethylammonium (TEA+) were detected. Vanadate and KCN, used to inhibit the H(+)-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11-26% after 1-2 min of BL. Input resistance of trichrome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H(+)-ATPase with subsequent transient activation of one or more types of ion channels.

  11. Membrane Protrusion Coarsening and Nanotubulation within Giant Unilamellar Vesicles

    KAUST Repository

    Węgrzyn, Ilona

    2011-11-16

    Hydrophobic side groups on a stimuli-responsive polymer, encapsulated within a single giant unilamellar vesicle, enable membrane attachment during compartment formation at elevated temperatures. We thermally modulated the vesicle through implementation of an IR laser via an optical fiber, enabling localized directed heating. Polymer-membrane interactions were monitored using confocal imaging techniques as subsequent membrane protrusions occurred and lipid nanotubes formed in response to the polymer hydrogel contraction. These nanotubes, bridging the vesicle membrane to the contracting hydrogel, were retained on the surface of the polymer compartment, where they were transformed into smaller vesicles in a process reminiscent of cellular endocytosis. This development of a synthetic vesicle system containing a stimuli-responsive polymer could lead to a new platform for studying inter/intramembrane transport through lipid nanotubes. © 2011 American Chemical Society.

  12. Tension-induced fusion of bilayer membranes and vesicles

    Science.gov (United States)

    Shillcock, Julian C.; Lipowsky, Reinhard

    2005-03-01

    Maintaining the integrity of their protective plasma membrane is a primary requirement of cells. Accordingly, cellular events that breach the membrane are tightly regulated. Artificial vesicles used in drug delivery must also stay intact until they have reached the desired target. In both cases, the intrinsic resistance of the membrane to rupture must be overcome to allow the efflux of the vesicle's contents. Here, we use mesoscopic simulations to study the fusion of 28-nm-diameter vesicles to 50 × 50 nm2 planar membrane patches over 2 μs. We monitor the time evolution of 93 different fusion attempts. This allows us to construct a global morphology diagram, using the initial tensions of the vesicle and the planar membrane patch as control parameters, and to determine the corresponding fusion statistics. All successful fusion events are observed to occur within 350 ns, which reflects the presence of alternative pathways for the tension relaxation.

  13. Mating-reactive membrane vesicles from cilia of Paramecium caudatum

    Science.gov (United States)

    1976-01-01

    Membrane vesicles with a high mating reactivity were obtained from cilia of Paramecium caudatum by treatment with a solution containing 2 M urea and 0.1 mM Na2-EDTA. All processes of conjugation were induced in cells of the complementary mating type by approximately 10 mug/ml proteins of the vesicles. Electron microscope observation showed that the membrane vesicles have a diameter of 100-150 nm. Electrophoretic analysis on SDS polyacrylamide gel revealed no significant difference in polypeptide patterns of the particles from the two complementary mating types. PMID:818093

  14. Asymmetric osmotic water permeation through a vesicle membrane

    Science.gov (United States)

    Su, Jiaye; Zhao, Yunzhen; Fang, Chang; Shi, Yue

    2017-05-01

    Understanding the water permeation through a cell membrane is of primary importance for biological activities and a key step to capture its shape transformation in salt solution. In this work, we reveal the dynamical behaviors of osmotically driven transport of water molecules across a vesicle membrane by molecular dynamics simulations. Of particular interest is that the water transport in and out of vesicles is highly distinguishable given the osmotic force are the same, suggesting an asymmetric osmotic transportation. This asymmetric phenomenon exists in a broad range of parameter space such as the salt concentration, temperature, and vesicle size and can be ascribed to the similar asymmetric potential energy of lipid-ion, lipid-water, lipid-solution, lipid-lipid, and the lipid-lipid energy fluctuation. Specifically, the water flux has a linear increase with the salt concentration, similar to the prediction by Nernst-Planck equation or Fick's first law. Furthermore, due to the Arrhenius relation between the membrane permeability and temperature, the water flux also exhibits excellent Arrhenius dependence on the temperature. Meanwhile, the water flux shows a linear increase with the vesicle surface area since the flux amount across a unit membrane area should be a constant. Finally, we also present the anonymous diffusion behaviors for the vesicle itself, where transitions from normal diffusion at short times to subdiffusion at long times are identified. Our results provide significant new physical insights for the osmotic water permeation through a vesicle membrane and are helpful for future experimental studies.

  15. Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

    Directory of Open Access Journals (Sweden)

    Azusa Oshima

    2017-09-01

    Full Text Available The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs to control the fusion process. We combined large unilamellar vesicles (LUVs containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO2 substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.

  16. Yeast Membrane Vesicles: Isolation and General Characteristics1

    Science.gov (United States)

    Christensen, Michael S.; Cirillo, Vincent P.

    1972-01-01

    Yeast membrane vesicles are formed when packed yeast are ground manually in a porcelain mortar and pestle with glass beads (0.2 mm diameter). These vesicles can be separated from the other components of the grinding mixture by a combination of centrifugation steps and elution from a column of the same glass beads (0.2 mm diameter). Isolated vesicles are osmotically sensitive, contain cytoplasmic components, and have energy-independent transport function. They are unable to metabolize glucose, but have respiratory function which is thought to be associated with intravesicular mitochondria. Invertase and oligomycin-insensitive adenosine triphosphatase are present in lysed vesicle preparations, and the appropriateness of these enzyme activities as membrane markers is discussed. Images PMID:4337848

  17. Glycosylation-induced depolarization facilitates subthreshold membrane oscillation in injured primary sensory neurons.

    Science.gov (United States)

    Li, Chen-Xu; Jing, Yan-Ling; Xie, Yi-Kuan

    2007-03-30

    Subthreshold membrane potential oscillations (SMPO) in the injured dorsal root ganglion (DRG) neurons are involved in the generation of spontaneous activity, which can directly evoke neuropathic pain. Nerve injury usually triggers the synthesis of large quantities of membrane protein in nerve injured DRG neurons. Membrane proteins are glycosylated by addition of sugars, especially negatively charged sialic acid residues, which may depolarize the resting membrane potential (Vm), open voltage-gated channels in injured neurons, and cause spontaneous activity. In the present study, we aimed to determine if increased negative charge on the cell surface, carried by the sialic acid residues, could contribute to the generation of SMPO in injured DRG neurons. Intracellular recording was performed in DRG neurons following chronic constrictive injury (CCI) of the sciatic nerve. Results indicated that both A- and C-type injured DRG neurons exhibited a higher incidence of SMPO and more depolarized Vm than those of the control neurons. Ca(2+), Mg(2+), Mn(2+), or poly-lysine, a positively charged organic compound, when topically applied to the DRG, not only reduced SMPO but also caused a rapid hyperpolarizing shift in Vm. Topical application of neuraminidase to selectively remove sialic acid residues on the extracellular membrane normalized the depolarized Vm and inhibited both spontaneous and evoked SMPO. However, application of Ca(2+), Mg(2+), Mn(2+) or neuraminidase had no effect on excitability and Vm in normal neurons. The results demonstrated that the increase in negatively charged sialic acid residues on the extracellular membrane of neuronal somata is a critical factor in the generation of SMPO and hyperexcitability in injured sensory neurons.

  18. Large plasma-membrane depolarization precedes rapid blue-light-induced growth inhibition in cucumber

    Science.gov (United States)

    Spalding, E. P.; Cosgrove, D. J.

    1989-01-01

    Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls began after a 30-s lag time, which was halved by increasing the fluence rate from 10 to 100 micromoles m-2 s-1. Prior to the growth suppression, the plasma-membrane of the irradiated cells depolarized by as much as 100 mV, then returned within 2-3 min to near its initial value. The potential difference measured with surface electrodes changed with an identical time course but opposite polarity. The lag time for the change in surface potential showed an inverse dependence on fluence rate, similar to the lag for the growth inhibition. Green light and red light caused neither the electrical response nor the rapid inhibition of growth. The depolarization by BL did not propagate to nonirradiated regions and exhibited a refractory period of about 10 min following a BL pulse. Fluence-response relationships for the electrical and growth responses provide correlational evidence that the plasma-membrane depolarization reflects an event in the transduction chain of this light-growth response.

  19. Curvature-Mediated Assembly of Janus Nanoparticles on Membrane Vesicles.

    Science.gov (United States)

    Bahrami, Amir Houshang; Weikl, Thomas R

    2018-01-08

    Besides direct particle-particle interactions, nanoparticles adsorbed to biomembranes experience indirect interactions that are mediated by the membrane curvature arising from particle adsorption. In this Letter, we show that the curvature-mediated interactions of adsorbed Janus particles depend on the initial curvature of the membrane prior to adsorption, that is, on whether the membrane initially bulges toward or away from the particles in our simulations. The curvature-mediated interaction can be strongly attractive for Janus particles adsorbed to the outside of a membrane vesicle, which initially bulges away from the particles. For Janus particles adsorbed to the vesicle inside, in contrast, the curvature-mediated interactions are repulsive. We find that the area fraction of the adhesive Janus particle surface is an important control parameter for the curvature-mediated interaction and assembly of the particles, besides the initial membrane curvature.

  20. Biogenesis and function of Porphyromonas gingivalis outer membrane vesicles

    Science.gov (United States)

    Xie, H

    2015-01-01

    Porphyromonas gingivalis is one of the keystone pathogens associated with chronic periodontitis. All P. gingivalis strains examined thus far produce outer membrane vesicles. Recent studies have found that vesicles possess some well-known virulence factors of P. gingivalis such as adhesins, toxins and proteolytic enzymes. Carrying most of the characteristic features of their parent P. gingivalis cells, vesicles communicate with host cells and other members of microbial biofilms, resulting in the transmission of virulence factors into these host cells and the formation of pathogenic bacteria-dominated microbial communities. An in-depth understanding of both the nature and role of vesicles in the pathogenicity of P. gingivalis is both important and timely, particularly when speaking of periodontitis and its related systemic effects. PMID:26343879

  1. Paeonia lactiflora Inhibits Cell Wall Synthesis and Triggers Membrane Depolarization in Candida albicans.

    Science.gov (United States)

    Lee, Heung-Shick; Kim, Younhee

    2017-02-28

    Fungal cell walls and cell membranes are the main targets of antifungals. In this study, we report on the antifungal activity of an ethanol extract from Paeonia lactiflora against Candida albicans, showing that the antifungal activity is associated with the synergistic actions of preventing cell wall synthesis, enabling membrane depolarization, and compromising permeability. First, it was shown that the ethanol extract from P. lactiflora was involved in damaging the integrity of cell walls in C. albicans. In isotonic media, cell bursts of C. albicans by the P. lactiflora ethanol extract could be restored, and the minimum inhibitory concentration (MIC) of the P. lactiflora ethanol extract against C. albicans cells increased 4-fold. In addition, synthesis of (1,3)-β-D-glucan polymer was inhibited by 87% and 83% following treatment of C. albicans microsomes with the P. lactiflora ethanol extract at their 1× MIC and 2× MIC, respectively. Second, the ethanol extract from P. lactiflora influenced the function of C. albicans cell membranes. C. albicans cells treated with the P. lactiflora ethanol extract formed red aggregates by staining with a membrane-impermeable dye, propidium iodide. Membrane depolarization manifested as increased fluorescence intensity by staining P. lactiflora-treated C. albicans cells with a membrane-potential marker, DiBAC4(3) ((bis-1,3-dibutylbarbituric acid) trimethine oxonol). Membrane permeability was assessed by crystal violet assay, and C. albicans cells treated with the P. lactiflora ethanol extract exhibited significant uptake of crystal violet in a concentration-dependent manner. The findings suggest that P. lactiflora ethanol extract is a viable and effective candidate for the development of new antifungal agents to treat Candida-associated diseases.

  2. Durable vesicles for reconstitution of membrane proteins in biotechnology.

    Science.gov (United States)

    Beales, Paul A; Khan, Sanobar; Muench, Stephen P; Jeuken, Lars J C

    2017-02-08

    The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with. © 2017 The Author(s).

  3. Numerical computations of the dynamics of fluidic membranes and vesicles

    CERN Document Server

    Barrett, John W; Nürnberg, Robert

    2015-01-01

    Vesicles and many biological membranes are made of two monolayers of lipid molecules and form closed lipid bilayers. The dynamical behaviour of vesicles is very complex and a variety of forms and shapes appear. Lipid bilayers can be considered as a surface fluid and hence the governing equations for the evolution include the surface (Navier--)Stokes equations, which in particular take the membrane viscosity into account. The evolution is driven by forces stemming from the curvature elasticity of the membrane. In addition, the surface fluid equations are coupled to bulk (Navier--)Stokes equations. We introduce a parametric finite element method to solve this complex free boundary problem, and present the first three dimensional numerical computations based on the full (Navier--)Stokes system for several different scenarios. For example, the effects of the membrane viscosity, spontaneous curvature and area difference elasticity (ADE) are studied. In particular, it turns out, that even in the case of no viscosit...

  4. A Pathogenic Potential of Acinetobacter baumannii-Derived Membrane Vesicles

    Directory of Open Access Journals (Sweden)

    Jong Suk Jin

    2011-12-01

    Full Text Available Acinetobacter baumannii secretes outer membrane vesicles (OMVs. A. baumannii OMVs deliver many virulence factors to host cells and then induce cytotoxicity and innate immune response. OMVs secreted from bacteria contribute directly to host pathology during A. baumannii infection.

  5. Association of Randall's Plaques with Collagen Fibers and Membrane Vesicles

    Science.gov (United States)

    Khan, Saeed R.; Rodriguez, Douglas E.; Gower, Laurie B.; Monga, Manoj

    2013-01-01

    Background Idiopathic calcium oxalate (CaOx) kidney stones develop by deposition of CaOx crystals on Randall's plaques (RP). Mechanisms involved in RP formation are still unclear. Objective It is our hypotheses that RP formation is similar to vascular calcification involving components of extracellular matrix including membrane bound vesicles (MV) and collagen fibers. In order to verify our hypothesis we critically examined renal papillary tissue from stone patients. Methods 4 mm cold-cup biopies of renal papillae were performed on fifteen idiopathic stone patients undergoing PCNL. Tissue was immediately fixed and processed for analyses by various light and electron microscopic techniques. Results and Limitations Spherulitic CaP crystals, the hallmark of RP's, were seen in all samples examined. They were seen in interstitium as well as laminated basement membrane of tubular epithelia. Large crystalline deposits comprised of dark elongated strands mixed with spherulites. Strands showed banded patterns similar to collagen. Crystal deposits were surrounded by collagen fibers and membrane bound vesicles. Energy dispersive x-ray microanalyses (EDX) and electron diffraction identified the crystals as hydroxyapatite. The number of kidneys examined is small and urinary data was not available for all the patients. Conclusions Results presented here show that crystals in the Randall's plaques are associated with both the collagen as well as MV. Collagen fibers appeared calcified and vesicles contained crystals. We conclude that crystal deposition in renal papillae may have started with membrane vesicle induced nucleation and grew by addition of crystals on the periphery within a collagen framework. PMID:22266007

  6. Membrane depolarization was required to induce DNA synthesis in murine macrophage cell line PU5-1.8.

    Science.gov (United States)

    Kong, S K; Suen, Y K; Choy, Y M; Fung, K P; Lee, C Y

    1991-01-01

    The role of membrane potential (Em) on the initiation of DNA synthesis in murine macrophage cell line PU5-1.8 was investigated with fluorescent probes bis-oxonol and diS-C3-(5). Incubation of PU5-1.8 cells in high K(+)-HEPES buffer or with gramicidin at 37 degrees C for 1h that depolarized the membrane induced [3H]-thymidine incorporation and expression of early response gene such as c-myc and c-fos. When PU5-1.8 cells were treated with a number of agents including fetal calf serum (FCS), lipopolysaccharide (LPS), epidermal growth factor (EGF), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and bradykinin (BK), only FCS caused DNA synthesis and membrane depolarization. Other agents had no effect on these events. The FCS-mediated DNA synthesis in PU5-1.8 cells was inhibited by clamping the membrane potential with valinomycin. Moreover, intracellular alkalinization induced by nigericin at pH 7.9, which is believed to be a permissive signal for mitogenesis, caused membrane depolarization. On the other hand, challenge of cells with phorbol 12-myristate 13 acetate (PMA) suppressed the K(+)-mediated DNA synthesis. However, the treatment of cells with PMA did not change the membrane potential but suppressed the gramicidin-mediated membrane depolarization. These observations suggest that there is a correlation between membrane depolarization and initiation of DNA synthesis in PU5-1.8 cells. PKC may be acting as a modulator in this transducing pathway.

  7. Origin of life: LUCA and extracellular membrane vesicles (EMVs)

    Science.gov (United States)

    Gill, S.; Forterre, P.

    2016-01-01

    Cells from the three domains of life produce extracellular membrane vesicles (EMVs), suggesting that EMV production is an important aspect of cellular physiology. EMVs have been implicated in many aspects of cellular life in all domains, including stress response, toxicity against competing strains, pathogenicity, detoxification and resistance against viral attack. These EMVs represent an important mode of inter-cellular communication by serving as vehicles for transfer of DNA, RNA, proteins and lipids between cells. Here, we review recent progress in the understanding of EMV biology and their various roles. We focus on the role of membrane vesicles in early cellular evolution and how they would have helped shape the nature of the last universal common ancestor. A membrane-protected micro-environment would have been a key to the survival of spontaneous molecular systems and efficient metabolic reactions. Interestingly, the morphology of EMVs is strongly reminiscent of the morphology of some virions. It is thus tempting to make a link between the origin of the first protocell via the formation of vesicles and the origin of viruses.

  8. Ba2+-inhibitable /sup 86/Rb+ fluxes across membranes of vesicles from toad urinary bladder

    Energy Technology Data Exchange (ETDEWEB)

    Garty, H.; Civan, M.M.

    1987-01-01

    /sup 86/Rb+ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba2+ required for half-maximal inhibition was 0.6 mM. The effects of externally added cations on /sup 86/Rb+ influx and efflux have established that this pathway is conductive, with a selectivity for K+, Rb+ and Cs+ over Na+ and Li+. The Rb+ uptake is inversely dependent on external pH, but not significantly affected by internal Ca2+ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb+ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K+ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na+ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca2+ activity directly blocks apical Na+ entry.

  9. Redox-active nanoceria depolarize mitochondrial membrane of human colon cancer cells

    Science.gov (United States)

    Jana, Saikat Kumar; Banerjee, Priyanka; Das, Soumen; Seal, Sudipta; Chaudhury, Koel

    2014-06-01

    Nanotherapeutics is emerging as a promising option to the various limitations and side effects associated with conventional chemotherapy. The present study investigates the cytotoxic effect of redox-active cerium oxide nanoparticles (nanoceria) on human colorectal adenocarcinoma-derived cell line (HCT 15). Exposure of these cells to nanoceria for 24 h with concentration ranging between 10 and 100 μM resulted in a significant reduction of cell viability in a dose-dependent manner. Further, at a concentration of 10 µM, nanoceria exhibited time-dependent cytotoxic effect when exposed to the cells for 24, 48, and 72 h. Upon treatment of the cells with nanoceria, reactive oxygen species (ROS) and lipid peroxidation which are indicators of oxidative stress and cytotoxicity increased significantly, in a dose-dependent manner. Nanoceria was also found to depolarize the mitochondrial membrane, thereby collapsing the membrane potential and leading to initiation of apoptosis. Scanning electron microscopic study of nanoceria-treated HCT 15 cells showed morphological changes and loss of filopodia and lamellipodia, indicating arrest of metastatic spread. Summarizing, when cultured HCT 15 cells are exposed to nanoceria, a dose-dependent cytotoxic effect mediated by ROS generation is observed.

  10. Excimer fluorescence compared to depolarization in the flow cytometric characterization of lateral membrane mobility in platelets

    Science.gov (United States)

    Rothe, Gregor; Schaefer, Buerk; Wimmer, Martin S.; Schmitz, Gerd

    1998-04-01

    An altered cellular membrane fluidity secondary to changes of cholesterol metabolism is a potentially important mechanism in the pathogenesis of atherosclerosis. Especially in blood platelets an increased sensitivity for stimulation dependent aggregation which is a risk factor for thrombosis has been experimentally linked to disorders of lipid and lipoprotein metabolism. The goal of this study was the development of a flow cytometric assay for the direct analysis of cellular membrane microviscosity in correlation to activation associated phenotypic changes of platelets in vitro. The analysis of fluorescence polarization following the staining of hydrophobic lipid regions of cell membranes with the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is a well established method for the analysis of membrane fluidity. The extent of fluorescence anisotropy dependent on the rotational mobility of this fluorochrome is indirectly proportional to the microviscosity of the stained membrane subcompartment. In this study, an alternative and more simple method based on the diffusion dependent excimer formation of pyrenedecanoic acid (PDA) (J. Immunol. Methods 96:225-31, 1987) was characterized in comparison to the DPH method as a reference. Human platelets showed a rapid uptake of both DPH and PDA resulting in the staining primarily of the plasma membrane after up to 30 min of incubation. Staining analyzed at 351 nm excitation resulted in a saturation of the depolarization coefficient of DPH at 20 (mu) M but an increase of the excimer to monomer ratio of PDA with increasing dye concentration. A 'membrane fluidity coefficient' which saturated at 5 (mu) M PDA was calculated as the excimer fluorescence divided through the square of monomer fluorescence thereby correcting for the influence of dye concentration on excimer formation. The temperature dependent changes of membrane viscosity were further used as a model for the comparison of both methods. Cells analyzed at temperatures

  11. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

    Science.gov (United States)

    Prada, Ilaria; Meldolesi, Jacopo

    2016-08-09

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

  12. Apricot melanoidins prevent oxidative endothelial cell death by counteracting mitochondrial oxidation and membrane depolarization.

    Directory of Open Access Journals (Sweden)

    Annalisa Cossu

    Full Text Available The cardiovascular benefits associated with diets rich in fruit and vegetables are thought to be due to phytochemicals contained in fresh plant material. However, whether processed plant foods provide the same benefits as unprocessed ones is an open question. Melanoidins from heat-processed apricots were isolated and their presence confirmed by colorimetric analysis and browning index. Oxidative injury of endothelial cells (ECs is the key step for the onset and progression of cardiovascular diseases (CVD, therefore the potential protective effect of apricot melanoidins on hydrogen peroxide-induced oxidative mitochondrial damage and cell death was explored in human ECs. The redox state of cytoplasmic and mitochondrial compartments was detected by using the redox-sensitive, fluorescent protein (roGFP, while the mitochondrial membrane potential (MMP was assessed with the fluorescent dye, JC-1. ECs exposure to hydrogen peroxide, dose-dependently induced mitochondrial and cytoplasmic oxidation. Additionally detected hydrogen peroxide-induced phenomena were MMP dissipation and ECs death. Pretreatment of ECs with apricot melanoidins, significantly counteracted and ultimately abolished hydrogen peroxide-induced intracellular oxidation, mitochondrial depolarization and cell death. In this regard, our current results clearly indicate that melanoidins derived from heat-processed apricots, protect human ECs against oxidative stress.

  13. Synchronism in mitochondrial ROS flashes, membrane depolarization and calcium sparks in human carcinoma cells.

    Science.gov (United States)

    Kuznetsov, Andrey V; Javadov, Sabzali; Saks, Valdur; Margreiter, Raimund; Grimm, Michael

    2017-06-01

    Mitochondria are major producers of reactive oxygen species (ROS) in many cells including cancer cells. However, complex interrelationships between mitochondrial ROS (mitoROS), mitochondrial membrane potential (ΔΨm) and Ca2+ are not completely understood. Using human carcinoma cells, we further highlight biphasic ROS dynamics: - gradual mitoROS increase followed by mitoROS flash. Also, we demonstrate heterogeneity in rates of mitoROS generation and flash initiation time. Comparing mitochondrial and near-extra-mitochondrial signals, we show that mechanisms of mitoROS flashes in single mitochondria, linked to mitochondrial permeability transition pore opening (ΔΨm collapse) and calcium sparks, may involve flash triggering by certain levels of external ROS released from the same mitochondria. In addition, mitochondria-mitochondria interactions can produce wave propagations of mitoROS flashes and ΔΨm collapses in cancer cells similar to phenomena of ROS-induced ROS release (RIRR). Our data suggest that in cancer cells RIRR, activation of mitoROS flashes and mitochondrial depolarization may involve participation of extramitochondrial-ROS produced either by individual mitochondria and/or by neighboring mitochondria. This could represent general mechanisms in ROS-ROS signaling with suggested role in both mitochondrial and cellular physiology and signaling. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Salmonella Choleraesuis outer membrane vesicles: Proteomics and immunogenicity.

    Science.gov (United States)

    Liu, Qiong; Yi, Jie; Liang, Kang; Zhang, Xiangmin; Liu, Qing

    2017-10-01

    Salmonella enterica serotype Choleraesuis (S. Choleraesuis), Gram-negative facultative intracellular pathogen is capable of inducing the cholera in pigs whose symptoms manifest as fever, depression, septicemia, arthritis, and diarrhea. Infections with S. Choleraesuis has resulted in great economic loss for the swine breeding operations. Bacterial outer membrane vesicles (OMVs) play an important role in pathogenicity and host-pathogen interaction. In this study, we purified OMVs released by S. Choleraesuis strain χ3545 and characterized their lipopolysaccharide (LPS) profile. The OMVs contained intact LPS molecules. By using LC-MS/MS, we identified 192 proteins in the OMVs. In addition, the subcellular location and biological functions of the vesicles was predicted. The proteins were mainly derived from outer membranes and cytoplasm. Several proteins were immunoreactive and associated with the secretion pathway. Some putative multi-drug resistance-associated proteins were also identified. Furthermore, immunization experiment via intranasal or intraperitoneal route in mice demonstrated that S. Choleraesuis OMVs could elicit strong humoral and mucosal immune responses. Although OMVs as vaccine did not provide strong protection against clinical strain of wild-type S. Choleraesuis, immunization of OMVs still prolonged the survival time of vaccinated mice after high dose of S. Choleraesuis infection. Overall, this study provides valuable fundamental information toward elucidating the pathogenicity and functions of OMVs secreted from S. Choleraesuis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Association of Extracellular Membrane Vesicles with Cutaneous Wound Healing

    Directory of Open Access Journals (Sweden)

    Uyen Thi Trang Than

    2017-05-01

    Full Text Available Extracellular vesicles (EVs are membrane-enclosed vesicles that are released into the extracellular environment by various cell types, which can be classified as apoptotic bodies, microvesicles and exosomes. EVs have been shown to carry DNA, small RNAs, proteins and membrane lipids which are derived from the parental cells. Recently, several studies have demonstrated that EVs can regulate many biological processes, such as cancer progression, the immune response, cell proliferation, cell migration and blood vessel tube formation. This regulation is achieved through the release and transport of EVs and the transfer of their parental cell-derived molecular cargo to recipient cells. This thereby influences various physiological and sometimes pathological functions within the target cells. While intensive investigation of EVs has focused on pathological processes, the involvement of EVs in normal wound healing is less clear; however, recent preliminarily investigations have produced some initial insights. This review will provide an overview of EVs and discuss the current literature regarding the role of EVs in wound healing, especially, their influence on coagulation, cell proliferation, migration, angiogenesis, collagen production and extracellular matrix remodelling.

  16. Bicarbonate sulfate exchange in canalicular rat liver plasma membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Meier, P.J.; Valantinas, J.; Hugentobler, G.; Rahm, I. (University Hospital, Zurich (Switzerland))

    1987-10-01

    The mechanism(s) and driving forces for biliary excretion of sulfate were investigated in canalicular rat liver plasma membrane vesicles (cLPM). Incubation of cLPM vesicles in the presence of an inside-to-outside (in, out) bicarbonate gradient but not pH or out-to-in sodium gradients, stimulated sulfate uptake 10-fold compared with the absence of bicarbonate and approximately 2-fold above sulfate equilibrium (overshoot). Initial rates of this bicarbonate gradient-driven ({sup 35}S)-sulfate uptake were saturable with increasing concentrations of sulfate and could be inhibited by probenecid, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate, acetazolamide, furosemide, 4-acetamideo-4{prime}-isothiocyanostilbene-2,2{prime}-disulfonic acid, and 4,4{prime}-diisothiocyanostilbene-2,2{prime}-disulfonic acid (IC{sub 50}, {approximately}40 {mu}M). Cisinhibition of initial bicarbonate gradient-stimulated sulfate uptake and transstimulation of sulfate uptake in the absence of bicarbonate were observed with sulfate, thiosulfate, and oxalate but not with chloride, nitrate, phosphate, acetate, lactate, glutamate, aspartate, cholate, taurocholate, dehydrocholate, taurodehydrocholate, and reduced or oxidized glutathione. These findings indicate the presence of a sulfate (oxalate)-bicarbonate anion exchange system in canalicular rat liver plasma membranes. These findings support the concept that bicarbonate-sensitive transport system might play an important role in bile acid-independent canalicular bile formation.

  17. Monosaccharide transport in protein-depleted vesicles from erythrocyte membranes

    National Research Council Canada - National Science Library

    M A Zoccoli; G E Lienhard

    1977-01-01

    .... Based on comparisons between erythrocytes and vesicles with regard to specificity, temparture dependence, and effects of inhibitors, we conclude that sorbose uptake into the vesicles occurs by way...

  18. The stochastic diffusion models of nerve membrane depolarization and interspike interval generation.

    Science.gov (United States)

    Lánský, P; Sato, S

    1999-01-01

    The first step to make the theory of stochastic diffusion processes that arise in connection with single neuron description more understandable is reviewing the deterministic leaky-integrator model. After this step the general principles of simple stochastic models are summarized which clearly reveal that two different sources of noise, intrinsic and external, can be identified. Many possible strategies of neuronal coding exist and one of these, the rate coding, for which the stochastic modeling is relevant, is pursued further. The rate coding is reflected, in experimental as well as theoretical studies, by an input-output curve and its properties are reviewed for the most common stochastic diffusion models. The results for the simplest stochastic diffusion model, the Wiener process, are presented and from them strong limitations of this model can be understood. The most common diffusion model is the Ornstein-Uhlenbeck process, which is one substantial step closer to reality since the spontaneous changes of the membrane potential are included in the model. Both these models are characterized by an additive noise. Taking into account the state dependency of the changes caused by neuronal inputs, we derive models where the noise has a multiplicative effect on the membrane depolarization. Two of these models are compared with the Wiener and Ornstein-Uhlenbeck models. How to identify the parameters of the models, which is an unavoidable task for the models verification, is investigated. The time-variable input is taken into account in the last part of the paper. An intuitive approach is stressed throughout the review.

  19. A novel isolation strategy for obtaining crude membrane vesicles from bovine skim milk

    DEFF Research Database (Denmark)

    Blans, Kristine; Larsen, Lotte Bach; Wiking, Lars

    2014-01-01

    Bovine milks content of phospholipid membranes have largely been explored in the cream fraction, and known as the milk fat globule membrane that surrounds fat droplets. In skim milk, the population of phospholipid membranes is reported to constitute membrane vesicles with a soluble content known...... components. Here we present a novel strategy for a short, gentle and non-denaturing isolation of skim milk-derived membrane vesicles. Methods: Untreated fresh bovine milk was defatted to remove milk fat globules. The resulting skim milk was subjected to ultracentrifugation. The resulting ochre...... fraction can be obtained from skim milk by ultracentrifugation. Casein micelle remnants as well as smaller protein components in the crude vesicle fraction can be successfully removed by size chromatography. Electron microscopy of the vesicle isolate reveals circular structures with membrane vesicle...

  20. Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles

    Directory of Open Access Journals (Sweden)

    Nunzio Iraci

    2016-02-01

    Full Text Available Extracellular vesicles (EVs are a heterogeneous population of secreted membrane vesicles, with distinct biogenesis routes, biophysical properties and different functions both in physiological conditions and in disease. The release of EVs is a widespread biological process, which is conserved across species. In recent years, numerous studies have demonstrated that several bioactive molecules are trafficked with(in EVs, such as microRNAs, mRNAs, proteins and lipids. The understanding of their final impact on the biology of specific target cells remains matter of intense debate in the field. Also, EVs have attracted great interest as potential novel cell-free therapeutics. Here we describe the proposed physiological and pathological functions of EVs, with a particular focus on their molecular content. Also, we discuss the advances in the knowledge of the mechanisms regulating the secretion of EV-associated molecules and the specific pathways activated upon interaction with the target cell, highlighting the role of EVs in the context of the immune system and as mediators of the intercellular signalling in the brain.

  1. Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles.

    Science.gov (United States)

    Iraci, Nunzio; Leonardi, Tommaso; Gessler, Florian; Vega, Beatriz; Pluchino, Stefano

    2016-02-06

    Extracellular vesicles (EVs) are a heterogeneous population of secreted membrane vesicles, with distinct biogenesis routes, biophysical properties and different functions both in physiological conditions and in disease. The release of EVs is a widespread biological process, which is conserved across species. In recent years, numerous studies have demonstrated that several bioactive molecules are trafficked with(in) EVs, such as microRNAs, mRNAs, proteins and lipids. The understanding of their final impact on the biology of specific target cells remains matter of intense debate in the field. Also, EVs have attracted great interest as potential novel cell-free therapeutics. Here we describe the proposed physiological and pathological functions of EVs, with a particular focus on their molecular content. Also, we discuss the advances in the knowledge of the mechanisms regulating the secretion of EV-associated molecules and the specific pathways activated upon interaction with the target cell, highlighting the role of EVs in the context of the immune system and as mediators of the intercellular signalling in the brain.

  2. Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

    Science.gov (United States)

    Kamiya, Koki; Kawano, Ryuji; Osaki, Toshihisa; Akiyoshi, Kazunari; Takeuchi, Shoji

    2016-09-01

    Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

  3. Chromosomal beta-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Ciofu, O; Beveridge, T J; Kadurugamuwa, J

    2000-01-01

    Membrane vesicles were isolated from one beta-lactam-sensitive and three beta-lactam-resistant Pseudomonas aeruginosa clinical isolates from patients with cystic fibrosis. The presence of the chromosomally encoded beta-lactamase in the membrane vesicles was shown by electron microscopy and enzyma...... and enzymatic studies. This is the first report of extracellular secretion of beta-lactamase in P. aeruginosa and it seems that the enzyme is packaged into membrane vesicles.......Membrane vesicles were isolated from one beta-lactam-sensitive and three beta-lactam-resistant Pseudomonas aeruginosa clinical isolates from patients with cystic fibrosis. The presence of the chromosomally encoded beta-lactamase in the membrane vesicles was shown by electron microscopy...

  4. Spheres of influence: Porphyromonas gingivalis outer membrane vesicles.

    Science.gov (United States)

    Gui, M J; Dashper, S G; Slakeski, N; Chen, Y-Y; Reynolds, E C

    2016-10-01

    Outer membrane vesicles (OMVs) are asymmetrical single bilayer membranous nanostructures produced by Gram-negative bacteria important for bacterial interaction with the environment. Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces OMVs that act as a virulence factor secretion system contributing to its pathogenicity. Despite their biological importance, the mechanisms of OMV biogenesis have not been fully elucidated. The ~14 times more curvature of the OMV membrane than cell outer membrane (OM) indicates that OMV biogenesis requires energy expenditure for significant curvature of the OMV membrane. In P. gingivalis, we propose that this may be achieved by upregulating the production of certain inner or outer leaflet lipids, which causes localized outward curvature of the OM. This results in selection of anionic lipopolysaccharide (A-LPS) and associated C-terminal domain (CTD) -family proteins on the outer surface due to their ability to accommodate the curvature. Deacylation of A-LPS may further enable increased curvature leading to OMV formation. Porphyromonas gingivalis OMVs that are selectively enriched in CTD-family proteins, largely the gingipains, can support bacterial coaggregation, promote biofilm development and act as an intercessor for the transport of non-motile bacteria by motile bacteria. The P. gingivalis OMVs are also believed to contribute to host interaction and colonization, evasion of immune defense mechanisms, and destruction of periodontal tissues. They may be crucial for both micro- and macronutrient capture, especially heme and probably other assimilable compounds for its own benefit and that of the wider biofilm community. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. A novel isolation strategy for obtaining crude membrane vesicles from bovine skim milk

    DEFF Research Database (Denmark)

    Blans, Kristine; Larsen, Lotte Bach; Wiking, Lars

    Bovine milks content of phospholipid membranes have largely been explored in the cream fraction, and known as the milk fat globule membrane that surrounds fat droplets. In skim milk, the population of phospholipid membranes is reported to constitute membrane vesicles with a soluble content known...... as exosomes and microvesicles. These vesicles contain various types of RNAs and proteins, suggested to transfer health-promoting messages from mother to offspring. However, the variety of the vesicles in milk is less understood and, additionally, complicated by the complexity of more pronounced milk...

  6. Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles

    Science.gov (United States)

    Roier, Sandro; Fenninger, Judith C.; Leitner, Deborah R.; Rechberger, Gerald N.; Reidl, Joachim; Schild, Stefan

    2013-01-01

    Pasteurella multocida is able to cause disease in humans and in a wide range of animal hosts, including fowl cholera in birds, atrophic rhinitis in pigs, and snuffles in rabbits. Together with Mannheimia haemolytica, P. multocida also represents a major bacterial causative agent of bovine respiratory disease (BRD), which is one of the most important causes for economic losses for the cattle backgrounding and feedlot industry. Commercially available vaccines only partially prevent infections caused by P. multocida and M. haemolytica. Thus, this study characterized the immunogenicity of P. multocida and M. haemolytica outer membrane vesicles (OMVs) upon intranasal immunization of BALB/c mice. Enzyme-linked immunosorbent assays (ELISA) revealed that OMVs derived from P. multocida or M. haemolytica are able to induce robust humoral and mucosal immune responses against the respective donor strain. In addition, also significant cross-immunogenic potential was observed for both OMV types. Colonization studies showed that a potential protective immune response against P. multocida is not only achieved by immunization with P. multocida OMVs, but also by immunization with OMVs derived from M. haemolytica. Immunoblot and immunoprecipitation analyses demonstrated that M. haemolytica OMVs induce a more complex immune response compared to P. multocida OMVs. The outer membrane proteins OmpA, OmpH, and P6 were identified as the three major immunogenic proteins of P. multocida OMVs. Amongst others, the serotype 1-specific antigen, an uncharacterized outer membrane protein, as well as the outer membrane proteins P2 and OmpA were found to be the most important antigens of M. haemolytica OMVs. These findings are useful for the future development of broad-spectrum OMV based vaccines against BRD and other infections caused by P. multocida or M. haemolytica. PMID:23731905

  7. The Lethal Cargo of Myxococcus xanthus Outer Membrane Vesicles

    Directory of Open Access Journals (Sweden)

    James E Berleman

    2014-09-01

    Full Text Available Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including MXAN_3564 (mepA, an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is the long-predicted (yet to date unknown primary extracellular protease in M. xanthus.

  8. Outer membrane vesicles of Pasteurella multocida contain virulence factors

    Science.gov (United States)

    Fernández-Rojas, Miguel A; Vaca, Sergio; Reyes-López, Magda; de la Garza, Mireya; Aguilar-Romero, Francisco; Zenteno, Edgar; Soriano-Vargas, Edgardo; Negrete-Abascal, Erasmo

    2014-01-01

    Pasteurella multocida (Pm) is a gram-negative bacterium able to infect different animal species, including human beings. This bacterium causes economic losses to the livestock industry because of its high morbidity and mortality in animals. In this work, we report the characterization of outer membrane vesicles (OMVs) released into the culture medium by different Pm serogroups. Purified OMVs in the range of 50–300 nm were observed by electron microscopy. Serum obtained from chickens infected with Pm recognized several proteins from Pm OMVs. Additionally, rabbit antiserum directed against a secreted protease from Actinobacillus pleuropneumoniae recognized a similar protein in the Pm OVMs, suggesting that OMVs from these bacterial species contain common immunogenic proteins. OmpA, a multifunctional protein, was identified in OMVs from different Pm serogroups, and its concentration was twofold higher in OMVs from Pm serogroups B and D than in OMVs from other serogroups. Three outer membrane proteins were also identified: OmpH, OmpW, and transferrin-binding protein. Three bands of 65, 110, and 250 kDa with proteolytic activity were detected in Pm OMVs of serogroups A and E. Additionally, β-lactamase activity was detected only in OMVs from Pm 12945 Ampr (serogroup A). Pm OMVs may be involved in different aspects of disease pathogenesis. PMID:25065983

  9. Outer membrane vesicles of Pasteurella multocida contain virulence factors.

    Science.gov (United States)

    Fernández-Rojas, Miguel A; Vaca, Sergio; Reyes-López, Magda; de la Garza, Mireya; Aguilar-Romero, Francisco; Zenteno, Edgar; Soriano-Vargas, Edgardo; Negrete-Abascal, Erasmo

    2014-10-01

    Pasteurella multocida (Pm) is a gram-negative bacterium able to infect different animal species, including human beings. This bacterium causes economic losses to the livestock industry because of its high morbidity and mortality in animals. In this work, we report the characterization of outer membrane vesicles (OMVs) released into the culture medium by different Pm serogroups. Purified OMVs in the range of 50-300 nm were observed by electron microscopy. Serum obtained from chickens infected with Pm recognized several proteins from Pm OMVs. Additionally, rabbit antiserum directed against a secreted protease from Actinobacillus pleuropneumoniae recognized a similar protein in the Pm OVMs, suggesting that OMVs from these bacterial species contain common immunogenic proteins. OmpA, a multifunctional protein, was identified in OMVs from different Pm serogroups, and its concentration was twofold higher in OMVs from Pm serogroups B and D than in OMVs from other serogroups. Three outer membrane proteins were also identified: OmpH, OmpW, and transferrin-binding protein. Three bands of 65, 110, and 250 kDa with proteolytic activity were detected in Pm OMVs of serogroups A and E. Additionally, β-lactamase activity was detected only in OMVs from Pm 12945 Amp(r) (serogroup A). Pm OMVs may be involved in different aspects of disease pathogenesis. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  10. Calreticulin from suboolemmal vesicles affects membrane regulation of polyspermy.

    Science.gov (United States)

    Saavedra, María Dolores; Mondéjar, Irene; Coy, Pilar; Betancourt, Miguel; González-Márquez, Humberto; Jiménez-Movilla, María; Avilés, Manuel; Romar, Raquel

    2014-03-01

    This study was designed to determine whether calreticulin (CRT), a chaperone protein, is present in in vitro-matured (IVM) pig oocytes and to study its potential role in the block to polyspermy. Western blot analysis, using an anti-CRT antibody, of oocyte lysate showed an immunoreactive band of ∼60  kDa. Simultaneous labeling of IVM oocytes with anti-CRT antibody and peanut agglutinin lectin (PNA lectin, a porcine cortical granules (CG)-specific binding lectin) revealed localization of CRT in the subplasmalemmal region with a 27.7% colocalization with PNA staining. After IVF, PNA labeling was not observed and anti-CRT labeling decreased significantly in zygotes and disappeared in two-cell embryos. Western blot analysis of oocyte exudate obtained from zona pellucida (ZP)-free oocytes activated with calcium ionophore confirmed the presence of a band that reacted with an anti-CRT antibody. Anti-CRT antibody and PNA labeling were not observed in activated oocytes despite being detectable in non-activated oocytes. The presence of CRT in vesicles located under the oolemma was demonstrated using immunogold cytochemistry at the ultrastructural level. To study the role of CRT in fertilization, ZP-enclosed and ZP-free oocytes were incubated with exogenous CRT and then inseminated. Whereas ZP-free oocytes showed fewer penetrating sperm and lower polyspermy rates than untreated oocytes, the opposite effect was observed in ZP-enclosed oocytes. In conclusion, CRT is confined to subplasmalemmal vesicles partially overlapping with CG contents. Its exocytosis after the oocyte activation seems to participate in the membrane block to polyspermy in pigs but is not involved in the ZP block.

  11. Structure formation of lipid membranes: Membrane self-assembly and vesicle opening-up to octopus-like micelles

    Science.gov (United States)

    Noguchi, Hiroshi

    2013-02-01

    We briefly review our recent studies on self-assembly and vesicle rupture of lipid membranes using coarse-grained molecular simulations. For single component membranes, lipid molecules self-assemble from random gas states to vesicles via disk-shaped clusters. Clusters aggregate into larger clusters, and subsequently the large disks close into vesicles. The size of vesicles are determined by kinetics than by thermodynamics. When a vesicle composed of lipid and detergent types of molecules is ruptured, a disk-shaped micelle called bicelle can be formed. When both surfactants have negligibly low critical micelle concentration, it is found that bicelles connected with worm-like micelles are also formed depending on the surfactant ratio and spontaneous curvature of the membrane monolayer.

  12. A novel isolation strategy for obtaining crude membrane vesicles from bovine skim milk

    DEFF Research Database (Denmark)

    Blans, Kristine; Larsen, Lotte Bach; Wiking, Lars

    2014-01-01

    Bovine milks content of phospholipid membranes have largely been explored in the cream fraction, and known as the milk fat globule membrane that surrounds fat droplets. In skim milk, the population of phospholipid membranes is reported to constitute membrane vesicles with a soluble content known...... as exosomes and microvesicles. These vesicles contain various types of RNAs and proteins, suggested to transfer health-promoting messages from mother to offspring. However, the variety of the vesicles in milk is less understood and, additionally, complicated by the complexity of more pronounced milk...... components. Here we present a novel strategy for a short, gentle and non-denaturing isolation of skim milk-derived membrane vesicles. Methods: Untreated fresh bovine milk was defatted to remove milk fat globules. The resulting skim milk was subjected to ultracentrifugation. The resulting ochre...

  13. PI3K and PKC contribute to membrane depolarization mediated by α2-adrenoceptors in the canine isolated mesenteric vein

    Directory of Open Access Journals (Sweden)

    Mutafova-Yambolieva Violeta N

    2005-06-01

    Full Text Available Abstract Background Norepinephrine (NE, a classic neurotransmitter in the sympathetic nervous system, induces vasoconstriction of canine isolated mesenteric vein that is accompanied by a sustained membrane depolarization. The mechanisms underlying the NE-elicited membrane depolarization remain undefined. In the present study we hypothesized that phosphatidylinositol 3-kinase (PI3K and protein kinase C (PKC are involved in the electrical field stimulation (EFS-induced slow membrane depolarization (SMD in canine isolated mesenteric vein. EFS (0.1–2 Hz, 0.1 ms, 15V, 10 s-induced changes in the membrane potential were recorded with a conventional intracellular microelectrode technique and evaluated in the absence and presence of inhibitors of neuronal activity, α-adrenoceptors, membrane ion channels, PI3K, inositol 1,4,5-triphosphate (InsP3 receptors, and PKC. Activation of PI3Kγ and PKCζ in response to exogenous NE and clonidine in the absence and presence of receptor and kinase inhibitors were also determined. Results Contractile responses to NE and clonidine (0.05 – 10 μM were significantly diminished in the presence of yohimbine (0.1 μM. Exogenous NE (0.1 μM and clonidine (1 μM elicited SMD. The resting membrane potential of canine mesenteric vein smooth muscle cells was -68.8 ± 0.8 mV. EFS elicited a biphasic depolarization comprised of excitatory junction potentials and SMD that are purinergic and adrenergic in nature, respectively. The magnitude of the SMD in response to EFS at 0.5 Hz was 9.4 ± 0.7 mV. This response was reduced by 65–98% by the fast Na+ channel inhibitor tetrodotoxin (1 μM, by the inhibitor of N-type Ca2+ channels ω-conotoxin GVIA (5 nM, the non-selective α-adrenoceptor blocker phentolamine (1 μM, the selective α2-adrenoceptor blocker yohimbine (0.1 μM, the ion channel inhibitors niflumic acid (NFA, 100 μM, 5-nitro-2-(3-phenylpropylamino benzoic acid (NPPB, 30 μM, 4,4'-diisothiocyanatostilbene-2

  14. Membrane vesicle production by Chlamydia trachomatis as an adaptive response

    Directory of Open Access Journals (Sweden)

    Kyla eFrohlich

    2014-06-01

    Full Text Available Bacteria have evolved specific adaptive responses to cope with changing environments. These adaptations include stress response phenotypes with dynamic modifications of the bacterial cell envelope and generation of membrane vesicles (MVs. The obligate intracellular bacterium, Chlamydia trachomatis, typically has a biphasic lifestyle, but can enter into an altered growth state typified by morphologically aberrant chlamydial forms, termed persistent growth forms, when induced by stress in vitro. How C. trachomatis can adapt to a persistent growth state in host epithelial cells in vivo is not well understood, but is an important question, since it extends the host-bacterial relationship in vitro and has thus been indicated as a survival mechanism in chronic chlamydial infections. Here, we review recent findings on the mechanistic aspects of bacterial adaptation to stress with a focus on how C. trachomatis remodels its envelope, produces MVs, and the potential important consequences of MV production with respect to host-pathogen interactions. Emerging data suggest that the generation of MVs may be an important mechanism for C. trachomatis intracellular survival of stress, and thus may aid in the establishment of a chronic infection in human genital epithelial cells.

  15. Secretable Small RNAs via Outer Membrane Vesicles in Periodontal Pathogens.

    Science.gov (United States)

    Choi, J-W; Kim, S-C; Hong, S-H; Lee, H-J

    2017-04-01

    MicroRNAs (miRNAs) have been shown to be major regulators of eukaryotic gene expression. However, bacterial RNAs comparable in size to eukaryotic miRNAs (18-22 nucleotides) have received little attention. Recently, a novel class of small RNAs similar in size to miRNAs (miRNA-size, small RNAs or msRNAs) have also been found in several bacteria. Like miRNAs, msRNAs are approximately 15 to 25 nucleotides in length, and their precursors are predicted to form a hairpin loop secondary structure. Here, we identified msRNAs in the periodontal pathogens Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Treponema denticola. We examined these msRNAs using a deep sequencing method and characterized dozens of msRNAs through bioinformatic analysis. Highly expressed msRNAs were selected for further validation. The findings suggest that this class of small RNAs is well conserved across the domains of life. Indeed, msRNAs secreted via bacterial outer membrane vesicles (OMVs) were detected. The ability of bacterial OMVs to deliver RNAs into eukaryotic cells was also observed. These msRNAs in OMVs allowed us to identify their potential human immune-related target genes. Furthermore, we found that exogenous msRNAs could suppress expression of certain cytokines in Jurkat T cells. We propose msRNAs may function as novel bacterial signaling molecules that mediate bacteria-to-human interactions. Furthermore, this study may provide fresh insight into bacterial pathogenic mechanisms of periodontal diseases.

  16. Vibrio fischeri-derived outer membrane vesicles trigger host development.

    Science.gov (United States)

    Aschtgen, Marie-Stephanie; Wetzel, Keith; Goldman, William; McFall-Ngai, Margaret; Ruby, Edward

    2016-04-01

    Outer membrane vesicles (OMV) are critical elements in many host-cell/microbe interactions. Previous studies of the symbiotic association between Euprymna scolopes and Vibrio fischeri had shown that within 12 h of colonizing crypts deep within the squid's light organ, the symbionts trigger an irreversible programme of tissue development in the host. Here, we report that OMV produced by V. fischeri are powerful contributors to this process. The first detectable host response to the OMV is an increased trafficking of macrophage-like cells called haemocytes into surface epithelial tissues. We showed that exposing the squid to other Vibrio species fails to induce this trafficking; however, addition of a high concentration of their OMV, which can diffuse into the crypts, does. We also provide evidence that tracheal cytotoxin released by the symbionts, which can induce haemocyte trafficking, is not part of the OMV cargo, suggesting two distinct mechanisms to induce the same morphogenesis event. By manipulating the timing and localization of OMV signal delivery, we showed that haemocyte trafficking is fully induced only when V. fischeri, the sole species able to reach and grow in the crypts, succeeds in establishing a sustained colonization. Further, our data suggest that the host's detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis. © 2015 John Wiley & Sons Ltd.

  17. Next-generation outer membrane vesicle vaccines from concept to clinical trials

    NARCIS (Netherlands)

    Waterbeemd, van de B.

    2013-01-01

    Only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. The OMV vaccines, however, provide limited coverage and are difficult to produce. This is caused by an obligatory detergent treatment, which removes lipopolysaccharide

  18. Membrane potential depolarization causes alterations in neuron arrangement and connectivity in cocultures

    OpenAIRE

    ?zkucur, Nurdan; Quinn, Kyle P.; Pang, Jin C; Du, Chuang; Georgakoudi, Irene; Miller, Eric; Levin, Michael; Kaplan, David L.

    2014-01-01

    Background The disruption of neuron arrangement is associated with several pathologies. In contrast to action potentials, the role of resting potential (Vmem) in regulating connectivity remains unknown. Methods Neuron assemblies were quantified when their Vmem was depolarized using ivermectin (Ivm), a drug that opens chloride channels, for 24?h in cocultures with astrocytes. Cell aggregation was analyzed using automated cluster analysis methods. Neural connectivity was quantified based on the...

  19. ATP-driven calcium transport in membrane vesicles of Streptococcus sanguis. [Streptococcus sanguis; Streptococcus faecalis; Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Houng, H.; Lynn, A.R.; Rosen, B.P.

    1986-11-01

    Calcium transport was investigated in membrane vesicles prepared from the oral bacterium Streptococcus sanguis. Procedures were devised for the preparation of membrane vesicles capable of accumulation /sup 45/Ca/sup 2 +/. Uptake was ATP dependent and did not require a proton motive force. Calcium transport in these vesicles was compared with /sup 45/Ca/sup 2 +/ accumulation in membrane vesicles from Streptococcus faecalis and Escherichia coli. The data support the existence of an ATP-driven calcium pump in S. sanguis similar to that in S. faecalis. This pump, which catalyzes uptake into membrane vesicles, would be responsible for extrusion of calcium from intact cells.

  20. The effect of vesicle shape, line tension, and lateral tension on membrane-binding proteins

    Science.gov (United States)

    Hutchison, Jaime B.

    Model membranes allow for the exploration of complex biological phenomena with simple, controllable components. In this thesis we employ model membranes to determine the effect of vesicle properties such as line tension, lateral tension, and shape on membrane-binding proteins. We find that line tension at the boundary between domains in a phase separated vesicle can accumulate model membrane-binding proteins (green fluorescent protein with a histidine tag), and that those proteins can, in turn, alter vesicle shape. These results suggest that domains in biological membranes may enhance the local concentration of membrane-bound proteins and thus alter protein function. We also explore how membrane mechanical and chemical properties alter the function of the N-BAR domain of amphiphysin, a membrane-binding protein implicated in endocytosis. We find that negatively charged lipids are necessary for N-BAR binding to membranes at detectable levels, and that, at least for some lipid species, binding may be cooperative. Measurements of N-BAR binding as a function of vesicle tension reveal that modest membrane tension of around 2 mN/m, corresponding to a strain of around 1%, strongly increases N-BAR binding. We attribute this increase in binding with tension to the insertion of N-BAR's N-terminal amphipathic helix into the membrane which increases the membrane area. We propose that N-BAR, which was previously described as being able to sense membrane curvature, may be sensing strain instead. Measurements of membrane deformation by N-BAR as a function of membrane tension reveal that tension can hinder membrane deformation. Thus, tension may favor N-BAR binding yet suppress membrane deformation/tubulation, which requires work against tension. These results suggest that membrane tension, a parameter that is often not controlled in model membranes but is tightly controlled in biological cells, may be important in regulating protein binding and assembly and, hence, protein

  1. Unconditionally energy stable numerical schemes for phase-field vesicle membrane model

    Science.gov (United States)

    Guillén-González, F.; Tierra, G.

    2018-02-01

    Numerical schemes to simulate the deformation of vesicles membranes via minimizing the bending energy have been widely studied in recent times due to its connection with many biological motivated problems. In this work we propose a new unconditionally energy stable numerical scheme for a vesicle membrane model that satisfies exactly the conservation of volume constraint and penalizes the surface area constraint. Moreover, we extend these ideas to present an unconditionally energy stable splitting scheme decoupling the interaction of the vesicle with a surrounding fluid. Finally, the well behavior of the proposed schemes are illustrated through several computational experiments.

  2. The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion.

    Directory of Open Access Journals (Sweden)

    Chengliang Zhang

    Full Text Available Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4 and Endophilin B1 (Endo B1 that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺-ATPases (V-ATPases to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA, producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.

  3. Selective depolarization of the muscle membrane in frog nerve-muscle preparations by a chromatographically purified extract of the dinoflagellate Ostreopsis lenticularis.

    Science.gov (United States)

    Meunier, F A; Mercado, J A; Molgó, J; Tosteson, T R; Escalona de Motta, G

    1997-07-01

    1. The actions of a chromatographically identified extract of the marine dinoflagellate Ostreopsis lenticularis, named ostreotoxin-3 (OTX-3), were studied on frog isolated neuromuscular preparations. 2. OTX-3 (1-10 microg ml(-1)) applied to cutaneous pectoris nerve-muscle preparations depolarized skeletal muscle fibres and caused spontaneous contractions. The depolarization was neither reversed by prolonged washing nor by (+)-tubocurarine. 3. OTX-3 decreased the amplitude of miniature end plate potentials (m.e.p.ps) but did not affect their frequency. 4. Extracellular recording of compound action potentials revealed that OTX-3 affected neither excitability nor conduction along intramuscular nerve branches. 5. End-plate potentials (e.p.ps) elicited by nerve stimulation were reduced in amplitude by OTX-3 and even showed reversed polarity in junctions deeply depolarized by the toxin. 6. Membrane depolarization induced by OTX-3 was decreased about 70% in muscles pretreated for 30 min with 10 microM tetrodotoxin. In contrast, muscles pretreated with 5 microM mu-conotoxin GIIIA were completely insensitive to OTX-3-induced depolarization. 7. OTX-3 did not affect e.p.p. amplitude and the quantal content of e.p.ps in junctions in which muscle depolarization was abolished by mu-conotoxin GIIIA. 8. OTX-3 is a novel type of sodium-channel activating toxin that discriminates between nerve and skeletal muscle membranes.

  4. Extracellular Membrane Vesicles and Phytopathogenicity of Acholeplasma laidlawii PG8

    Directory of Open Access Journals (Sweden)

    Vladislav M. Chernov

    2012-01-01

    Full Text Available For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 (a ubiquitous mycoplasma that is one of the five common species of cell culture contaminants and is a causative agent for phytomycoplasmoses in Oryza sativa L. plants was studied. Data on the ability of extracellular vesicles of Acholeplasma laidlawii PG8 to penetrate from the nutrient medium into overground parts of Oryza sativa L. through the root system and to cause alterations in ultrastructural organization of the plants were presented. As a result of the analysis of ultrathin leaf sections of plants grown in medium with A. laidlawii PG8 vesicles, we detected significant changes in tissue ultrastructure characteristic to oxidative stress in plants as well as their cultivation along with bacterial cells. The presence of nucleotide sequences of some mycoplasma genes within extracellular vesicles of Acholeplasma laidlawii PG8 allowed a possibility to use PCR (with the following sequencing to perform differential detection of cells and bacterial vesicles in samples under study. The obtained data may suggest the ability of extracellular vesicles of the mycoplasma to display in plants the features of infection from the viewpoint of virulence criteria—invasivity, infectivity—and toxigenicity—and to favor to bacterial phytopathogenicity.

  5. Extracellular membrane vesicles in blood products-biology and clinical relevance

    Directory of Open Access Journals (Sweden)

    Emilija Krstova Krajnc

    2016-01-01

    Full Text Available Extracellular membrane vesicles are fragments shed from plasma membranes off all cell types that are undergoing apoptosis or are being subjected to various types of stimulation or stress.  Even in the process of programmed cell death (apoptosis, cell fall apart of varying size vesicles. They expose phosphatidylserine (PS on the outer leaflet of their membrane, and bear surface membrane antigens reflecting their cellular origin. Extracellular membrane vesicles have been isolated from many types of biological fluids, including serum, cerebrospinal fluid, urine, saliva, tears and conditioned culture medium. Flow cytometry is one of the many different methodological approaches that have been used to analyze EMVs. The method attempts to characterize the EMVs cellular origin, size, population, number, and structure. EMVs are present and accumulate in blood products (erythrocytes, platelets as well as in fresh frozen plasma during storage. The aim of this review is to highlight the importance of extracellular vesicles as a cell-to-cell communication system and the role in the pathogenesis of different diseases. Special emphasis will be given to the implication of extracellular membrane vesicles in blood products and their clinical relevance. Although our understanding of the role of  EMVs in disease is far from comprehensive, they display promise as biomarkers for different diseases in the future and also as a marker of quality and safety in the quality control of blood products.

  6. Preparation of pure and intact Plasmodium falciparum plasma membrane vesicles and partial characterisation of the plasma membrane ATPase

    Directory of Open Access Journals (Sweden)

    Smith Pete J

    2002-04-01

    Full Text Available Abstract Background In host erythrocytes, the malaria parasite must contend with ion and drug transport across three membranes; its own plasma membrane, the parasitophorous membrane and the host plasma membrane. Isolation of pure and intact Plasmodium falciparum plasma membrane would provide a suitable model to elucidate the possible role played by the parasite plasma membrane in ion balance and drug transport. Results This study describes a procedure for isolating parasite plasma membrane from P. falciparum-infected erythrocytes. With this method, the trophozoites released by saponin treatment were cleansed of erythrocyte membranes using anti-erythrocyte antibodies fixed to polystyrene beads. These trophozoites were then biotinylated and the parasite plasma membrane was disrupted by nitrogen cavitation. This process allows the membranes to reform into vesicles. The magnetic streptavidin beads bind specifically to the biotinylated parasite plasma membrane vesicles facilitating their recovery with a magnet. These vesicles can then be easily released from the magnetic beads by treatment with dithiotreithol. The parasite plasma membrane showed optimal ATPase activity at 2 mM ATP and 2 mM Mg2+. It was also found that Ca2+ could not substitute for Mg2+ ATPase activity in parasite plasma membranes whereas activity was completely preserved when Mn2+ was used instead of Mg2+. Other nucleoside triphosphates tested were hydrolysed as efficiently as ATP, while the nucleoside monophosphate AMP was not. Conclusions We have described the successful isolation of intact P. falciparum plasma membrane vesicles free of contaminating organelles and determined the experimental conditions for optimum ATPase activity.

  7. A study on the membrane depolarization of skeletal muscles caused by a scorpion toxin, sea anemone toxin II and crotamine and the interaction between toxins.

    Science.gov (United States)

    Chang, C. C.; Hong, S. J.; Su, M. J.

    1983-01-01

    Quinquestriatus toxin (QTX) isolated from the venom of a scorpion (Leiurus quinquestriatus) and sea anemone (Anemonia sulcata) toxin II enhanced the twitch response of the rat and mouse diaphragms and like crotamine (isolated from the venom of Crotalus durissus terrificus) caused spontaneous fasciculation of the muscle. Trains of action potentials in muscles at 70-250 Hz, which could not be antagonized by (+)-tubocurarine, were triggered by single stimulation or occurred spontaneously after treatment with these toxins. QTX and toxin II prolonged the rat muscle action potential 3 to 4 fold whereas crotamine prolonged the action potential by only 30%. The membrane potential was depolarized from about -82 mV to -55 mV by crotamine 2 micrograms ml-1, -41 mV by toxin II 5 micrograms ml-1 and to -50 mV by QTX 1 microgram ml-1. The concentrations to induce 50% maximal depolarization (K0.5) were 0.07, 0.15 and greater than 0.4 microgram ml-1, respectively, for QTX, crotamine and toxin II, whereas the rates of depolarization were in the order toxin II greater than or equal to crotamine greater than QTX. The depolarizing effects of crotamine and QTX, but not of toxin II, were saturable. The depolarizing effects of all three toxins were irreversible whereas the membrane potential could be restored by tetrodotoxin non-competitively. Simultaneous treatment with crotamine and QTX or crotamine and toxin II at concentrations below K0.5 caused only additive effects on depolarization. When the muscle was depolarized by pretreating with a saturating concentration of crotamine, the onset of depolarization by QTX was greatly retarded whereas that by toxin II was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6197125

  8. Dihydropyridine-sensitive ion currents and charge movement in vesicles derived from frog skeletal muscle plasma membranes.

    Science.gov (United States)

    Camacho, J; Carapia, A; Calvo, J; García, M C; Sánchez, J A

    1999-10-01

    1. Whole-cell voltage clamp experiments were performed in vesicles derived from frog skeletal muscle plasma membranes to characterize the electrophysiological properties of dihydropyridine (DHP) receptors. This preparation allows control of the composition of the internal medium and the recording of currents, without the influence of the sarcoplasmic reticulum (SR). 2. In solutions containing Ba2+, Bay K 8644-sensitive, L-type inward currents were recorded. Peak Ba2+ currents (IBa) averaged 3.0 microA microF-1 and inactivated in a voltage-dependent manner. Half-maximal steady-state inactivation occurred at -40 mV. No major facilitation of tail currents was observed. 3. The time course of activation of L-type Ca2+ channels was voltage dependent and 10 times faster than that in muscle fibres; the current density values were also much lower. 4. Lowering [Mg2+]i from 2 to 0.1 mM shifted the time to peak of IBa versus voltage relation by -13 mV. 5. In solutions that contained mostly impermeant ions, non-linear capacitive currents were recorded. Charge movement with properties resembling charge 1 was observed in polarized vesicles. The charge movement depended on voltage with Boltzmann parameters: Qmax (maximum charge), 45.6 nC microF-1; V (potential at which Q = 0.5 Qmax), -58.4 mV; and k (slope factor), 22. 3 mV. There was no indication of the presence of Qgamma (the 'hump' component of charge movement). 6. In depolarized vesicles, non-linear currents were observed during hyperpolarizing pulses. The currents produced an excessive charge during 'on' transients only. Charge during 'off' transients was linear from -180 to +60 mV. There was no evidence of the presence of charge 2.

  9. pH-induced proton permeability changes of plasma membrane vesicles

    NARCIS (Netherlands)

    Miedema, H; Prins, HBA; Staal, H.

    In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles.

  10. Uniport of monoanionic L-malate in membrane vesicles from Leuconostoc oenos

    NARCIS (Netherlands)

    SALEMA, M; POOLMAN, B; LOLKEMA, JS; DIAS, MCL; KONINGS, WN

    1994-01-01

    L-malate transport was studied in membrane vesicles from Leuconostoc oenos MLE(-) (mutant lacking malolactic enzyme) which were fused with liposomes containing beef heart cytochrome c oxidase as a proton-motive-force-generating system. In these hybrid membranes, accumulation of L-malate was observed

  11. Statistical thermodynamics of association colloids : the equilibrium structure of micelles, vesicles, and bilayer membranes

    NARCIS (Netherlands)

    Leermakers, F.A.M.

    1988-01-01

    The aim of the present study was to unravel the general equilibrium physical properties of lipid bilayer membranes. We consider four major questions:
    1. What determines the morphology of the association colloids (micelles, membranes, vesicles) in general?
    2. Do the

  12. Membrane-elasticity model of Coatless vesicle budding induced by ESCRT complexes.

    Directory of Open Access Journals (Sweden)

    Bartosz Różycki

    Full Text Available The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.

  13. Endocytic proteins drive vesicle growth via instability in high membrane tension environment

    CERN Document Server

    Walani, Nikhil; Agrawal, Ashutosh

    2015-01-01

    Clathrin-mediated endocytosis (CME) is a key pathway for transporting cargo into cells via membrane vesicles. It plays an integral role in nutrient import, signal transduction, neurotransmission and cellular entry of pathogens and drug-carrying nanoparticles. As CME entails substantial local remodeling of the plasma membrane, the presence of membrane tension offers resistance to bending and hence, vesicle formation. Experiments show that in such high tension conditions, actin dynamics is required to carry out CME successfully. In this study, we build upon these pioneering experimental studies to provide fundamental mechanistic insights into the roles of two key endocytic proteins, namely, actin and BAR proteins in driving vesicle formation in high membrane tension environment. Our study reveals a new actin force induced `snap-through instability' that triggers a rapid shape transition from a shallow invagination to a highly invaginated tubular structure. We show that the association of BAR proteins stabilizes...

  14. Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Hasan, Nazarul [Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 319 Abraham Flexner Way, Room 515, Louisville, KY 40202 (United States); Hu, Chuan, E-mail: chuan.hu@louisville.edu [Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 319 Abraham Flexner Way, Room 515, Louisville, KY 40202 (United States)

    2010-01-01

    Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

  15. Electrogenic Na+-independent Pi transport in canine renal basolateral membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Schwab, S.J.; Hammerman, M.R.

    1986-03-01

    To define the mechanism by which Pi exists from the renal proximal tubular cell across the basolateral membrane, we measured 32Pi uptake in basolateral membrane vesicles from dog kidney in the absence of Na+. Preloading of basolateral vesicles with 2 mM Pi transstimulated 32Pi uptake, which is consistent with counterflow. We used measurements of transstimulation to quantitate the transport component of 32Pi uptake. Transstimulation of 32Pi uptake was inhibited less than 30% by concentrations of probenecid as high as 50 mM. In contrast, transstimulation of 35SO4(2-) uptake by intravesicular SO4(2-) was inhibited 92% by 5 mM probenecid. Preloading basolateral vesicles with SO4(2-) did not result in transstimulation of 32Pi uptake. Accumulation of 32Pi in basolateral vesicles above steady state was driven by a membrane potential (intravesicular positive), consistent with Na+-independent Pi transport being accompanied by the net transfer of negative charge across the membrane. We conclude that carrier-mediated, electrogenic Na+-independent 32Pi transport can be demonstrated in basolateral vesicles from dog kidney. This process appears to be mediated, at least in part, via a mechanism different from that by which SO4(2-) is transported. Electrogenic Na+-independent Pi transport may reflect one means by which Pi reabsorbed across the luminal membrane exists from the proximal tubular cell down an electrochemical gradient.

  16. Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages.

    Science.gov (United States)

    Athman, Jaffre J; Wang, Ying; McDonald, David J; Boom, W Henry; Harding, Clifford V; Wearsch, Pamela A

    2015-08-01

    Mycobacterium tuberculosis is an intracellular pathogen that infects lung macrophages and releases microbial factors that regulate host defense. M. tuberculosis lipoproteins and lipoglycans block phagosome maturation, inhibit class II MHC Ag presentation, and modulate TLR2-dependent cytokine production, but the mechanisms for their release during infection are poorly defined. Furthermore, these molecules are thought to be incorporated into host membranes and released from infected macrophages within exosomes, 40-150-nm extracellular vesicles that derive from multivesicular endosomes. However, our studies revealed that extracellular vesicles released from infected macrophages include two distinct, largely nonoverlapping populations: one containing host cell markers of exosomes (CD9, CD63) and the other containing M. tuberculosis molecules (lipoglycans, lipoproteins). These vesicle populations are similar in size but have distinct densities, as determined by separation on sucrose gradients. Release of lipoglycans and lipoproteins from infected macrophages was dependent on bacterial viability, implicating active bacterial mechanisms in their secretion. Consistent with recent reports of extracellular vesicle production by bacteria (including M. tuberculosis), we propose that bacterial membrane vesicles are secreted by M. tuberculosis within infected macrophages and subsequently are released into the extracellular environment. Furthermore, extracellular vesicles released from M. tuberculosis-infected cells activate TLR2 and induce cytokine responses by uninfected macrophages. We demonstrate that these activities derive from the bacterial membrane vesicles rather than exosomes. Our findings suggest that bacterial membrane vesicles are the primary means by which M. tuberculosis exports lipoglycans and lipoproteins to impair effector functions of infected macrophages and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected

  17. Membrane topology of insulin receptors reconstituted into lipid vesicles

    DEFF Research Database (Denmark)

    Tranum-Jensen, Jørgen; Christiansen, K.; Carlsen, Jens

    1994-01-01

    Anatomy, insulin receptors, membrane reconstitution, electron microscopy, quaternary structure, immunogold labeling......Anatomy, insulin receptors, membrane reconstitution, electron microscopy, quaternary structure, immunogold labeling...

  18. Sites of glucose transporter-4 vesicle fusion with the plasma membrane correlate spatially with microtubules.

    Directory of Open Access Journals (Sweden)

    Jennine M Dawicki-McKenna

    Full Text Available In adipocytes, vesicles containing glucose transporter-4 (GLUT4 redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin, preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion.

  19. Sites of Glucose Transporter-4 Vesicle Fusion with the Plasma Membrane Correlate Spatially with Microtubules

    Science.gov (United States)

    Dawicki-McKenna, Jennine M.; Goldman, Yale E.; Ostap, E. Michael

    2012-01-01

    In adipocytes, vesicles containing glucose transporter-4 (GLUT4) redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF) microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin), preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion. PMID:22916292

  20. Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

    Directory of Open Access Journals (Sweden)

    Campbell Kevin P

    2007-08-01

    Full Text Available Abstract Background Polymorphonuclear neutrophils (PMN constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. Results To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP and dysferlin were further validated by immunoblot analysis. Conclusion Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.

  1. Quantitative optical microscopy and micromanipulation studies on the lipid bilayer membranes of giant unilamellar vesicles

    DEFF Research Database (Denmark)

    Bagatolli, Luis; Needham, David

    2014-01-01

    experimentation. For instance, using different fluorescent reporters, fluorescence microscopy allows strategies to study membrane lateral structure/dynamics at the level of single vesicles of diverse compositions. The micropipette manipulation technique on the other hand, uses Hoffman modulation contrast...... of well-defined stresses to a single membrane or observe the effects of adhesive spreading. Knowledge gained by these studies has informed several applications of lipid membranes including their use as lung surfactants and drug delivery systems for cancer....

  2. Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

    DEFF Research Database (Denmark)

    Vind-Kezunovic, D.; Nielsen, C.H.; Wojewodzka, U.

    2008-01-01

    Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (L(o)) and liquid-disordered (L(d)) phases. Here, we show mum-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the ...... mum-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events Udgivelsesdato: 2008/11....... Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total 'L(o)/L(d)' boundary line tension arising from the coalescence of smaller L(d)-like domains makes it energetically favourable for L(d)-like domains to bend from flat...

  3. Acinetobacter baumannii outer membrane vesicles elicit a potent innate immune response via membrane proteins.

    Science.gov (United States)

    Jun, So Hyun; Lee, Jung Hwa; Kim, Bo Ra; Kim, Seung Il; Park, Tae In; Lee, Je Chul; Lee, Yoo Chul

    2013-01-01

    Acinetobacter baumannii is increasingly becoming a major nosocomial pathogen. This opportunistic pathogen secretes outer membrane vesicles (OMVs) that interact with host cells. The aim of this study was to investigate the ability of A. baumannii OMVs to elicit a pro-inflammatory response in vitro and the immunopathology in response to A. baumannii OMVs in vivo. OMVs derived from A. baumannii ATCC 19606(T) induced expression of pro-inflammatory cytokine genes, interleukin (IL)-1β and IL-6, and chemokine genes, IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1, in epithelial cells in a dose-dependent manner. Disintegration of OMV membrane with ethylenediaminetetraacetic acid resulted in low expression of pro-inflammatory cytokine genes, as compared with the response to intact OMVs. In addition, proteinase K-treated A. baumannii OMVs did not induce significant increase in expression of pro-inflammatory cytokine genes above the basal level, suggesting that the surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory response. Early inflammatory processes, such as vacuolization and detachment of epithelial cells and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs. Our data demonstrate that OMVs produced by A. baumannii elicit a potent innate immune response, which may contribute to immunopathology of the infected host.

  4. Fimbriae-mediated outer membrane vesicle production and invasion of Porphyromonas gingivalis

    Science.gov (United States)

    Mantri, Chinmay K; Chen, Chin-Ho; Dong, Xinhong; Goodwin, Jeffery Shawn; Pratap, Siddharth; Paromov, Victor; Xie, Hua

    2015-01-01

    Porphyromonas gingivalis is a keystone periopathogen that plays an essential role in the progress of periodontitis. Like other gram-negative bacteria, the ability of P. gingivalis to produce outer membrane vesicles is a strategy used to interact with, and survive within its biological niches. Here we compared the protein components associated with vesicles derived from a fimbriated strain (33277) and an afimbriated strain (W83) of P. gingivalis using proteomic analyses. Some well-known virulence factors were identified in vesicles from both strains, such as gingipains and hemagglutinin. In contrast, FimC, FimD, and FimE, minor components of long fimbriae were found exclusively in 33277 vesicles, while proteins with a tetratricopeptide repeat (TPR) domain were unique to W83 vesicles. We found that significantly more 33277 than W83 vesicles were internalized into human oral keratinocytes and gingival fibroblasts. Interestingly, FimA, a well-known adhesin responsible for the attachment and invasion of P. gingivalis into host cells, was not essential for the invasive capabilities of P. gingivalis vesicles. Rather minor components of long fimbriae were required for an efficient invasive activity of vesicles. The most striking finding was that P. gingivalis strains lacking or having a reduced FimA expression showed a significant reduction in vesiculation. These results suggest that production and pathogenicity of P. gingivalis vesicles may largely depend on expression of the fim locus, and that the integration of vesicle production and pathogenicity with fimbrial expression may allow P. gingivalis to confer upon itself certain functional advantages. PMID:25524808

  5. Fimbriae-mediated outer membrane vesicle production and invasion of Porphyromonas gingivalis.

    Science.gov (United States)

    Mantri, Chinmay K; Chen, Chin-Ho; Dong, Xinhong; Goodwin, Jeffery Shawn; Pratap, Siddharth; Paromov, Victor; Xie, Hua

    2015-02-01

    Porphyromonas gingivalis is a keystone periopathogen that plays an essential role in the progress of periodontitis. Like other gram-negative bacteria, the ability of P. gingivalis to produce outer membrane vesicles is a strategy used to interact with, and survive within its biological niches. Here we compared the protein components associated with vesicles derived from a fimbriated strain (33277) and an afimbriated strain (W83) of P. gingivalis using proteomic analyses. Some well-known virulence factors were identified in vesicles from both strains, such as gingipains and hemagglutinin. In contrast, FimC, FimD, and FimE, minor components of long fimbriae were found exclusively in 33277 vesicles, while proteins with a tetratricopeptide repeat (TPR) domain were unique to W83 vesicles. We found that significantly more 33277 than W83 vesicles were internalized into human oral keratinocytes and gingival fibroblasts. Interestingly, FimA, a well-known adhesin responsible for the attachment and invasion of P. gingivalis into host cells, was not essential for the invasive capabilities of P. gingivalis vesicles. Rather minor components of long fimbriae were required for an efficient invasive activity of vesicles. The most striking finding was that P. gingivalis strains lacking or having a reduced FimA expression showed a significant reduction in vesiculation. These results suggest that production and pathogenicity of P. gingivalis vesicles may largely depend on expression of the fim locus, and that the integration of vesicle production and pathogenicity with fimbrial expression may allow P. gingivalis to confer upon itself certain functional advantages. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  6. Single-vesicle detection and analysis of peptide-induced membrane permeabilization

    DEFF Research Database (Denmark)

    Kristensen, Kasper; Ehrlich, Nicky; Henriksen, Jonas Rosager

    2015-01-01

    methods as alternatives to the quenching-based assays for studying peptide-induced leakage from large unilamellar lipid vesicles. Specifically, we use fluorescence correlation spectroscopy (FCS) to study the leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles...... of the two methods; accordingly, it can be assumed that the two methods provide complementary information about the same leakage process. We use the two methods to investigate the membrane-permeabilizing activities of three well-studied cationic membrane-active peptides: mastoparan X, melittin, and magainin...

  7. Inositol 1,4,5-trisphosphate-induced calcium release from platelet plasma membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Rengasamy, A.; Feinberg, H.

    1988-02-15

    A platelet membrane preparation, enriched in plasma membrane markers, took up /sup 45/Ca/sup 2 +/ in exchange for intravesicular Na+ and released it after the addition of inositol 1,4,5-trisphosphate (IP3). The possibility that contaminating dense tubular membrane (DTS) vesicles contributed the Ca/sup 2 +/ released by IP3 was eliminated by the addition of vanadate to inhibit Ca/sup +/-ATPase-mediated DTS Ca/sup 2 +/ sequestration and by the finding that only plasma membrane vesicles exhibit Na/sup +/-dependent Ca/sup 2 +/ uptake. Ca/sup 2 +/ released by IP3 was dependent on low extravesicular Ca/sup 2 +/ concentrations. IP3-induced Ca/sup 2 +/ release was additive to that released by Na/sup +/ addition while GTP or polyethylene glycol (PEG) had no effect. These results strongly suggest that IP3 facilitates extracellular Ca/sup 2 +/ influx in addition to release from DTS membranes.

  8. The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles.

    Science.gov (United States)

    Biagini, Massimiliano; Garibaldi, Manuela; Aprea, Susanna; Pezzicoli, Alfredo; Doro, Francesco; Becherelli, Marco; Taddei, Anna Rita; Tani, Chiara; Tavarini, Simona; Mora, Marirosa; Teti, Giuseppe; D'Oro, Ugo; Nuti, Sandra; Soriani, Marco; Margarit, Immaculada; Rappuoli, Rino; Grandi, Guido; Norais, Nathalie

    2015-08-01

    Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Secretion of Catecholamines from Adrenal Gland by a Single Electrical Shock: Electrotonic Depolarization of Medullary Cell Membrane

    Science.gov (United States)

    Wakade, Arun R.; Wakade, Taruna D.

    1982-05-01

    Transmural stimulation of the isolated adrenal gland of the rat and guinea pig results in secretion of catecholamines. The secretion is due to activation of cholinergic receptors of the adrenal medulla by acetylcholine released from splanchnic nerve terminals after transmural stimulation. Our aim was to see whether the same experimental technique could be used to directly excite the adrenal medullary cell membrane by electrical stimulation and whether such stimulation would result in secretion of catecholamines. We demonstrate here that a single electrical shock to the perfused adrenal gland of the rat results in massive secretion of epinephrine and norepinephrine. The secretion is directly related to the strength and duration of the applied stimulus over a wide range. Catecholamine secretion is unaffected by tetrodotoxin or hexamethonium/atropine but is abolished by Ca2+ lack or 3 mM Mn2+. We suggest that the adrenal medullary membrane undergoes nonpropagated electrotonic depolarization on electrical stimulation and thereby voltage-dependent Ca2+ channels are opened to initiate secretion.

  10. Biochemical and immunochemical analyses of detergent solubilized antigens from membrane vesicles of Aspergillus fumigatus.

    Science.gov (United States)

    Piechura, J E; Riefel, R S; Daft, L J

    1987-11-01

    A membrane vesicle fraction isolated from exponentially growing Aspergillus fumigatus strain Ag 507 cultures was obtained by mechanical disruption of intact Aspergillus cells under specific osmotic conditions followed by a pH fractionation technique. Electron micrographs of the membrane vesicles indicated unit membrane structures free from cell wall material. High glucose-6-phosphatase and low lactate dehydrogenase activities verified the relative purity of the membrane vesicle fraction. Allergic bronchopulmonary aspergillosis (ABPA) patient and normal human sera were incubated with the membrane vesicle fraction followed by colloidal gold tagged rabbit antiserum to human IgG or IgE. Electron micrographs indicated ABPA patient sera possessed specific IgG and IgE antibodies to membranous components. The detergent octyl-beta-D-glucopyranoside was used to extract membrane vesicle components (MC). The enzyme profile of MC compared with cell sap components (CS) showed differences in types of enzymes. Two-dimensional polyacrylamide gel electrophoretic analyses of MC and CS detected components shared as well as unique to each fraction. In crossed immunoelectrophoresis using both rabbit antisera raised to MC and ABPA patient sera, 5 peaks were detected, while analysis of CS using rabbit antisera raised to CS produced 20 major peaks. Immunoelectrophoresis and double immunodiffusion data supported the crossed immunoelectrophoretic data: MC differed from CS. Enzyme-linked immunosorbent assay indicated high specific IgG and IgE antibody levels to MC in ABPA patient sera. Crossed immuno-affinoelectrophoresis with concanavalin A partially characterized the MC, which consist of components which have glycoprotein elements (i.e., containing alpha-D-glucose or alpha-D-mannose).

  11. pH Dependent Elasticity of Polystyrene-blockpoly(acrylic acid) Vesicle Shell Membranes by Atomic Force Microscopy

    NARCIS (Netherlands)

    Chen, Qi; Vancso, Gyula J.

    2011-01-01

    We assess the elastic properties of PS-b-PAA vesicle membranes under different pH values by AFM force measurements. We find that based on the shell deformation theory, the values of the estimated apparent Young's modulus of the vesicle membranes decrease as the pH of the solution increases. The

  12. Membrane vesicle release in bacteria, eukaryotes, and archaea: a conserved yet underappreciated aspect of microbial life.

    Science.gov (United States)

    Deatherage, Brooke L; Cookson, Brad T

    2012-06-01

    Interaction of microbes with their environment depends on features of the dynamic microbial surface throughout cell growth and division. Surface modifications, whether used to acquire nutrients, defend against other microbes, or resist the pressures of a host immune system, facilitate adaptation to unique surroundings. The release of bioactive membrane vesicles (MVs) from the cell surface is conserved across microbial life, in bacteria, archaea, fungi, and parasites. MV production occurs not only in vitro but also in vivo during infection, underscoring the influence of these surface organelles in microbial physiology and pathogenesis through delivery of enzymes, toxins, communication signals, and antigens recognized by the innate and adaptive immune systems. Derived from a variety of organisms that span kingdoms of life and called by several names (membrane vesicles, outer membrane vesicles [OMVs], exosomes, shedding microvesicles, etc.), the conserved functions and mechanistic strategies of MV release are similar, including the use of ESCRT proteins and ESCRT protein homologues to facilitate these processes in archaea and eukaryotic microbes. Although forms of MV release by different organisms share similar visual, mechanistic, and functional features, there has been little comparison across microbial life. This underappreciated conservation of vesicle release, and the resulting functional impact throughout the tree of life, explored in this review, stresses the importance of vesicle-mediated processes throughout biology.

  13. Multivalent ligand-receptor-mediated interaction of small filled vesicles with a cellular membrane

    Science.gov (United States)

    Zhdanov, Vladimir P.

    2017-07-01

    The ligand-receptor-mediated contacts of small sub-100-nm-sized lipid vesicles (or nanoparticles) with the cellular membrane are of interest in the contexts of cell-to-cell communication, endocytosis of membrane-coated virions, and drug (RNA) delivery. In all these cases, the interior of vesicles is filled by biologically relevant content. Despite the diversity of such systems, the corresponding ligand-receptor interaction possesses universal features. One of them is that the vesicle-membrane contacts can be accompanied by the redistribution of ligands and receptors between the contact and contact-free regions. In particular, the concentrations of ligands and receptors may become appreciably higher in the contact regions and their composition may there be different compared to that in the suspended state in the solution. A statistical model presented herein describes the corresponding distribution of various ligands and receptors and allows one to calculate the related change of the free energy with variation of the vesicle-engulfment extent. The results obtained are used to clarify the necessary conditions for the vesicle-assisted pathway of drug delivery.

  14. Vesicle fission of giant unilamellar vesicles of liquid-ordered-phase membranes induced by amphiphiles with a single long hydrocarbon chain.

    Science.gov (United States)

    Inaoka, Yasuyuki; Yamazaki, Masahito

    2007-01-16

    Vesicle fissions are very important processes of biomembranes in cells, but their mechanisms are not clear and are controversial. Using the single giant unilamellar vesicle (GUV) method, we recently found that low concentrations (less than the critical micelle concentration (CMC)) of lysophosphatidylcholine (lyso-PC) induced the vesicle fission of GUVs of dipalmitoylphosphatidylcholine/cholesterol(6/4) (DPPC/chol(6/4)) membranes and sphingomyelin/cholesterol membranes (6/4) in the liquid-ordered (lo) phase. In this report, to elucidate its mechanism, we have investigated the effect of low concentrations (much less than their CMC) of other amphiphiles with a single long hydrocarbon chain (i.e., single long chain amphiphiles) on DPPC/chol(6/4) GUVs as well as the effect of the membrane composition on the lyso-PC-induced vesicle fission. We found that low concentrations of single long chain amphiphiles (lyosophosphatidic acid, octylglucoside, and sodium dodecyl sulfate) induced the shape change from a prolate to two spheres connected by a very narrow neck, indicating that the single long chain amphiphiles can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. As the single long chain amphiphile concentrations were increased, all of them induced vesicle fission of DPPC/chol(6/4) GUVs above their threshold concentrations. To elucidate the role of cholesterol in the single long chain amphiphile-induced vesicle fission, we investigated the effect of lyso-PC on GUVs of dioleoyl-PC (DOPC)/chol(6/4) membranes in the Lalpha phase; no vesicle fission occurred, indicating that cholesterol in itself did not play an important role in the vesicle fission. Finally, to elucidate the effect of the inclusion of DOPC in the lo-phase membrane of GUVs on the lyso-PC-induced vesicle fission of the DPPC/chol(6/4) GUV, we investigated the effect of low concentrations of lyso-PC on GUVs of DPPC/DOPC/chol membranes. With an increase in DOPC

  15. Polymeric capsule-cushioned leukocyte cell membrane vesicles as a biomimetic delivery platform

    Science.gov (United States)

    Gao, Changyong; Wu, Zhiguang; Lin, Zhihua; Lin, Xiankun; He, Qiang

    2016-02-01

    We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural cell membrane camouflaged polymeric multilayer capsules with the immunosuppressive and tumor-recognition functionalities of natural leukocytes provide a new biomimetic delivery platform for disease therapy.We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural

  16. Improved Production Process for Native Outer Membrane Vesicle Vaccine against Neisseria meningitidis

    NARCIS (Netherlands)

    Waterbeemd, van de B.; Wijffels, R.H.; Zomer, G.; Kaaijk, P.; Ruiterkamp, N.; Dobbelsteen, van den G.J.M.; Pol, van der L.A.

    2013-01-01

    An improved detergent-free process has been developed to produce vaccine based on native outer membrane vesicles (NOMV) against Neisseria meningitidis serogroup B. Performance was evaluated with the NonaMen vaccine concept, which provides broad coverage based on nine distinct PorA antigens. Scalable

  17. Transport of Basic Amino Acids by Membrane Vesicles of Lactococcus lactis

    NARCIS (Netherlands)

    Driessen, Arnold J.M.; Leeuwen, Cornelis van; Konings, Wilhelmus

    The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In

  18. SODIUM ION-DEPENDENT AMINO-ACID-TRANSPORT IN MEMBRANE-VESICLES OF BACILLUS-STEAROTHERMOPHILUS

    NARCIS (Netherlands)

    HEYNE, RIR; DEVRIJ, W; CRIELAARD, W; KONINGS, WN

    Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (K(t) = 1.0 mM) and L-leucine (K(t) = 0.4 mM). In contrast, the Na+-H+-L-glutamate transport system has a high affinity for sodium

  19. The proteome of red cell membranes and vesicles during storage in blood bank conditions.

    NARCIS (Netherlands)

    Bosman, G.J.C.G.M.; Lasonder, E.; Luten, M.; Roerdinkholder-Stoelwinder, B.; Novotny, V.M.J.; Bos, H.; Grip, W.J. de

    2008-01-01

    BACKGROUND: During storage of red cells (RBCs) for transfusion, RBCs undergo a number of biochemical and morphologic changes. To be able to identify the mechanisms underlying these storage lesions, a proteomic analysis of the membranes of RBCs and their vesicles was performed during various periods

  20. Staphylococcus aureus α-toxin-dependent induction of host cell death by membrane-derived vesicles.

    Directory of Open Access Journals (Sweden)

    Bernard Thay

    Full Text Available Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs, which analogously to outer membrane vesicles (OMVs of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity.

  1. Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

    DEFF Research Database (Denmark)

    Vind-Kezunovic, Dina; Helix Nielsen, Claus; Wojewodzka, Urszula

    2008-01-01

    on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total 'L-o/L-d' boundary line tension arising from the coalescence of smaller L-d-like domains makes it energetically favourable for L-d-like domains to bend from flat mu m-sized surfaces...... to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events....

  2. Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells

    Directory of Open Access Journals (Sweden)

    Gábor J. Szebeni

    2017-10-01

    Full Text Available Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549, hepatocellular carcinoma (HepG2 and pancreatic cancer cell line (PANC-1. Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER and the up-regulation of ER stress-related unfolded protein response (UPR genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

  3. Mannheimia haemolytica A2 secretes different proteases into the culture medium and in outer membrane vesicles.

    Science.gov (United States)

    Ramírez Rico, Gerardo; Martínez-Castillo, Moisés; González-Ruíz, Cynthia; Luna-Castro, Sarahí; de la Garza, Mireya

    2017-12-01

    Respiratory diseases in ruminants have a significantly negative impact on the worldwide economy. The bacterium Mannheimia haemolytica is involved in pneumonic infections in bovine and ovine. In gram-negative bacteria, six secretion systems related to the colonization process and host tissue damage have been reported. In addition, in the last two decades, the production of outer membrane vesicles has been studied as a different bacterial strategy to release virulence factors, such as exotoxins, lipopolysaccharides, and proteases. However, in M. haemolytica serotype A2, protease secretion and release in vesicles have not been reported as virulence mechanisms. The aim of this work was to identify proteases released into the culture supernatant and in vesicles of M. haemolytica A2. Our results showed evident differences in the molecular mass and activity of proteases present in culture supernatants and outer membrane vesicles based on zymography assays. The biochemical characterization of M. haemolytica proteases revealed that the main types were cysteine and metalloproteases. A specific metalloprotease of 100 kDa was active in the culture supernatants, but it was not active and was found in low quantities in vesicles. Proteases could be an important virulence factor during the infectious pneumonic process led by M. haemolytica. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Raman spectroscopy of single extracellular vesicles reveals subpopulations with varying membrane content (Conference Presentation)

    Science.gov (United States)

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit S.; Saari, Heikki; Lazaro Ibañez, Elisa; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2016-03-01

    Exosomes are small (~100nm) membrane bound vesicles excreted by cells as part of their normal biological processes. These extracellular vesicles are currently an area of intense research, since they were recently found to carry functional mRNA that allows transfer of proteins and other cellular instructions between cells. Exosomes have been implicated in a wide range of diseases, including cancer. Cancer cells are known to have increased exosome production, and may use those exosomes to prepare remote environments for metastasis. Therefore, there is a strong need to develop characterization methods to help understand the structure and function of these vesicles. However, current techniques, such as proteomics and genomics technologies, rely on aggregating a large amount of exosome material and reporting on chemical content that is averaged over many millions of exosomes. Here we report on the use of laser-tweezers Raman spectroscopy (LTRS) to probe individual vesicles, discovering distinct heterogeneity among exosomes both within a cell line, as well as between different cell lines. Through principal components analysis followed by hierarchical clustering, we have identified four "subpopulations" of exosomes shared across seven cell lines. The key chemical differences between these subpopulations, as determined by spectral analysis of the principal component loadings, are primarily related to membrane composition. Specifically, the differences can be ascribed to cholesterol content, cholesterol to phospholipid ratio, and surface protein expression. Thus, we have shown LTRS to be a powerful method to probe the chemical content of single extracellular vesicles.

  5. Depolarization of mitochondrial membrane potential is the initial event in non-nucleoside reverse transcriptase inhibitor efavirenz induced cytotoxicity.

    Science.gov (United States)

    Ganta, Krishna Kumar; Mandal, Anirban; Chaubey, Binay

    2017-02-01

    Efavirenz is a non-nucleoside reverse transcriptase inhibitor (NNRTI) and an active constituent of the highly active antiretroviral therapy regime. It has significantly contributed in control and management of human immunodeficiency virus propagation. However, EFV administration has also led to severe adverse effects, several reports highlighted the role of EFV in mitochondrial dysfunction and toxicity but the molecular mechanism has been poorly understood. In present study, human hepatoma cells Huh 7.5 were treated with clinically relevant concentrations of EFV and parameters like cytotoxicity, mitochondrial transmembrane potential, mitochondrial morphology, cytochrome c release, mitochondria-mediated apoptosis, mtDNA and mtRNA levels and EFV distribution into mitochondrial compartment were evaluated to understand sequence of events leading to cell death in EFV-treated cells. EFV at its clinically relevant concentration was significantly toxic after 48 and 72 h of treatments. EFV-mediated toxicity is initiated with the permeabilization of mitochondrial outer membrane and change in mitochondrial membrane potential (Δψm) which triggers a series of events like cytochrome c release, alteration in mitochondrial morphology and mitochondria-mediated apoptosis. Total mitochondrial content is reduced after 48 h of EFV treatment at IC50 concentration which is also reflected in reduced mitochondrial DNA and RNA levels. After detecting EFV in mitochondrial compartment after 12 h of incubation with EFV, we hypothesize that EFV being a lipophilic molecule is internalized into the mitochondrial compartment causing depolarization of Δψm which subsequently leads to a cascade of events causing cell death.

  6. Millimeter Wave Radiations Affect Membrane Hydration in Phosphatidylcholine Vesicles

    Directory of Open Access Journals (Sweden)

    Giuseppe Chidichimo

    2013-07-01

    Full Text Available A clear understanding of the response of biological systems to millimeter waves exposure is of increasing interest for the scientific community due to the recent convincing use of these radiations in the ultrafast wireless communications. Here we report a deuterium nuclear magnetic resonance spectroscopy (2H-NMR investigation on the effects of millimeter waves in the 53–78 GHz range on phosphocholine bio-mimetic membranes. Millimeter waves significantly affect the polar interface of the membrane causing a decrease of the heavy water quadrupole splitting. This effect is as important as inducing the transition from the fluid to the gel phase when the membrane exposure occurs in the neighborhood of the transition point. On the molecular level, the above effect can be well explained by membrane dehydration induced by the radiation.

  7. Fundamental Studies of Assembly and Mechanical Properties of Lipid Bilayer Membranes and Unilamellar Vesicles

    Science.gov (United States)

    Wang, Xi

    This dissertation work focuses on: (i) obtaining a phospholipid bilayer membrane (LBM)/conducting electrode system with low defect density and optimized rigidity; (ii) investigating vesicle stability and mechanical properties. LBM is a simplified yet representative cell membrane model. LBMs assembled on conductive surfaces can probe protein-LBM interactions activities electrochemically. Sterically stabilized vesicles could be used as cell models or for drug delivery. The main challenges for LBM assembly on gold are vesicles do not spontaneously rupture to form LBMs on gold and the roughness of the gold substrate has considerable influence on molecular film defect density. In this study, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles were functionalized with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-poly(ethylene glycol)-2000-N-[3-(2-pyridyldithio)propionate] (DSPE-PEG-PDP) to yield stable LBMs on gold without surface modification. A template-stripping method was used to obtain atomically flat and pristine gold surfaces. The critical force to initiate vesicle rupture decreases with increasing DSPE-PEG-PDP concentration, indicating that gold-thiolate bonding between DSPE-PEG-PDP and gold substrates promotes LBM formation. Mechanical properties of LBMs and vesicles were investigated as a function of DSPE-PEG-PDP concentration via Atomic Force Microscopy. The elastic moduli of LBMs were determined with DSPE-PEG-PDP concentration ranging from 0mol% to 24mol% and were found to depend on PEG chain conformation. Incorporating DSPE-PEG-PDP molecules with PEG in mushroom conformation results in a decrease of LBM rigidity, while incorporating PEG in brush conformation leads to LBM stiffening. Contrarily, mechanical properties of functionalized vesicles did not vary significantly by varying DSPE-PEG-PDP concentration. LBM with tunable rigidity by adjusting DSPE-PEG-PDP concentration provides a versatile cell membrane model for studying protein or

  8. [-Na(+)-K+ ATPase activity in vesicles of plasmatic membrane of breast cancer].

    Science.gov (United States)

    Salazar Esquivel, Edith Lucía; Calzada Sánchez, Leobardo

    2005-03-01

    -Na(+)-K+ ATPase is a useful marker which determines the origin of breast cancer cells. -Na(+)-K+ ATPase activity, as well as viability of plasma membrane vesicles isolated from breast carcinoma tissues were demonstrated by histochemical detection. Breast carcinoma tissue samples of patients who attended consultation in the oncology service at Hospital de Ginecoobstetricia Núm. 4 Dr. Luis Castelazo Ayala, IMSS were examined. Tissue samples from adenocarcinoma were homogenized in 4 volumes of TED solution at 4 degrees C (Tris-HCI 0.01 M, EDTA 0.0015 M, dithiothreitol 0.001 M, pH 7.4) and subsequently centrifuged. The collected sample was homogenized and stratified in a discontinuous sucrose gradient (20 to 50%) and then centrifuged for 60 min at 30,000 xg. In order to determine -Na(+)-K+ ATPase activity in plasma membrane vesicles, suspension was incubated at Tris-maleate 0.04 M, adenosine triphosphate (ATP) 0.004 M, Mgcl 20.004 M, NaCI 0.1 M y Pb (NO3) 20.005 M, pH 7.0. Reactions were carried out for 15 min at 37 degrees C in prefixed vesicles in 3% glutaraldehyde in 0.1 M, cacodylate buffer stock, pH7.4 for 60 min. Histochemical detection demonstrated membrane vesicles from breast carcinoma tissues and proved their viability after tumoral progression.

  9. Inhibition of myelin membrane sheath formation by oligodendrocyte-derived exosome-like vesicles.

    Science.gov (United States)

    Bakhti, Mostafa; Winter, Christine; Simons, Mikael

    2011-01-07

    Myelin formation is a multistep process that is controlled by a number of different extracellular factors. During the development of the central nervous system (CNS), oligodendrocyte progenitor cells differentiate into mature oligodendrocytes that start to enwrap axons with myelin membrane sheaths after receiving the appropriate signal(s) from the axon or its microenvironment. The signals required to initiate this process are unknown. Here, we show that oligodendrocytes secrete small membrane vesicles, exosome-like vesicles, into the extracellular space that inhibit both the morphological differentiation of oligodendrocytes and myelin formation. The inhibitory effects of exosome-like vesicles were prevented by treatment with inhibitors of actomyosin contractility. Importantly, secretion of exosome-like vesicles from oligodendrocytes was dramatically reduced when cells were incubated by conditioned neuronal medium. In conclusion, our results provide new evidence for small and diffusible oligodendroglial-derived vesicular carriers within the extracellular space that have inhibitory properties on cellular growth. We propose that neurons control the secretion of autoinhibitory oligodendroglial-derived exosomes to coordinate myelin membrane biogenesis.

  10. Formation of Mitochondrial Outer Membrane Derived Protrusions and Vesicles in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Akihiro Yamashita

    Full Text Available Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (mitochondrial outer-membrane protrusions; MOPs, while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (mitochondrial-derived vesicles. Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN. The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence.

  11. Giant Unilamellar Vesicle Electroformation: From Lipid Mixtures to Native Membranes Under Physiological Conditions

    DEFF Research Database (Denmark)

    Meleard, Philippe; Bagatolli, Luis; Pott, Tanja

    2009-01-01

    . Finally, we provide firm evidence that electroformation is a method of choice to produce giant vesicles from native cell membranes. This is illustrated with the example of GUV electroformation from red blood cell ghosts in a physiologically pertinent buffer. GUVs obtained in this manner maintain......Giant unilamellar vesicles (GUVs) are well-known model systems, especially because they are easily observable using optical microscopy. In this chapter, we revisit in detail the versatile GUV electroformation protocol. We demonstrate how GUV electroformation can be adapted to various membrane...... systems including synthetic lipid mixtures, natural lipid extracts, and bilayers containing membrane proteins. Further, we show how to adjust this protocol to a given aqueous environment and prove that GUVs can be obtained under physiologically relevant conditions, that is, in the presence of electrolytes...

  12. Spontaneous complement activation on human B cells results in localized membrane depolarization and the clustering of complement receptor type 2 and C3 fragments

    DEFF Research Database (Denmark)

    Løbner, Morten; Leslie, Robert G Q; Prodinger, Wolfgang M

    2009-01-01

    While our previous studies have demonstrated that complement activation induced by complement receptors type 2 (CR2/CD21) and 1 (CR1/CD35) results in C3-fragment deposition and membrane attack complex (MAC) formation in human B cells, the consequences of these events for B-cell functions remain...... requires activation of complement via the alternative pathway, as indicated by total inhibition upon neutralization of factor D, and is abrogated by combined blockade of CR1 and CR2, but not of either receptor alone. The membrane depolarization is not associated with the apoptosis of B cells, as examined...... by co-staining with APO-2.7 or by the TdT-mediated biotin-dUTP nick-end labelling (TUNEL) assay. Confocal microscopy revealed that depolarization and C3 deposition, unlike MAC deposition, are limited to restricted areas on the B-cell surface. Double staining revealed a close association between the C3...

  13. Vesicle Photonics

    Science.gov (United States)

    Vasdekis, A. E.; Scott, E. A.; Roke, S.; Hubbell, J. A.; Psaltis, D.

    2013-07-01

    Amphiphiles, under appropriate conditions, can self-assemble into nanoscale thin membrane vessels (vesicles) that encapsulate and hence protect and transport molecular payloads. Vesicles assemble naturally within cells but can also be artificially synthesized. In this article, we review the mechanisms and applications of light-field interactions with vesicles. By being associated with light-emitting entities (e.g., dyes, fluorescent proteins, or quantum dots), vesicles can act as imaging agents in addition to cargo carriers. Vesicles can also be optically probed on the basis of their nonlinear response, typically from the vesicle membrane. Light fields can be employed to transport vesicles by using optical tweezers (photon momentum) or can directly perturb the stability of vesicles and hence trigger the delivery of the encapsulated payload (photon energy). We conclude with emerging vesicle applications in biology and photochemical microreactors.

  14. Cdc42 interaction with N-WASP and Toca-1 regulates membrane tubulation, vesicle formation and vesicle motility: implications for endocytosis.

    Directory of Open Access Journals (Sweden)

    Wenyu Bu

    Full Text Available Transducer of Cdc42-dependent actin assembly (Toca-1 consists of an F-BAR domain, a Cdc42 binding site and an SH3 domain. Toca-1 interacts with N-WASP, an activator of actin nucleation that binds Cdc42. Cdc42 may play an important role in regulating Toca-1 and N-WASP functions. We report here that the cellular expression of Toca-1 and N-WASP induces membrane tubulation and the formation of motile vesicles. Marker and uptake analysis suggests that the tubules and vesicles are associated with clathrin-mediated endocytosis. Forster resonance energy transfer (FRET and Fluorescence Lifetime Imaging Microscopy (FLIM analysis shows that Cdc42, N-WASP and Toca-1 form a trimer complex on the membrane tubules and vesicles and that Cdc42 interaction with N-WASP is critical for complex formation. Modulation of Cdc42 interaction with Toca-1 and/or N-WASP affects membrane tubulation, vesicle formation and vesicle motility. Thus Cdc42 may influence endocytic membrane trafficking by regulating the formation and activity of the Toca-1/N-WASP complex.

  15. Saccharomyces cerevisiae cells lacking Pex3 contain membrane vesicles that harbor a subset of peroxisomal membrane proteins.

    Science.gov (United States)

    Wróblewska, Justyna P; Cruz-Zaragoza, Luis Daniel; Yuan, Wei; Schummer, Andreas; Chuartzman, Silvia G; de Boer, Rinse; Oeljeklaus, Silke; Schuldiner, Maya; Zalckvar, Einat; Warscheid, Bettina; Erdmann, Ralf; van der Klei, Ida J

    2017-10-01

    Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Dynamics of Crowded Vesicles: Local and Global Responses to Membrane Composition.

    Directory of Open Access Journals (Sweden)

    Daniel A Holdbrook

    Full Text Available The bacterial cell envelope is composed of a mixture of different lipids and proteins, making it an inherently complex organelle. The interactions between integral membrane proteins and lipids are crucial for their respective spatial localization within bacterial cells. We have employed microsecond timescale coarse-grained molecular dynamics simulations of vesicles of varying sizes and with a range of protein and lipid compositions, and used novel approaches to measure both local and global system dynamics, the latter based on spherical harmonics analysis. Our results suggest that both hydrophobic mismatch, enhanced by embedded membrane proteins, and curvature based sorting, due to different modes of undulation, may drive assembly in vesicular systems. Interestingly, the modes of undulation of the vesicles were found to be altered by the specific protein and lipid composition of the vesicle. Strikingly, lipid dynamics were shown to be coupled to proteins up to 6 nm from their surface, a substantially larger distance than has previously been observed, resulting in multi-layered annular rings enriched with particular types of phospholipid. Such large protein-lipid complexes may provide a mechanism for long-range communication. Given the complexity of bacterial membranes, our results suggest that subtle changes in lipid composition may have major implications for lipid and protein sorting under a curvature-based membrane-sorting model.

  17. Model Systems of Precursor Cellular Membranes: Long-Chain Alcohols Stabilize Spontaneously Formed Oleic Acid Vesicles

    Science.gov (United States)

    Rendón, Adela; Carton, David Gil; Sot, Jesús; García-Pacios, Marcos; Montes, Ruth; Valle, Mikel; Arrondo, José-Luis R.; Goñi, Felix M.; Ruiz-Mirazo, Kepa

    2012-01-01

    Oleic acid vesicles have been used as model systems to study the properties of membranes that could be the evolutionary precursors of more complex, stable, and impermeable phospholipid biomembranes. Pure fatty acid vesicles in general show high sensitivity to ionic strength and pH variation, but there is growing evidence that this lack of stability can be counterbalanced through mixtures with other amphiphilic or surfactant compounds. Here, we present a systematic experimental analysis of the oleic acid system and explore the spontaneous formation of vesicles under different conditions, as well as the effects that alcohols and alkanes may have in the process. Our results support the hypothesis that alcohols (in particular 10- to 14-C-atom alcohols) contribute to the stability of oleic acid vesicles under a wider range of experimental conditions. Moreover, studies of mixed oleic-acid-alkane and oleic-acid-alcohol systems using infrared spectroscopy and Langmuir trough measurements indicate that precisely those alcohols that increased vesicle stability also decreased the mobility of oleic acid polar headgroups, as well as the area/molecule of lipid. PMID:22339864

  18. A Bcl-xL-Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis.

    Science.gov (United States)

    Li, Hongmei; Alavian, Kambiz N; Lazrove, Emma; Mehta, Nabil; Jones, Adrienne; Zhang, Ping; Licznerski, Pawel; Graham, Morven; Uo, Takuma; Guo, Junhua; Rahner, Christoph; Duman, Ronald S; Morrison, Richard S; Jonas, Elizabeth A

    2013-07-01

    Following exocytosis, the rate of recovery of neurotransmitter release is determined by vesicle retrieval from the plasma membrane and by recruitment of vesicles from reserve pools within the synapse, which is dependent on mitochondrial ATP. The anti-apoptotic Bcl-2 family protein Bcl-xL also regulates neurotransmitter release and recovery in part by increasing ATP availability from mitochondria. We now find, that Bcl-xL directly regulates endocytic vesicle retrieval in hippocampal neurons through protein-protein interaction with components of the clathrin complex. Our evidence suggests that, during synaptic stimulation, Bcl-xL translocates to clathrin-coated pits in a calmodulin-dependent manner and forms a complex with the GTPase Drp1, Mff and clathrin. Depletion of Drp1 produces misformed endocytic vesicles. Mutagenesis studies suggest that formation of the Bcl-xL-Drp1 complex is necessary for the enhanced rate of vesicle endocytosis produced by Bcl-xL, thus providing a mechanism for presynaptic plasticity.

  19. Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form

    Directory of Open Access Journals (Sweden)

    Oscarsson Jan

    2008-01-01

    Full Text Available Abstract Background Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles. Results The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S and in its spontaneous laboratory variant (D7SS resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 μm, AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05–0.2 μm were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL. Conclusion Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.

  20. The Xylella fastidiosa PD1063 protein is secreted in association with outer membrane vesicles.

    Science.gov (United States)

    Pierce, Brittany K; Voegel, Tanja; Kirkpatrick, Bruce C

    2014-01-01

    Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa.

  1. Interfacial effects on droplet electrohydrodynamics: particle vortices, patchy membranes, and vesicle drums

    Science.gov (United States)

    Vlahovska, Petia

    2014-11-01

    The analytical work by Acrivos group on drop dynamics in linear flows and rheology of dilute emulsions (papers with Frankel and Barthes-Biesel) have provided solid basis for more than 40 years of research on drops and capsules. These classical papers have inspired my research on drops with ``complex'' interfaces - surfactant-laden and particle-covered drops, and vesicles (drops encapsulated with lipid bilayer membranes). I will present some of our recent experimental observations on these systems in uniform DC and AC electric fields, where the coupling of the electric-field-induced flow and complex mechanics of the interface drives peculiar (and yet to be explained) behaviors: drum-like and asymmetric dumbbell shapes of vesicles; domains formation and motion in multicomponent membranes; particle assembly in dynamic vortices; drop kayaking. Possible implications of our findings to the design of patchy particles and electrorheology of emulsions will be discussed. Supported by NSF-CBET 1132614.

  2. Plesiomonas shigelloides exports a lethal cytotoxic-enterotoxin (LCE by membrane vesicles

    Directory of Open Access Journals (Sweden)

    Marilucia Santos Ludovico

    2016-11-01

    Full Text Available Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50 kDa and isoelectric point (pI around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE. The presence of membrane vesicles (MVs on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM. From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.

  3. Calcium uptake by brush-border and basolateral membrane vesicles in chick duodenum

    Energy Technology Data Exchange (ETDEWEB)

    Takito, J.; Shinki, T.; Sasaki, T.; Suda, T. (Showa Univ., Tokyo (Japan))

    1990-01-01

    Calcium uptake was compared between duodenal brush-border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from vitamin D-deficient chicks and those injected with 625 ng of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). The uptake by BBMV in the 1 alpha,25-(OH)2D3-treated birds attained a maximum (280% of the control) at 12 h and was maintained at an elevated level (210%) at 24 h after the injection of the vitamin. In contrast, ATP-dependent calcium uptake by BLMV reached a maximum (185% of the control) at 6 h and decreased to the control level at 24 h. The kinetic analysis revealed that 1 alpha,25(OH)2D3 increased Vmax values without any changes in apparent Km values in both BBMV and BLMV. The activity of ATP-dependent calcium uptake was localized exclusively in the basolateral membrane, and the activity was inhibited by vanadate (IC50, 1 microM), but not by oligomycin, theophylline, calmodulin, trifluoperazine, or calbindin D28K. These results indicate that calcium transport through both the brush-border and basolateral membranes is involved in the 1 alpha,25(OH)2D3-dependent intestinal calcium absorption. The initiation of calcium absorption by 1 alpha,25(OH)2D3 appears to be due to an increase in the rate of calcium efflux at the basolateral membrane rather than the rate at the brush-border membrane.

  4. Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

    Science.gov (United States)

    Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

    2017-11-09

    Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

  5. Gentamicin Delivery to Burkholderia cepacia Group IIIa Strains via Membrane Vesicles from Pseudomonas aeruginosa PAO1

    OpenAIRE

    Allan, Nick D.; Beveridge, Terry J.

    2003-01-01

    When Pseudomonas aeruginosa PAO1 is treated with gentamicin, it releases membrane vesicles containing gentamicin (g-MVs) and peptidoglycan hydrolase, which makes the MVs bactericidal. We evaluate the ability of g-MVs to deliver gentamicin past the intrinsic permeability barrier of group IIIa Burkholderia cepacia and show that strain CEP0248 with low resistance to gentamicin is killed but the highly resistant strain C5424 is not. Immunoelectron microscopy revealed that gentamicin was delivered...

  6. Membrane Vesicle Release in Bacteria, Eukaryotes, and Archaea: a Conserved yet Underappreciated Aspect of Microbial Life

    OpenAIRE

    Deatherage, Brooke L.; Cookson, Brad T.

    2012-01-01

    Interaction of microbes with their environment depends on features of the dynamic microbial surface throughout cell growth and division. Surface modifications, whether used to acquire nutrients, defend against other microbes, or resist the pressures of a host immune system, facilitate adaptation to unique surroundings. The release of bioactive membrane vesicles (MVs) from the cell surface is conserved across microbial life, in bacteria, archaea, fungi, and parasites. MV production occurs not ...

  7. Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis Implications for Vaccine Development

    NARCIS (Netherlands)

    Waterbeemd, van de B.; Zomer, G.; IJssel, van den J.; Keulen, van L.; Eppink, M.H.M.; Ley, de P.; Pol, van der L.A.

    2013-01-01

    Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use

  8. Membrane-shed vesicles from the parasite Trichomonas vaginalis: characterization and their association with cell interaction.

    Science.gov (United States)

    Nievas, Yesica R; Coceres, Veronica M; Midlej, Victor; de Souza, Wanderley; Benchimol, Marlene; Pereira-Neves, Antonio; Vashisht, Ajay A; Wohlschlegel, James A; Johnson, Patricia J; de Miguel, Natalia

    2017-12-08

    Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor-ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.

  9. Epoxide-mediated differential packaging of Cif and other virulence factors into outer membrane vesicles.

    Science.gov (United States)

    Ballok, Alicia E; Filkins, Laura M; Bomberger, Jennifer M; Stanton, Bruce A; O'Toole, George A

    2014-10-01

    Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. On the interaction between chromaffin granule membranes and intragranular vesicles--theory and analysis of freeze-fracture micrographs.

    Science.gov (United States)

    Engel, J; Pastushenko, V F; Richter, W; Donath, E

    1991-01-01

    We present a model for the calculation of intragranular vesicle adhesion energy in a two-vesicle system consisting of an external secretory vesicle (chromaffin granule) and an intragranular vesicle (IGV) that adheres from the inside to the granule membrane. The geometrical parameters characterizing the granule-IGV systems were derived from freeze-fracture electron micrographs. Adhesion is brought about by incubation of the granules in hyperosmolar sucrose solutions. It is accompanied by a deformation of the granule because the intragranular vesicle bulges it outwards, and by segregation of intramembraneous particles from the adherent part of the granule membrane. Adhesion prevents the deformed granules from osmotic reexpansion and, therefore, causes hyperosmotic relaxation lysis. We estimated specific adhesion energy at -3 erg/cm2, a value which is 10 - 1000 times larger than the energy of van der Waals interaction between membranes. This large interaction energy probably results from changes of the granule core induced by dehydration. A minimization of the interface between the granule core and adjacent membranes could exclude intragranular vesicles from the core and squeeze them towards the granule membrane. This might induce a new kind of interaction between both membranes, which is irreversible and causes lysis upon osmotic relaxation.

  11. Evidence for carrier-mediated chloride/bicarbonate exchange in canalicular rat liver plasma membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Meier, P.J.; Knickelbein, R.; Moseley, R.H.; Dobbins, J.W.; Boyer, J.L.

    1985-04-01

    To determine whether anion exchangers might play a role in hepatic bile formation, the authors looked for the presence of Cl/sup -/:OH/sup -/ and Cl/sup -/:HCO3/sup -/ exchange in highly purified canalicular (c) and basolateral (bl) rat liver plasma membrane (LPM) vesicles. In cLPM vesicles, a pH gradient stimulated /sup 36/Cl- uptake twofold above values obtained during pH-equilibrated conditions. When 50 mM HCO3/sup -/ was also present inside the vesicles, the same pH gradient resulted in Cl/sup -/ uptake to levels fourfold above pH- and HCO3--equilibrated controls and two- to threefold above Cl- equilibrium. Initial rates of both pH and HCO3/sup -/ gradient-stimulated Cl/sup -/ uptake were completely inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). A valinomycin-induced K/sup +/ diffusion potential (inside positive) also stimulated Cl/sup -/ uptake in cLPM, but this conductive Cl- pathway was insensitive to DIDS. The DIDS-sensitive, pH and HCO3- gradient-stimulated Cl/sup -/ uptake demonstrated: saturation with Cl/sup -/; partial inhibition by bumetanide (26%), furosemide (33%), probenecid (37%), and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (49%); cis-inhibition by chloride and nitrate but not by sulfate and various organic anions, and independence from the membrane potential. These data demonstrate the presence of an electroneutral Cl/sup -/:OH/sup -/ and Cl/sup -/:HCO3/sup -/ exchanger in rat liver canalicular membranes that favors Cl/sup -/:HCO3/sup -/ exchange. In contrast, no evidence was found for the presence of a Cl/sup -/:HCO3/sup -/ (OH/sup -/) exchange system in blLPM vesicles.

  12. Characterization of membrane-shed micro-vesicles from cytokine-stimulated beta-cells using proteomics strategies

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Jensen, Soren Skov; Le Bihan, Marie Catherine

    2012-01-01

    Micro-particles and exosomes are two of the most well characterized membrane-derived micro-vesicles released either directly from the plasma membrane or released through the fusion of intracellular multi-vesicular bodies with the plasma membrane, respectively. They are thought to be involved...... in many significant biological processes such as cell-to-cell communication, rescue from apoptosis and immunological responses. Here we report for the first time a quantitative study of proteins from beta-cell-derived micro-vesicles generated after cytokine induced apoptosis using stable-isotope labeled...... amino acids in cell culture (SILAC) combined with mass spectrometry. We identified and quantified a large number of beta-cell specific proteins and proteins previously described in micro-vesicles from other cell types in addition to new proteins located to these vesicles. In addition, we quantified...

  13. Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes.

    Science.gov (United States)

    Hrynevich, Sviatlana V; Pekun, Tatyana G; Waseem, Tatyana V; Fedorovich, Sergei V

    2015-06-01

    Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

  14. The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition.

    Science.gov (United States)

    Hussain, Sunyia; Bernstein, Harris D

    2018-01-08

    Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "β-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively, but often requires non-physiological conditions and time scales and is strongly influenced by properties of the lipid bilayer including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the β-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA and another model protein (OmpG) folded at significantly different rates and that an α-helix embedded inside the EspP β-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Maturational Changes in Rabbit Renal Brush Border Membrane Vesicle Urea Permeability

    Science.gov (United States)

    Quigley, Raymond; Flynn, Marisa; Baum, Michel

    2014-01-01

    Urea transport in the proximal tubule is thought to occur by passive diffusion through the lipid bilayers of the cell membranes. The lipid composition of cell membranes changes during maturation and may directly affect urea permeability of proximal tubule membranes. The present study examined the maturation of urea transport in rabbit renal brush border membrane vesicles (BBMV). BBMV from adult and neonatal (9- to 11-d-old) New Zealand white rabbits were loaded with 500 mM urea and mixed with an iso-osmotic mannitol solution using a stop-flow instrument. Vesicle shrinkage, due to efflux of urea, was followed with light scattering and urea permeability was calculated from an exponential fit of the data. Urea permeability was significantly lower in the neonatal BBMV than the adult at 25°C (0.34 ± 0.04 × 10−6 versus 0.56 ± 0.03 × 10−6 cm/sec; p excretion. This may also play a role in developing and maintaining a high medullary urea concentration and thus the ability to concentrate the urine during renal maturation. PMID:9890623

  16. Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets

    Directory of Open Access Journals (Sweden)

    Tseday Z. Tegegn

    2016-05-01

    Full Text Available Background: Freezing is promising for extended platelet (PLT storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs. PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA of CPPs. Methods: CPPs and corresponding liquid-stored PLTs (LSPs were characterized by flow cytometry (FC, fluorescence polarization (FP, nanoparticle tracking analysis (NTA, electron microscopy (SEM, TEM, atomic force microscopy (AFM and thrombin-generation (TG test. Results: SEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing–thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a+ PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent. Conclusions: Major portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing–thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have

  17. Characterization of procoagulant extracellular vesicles and platelet membrane disintegration in DMSO-cryopreserved platelets.

    Science.gov (United States)

    Tegegn, Tseday Z; De Paoli, Silvia H; Orecna, Martina; Elhelu, Oumsalama K; Woodle, Samuel A; Tarandovskiy, Ivan D; Ovanesov, Mikhail V; Simak, Jan

    2016-01-01

    Freezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs. CPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test. SEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing-thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a(+) PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent. Major portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing-thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant contribution to PCA of CPPs.

  18. Bolaform surfactants with polyoxometalate head groups and their assembly into ultra-small monolayer membrane vesicles

    Science.gov (United States)

    Landsmann, Steve; Luka, Martin; Polarz, Sebastian

    2012-01-01

    Surfactants are indispensable in established technologies as detergents or emulsification agents, and also in recent studies for controlling the growth of nanoparticles or for creating nanocarriers. Although the properties of conventional, organic surfactants are thoroughly explored, strong interest persists in surfactants that possess unique features inaccessible for ordinary systems. Here we present dipolar, bolaform surfactants with a head group comprising of 11 tungsten atoms. These novel compounds are characterized by an exceptionally low critical self-organization concentration, which leads to monolayer vesicles with a diameter of only 15 nm, that is, substantially smaller than for any other system. The membrane of the vesicles is impermeable for water-soluble and oil-soluble guests. Control over release kinetics, which can be followed via the quantitative fluorescence quenching of confined fluorophores, is gained by means of pH adjustments. PMID:23250429

  19. Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration.

    Science.gov (United States)

    Assani, Akym; Moundanga, Sylvie; Beney, Laurent; Gervais, Patrick

    2009-12-01

    Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium. Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration-rehydration. The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock. It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock.

  20. Characterisation of surface blebbing and membrane vesicles produced by Flavobacterium psychrophilum

    DEFF Research Database (Denmark)

    Møller, Jeannette Dan; Barnes, A.C.; Dalsgaard, Inger

    2005-01-01

    The surface of Flavobacterium psychrophilum was examined by electron microscopy to determine if previous findings of haemagglutination positive (HA+) and haemagglutination negative (HA-) abilities could be correlated with expression of pili or of a capsular layer. A thin capsular layer was observ...... in releasing nutrients from its surrounding environment as well as playing a role in impeding the immune response of its host...... in both HA+ and HA- strains but typical pili were absent. However, long, tubular blebs that released membrane vesicles (MVs) into the supernatant were observed on up to 94% of cells within 1 sample. The surface blebbing was increased for 1 strain following growth on media with restricted iron availability....... The MVs had an intact membrane bilayer and were released from blebbing cells of both strains. The protein profiles of MVs, while containing some banding similarity with the profile of outer membrane preparations (OMPs) and of lysed whole cells (WCs), showed several bands that reacted strongly with rabbit...

  1. Extracellular vesicles: a platform for the structure determination of membrane proteins by Cryo-EM.

    Science.gov (United States)

    Zeev-Ben-Mordehai, Tzviya; Vasishtan, Daven; Siebert, C Alistair; Whittle, Cathy; Grünewald, Kay

    2014-11-04

    Membrane protein-enriched extracellular vesicles (MPEEVs) provide a platform for studying intact membrane proteins natively anchored with the correct topology in genuine biological membranes. This approach circumvents the need to conduct tedious detergent screens for solubilization, purification, and reconstitution required in classical membrane protein studies. We have applied this method to three integral type I membrane proteins, namely the Caenorhabditis elegans cell-cell fusion proteins AFF-1 and EFF-1 and the glycoprotein B (gB) from Herpes simplex virus type 1 (HSV1). Electron cryotomography followed by subvolume averaging allowed the 3D reconstruction of EFF-1 and HSV1 gB in the membrane as well as an analysis of the spatial distribution and interprotein interactions on the membrane. MPEEVs have many applications beyond structural/functional investigations, such as facilitating the raising of antibodies, for protein-protein interaction assays or for diagnostics use, as biomarkers, and possibly therapeutics. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Electrostatically Driven Assembly of Charged Amphiphiles Forming Crystallized Membranes, Vesicles and Nanofiber Arrays

    Science.gov (United States)

    Leung, Cheuk Yui Curtis

    Charged amphiphilic molecules can self-assemble into a large variety of objects including membranes, vesicles and fibers. These micro to nano-scale structures have been drawing increasing attention due to their broad applications, especially in biotechnology and biomedicine. In this dissertation, three self-assembled systems were investigated: +3/-1 self-assembled catanionic membranes, +2/-1 self-assembled catanionic membranes and +1 self-assembled nanofibers. Transmission electron microscopy (TEM) combined with synchrotron small and wide angle x-ray scattering (SAXS and WAXS) were used to characterize the coassembled structures from the mesoscopic to nanometer scale. We designed a system of +3 and -1 ionic amphiphiles that coassemble into crystalline ionic bilayer vesicles with large variety of geometries that resemble polyhedral cellular crystalline shells and archaea wall envelopes. The degree of ionization of the amphiphiles and their intermolecular electrostatic interactions can be controlled by varying pH. The molecular packing of these membranes showed a hexagonal to rectangular-C to hexagonal phase transition with increasing pH, resulting in significant changes to the membrane morphology. A similar mixture of +2 and -1 ionic amphiphiles was also investigated. In addition to varying pH, which controls the headgroup attractions, we also adjust the tail length of the amphiphiles to control the van der Waals interactions between the tails. A 2D phase diagram was developed to show how pH and tail length can be used to control the intermolecular packing within the membranes. Another system of self-assembled nanofiber network formed by positively charged amphiphiles was also studied. These highly charged fibers repel each other and are packed in hexagonal lattice with lattice constant at least eight times of the fiber diameter. The d-spacing and the crystal structure can be controlled by varying the solution concentration and temperature.

  3. Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae.

    Science.gov (United States)

    Manabe, Kunio; Kawano-Kawada, Miyuki; Ikeda, Koichi; Sekito, Takayuki; Kakinuma, Yoshimi

    2016-06-01

    The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.

  4. Coupled transport of p-aminohippurate by rat kidney basolateral membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Pritchard, J.B. (National Institutes of Health, Research Triangle Park, NC (USA))

    1988-10-01

    p-Aminohippuric acid (PAH) transport by basolateral membrane (BLM) vesicles isolated from rat renal cortex was stimulated very little by a Na{sup +} gradient (out > in). However, when micromolar concentrations of glutaric acid or {alpha}-ketoglutaric acid were added in the presence of a out > in Na{sup +} gradient, PAH uptake was accelerated >20-fold and an overshoot of greater than fivefold was produced. Other anions, e.g., fumarate, stimulated PAH uptake very modestly under these conditions, and that stimulation was totally prevented by short circuiting, i.e., with K{sup +} (in = out) and valinomycin. Glutarate-stimulated uptake was inhibited by 4-acetamide-4{prime}-({sup 14}C)-isothiocyanostilbene-2,2{prime}-disulfonic acid (SITS) and probenecid and was slightly stimulated by the imposition of an inside-negative membrane potential. Furthermore, even in the absence of a Na{sup +} gradient, glutarate-loaded vesicles exhibited a marked acceleration of ({sup 3}H)-PAH uptake (5-fold) and a modest overshoot (2.5-fold). These results suggest an indirect coupling of BLM PAH uptake to the Na{sup +} gradient by a cyclic accumulation (Na{sup +}-dependent) of glutarate followed by its efflux from the vesicle in exchange for PAH. This coupled system was absent in apical membranes. Thus net secretory transport of PAH may entail Na{sup +}-dependent, glutarate-driven PAH uptake at the BLM, followed by the exit of PAH into the lumen down its electrochemical gradient, probably in exchange for other anions, e.g., {sup 36}Cl{sup {minus}}, HCO{sub 3}{sup {minus}}, or OH{sup {minus}}.

  5. In vitro study of interaction of synaptic vesicles with lipid membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, S K; Castorph, S; Salditt, T [Institute for X-ray Physics, University of Goettingen, 37077 Goettingen (Germany); Konovalov, O [European Synchrotron Radiation Facility, 38043 Grenoble Cedex (France); Jahn, R; Holt, M, E-mail: sghosh1@gwdg.d, E-mail: mholt@gwdg.d, E-mail: tsaldit@gwdg.d [Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Goettingen (Germany)

    2010-10-15

    The fusion of synaptic vesicles (SVs) with the plasma membrane in neurons is a crucial step in the release of neurotransmitters, which are responsible for carrying signals between nerve cells. While many of the molecular players involved in this fusion process have been identified, a precise molecular description of their roles in the process is still lacking. A case in point is the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}). Although PIP{sub 2} is known to be essential for vesicle fusion, its precise role in the process remains unclear. We have re-investigated the role of this lipid in membrane structure and function using the complementary experimental techniques of x-ray reflectivity, both on lipid monolayers at an air-water interface and bilayers on a solid support, and grazing incidence x-ray diffraction on lipid monolayers. These techniques provide unprecedented access to structural information at the molecular level, and detail the profound structural changes that occur in a membrane following PIP{sub 2} incorporation. Further, we also confirm and extend previous findings that the association of SVs with membranes is enhanced by PIP{sub 2} incorporation, and reveal the structural changes that underpin this phenomenon. Further, the association is further intensified by a physiologically relevant amount of Ca{sup 2+} ions in the subphase of the monolayer, as revealed by the increase in interfacial pressure seen with the lipid monolayer system. Finally, a theoretical calculation concerning the products arising from the fusion of these SVs with proteoliposomes is presented, with which we aim to illustrate the potential future uses of this system.

  6. Elevated hydrostatic pressures induce apoptosis and oxidative stress through mitochondrial membrane depolarization in PC12 neuronal cells: A cell culture model of glaucoma.

    Science.gov (United States)

    Tök, Levent; Nazıroğlu, Mustafa; Uğuz, Abdülhadi Cihangir; Tök, Ozlem

    2014-10-01

    Despite the importance of oxidative stress and apoptosis through mitochondrial depolarization in neurodegenerative diseases, their roles in etiology of glaucoma are poorly understood. We aimed to investigate whether oxidative stress and apoptosis formation are altered in rat pheochromocytoma-derived cell line-12 (PC12) neuronal cell cultures exposed to elevated different hydrostatic pressures as a cell culture model of glaucoma. Cultured PC12 cells were subjected to 0, 15 and 70 mmHg hydrostatic pressure for 1 and 24 h. Then, the following values were analyzed: (a) cell viability; (b) lipid peroxidation and intracellular reactive oxygen species production; (c) mitochondrial membrane depolarization; (d) cell apoptosis; (e) caspase-3 and caspase-9 activities; (f) reduced glutathione (GSH) and glutathione peroxidase (GSH-Px). The hydrostatic pressures (15 and 70 mmHg) increased oxidative cell damage through a decrease of GSH and GSH-Px values, and increasing mitochondrial membrane potential. Additionally, 70 mmHg hydrostatic pressure for 24 h indicated highest apoptotic effects, as demonstrated by plate reader analyses of apoptosis, caspase-3 and -9 values. The present data indicated oxidative stress, apoptosis and mitochondrial changes in PC12 cell line during different hydrostatic pressure as a cell culture model of glaucoma. This findings support the view that mitochondrial oxidative injury contributes early to glaucomatous optic neuropathy.

  7. Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

    Science.gov (United States)

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2015-08-18

    Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic

  8. Sodium-stimulated glutamate transport in osmotically shocked cells and membrane vesicles of Escherichia coli.

    Science.gov (United States)

    Miner, K M; Frank, L

    1974-03-01

    Three phenotypically distinct strains of Escherichia coli B were studied: one in which the transport of glutamate was strongly stimulated by sodium, one in which the transport was relatively independent of sodium, and one which did not transport glutamate. Membrane vesicle preparations from the three strains followed the behavior of whole cells with respect to sodium-stimulated transport. Although glutamate-binding material could be released from cells by osmotic shock, its affinity for glutamate was not significantly influenced by sodium. Furthermore, the shocked cells retained sodium-stimulated transport. The accumulated results suggest that the sodium-activated glutamate transport system resides in the cytoplasmic membrane and that releasable binding protein(s) is not intimately involved in its function.

  9. Vesicles between plasma membrane and cell wall prior to visible senescence of Iris and Dendrobium flowers.

    Science.gov (United States)

    Kamdee, Channatika; Kirasak, Kanjana; Ketsa, Saichol; van Doorn, Wouter G

    2015-09-01

    Cut Iris flowers (Iris x hollandica, cv. Blue Magic) show visible senescence about two days after full opening. Epidermal cells of the outer tepals collapse due to programmed cell death (PCD). Transmission electron microscopy (TEM) showed irregular swelling of the cell walls, starting prior to cell collapse. Compared to cells in flowers that had just opened, wall thickness increased up to tenfold prior to cell death. Fibrils were visible in the swollen walls. After cell death very little of the cell wall remained. Prior to and during visible wall swelling, vesicles (paramural bodies) were observed between the plasma membrane and the cell walls. The vesicles were also found in groups and were accompanied by amorphous substance. They usually showed a single membrane, and had a variety of diameters and electron densities. Cut Dendrobium hybrid cv. Lucky Duan flowers exhibited visible senescence about 14 days after full flower opening. Paramural bodies were also found in Dendrobium tepal epidermis and mesophyll cells, related to wall swelling and degradation. Although alternative explanations are well possible, it is hypothesized that paramural bodies carry enzymes involved in cell wall breakdown. The literature has not yet reported such bodies in association with senescence/PCD. Copyright © 2015 Elsevier GmbH. All rights reserved.

  10. The formation of endosymbiotic membrane compartments: membrane identity markers and the regulation of vesicle trafficking

    NARCIS (Netherlands)

    Ivanov, S.

    2012-01-01

    In symbiosis of plants and arbuscular mycorrhizal fungi as well as in rhizobium-legume symbiosis the microbes are hosted intracellularly, inside specialized membrane compartments of the host. These membrane compartments are morphologically different but similar in function, since they control the

  11. On-chip light sheet illumination enables diagnostic size and concentration measurements of membrane vesicles in biofluids

    Science.gov (United States)

    Deschout, Hendrik; Raemdonck, Koen; Stremersch, Stephan; Maoddi, Pietro; Mernier, Guillaume; Renaud, Philippe; Jiguet, Sébastien; Hendrix, An; Bracke, Marc; van den Broecke, Rudy; Röding, Magnus; Rudemo, Mats; Demeester, Jo; de Smedt, Stefaan C.; Strubbe, Filip; Neyts, Kristiaan; Braeckmans, Kevin

    2014-01-01

    Cell-derived membrane vesicles that are released in biofluids, like blood or saliva, are emerging as potential non-invasive biomarkers for diseases, such as cancer. Techniques capable of measuring the size and concentration of membrane vesicles directly in biofluids are urgently needed. Fluorescence single particle tracking microscopy has the potential of doing exactly that by labelling the membrane vesicles with a fluorescent label and analysing their Brownian motion in the biofluid. However, an unbound dye in the biofluid can cause high background intensity that strongly biases the fluorescence single particle tracking size and concentration measurements. While such background intensity can be avoided with light sheet illumination, current set-ups require specialty sample holders that are not compatible with high-throughput diagnostics. Here, a microfluidic chip with integrated light sheet illumination is reported, and accurate fluorescence single particle tracking size and concentration measurements of membrane vesicles in cell culture medium and in interstitial fluid collected from primary human breast tumours are demonstrated.Cell-derived membrane vesicles that are released in biofluids, like blood or saliva, are emerging as potential non-invasive biomarkers for diseases, such as cancer. Techniques capable of measuring the size and concentration of membrane vesicles directly in biofluids are urgently needed. Fluorescence single particle tracking microscopy has the potential of doing exactly that by labelling the membrane vesicles with a fluorescent label and analysing their Brownian motion in the biofluid. However, an unbound dye in the biofluid can cause high background intensity that strongly biases the fluorescence single particle tracking size and concentration measurements. While such background intensity can be avoided with light sheet illumination, current set-ups require specialty sample holders that are not compatible with high-throughput diagnostics

  12. Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

    Science.gov (United States)

    Sthanam, Lakshmi Kavitha; Srivastava, Rohit; Basu, Bhakti; Dutta, Suryendu; Sen, Shamik; Modi, Deepak

    2016-01-01

    Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death. PMID:27583406

  13. Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth.

    Directory of Open Access Journals (Sweden)

    Manalee Vishnu Surve

    2016-09-01

    Full Text Available Infection of the genitourinary tract with Group B Streptococcus (GBS, an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.

  14. Vesicle Pools: Lessons from Adrenal Chromaffin Cells

    Directory of Open Access Journals (Sweden)

    David R Stevens

    2011-02-01

    Full Text Available The adrenal chromaffin cell serves as a model system to study fast Ca2+-dependent exocytosis. Membrane capacitance measurements in combination with Ca2+ uncaging offers a temporal resolution in the millisecond range and reveals that catecholamine release occurs in three distinct phases. Release of a readily releasable (RRP and a slowly releasable (SRP pool are followed by sustained release, due to maturation and release of vesicles which were not release-ready at the start of the stimulus. Trains of depolarizations, a more physiological stimulus, induce release from a small immediately releasable pool of vesicles residing adjacent to calcium channels, as well as from the RRP. The SRP is poorly activated by depolarization. A sequential model, in which non-releasable docked vesicles are primed to a slowly releasable state, and then further mature to the readily releasable state, has been proposed. The docked state, dependent on membrane proximity, requires SNAP-25, synaptotagmin and syntaxin. The ablation or modification of SNAP-25 and syntaxin, components of the SNARE complex, as well as of synaptotagmin, the calcium sensor, and modulators such complexins and Snapin alter the properties and/or magnitudes of different phases of release, and in particular can ablate the RRP. These results indicate that the composition of the SNARE complex and its interaction with modulatory molecules drives priming and provides a molecular basis for different pools of releasable vesicles.

  15. Effect of vanadate on glucose transporter (GLUT4) intrinsic activity in skeletal muscle plasma membrane giant vesicles

    DEFF Research Database (Denmark)

    Kristiansen, S; Youn, J; Richter, Erik

    1996-01-01

    Maximally effective concentrations of vanadate (a phosphotyrosine phosphatase inhibitor) increase glucose transport in muscle less than maximal insulin stimulation. This might be due to vanadate-induced decreased intrinsic activity of GLUT4 accompanying GLUT4 translocation. Thus, the effect...... of vanadate (NaVO3) on glucose transporter (GLUT4) intrinsic activity (V(max) = intrinsic activity x [GLUT4 protein]) was studied in muscle plasma membrane giant vesicles. Giant vesicles (average diameter 7.6 microns) were produced by collagenase treatment of rat skeletal muscle. The vesicles were incubated......-induced decrease in glucose uptake was not due to a decrease in number of vesicles. To further verify the apparent vanadate-induced decrease in GLUT4 intrinsic activity, the kinetics of glucose transport were also examined. In the presence of 10 mmol l-1 vanadate the V(max) and K(m) were decreased (P

  16. Electroformation of Giant Unilamellar Vesicles from Native Membranes and Organic Lipid Mixtures for the Study of Lipid Domains under Physiological Ionic-Strength Conditions

    DEFF Research Database (Denmark)

    Montes, Ruth; Ahyayauch, Hasna; Ibarguren, Maitane

    2010-01-01

    Giant unilamellar vesicles (GUVs) constitute a cell-sized model membrane system that allows direct visualization of particular membrane-related phenomena, such as domain formation, at the level of single vesicles using fluorescence microscopy-related techniques. Currently available protocols for ...

  17. Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

    Science.gov (United States)

    Jäger, Jens; Keese, Susanne; Roessle, Manfred; Steinert, Michael; Schromm, Andra B

    2015-05-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells. © 2014 John Wiley & Sons Ltd.

  18. An extended model of vesicle fusion at the plasma membrane to estimate protein lateral diffusion from TIRF microscopy images.

    Science.gov (United States)

    Basset, Antoine; Bouthemy, Patrick; Boulanger, Jérôme; Waharte, François; Salamero, Jean; Kervrann, Charles

    2017-07-24

    Characterizing membrane dynamics is a key issue to understand cell exchanges with the extra-cellular medium. Total internal reflection fluorescence microscopy (TIRFM) is well suited to focus on the late steps of exocytosis at the plasma membrane. However, it is still a challenging task to quantify (lateral) diffusion and estimate local dynamics of proteins. A new model was introduced to represent the behavior of cargo transmembrane proteins during the vesicle fusion to the plasma membrane at the end of the exocytosis process. Two biophysical parameters, the diffusion coefficient and the release rate parameter, are automatically estimated from TIRFM image sequences, to account for both the lateral diffusion of molecules at the membrane and the continuous release of the proteins from the vesicle to the plasma membrane. Quantitative evaluation on 300 realistic computer-generated image sequences demonstrated the efficiency and accuracy of the method. The application of our method on 16 real TIRFM image sequences additionally revealed differences in the dynamic behavior of Transferrin Receptor (TfR) and Langerin proteins. An automated method has been designed to simultaneously estimate the diffusion coefficient and the release rate for each individual vesicle fusion event at the plasma membrane in TIRFM image sequences. It can be exploited for further deciphering cell membrane dynamics.

  19. Detection and Physicochemical Characterization of Membrane Vesicles (MVs of Lactobacillus reuteri DSM 17938

    Directory of Open Access Journals (Sweden)

    Rossella Grande

    2017-06-01

    Full Text Available Membrane vesicles (MVs are bilayer structures which bleb from bacteria, and are important in trafficking biomolecules to other bacteria or host cells. There are few data about MVs produced by the Gram-positive commensal-derived probiotic Lactobacillus reuteri; however, MVs from this species may have potential therapeutic benefit. The aim of this study was to detect and characterize MVs produced from biofilm (bMVs, and planktonic (pMVs phenotypes of L. reuteri DSM 17938. MVs were analyzed for structure and physicochemical characterization by Scanning Electron Microscope (SEM and Dynamic Light Scattering (DLS. Their composition was interrogated using various digestive enzyme treatments and subsequent Transmission Electron Microscopy (TEM analysis. eDNA (extracellular DNA was detected and quantified using PicoGreen. We found that planktonic and biofilm of L. reuteri cultures generated MVs with a broad size distribution. Our data also showed that eDNA was associated with pMVs and bMVs (eMVsDNA. DNase I treatment demonstrated no modifications of MVs, suggesting that an eDNA-MVs complex protected the eMVsDNA. Proteinase K and Phospholipase C treatments modified the structure of MVs, showing that lipids and proteins are important structural components of L. reuteri MVs. The biological composition and the physicochemical characterization of MVs generated by the probiotic L. reuteri may represent a starting point for future applications in the development of vesicles-based therapeutic systems.

  20. Cimetidine transport in rabbit renal cortical brush-border membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    McKinney, T.D.; Kunnemann, M.E.

    1987-03-01

    Cimetidine is an organic cation and commonly prescribed drug that is eliminated primarily by proximal renal tubular secretion. The present studies evaluated cimetidine transport in rabbit renal cortical brush-border membrane vesicles (BBMV). (/sup 3/H)Cimetidine uptake varied inversely with media osmolarity and was stimulated with uphill transport above equilibrium values (overshoot) produced by an initial proton gradient directed from the vesicle interior outwardly. Uphill transport occurred earlier and was of greater magnitude at 25/sup 0/C than at 5/sup 0/C. pH-stimulated (/sup 3/H)cimetidine uptake was inhibited by excess nonradiolabeled cimetidine, quinidine, and procainamide but was affected little by probenecid. Tetraethylammonium inhibited cimetidine uptake in the presence and absence of an initial proton gradient, indicating that nonionic diffusion and simple diffusion cannot totally account for cimetidine transport in BBMV. Preloading BBMV with an excess of procainamide enhanced cimetidine uptake. These results are consistent with the hypothesis that cimetidine is transported across BBMV by organic cation-proton exchange.

  1. Analysis of bacteria-derived outer membrane vesicles using tunable resistive pulse sensing

    Science.gov (United States)

    Bogomolny, Evgeny; Hong, Jiwon; Blenkiron, Cherie; Simonov, Denis; Dauros, Priscila; Swift, Simon; Phillips, Anthony; Willmott, Geoff R.

    2015-03-01

    Accurate characterization of submicron particles within biological fluids presents a major challenge for a wide range of biomedical research. Detection, characterization and classification are difficult due to the presence of particles and debris ranging from single molecules up to particles slightly smaller than cells. Especial interest arises from extracellular vesicles (EVs) which are known to play a pivotal role in cell-signaling in multicellular organisms. Tunable resistive pulse sensing (TRPS) is increasingly proving to be a useful tool for high throughput particle-by-particle analysis of EVs and other submicron particles. This study examines the capability of TRPS for characterization of EVs derived from bacteria, also called outer membrane vesicles (OMVs). Measurement of a size distribution (124 +/- 3 nm modal diameter) and concentration (lower bound 7.4 x 109 mL-1) are demonstrated using OMVs derived from uropathogenic Escherichia coli. Important aspects of measurement are discussed, including sample preparation and size selection. Application of TRPS to study EVs could assist the development of these particles in clinical diagnostics and therapeutics.

  2. Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.

    Science.gov (United States)

    Aschtgen, Marie-Stephanie; Lynch, Jonathan B; Koch, Eric; Schwartzman, Julia; McFall-Ngai, Margaret; Ruby, Edward

    2016-08-15

    Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. Determining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development

  3. Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

    Science.gov (United States)

    Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

    To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function. Published by Elsevier Masson SAS.

  4. Immunogold labelling is a quantitative method as demonstrated by studies on aminopeptidase N in microvillar membrane vesicles

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Wetterberg, L L; Sjöström, H

    1992-01-01

    Microvillar membrane vesicle preparations with varying content of aminopeptidase N were prepared from enterocytes of the pig small intestine. Postembedding immunogold labelling of aminopeptidase N was performed on these glutaraldehyde/paraformaldehyde-fixed, osmium tetroxide-treated and Epon-embe...... that postembedding immunogold labelling can be used quantitatively....

  5. Outer membrane vesicles of Gallibacterium anatis induce protective immunity in egg-laying hens

    DEFF Research Database (Denmark)

    Pors, Susanne Elisabeth; Pedersen, Ida Just; Skjerning, Ragnhild Bager

    2016-01-01

    Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have previo...

  6. Development of lysosome-mimicking vesicles to study the effect of abnormal accumulation of sphingosine on membrane properties

    OpenAIRE

    Carreira, Ana C; de Almeida, Rodrigo F.M.; Silva, Liana C.

    2017-01-01

    Synthetic systems are widely used to unveil the molecular mechanisms of complex cellular events. Artificial membranes are key examples of models employed to address lipid-lipid and lipid-protein interactions. In this work, we developed a new synthetic system that more closely resembles the lysosome ? the lysosome-mimicking vesicles (LMVs) ? displaying stable acid-to-neutral pH gradient across the membrane. To evaluate the advantages of this synthetic system, we assessed the distinct effects o...

  7. Characteristics of glutamine transport in dog jejunal brush-border membrane vesicles.

    Science.gov (United States)

    Bulus, N M; Abumrad, N N; Ghishan, F K

    1989-07-01

    The present study characterizes glutamine transport across brush-border membrane vesicles (BBMV) prepared from dog jejunum. The purity of these vesicles was demonstrated by a 20-fold enrichment of leucine aminopeptidase, a marker for BBM. Glutamine uptake was found to occur into an osmotically active space with no membrane binding and to exhibit temperature and pH dependence (optimal uptake at pH 7-7.5). Glutamine uptake was driven by an inwardly directed Na+ gradient with a distinct overshoot not observed under K+ gradient. Lithium could not substitute for Na+ as a stimulator of glutamine uptake. Na+-dependent glutamine uptake was not inhibited by methylaminoisobutyric acid, a typical substrate for system A, and was found to be electrogenic and saturable with a Km of 0.97 +/- 0.58 mM and a Vmax of 3.93 +/- 0.99 nmol.mg protein-1.10 s-1. A Na+-glutamine coupling ratio of 1:1 could be demonstrated by a plot of Hill transformation. Na+-independent glutamine uptake was found to be electroneutral and saturable with a Km of 3.70 +/- 0.66 mM and a Vmax of 2.70 +/- 1.55 nmol.mg protein-1.10 s-1. Inhibition studies confirmed the presence of a Na+-dependent as well as a Na+-independent carrier for glutamine uptake. We conclude that glutamine uptake across dog BBMV occurs via two transport systems: a Na+-dependent high-affinity system similar to the neutral brush-border system and a Na+-independent lower-affinity system similar to system L.

  8. [Bacterial outer membrane vesicles as nano carriers to study immunological activities].

    Science.gov (United States)

    Qi, Chen; Min, W U; Hongzhen, Bai; Zeling, Guo; Jun, Zhou; Qingqing, Wang; Guping, Tang

    2017-03-25

    Objective: To prepare a nano-carrier based on combining bacterial outer membrane vesicles (OMV) with three block polymer pluronic F127 (PEO 100 -PPO 65 -PEO 100 ) (OMV-F127) and to investigate its immunological activity. Methods: Attenuated salmonella (sal) was cultivated. OMV were separated by centrifugal ultrafiltration or ultrasonication, and OMV-F127 was prepared by mechanical extrudation method. The protein contents and compositions were tested with BCA and SDS-PAGE; the morphology of OMV, F127 and OMV-F127 were observed with FM and TEM; the particle sizes and their zeta potential were determined with DLS. Mouse macrophage RAW246.7 cells were treated with OMV-F127 (50 μg/mL, 100 μg/mL) in vitro, and the concentrations of IL-12, TNF-α and IFN-γ in culture supernatant were measured with ELISA kits. Results: The contents of protein in separated OMV by centrifugal ultrafiltration and ultrasonication were 2.8 mg/mL and 2.7 mg/mL, respectively. SDS-PAGE showed the marker protein OmpF/C in OMV. Under the FM and TEM, ball-like structure of F127 and OMV-F127 was observed. Size analysis revealed that the diameters of OMV, F127 and OMV-F127 were 72±2 nm, 90±3 nm and 92±2 nm, respectively. ELISA tests revealed that OMV-F127 significantly stimulated the secretion of IL-12, TNF-α and IFN-γ in RAW246.7 cells. Conclusion: A nano-carrier based on bacterial outer membrane vesicles has been prepared, which can stimulate the secretion of cytokines and may have immunomodulatory effects.

  9. Depolarization of sperm membrane potential is a common feature of men with subfertility and is associated with low fertilization rate at IVF.

    Science.gov (United States)

    Brown, Sean G; Publicover, Stephen J; Mansell, Steven A; Lishko, Polina V; Williams, Hannah L; Ramalingam, Mythili; Wilson, Stuart M; Barratt, Christopher L R; Sutton, Keith A; Da Silva, Sarah Martins

    2016-06-01

    Are significant abnormalities in outward (K(+)) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success? Negligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF. Sperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K(+) channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K(+) channels in human spermatozoa or the incidence and functional consequences of K(+) channel defects. Spermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study. Samples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca(2+) influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K(+) signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities. Patch clamp electrophysiology was used to assess outward (K

  10. Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis

    Directory of Open Access Journals (Sweden)

    Thais Gonçalves Ferreira

    Full Text Available BACKGROUND Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA, although their involvement in virulence is still not clear. OBJECTIVE The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM and immunoelectron microscopy (IM. To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS. FINDINGS TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS

  11. Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles.

    Science.gov (United States)

    Elhenawy, Wael; Debelyy, Mykhaylo O; Feldman, Mario F

    2014-03-11

    Outer membrane vesicles (OMV) are spherical membranous structures released from the outer membrane (OM) of Gram-negative bacteria. OMV have been proposed to play several different roles during both pathogenesis and symbiosis. Despite the fact that OMV were described several decades ago, their biogenesis is a poorly characterized process. Whether OMV are produced by an active mechanism or by passive disintegration of the OM is a still matter of controversy. Bacteroides fragilis and Bacteroides thetaiotaomicron are important members of the human microbiota. In this work, we determined and compared the protein compositions of OM and OMV from B. fragilis and B. thetaiotaomicron. SDS-PAGE analysis of both fractions revealed dramatically different protein profiles. Proteomic analysis of OM and OMV in B. fragilis identified more than 40 proteins found exclusively in OMV and more than 30 proteins detectable only in the OM. The OMV-specific proteome showed a high prevalence of glycosidases and proteases, some of which were shown to be active in vitro. Similar results were obtained for B. thetaiotaomicron. Most of the OMV-exclusive proteins were acidic. Based on these results, we propose that these species possess machinery devoted to selectively pack acidic proteins into the OMV. These OMV equipped with hydrolytic enzymes could help in securing nutrients for the benefit of the whole bacterial community present in the microbiota, uncovering a novel function for bacterial OMV. IMPORTANCE The members of genus Bacteroides are key players in the symbiosis between the human host and the gut microbiota. It is known for its ability to degrade a wide variety of glycans that are not substrates for human glycosidases. The cleaved glycans can be utilized by Bacteroides and other microbiota members, resulting in the production of short-chain fatty acids that are beneficial for the host. Although members of the genus Bacteroides are known to secrete different hydrolases, their secretion

  12. Ammonium ion substitutes for K/sup +/ in ATP-dependent Na/sup +/ transport by basolateral membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Towle, D.W.; Hoelleland, T.

    1987-03-01

    Ion-transporting cells from posterior gills of blue crabs (Callinectes sapidus) acclimated to low salinity were used as starting material for the preparation of microsomal membrane vesicles by density gradient centrifugation. The Na/sup +/-K/sup +/-adenosinetriphosphatase (ATPase)-enriched basolateral vesicles were loaded with KCl- or NH/sub 4//sup +/-containing medium by dilution and centrifugation, and initial rates of /sup 22/Na/sup +/ uptake into the vesicles were measured by a rapid filtration procedure. Varying the extravesicular sucrose concentration altered equilibrium uptake of /sup 22/Na/sup +/, indicating the existence of osmotically sensitive vesicles. Monensin, a sodium-specific ionophore, enhanced passive uptake of /sup 22/Na/sup +/ across the vesicle membrane in the absence of ATP. With 100 mM KCl in the intravesicular medium, addition of ATP to the extravesicular medium increased initial rates of /sup 22/Na/sup +/ uptake 10- to 20-fold over levels measured without ATP. A nonhydrolyzable ATP analog failed to stimulate /sup 22/Na/sup +/ uptake. Intravesicular K/sup +/ could be replaced by NH/sub 4//sup +/ but not by choline. With NH/sub 4//sup +/ as counterion, Na/sup +/ transport was inhibited by digitoxin, but valinomycin had no effect. A study of the kinetic effects of intravesicular K/sup +/ and NH/sub 4//sup +/ on initial rates of /sup 22/Na/sup +/ uptake indicated the existence of two classes of binding sites, one responding to counterion concentrations in the millimolar range and a second class responding to counterion concentrations over 50 mM. The results indicate that ATP-dependent /sup 22/Na/sup +/ uptake by membrane vesicles from Callinectes sapidus gill, mediated by Na/sup +/-K/sup +/-ATPase, can utilize either K/sup +/ or NH/sub 4//sup +/ as counterion.

  13. Increased Outer Membrane Vesicle Formation in a Helicobacter pylori tolB Mutant.

    Science.gov (United States)

    Turner, Lorinda; Praszkier, Judyta; Hutton, Melanie L; Steer, David; Ramm, Georg; Kaparakis-Liaskos, Maria; Ferrero, Richard L

    2015-08-01

    Multiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori. H. pylori ΔtolB, Δpal and ΔtolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively. H. pylori ΔtolB and Δpal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H. pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H. pylori ΔtolB and Δpal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, ΔtolB, Δpal and ΔtolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs. This work demonstrates that TolB and Pal are important for membrane integrity in H. pylori. Moreover, it shows how H. pylori tolB-pal genes may be manipulated to develop "hypervesiculating" strains for vaccine purposes. © 2015 John Wiley & Sons Ltd.

  14. Evidence for carrier-mediated Cl-SO/sub 4/ exchange in rabbit ileal basolateral membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Schron, C.M.; Knickelbein, R.G.; Aronson, P.S.; Dobbins, J.W.

    1987-09-01

    In rabbit ileal basolateral membrane (BLM) vesicles, an outwardly directed Cl gradient ((Cl) in/out = 60/6 mM) stimulated the initial velocity of /sup 35/SO/sub 4/ uptake compared with uptake in the absence of Cl. Under Cl gradient conditions, /sup 35/SO/sub 4/ was transiently accumulated at a concentration twice that found at equilibrium (overshoot). Chloride gradient-stimulated SO/sub 4/ uptake was markedly reduced by inhibitors of anion exchange and was saturable. SO/sub 4/ uptake by BLM vesicles was not stimulated by imposition of an inside-positive electrical potential, suggesting that the stimulation by a Cl gradient was not due to an induced electrical potential. Oxalate, nitrate, iodide, and bromide inhibited the initial velocity of Cl gradient-stimulated SO/sub 4/ uptake, whereas phosphate, ..beta..-hydroxybutyrate, lactate, and p-aminohippurate had not effect. When /sup 35/SO/sub 4/ uptake by BLM vesicles was compared with that of brush-border membrane vesicles, Cl gradient-stimulated SO/sub 4/ uptake was found predominantly in the BLM preparation. In conclusion, these findings provide evidence for a carrier on the ileal basolateral membrane that mediates Cl-SO/sub 4/ exchange.

  15. The influence of membrane stress on phase separation and domain shape in phospholipid vesicles

    Science.gov (United States)

    Chen, Dong; Santore, Maria

    2012-02-01

    Phase separation of mixed phospholipid bilayers is of interest due to the potential role of phospholipid rafts in cell adhesion and signaling. Studies of membrane dynamics and the phase diagram itself typically neglect the role of tension, though it is expected that imposition of moderate membrane tensions might mildly shift the phase separation temperature, as anticipated by Clausius Clapeyron. We show here, using a simple binary system (DOPC/DPPC), a more dramatic effect: The tension imposed on giant unilamellar phospholipid vesicles can alter the phase and the domain shape, completely changing the composition of the liquid and solid phases, their proportions, and the transition temperature. The example in this talk demonstrates how striped or patchy hexagonal phases can develop, depending on thermal history and tension. Different incorporation of tracers into the ordered phases suggests fundamental differences in their structure at the molecular level. Rapid quenching and low tensions favor hexagonal patches while increased tension and slower quenching favors a striped phase. For this reason it is believed that the patches contain corrugations such that the structure of the ordered phase is metastable.

  16. Outer Membrane Vesicles (OMVs) of Gram-negative Bacteria: A Perspective Update.

    Science.gov (United States)

    Jan, Arif Tasleem

    2017-01-01

    Outer Membrane Vesicles (OMVs) of Gram-negative bacteria are spherical membrane-enclosed entities of endocytic origin. Reported in the consortia of different bacterial species, production of OMVs into extracellular milieu seems essential for their survival. Enriched with bioactive proteins, toxins, and virulence factors, OMVs play a critical role in the bacteria-bacteria and bacteria-host interactions. Emergence of OMVs as distinct cellular entities helps bacteria in adaptating to diverse niches, in competing with other bacteria to protect members of producer species and more importantly play a crucial role in host-pathogen interaction. Composition of OMV, their ability to modulate host immune response, along with coordinated secretion of bacterial effector proteins, endows them with the armory, which can withstand hostile environments. Study of the OMV production under natural and diverse stress conditions has broadened the horizons, and also opened new frontiers in delineating the molecular machinery involved in disease pathogenesis. Playing diverse biological and pathophysiological functions, OMVs hold a great promise in enabling resurgence of bacterial diseases, in concomitance with the steep decline in the efficiency of antibiotics. Having multifaceted role, their emergence as a causative agent for a series of infectious diseases increases the probability for their exploitation in the development of effective diagnostic tools and as vaccines against diverse pathogenic species of Gram-negative origin.

  17. Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    Jennifer M Bomberger

    2009-04-01

    Full Text Available Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane-derived vesicles (OMV secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including beta-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP-mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.

  18. Rapid synaptic vesicle endocytosis in cone photoreceptors of salamander retina

    Science.gov (United States)

    Van Hook, Matthew J.; Thoreson, Wallace B.

    2013-01-01

    Following synaptic vesicle exocytosis, neurons retrieve the fused membrane by a process of endocytosis in order to provide a supply of vesicles for subsequent release and maintain the presynaptic active zone. Rod and cone photoreceptors use a specialized structure called the synaptic ribbon that enables them to sustain high rates of neurotransmitter release. They must also employ mechanisms of synaptic vesicle endocytosis capable of keeping up with release. While much is known about endocytosis at another retinal ribbon synapse, that of the goldfish Mb1 bipolar cell, less is known about endocytosis in photoreceptors. We used capacitance recording techniques to measure vesicle membrane fusion and retrieval in photoreceptors from salamander retinal slices. We found that application of brief depolarizing steps (endocytosis with a time constant ~250 ms. In some cases, the capacitance trace overshot the baseline, indicating excess endocytosis. Calcium had no effect on the time constant, but enhanced excess endocytosis resulting in a faster rate of membrane retrieval. Surprisingly, endocytosis was unaffected by blockers of dynamin, suggesting that cone endocytosis is dynamin-independent. This contrasts with synaptic vesicle endocytosis in rods, which was inhibited by the dynamin inhibitor dynasore and GTPγS introduced through the patch pipette, suggesting that the two photoreceptor types employ distinct pathways for vesicle retrieval. The fast kinetics of synaptic vesicle endocytosis in photoreceptors likely enables these cells to maintain a high rate of transmitter release, allowing them to faithfully signal changes in illumination to second-order neurons. PMID:23238726

  19. Photothermal Treatment of Human Pancreatic Cancer Using PEGylated Multi-Walled Carbon Nanotubes Induces Apoptosis by Triggering Mitochondrial Membrane Depolarization Mechanism.

    Science.gov (United States)

    Mocan, Teodora; Matea, Cristian T; Cojocaru, Iulia; Ilie, Ioana; Tabaran, Flaviu A; Zaharie, Florin; Iancu, Cornel; Bartos, Dana; Mocan, Lucian

    2014-01-01

    Pancreatic cancer (PC) is one of the most lethal solid tumor in humans, with an overall 5-year survival rate of less than 5%. Thermally active carbon nanotubes have already brought to light promising results in PC research and treatment. We report here the construct of a nano-biosystem based on multi-walled carbon nanotubes and polyethylene glycol (PEG) molecules validated through AFM, UV-Vis and DLS. We next studied the photothermal effect of these PEG-ylated multi-walled carbon nanotubes (5, 10 and 50 μg/mL, respectively) on pancreatic cancer cells (PANC-1) and further analyzed the molecular and cellular events involved in cell death occurrence. Using cell proliferation, apoptosis, membrane polarization and oxidative stress assays for ELISA, fluorescence microscopy and flow cytometry we show here that hyperthermia following MWCNTs-PEG laser mediated treatment (808 nm, 2W) leads to mitochondrial membrane depolarization that activates the flux of free radicals within the cell and the oxidative state mediate cellular damage in PC cells via apoptotic pathway. Our results are of decisive importance especially in regard with the development of novel nano-biosystems capable to target mitochondria and to synergically act both as cytotoxic drug as well as thermally active agents in order to overcome one of the most common problem met in oncology, that of intrinsic resistance to chemotherapeutics.

  20. Minocycline chelates Ca2+, binds to membranes, and depolarizes mitochondria by formation of Ca2+-dependent ion channels

    Science.gov (United States)

    Antonenko, Yuri N.; Rokitskaya, Tatyana I.; Cooper, Arthur J. L.

    2010-01-01

    Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca2+-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates Ca2+ ions. Secondly, minocycline, in a Ca2+-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show that minocycline, in the presence of Ca2+, induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca2+-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition. PMID:20180001

  1. Development of lysosome-mimicking vesicles to study the effect of abnormal accumulation of sphingosine on membrane properties.

    Science.gov (United States)

    Carreira, Ana C; de Almeida, Rodrigo F M; Silva, Liana C

    2017-06-21

    Synthetic systems are widely used to unveil the molecular mechanisms of complex cellular events. Artificial membranes are key examples of models employed to address lipid-lipid and lipid-protein interactions. In this work, we developed a new synthetic system that more closely resembles the lysosome - the lysosome-mimicking vesicles (LMVs) - displaying stable acid-to-neutral pH gradient across the membrane. To evaluate the advantages of this synthetic system, we assessed the distinct effects of sphingosine (Sph) accumulation in membrane structure and biophysical properties of standard liposomes (no pH gradient) and in LMVs with lipid composition tuned to mimic physiological- or NPC1-like lysosomes. Ternary 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/Sphingomyelin (SM)/Cholesterol (Chol) mixtures with, respectively, low and high Chol/SM levels were prepared. The effect of Sph on membrane permeability and biophysical properties was evaluated by fluorescence spectroscopy, electrophoretic and dynamic light scattering. The results showed that overall Sph has the ability to cause a shift in vesicle surface charge, increase membrane order and promote a rapid increase in membrane permeability. These effects are enhanced in NPC1- LMVs. The results suggest that lysosomal accumulation of these lipids, as observed under pathological conditions, might significantly affect lysosomal membrane structure and integrity, and therefore contribute to the impairment of cell function.

  2. Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons.

    Science.gov (United States)

    Villarreal, Seth; Lee, Sung Hoon; Wu, Ling-Gang

    2017-09-04

    During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

  3. Selective adhesion, lipid exchange and membrane-fusion processes between vesicles of various sizes bearing complementary molecular recognition groups.

    Science.gov (United States)

    Marchi-Artzner, V; Gulik-Krzywicki, T; Guedeau-Boudeville, M A; Gosse, C; Sanderson, J M; Dedieu, J C; Lehn, J M

    2001-06-18

    Equimolar mixtures of large unilamellar vesicles (LUVs) obtained from mixtures of egg lecithin and lipids containing complementary hydrogen bonding head groups (barbituric acid (BAR) and 2,4,6-triaminopyrimidine (TAP)) were shown to aggregate and fuse. These events have been studied in detail using electron microscopy and dynamic light scattering, and by fluorimetry using membrane or water-soluble fluorescence probes. It was shown that aggregation was followed by two competitive processes: a) lipid mixing leading to redispersion of the vesicles; b) fusion events generating much larger vesicles. In order to better understand the nature of the interaction, the effects of ionic strength and surface concentration of recognition lipids on the aggregation process were investigated by dynamic light scattering. Additionally, it was possible to inhibit the aggregation kinetics through addition of a soluble barbituric acid competitor. The study was extended to giant unilamellar vesicles (GUVs) to investigate the size effect and visualise the phenomena in situ. The interactions between complementary LUVs and GUVs or GUVs and GUVs were studied by optical microscopy using dual fluorescent labelling of both vesicle populations. A selective adhesion of LUVs onto GUVs was observed by electron and optical microscopies, whereas no aggregation took place in case of a GUV/GUV mixture. Furthermore, a fusion assay of GUV and LUV using the difference of size between GUV and LUV and calceine self-quenching showed that no mixing between the aqueous pools occured. © 2001 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.

  4. Effect of carbon black nanomaterial on biological membranes revealed by shape of human erythrocytes, platelets and phospholipid vesicles.

    Science.gov (United States)

    Pajnič, Manca; Drašler, Barbara; Šuštar, Vid; Krek, Judita Lea; Štukelj, Roman; Šimundić, Metka; Kononenko, Veno; Makovec, Darko; Hägerstrand, Henry; Drobne, Damjana; Kralj-Iglič, Veronika

    2015-03-28

    We studied the effect of carbon black (CB) agglomerated nanomaterial on biological membranes as revealed by shapes of human erythrocytes, platelets and giant phospholipid vesicles. Diluted human blood was incubated with CB nanomaterial and observed by different microscopic techniques. Giant unilamellar phospholipid vesicles (GUVs) created by electroformation were incubated with CB nanomaterial and observed by optical microscopy. Populations of erythrocytes and GUVs were analyzed: the effect of CB nanomaterial was assessed by the average number and distribution of erythrocyte shape types (discocytes, echinocytes, stomatocytes) and of vesicles in test suspensions, with respect to control suspensions. Ensembles of representative images were created and analyzed using computer aided image processing and statistical methods. In a population study, blood of 14 healthy human donors was incubated with CB nanomaterial. Blood cell parameters (concentration of different cell types, their volumes and distributions) were assessed. We found that CB nanomaterial formed micrometer-sized agglomerates in citrated and phosphate buffered saline, in diluted blood and in blood plasma. These agglomerates interacted with erythrocyte membranes but did not affect erythrocyte shape locally or globally. CB nanomaterial agglomerates were found to mediate attractive interaction between blood cells and to present seeds for formation of agglomerate - blood cells complexes. Distortion of disc shape of resting platelets due to incubation with CB nanomaterial was not observed. CB nanomaterial induced bursting of GUVs while the shape of the remaining vesicles was on the average more elongated than in control suspension, indicating indirect osmotic effects of CB nanomaterial. CB nanomaterial interacts with membranes of blood cells but does not have a direct effect on local or global membrane shape in physiological in vitro conditions. Blood cells and GUVs are convenient and ethically acceptable

  5. NaCl reflection coefficients in proximal tubule apical and basolateral membrane vesicles. Measurement by induced osmosis and solvent drag.

    Science.gov (United States)

    Pearce, D; Verkman, A S

    1989-01-01

    Two independent methods, induced osmosis and solvent drag, were used to determine the reflection coefficients for NaCl (sigma NaCl) in brush border and basolateral membrane vesicles isolated from rabbit proximal tubule. In the induced osmosis method, vesicles loaded with sucrose were subjected to varying inward NaCl gradients in a stopped-flow apparatus. sigma NaCl was determined from the osmolality of the NaCl solution required to cause no initial osmotic water flux as measured by light scattering (null point). By this method sigma NaCl was greater than 0.92 for both apical and basolateral membranes with best estimates of 1.0. sigma NaCl was determined by the solvent drag method using the Cl-sensitive fluorescent indicator, 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ), to detect the drag of Cl into vesicles by inward osmotic water movement caused by an outward osmotic gradient. sigma NaCl was determined by comparing experimental data with theoretical curves generated using the coupled flux equations of Kedem and Katchalsky. By this method we found that sigma NaCl was greater than 0.96 for apical and greater than 0.98 for basolateral membrane vesicles, with best estimates of 1.0 for both membranes. These results demonstrate that sigma NaCl for proximal tubule apical and basolateral membranes are near unity. Taken together with previous results, these data suggest that proximal tubule water channels are long narrow pores that exclude NaCl. PMID:2765660

  6. Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces.

    Science.gov (United States)

    Ionescu, Michael; Zaini, Paulo A; Baccari, Clelia; Tran, Sophia; da Silva, Aline M; Lindow, Steven E

    2014-09-16

    Outer membrane vesicles (OMVs) of Gram-negative bacteria have been studied intensively in recent years, primarily in their role in delivering virulence factors and antigens during pathogenesis. However, the near ubiquity of their production suggests that they may play other roles, such as responding to envelope stress or trafficking various cargoes to prevent dilution or degradation by other bacterial species. Here we show that OMVs produced by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, block its interaction with various surfaces such as the walls of xylem vessels in host plants. The release of OMVs was suppressed by the diffusible signal factor-dependent quorum-sensing system, and a X. fastidiosa ΔrpfF mutant in which quorum signaling was disrupted was both much more virulent to plants and less adhesive to glass and plant surfaces than the WT strain. The higher virulence of the ΔrpfF mutant was associated with fivefold higher numbers of OMVs recovered from xylem sap of infected plants. The frequency of attachment of X. fastidiosa to xylem vessels was 20-fold lower in the presence of OMVs than in their absence. OMV production thus is a strategy used by X. fastidiosa cells to adjust attachment to surfaces in its transition from adhesive cells capable of insect transmission to an "exploratory" lifestyle for systemic spread within the plant host which would be hindered by attachment. OMV production may contribute to the movement of other bacteria in porous environments by similarly reducing their contact with environmental constituents.

  7. CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

    Science.gov (United States)

    Miller, Sharon E; Mathiasen, Signe; Bright, Nicholas A; Pierre, Fabienne; Kelly, Bernard T; Kladt, Nikolay; Schauss, Astrid; Merrifield, Christien J; Stamou, Dimitrios; Höning, Stefan; Owen, David J

    2015-04-20

    The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Stenotrophomonas maltophilia outer membrane vesicles elicit a potent inflammatory response in vitro and in vivo.

    Science.gov (United States)

    Kim, Yoo Jeong; Jeon, Hyejin; Na, Seok Hyeon; Kwon, Hyo Il; Selasi, Gati Noble; Nicholas, Asiimwe; Park, Tae In; Lee, Sang Hwa; Lee, Je Chul

    2016-11-01

    Stenotrophomonas maltophilia has become one of the most prevalent opportunistic pathogens in hospitalized patients. This microorganism secretes outer membrane vesicles (OMVs), but the pathogenesis of S. maltophilia as it relates to OMVs has not been characterized. This study investigated the cytotoxic activity of S. maltophilia OMVs and their ability to induce inflammatory responses both in vitro and in vivo Stenotrophomonas maltophilia ATCC 13637 and two clinical isolates were found to secrete spherical OMVs during in vitro culture. OMVs from S. maltophilia ATCC 13637 were cytotoxic to human lung epithelial A549 cells. Stenotrophomonas maltophilia OMVs stimulated the expression of proinflammatory cytokine and chemokine genes, including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α and monocyte chemoattractant protein-1, in A549 cells. Early inflammatory responses such as congestion and neutrophilic infiltrations and profound expression of proinflammatory cytokine and chemokine genes were observed in the lungs of mice injected with S. maltophilia OMVs, and were similar to responses elicited by the bacteria. Our data demonstrate that S. maltophilia OMVs are important secretory nanocomplexes that elicit a potent inflammatory response that might contribute to S. maltophilia pathogenesis during infection. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Fluorescent BODIPY Rotor: Viscometer for Cellular Organelles and Membrane-Mimicking Vesicles

    Science.gov (United States)

    Kimball, J.; Raut, S.; Fudala, R.; Doan, H.; Maliwal, B.; Sabnis, N.; Lacko, A.; Gryczynski, I.; Dzyuba, S.; Gryczynski, Z.

    2015-03-01

    Many cellular processes, such as mass and signal transport, metabolism and protein-protein interactions are governed in part by diffusion, and thus affected by their local microviscosity. Changes in this microviscosity has also been linked to various diseases, including atherosclerosis, Alzheimer's disease and diabetes. Therefore, directly measuring the heterogeneous viscosity of cellular constitutes can lead to greater understanding of these processes. To this effect, a novel homodiemeric BODIPY dye was evaluated as a fluorescent rotor probe for this application. A linear dependence on viscosity in the range of typical cellular microviscosity was established for steady-state and time-resolved properties of the dye. It was then embedded in vitro to membrane-mimicking lipid vesicles (DPPC, POPC, and POPC plus cholesterol) and results indicated it to be a viable sensor for lifetime-based determination of microviscosity. The BODIPY dye was lastly endocytosed by SKOV3 cells and Fluorescence Lifetime Imaging Microscopy (FLIM) was performed, successfully mapping the viscosity of internal cell components. This work was supported by the NIH Grant R01EB12003, the NSF Grant CBET-1264608, and the INFOR Grant from TCU.

  10. Modulation of GABA-stimulated chloride influx into membrane vesicles from rat cerebral cortex by triazolobenzodiazepines

    Energy Technology Data Exchange (ETDEWEB)

    Obata, T.; Yamamura, H.I.

    1988-01-01

    The effects of triazolobenzodiazepines of GABA-stimulated /sup 36/Cl/sup -/ uptake by membrane vesicles from rat cerebral cortex were examined. Triazolam and alprazolam showed a significant enhancement of GABA-stimulated /sup 36/Cl/sup -/ uptake at 0.01-10 uM. On the other hand, adinazolam showed a small enhancement at 0.1-1 uM followed by a significant inhibition of GABA-stimulated /sup 36/Cl/sup -/ uptake at 100 uM. The enhancement of GABA-stimulated /sup 36/Cl/sup -/ uptake by 1 uM alprazolam was antagonized by Ro15-1788, a benzodiazepine antagonist, but the inhibition of this response by 30 uM adinazolam was not antagonized by Ro15-1788. These results indicate that triazolobenzodiazepines enhanced GABA-stimulated /sup 36/Cl/sup -/ uptake through benzodiazepine receptors. High concentrations of adinazolam inhibit GABA-stimulated /sup 36/Cl/sup -/ uptake which may be due to the direct blockade of GABA-gated chloride channel. 23 references, 4 figures.

  11. Isolation, Characterization and Biological Properties of Membrane Vesicles Produced by the Swine Pathogen Streptococcus suis.

    Directory of Open Access Journals (Sweden)

    Bruno Haas

    Full Text Available Streptococcus suis, more particularly serotype 2, is a major swine pathogen and an emerging zoonotic agent worldwide that mainly causes meningitis, septicemia, endocarditis, and pneumonia. Although several potential virulence factors produced by S. suis have been identified in the last decade, the pathogenesis of S. suis infections is still not fully understood. In the present study, we showed that S. suis produces membrane vesicles (MVs that range in diameter from 13 to 130 nm and that appear to be coated by capsular material. A proteomic analysis of the MVs revealed that they contain 46 proteins, 9 of which are considered as proven or suspected virulence factors. Biological assays confirmed that S. suis MVs possess active subtilisin-like protease (SspA and DNase (SsnA. S. suis MVs degraded neutrophil extracellular traps, a property that may contribute to the ability of the bacterium to escape the host defense response. MVs also activated the nuclear factor-kappa B (NF-κB signaling pathway in both monocytes and macrophages, inducing the secretion of pro-inflammatory cytokines, which may in turn contribute to increase the permeability of the blood brain barrier. The present study brought evidence that S. suis MVs may play a role as a virulence factor in the pathogenesis of S. suis infections, and given their composition be an excellent candidate for vaccine development.

  12. Diamines interfere with the transport of L-ornithine in Ehrlich-cell plasma-membrane vesicles.

    Science.gov (United States)

    Medina, M A; Urdiales, J L; Mates, J M; Núñez de Castro, I; Sánchez-Jiménez, F

    1991-12-15

    1. L-Ornithine transport by plasma-membrane vesicles isolated from Ehrlich cells is Na(+)-independent and shows a saturable and a diffusional component. 2. Putrescine, histamine, 5-hydroxytryptamine and 2,3-diaminopropane at 55 microM concentration significantly inhibit 0.5 mM-L-ornithine transport at least for the first 10 min of incubation. 3. There is a trans-stimulatory effect of putrescine on L-ornithine transport.

  13. The toolbox of vesicle sidedness determination

    NARCIS (Netherlands)

    Meszaros, Peter; Hoekstra, Dick; Kok, Jan Willem

    2012-01-01

    Vesicles prepared from cellular plasma membranes are widely used in science for different purposes. The outer membrane leaflet differs from the inner membrane leaflet of the vesicle, and during vesicle preparation procedures two types of vesicles will be generated: right-side-out vesicles, of which

  14. Light-dependent delta pH and membrane potential changes in halobacterial vesicles coupled to sodium transport

    Energy Technology Data Exchange (ETDEWEB)

    Kamo, N.; Racanelli, T.; Packer, L.

    1982-01-01

    Bacteriorhodopsin and Halorhodopsin present in Halobacterium halobium strains have been investigated in relation to Na/sup +//H/sup +/ exchange in isolated cell envelope vesicles. Upon illumination, these retinal proteins result in extrusion of sodium ions by either an electrogenic Na/sup +//Ha/sup +/ antiporter and/or a direct sodium pump. Since a molecular characterization of these mechanism(s) of sodium extrusion has not yet been realized, it was of interest to measure directly the light- and sodium-dependent changes in delta pH and membrane potential under nearly identical conditions in S9 and R1mR cell membrane vesicles to gain information on the relation of these retinal proteins to sodium extrusion. These activities were evaluated in terms of their dependence on light intensity, and on the inhibitory effect of chemical modifiers of carboxyl groups (carbodiimides); electroneutral exchanges (monensin and triphenyltin); digitoxin and some analogues; and phloretin. Under most of the conditions and treatments employed, light- and sodium-dependent delta pH led to similar effects in both membrane vesicle types. Hence, it is concluded that the delta pH and delta psi which arise from sodium transport occur by either a single mechanism or by one which shares common features.

  15. The transbilayer movement of phosphatidylcholine in vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

    NARCIS (Netherlands)

    Gerritsen, W.J.; Henricks, P.A.J.; Kruijff, B. de; Deenen, L.L.M. van

    1980-01-01

    Vesicles have been prepared from 18 : 1c/18 : 1c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose,

  16. Pannexin2 oligomers localize into endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane

    Directory of Open Access Journals (Sweden)

    Daniela eBoassa

    2015-02-01

    Full Text Available Pannexin2 (Panx2 is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS have been documented. Whereas Pannexin1 (Panx1 is fairly ubiquitous and Pannexin3 (Panx3 is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa and HEK293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the

  17. Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS

    DEFF Research Database (Denmark)

    Schneider, Falk; Waithe, Dominic; Clausen, Mathias P

    2017-01-01

    Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signalling, and they are suggested to be strongly associated with the actin cytoskeleton. Here, we utilise super-resolution STED microscopy combined with fluorescence correlation spectroscopy...... (STED-FCS) to access and compare the diffusion characteristics of fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the live cell plasma membrane and in actin cytoskeleton-free cell-derived giant plasma membrane vesicles (GPMVs). Hindered diffusion of phospholipids and sphingolipids...... forming immobile clusters, both of which disappear in GPMVs. Our data underline the crucial role of the actin cortex in maintaining hindered diffusion modes of many but not all of the membrane molecules, and highlight a powerful experimental approach to decipher specific influences on molecular plasma...

  18. Meningococcal Outer Membrane Vesicle Composition-Dependent Activation of the Innate Immune Response.

    Science.gov (United States)

    Zariri, Afshin; Beskers, Joep; van de Waterbeemd, Bas; Hamstra, Hendrik Jan; Bindels, Tim H E; van Riet, Elly; van Putten, Jos P M; van der Ley, Peter

    2016-10-01

    Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen-associated molecular patterns, including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an extremely potent hexa-acylated LPS, leading to adverse effects when its OMVs are applied as vaccines. To create safe OMV vaccines, detergent treatment is generally used to reduce the LPS content. While effective, this method also leads to loss of protective antigens such as lipoproteins. Alternatively, genetic modification of LPS can reduce its toxicity. In the present study, we have compared the effects of standard OMV isolation methods using detergent or EDTA with those of genetic modifications of LPS to yield a penta-acylated lipid A (lpxL1 and pagL) on the in vitro induction of innate immune responses. The use of detergent decreased both Toll-like receptor 4 (TLR4) and TLR2 activation by OMVs, while the LPS modifications reduced only TLR4 activation. Mutational removal of PorB or lipoprotein factor H binding protein (fHbp), two proteins known to trigger TLR2 signaling, had no effect, indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent-treated OMVs and lpxL1 OMVs showed similar reductions of cytokine profiles in the human monocytic cell line MM6 and human dendritic cells (DCs). OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of proinflammatory cytokines interleukin-6 (IL-6) and IL-1β but not of IP-10, a typical TRIF-dependent chemokine. Taken together, these data show that lipid A modification can be used to obtain OMVs with reduced activation of innate immunity, similar to what is found after detergent treatment. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

    Directory of Open Access Journals (Sweden)

    Anna Lena Jung

    2016-04-01

    Full Text Available The formation and release of outer membrane vesicles (OMVs is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila, a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.

  20. Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yong; Liu, Liguo; Fu, Hua; Wei, Candong, E-mail: weicando@ipbcams.ac.cn; Jin, Qi, E-mail: zdsys@vip.sina.com

    2014-10-31

    Highlights: • We utilized mTRAQ-based quantification to study protein changes in Congo red-induced OMVs. • A total of 148 proteins were identified in S. flexneri-derived OMVs. • Twenty-eight and five proteins are significantly up- and down-regulated in the CR-induced OMV, respectively. • The result implied that a special sorting mechanism of particular proteins into OMVs may exist. • Key node proteins in the protein interaction network might be important for pathogenicity. - Abstract: The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis. In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein–protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria.

  1. Leucine transport in brush border membrane vesicles from freshwater insect larvae.

    Science.gov (United States)

    Forcella, Matilde; Berra, Elisa; Giacchini, Roberto; Parenti, Paolo

    2006-11-01

    Leucine transport across brush border membrane vesicles prepared from four insect species common to European freshwater streams has been characterized. The species studied were: Ephemera danica (Ephemeroptera: Ephemeridae), Isoperla grammatica (Plecoptera: Perlodidae), Hydropsyche pellucidula (Trichoptera: Hydropsychidae), and Hybomitra bimaculata (Diptera: Tabanidae). The transport differed among the studied taxa for several features, including pH and sodium dependence, substrate affinity and specificity, and efficiency. In H. pellucidula and E. danica, leucine uptake was higher at pH 7.4 than at more alkaline or acidic pH values, whereas in I. grammatica and H. bimaculata, the uptake was rather constant when pH varied from 5.0 to 7.4, then strongly decreased at pH 8.8. All but E. danica displayed a transient intravescicular leucine accumulation in the presence of sodium, suggesting the existence of a cation-leucine symport mechanism. The sodium dependence ranged according to the following order: H. pellucidula > I. grammatica > H. bimaculata > E. danica. Moreover, in H. pellucidula and I. grammatica, the sodium-dependence was stronger at pH 8.8 than at pH 7.4. In E. danica, leucine uptake was sodium-independent at all pH values. The highest value of V(max) (45.3 pmol.s(-1).mg proteins(-1)) was in E. danica, which, however, displayed the lowest affinity (K(m) 137 muM) when compared to the kinetic parameters of other taxa. The V(max) and K(m) values were: 40 and 52.5, 32.1 and 12.5, and 4.5 and 230 for H. bimaculata, H. pellucidula, and I. grammatica, respectively. The obtained results are discussed within our current knowledge of amino acid transport systems in insects. (c) 2006 Wiley-Liss, Inc.

  2. Automatically Identifying Fusion Events between GLUT4 Storage Vesicles and the Plasma Membrane in TIRF Microscopy Image Sequences.

    Science.gov (United States)

    Wu, Jian; Xu, Yingke; Feng, Zhouyan; Zheng, Xiaoxiang

    2015-01-01

    Quantitative analysis of the dynamic behavior about membrane-bound secretory vesicles has proven to be important in biological research. This paper proposes a novel approach to automatically identify the elusive fusion events between VAMP2-pHluorin labeled GLUT4 storage vesicles (GSVs) and the plasma membrane. The differentiation is implemented to detect the initiation of fusion events by modified forward subtraction of consecutive frames in the TIRFM image sequence. Spatially connected pixels in difference images brighter than a specified adaptive threshold are grouped into a distinct fusion spot. The vesicles are located at the intensity-weighted centroid of their fusion spots. To reveal the true in vivo nature of a fusion event, 2D Gaussian fitting for the fusion spot is used to derive the intensity-weighted centroid and the spot size during the fusion process. The fusion event and its termination can be determined according to the change of spot size. The method is evaluated on real experiment data with ground truth annotated by expert cell biologists. The evaluation results show that it can achieve relatively high accuracy comparing favorably to the manual analysis, yet at a small fraction of time.

  3. Automatically Identifying Fusion Events between GLUT4 Storage Vesicles and the Plasma Membrane in TIRF Microscopy Image Sequences

    Directory of Open Access Journals (Sweden)

    Jian Wu

    2015-01-01

    Full Text Available Quantitative analysis of the dynamic behavior about membrane-bound secretory vesicles has proven to be important in biological research. This paper proposes a novel approach to automatically identify the elusive fusion events between VAMP2-pHluorin labeled GLUT4 storage vesicles (GSVs and the plasma membrane. The differentiation is implemented to detect the initiation of fusion events by modified forward subtraction of consecutive frames in the TIRFM image sequence. Spatially connected pixels in difference images brighter than a specified adaptive threshold are grouped into a distinct fusion spot. The vesicles are located at the intensity-weighted centroid of their fusion spots. To reveal the true in vivo nature of a fusion event, 2D Gaussian fitting for the fusion spot is used to derive the intensity-weighted centroid and the spot size during the fusion process. The fusion event and its termination can be determined according to the change of spot size. The method is evaluated on real experiment data with ground truth annotated by expert cell biologists. The evaluation results show that it can achieve relatively high accuracy comparing favorably to the manual analysis, yet at a small fraction of time.

  4. Effect of toluene on Pseudomonas stutzeri ST-9 morphology - plasmolysis, cell size, and formation of outer membrane vesicles.

    Science.gov (United States)

    Michael, Esti; Nitzan, Yeshayahu; Langzam, Yakov; Luboshits, Galia; Cahan, Rivka

    2016-08-01

    Isolated toluene-degrading Pseudomonas stutzeri ST-9 bacteria were grown in a minimal medium containing toluene (100 mg·L(-1)) (MMT) or glucose (MMG) as the sole carbon source, with specific growth rates of 0.019 h(-1) and 0.042 h(-1), respectively. Scanning (SEM) as well as transmission (TEM) electron microscope analyses showed that the bacterial cells grown to mid-log phase in the presence of toluene possess a plasmolysis space. TEM analysis revealed that bacterial cells that were grown in MMT were surrounded by an additional "material" with small vesicles in between. Membrane integrity was analyzed by leakage of 260 nm absorbing material and demonstrated only 7% and 8% leakage from cultures grown in MMT compared with MMG. X-ray microanalysis showed a 4.3-fold increase in Mg and a 3-fold increase in P in cells grown in MMT compared with cells grown in MMG. Fluorescence-activated cell sorting (FACS) analysis indicated that the permeability of the membrane to propidium iodide was 12.6% and 19.6% when the cultures were grown in MMG and MMT, respectively. The bacterial cell length increased by 8.5% ± 0.1% and 17% ± 2%, as measured using SEM images and FACS analysis, respectively. The results obtained in this research show that the presence of toluene led to morphology changes, such as plasmolysis, cell size, and formation of outer membrane vesicles. However, it does not cause significant damage to membrane integrity.

  5. Selective depolarization of the muscle membrane in frog nerve-muscle preparations by a chromatographically purified extract of the dinoflagellate Ostreopsis lenticularis

    OpenAIRE

    Meunier, Frédéric A.; Mercado, José A; Molgó, Jordi; Tosteson, Thomas R.; Escalona de Motta, Gladys

    1997-01-01

    The actions of a chromatographically identified extract of the marine dinoflagellate Ostreopsis lenticularis, named ostreotoxin-3 (OTX-3), were studied on frog isolated neuromuscular preparations.OTX-3 (1–10 μg ml−1) applied to cutaneous pectoris nerve-muscle preparations depolarized skeletal muscle fibres and caused spontaneous contractions. The depolarization was neither reversed by prolonged washing nor by (+)-tubocurarine.OTX-3 decreased the amplitude of miniature end plate potentials (m....

  6. Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

    Science.gov (United States)

    Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H; Sosinsky, Gina E

    2014-01-01

    Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

  7. Role of lipid phase separations and membrane hydration in phospholipid vesicle fusion.

    NARCIS (Netherlands)

    Hoekstra, D.

    1982-01-01

    The relationship between lipid phase separation and fusion of small unilamellar phosphatidylserine-containing vesicles was investigated. The kinetics of phase separation were monitored by following the increase of self-quenching of the fluorescent phospholipid analogue

  8. Exocytosis and Endocytosis of Small Vesicles across the Plasma Membrane in Saccharomyces cerevisiae

    Science.gov (United States)

    Stein, Kathryn; Chiang, Hui-Ling

    2014-01-01

    When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), phosphoenolpyruvate carboxykinase, isocitrate lyase, and malate dehydrogenase, as well as the non-gluconeogenic enzymes glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A, are secreted into the periplasm. In the extracellular fraction, these secreted proteins are associated with small vesicles that account for more than 90% of the total number of extracellular structures observed. When glucose is added to glucose-starved cells, FBPase is internalized and associated with clusters of small vesicles in the cytoplasm. Specifically, the internalization of FBPase results in the decline of FBPase and vesicles in the extracellular fraction and their appearance in the cytoplasm. The clearance of extracellular vesicles and vesicle-associated proteins from the extracellular fraction is dependent on the endocytosis gene END3. This internalization is regulated when cells are transferred from low to high glucose. It is rapidly occurring and is a high capacity process, as clusters of vesicles occupy 10%–20% of the total volume in the cytoplasm in glucose re-fed cells. FBPase internalization also requires the VPS34 gene encoding PI3K. Following internalization, FBPase is delivered to the vacuole for degradation, whereas proteins that are not degraded may be recycled. PMID:25192542

  9. Fluorescent Probe Study of AOT Vesicle Membranes and Their Alteration upon Addition of Aniline or the Aniline Dimer p-Aminodiphenylamine (PADPA).

    Science.gov (United States)

    Iwasaki, Fumihiko; Luginbühl, Sandra; Suga, Keishi; Walde, Peter; Umakoshi, Hiroshi

    2017-02-28

    Artificial vesicles formed from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in aqueous solution are used successfully as additives for enzymatic oligomerizations or polymerizations of aniline or the aniline dimer p-aminodiphenylamine (PADPA) under slightly acidic conditions (e.g., pH 4.3 with horseradish peroxidase and hydrogen peroxide as oxidants). In these systems, the reactions occur membrane surface-confined. Therefore, (i) the physicochemical properties of the vesicle membrane and (ii) the interaction of aniline or PADPA with the AOT membrane play crucial roles in the progress and final outcome of the reactions. For this reason, the properties of AOT vesicles with and without added aniline or PADPA were investigated by using two fluorescent membrane probes: 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-lauroyl-2-dimethylaminonaphthalene (Laurdan). DPH and Laurdan were used as "sensors" of the membrane fluidity, surface polarity, and membrane phase state. Moreover, the effect of hexanol, alone or in combination with aniline or PADPA, as a possible modifier of the AOT membrane, was also studied with the aim of evaluating whether the membrane fluidity and surface polarity is altered significantly by hexanol, which, in turn, may have an influence on the mentioned types of reactions. The data obtained indicate that the AOT vesicle membrane at room temperature and pH 4.3 (0.1 M NaH2PO4) is more fluid and has a more polar surface than in the case of fluid phospholipid vesicle membranes formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Furthermore, the fluorescence measurements indicate that mixed AOT-hexanol membranes are less fluid than pure AOT membranes and that they have a lower surface polarity than pure AOT membranes. PADPA strongly binds to AOT and to mixed AOT/hexanol membranes and leads to drastic changes in the membrane properties (decrease in fluidity and surface polarity), resulting in Laurdan fluorescence spectra, which are characteristic for

  10. Monocyte activation drives preservation of membrane thiols by promoting release of oxidised membrane moieties via extracellular vesicles.

    Science.gov (United States)

    Szabó-Taylor, K É; Tóth, E Á; Balogh, A M; Sódar, B W; Kádár, L; Pálóczi, K; Fekete, N; Németh, A; Osteikoetxea, X; Vukman, K V; Holub, M; Pállinger, É; Nagy, Gy; Winyard, P G; Buzás, E I

    2017-07-01

    The redox state of cellular exofacial molecules is reflected by the amount of available thiols. Furthermore, surface thiols can be considered as indicators of immune cell activation. One group of thiol containing proteins, peroxiredoxins, in particular, have been associated with inflammation. In this study, we assessed surface thiols of the U937 and Thp1 monocyte cell lines and primary monocytes in vitro upon inflammatory stimulation by irreversibly labelling the cells with a fluorescent derivative of maleimide. We also investigated exofacial thiols on circulating blood mononuclear cells in patients with rheumatoid arthritis and healthy controls. When analysing extracellular vesicles, we combined thiol labelling with the use of antibodies to specific CD markers to exclude extracellular vesicle mimicking signals from thiol containing protein aggregates. Furthermore, differential detergent lysis was applied to confirm the vesicular nature of the detected extracellular events in blood plasma. We found an increase in exofacial thiols on monocytes upon in vitro stimulation by LPS or TNF, both in primary monocytes and monocytic cell lines (pextracellular vesicles showed a decrease in their exofacial thiols compared with those from unstimulated cells (pextracellular vesicles of isolated CD14 + cells from rheumatoid arthritis patients had decreased thiol levels compared with healthy subjects (pextracellular vesicles was increased in rheumatoid arthritis blood plasma (pextracellular vesicle-enriched preparations from blood plasma. Our data show that cell surface thiols play a protective role and reflect oxidative stress resistance state in activated immune cells. Furthermore, they support a role of extracellular vesicles in the redox regulation of human monocytes, possibly representing an antioxidant mechanism. Copyright © 2017. Published by Elsevier Inc.

  11. Profiling the outer membrane proteome during growth and development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles.

    Science.gov (United States)

    Kahnt, Jörg; Aguiluz, Kryssia; Koch, Jürgen; Treuner-Lange, Anke; Konovalova, Anna; Huntley, Stuart; Hoppert, Michael; Søgaard-Andersen, Lotte; Hedderich, Reiner

    2010-10-01

    Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.

  12. Reconstituted proteolipid vesicles prepared from Mycoplasma fermentans membranes are able to bind and fuse with Molt-3 cells.

    Science.gov (United States)

    Rechnitzer, Hagai; Rottem, Shlomo

    2006-10-01

    We describe and characterize reconstituted proteolipid vesicles (rPLV) prepared from solubilized Mycoplasma fermentans membranes and studied their binding to and fusion with host Molt-3 cells. The rPLV were prepared following membrane solubilization by Triton X-100 and detergent removal by SM-2 resin beads. The vesicles thus obtained had a rather uniform diameter of about 1 microm and were sealed as monitored by measuring in an assay that measures the quenching by sodium dithionite of a hydrophobic fluorescent probe incorporated into the rPLV membrane. The rPLV adhered to Molt-3 cells and, based on measurements of lipid mixing, fused with the host cells at a similar rate and to about the same extent as intact M. fermentans. Preliminary experiments showed that a chimeric protein, GnRH-PE66, could be encapsulated within these rPLV, opening the way to develop a system for the transfer of high-molecular weight soluble molecules, encapsulated in the rPLV, to target eukaryotic cells.

  13. Protection from hemolytic uremic syndrome by eyedrop vaccination with modified enterohemorrhagic E. coli outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    Kyoung Sub Choi

    Full Text Available We investigated whether eyedrop vaccination using modified outer membrane vesicles (mOMVs is effective for protecting against hemolytic uremic syndrome (HUS caused by enterohemorrhagic E. coli (EHEC O157:H7 infection. Modified OMVs and waaJ-mOMVs were prepared from cultures of MsbB- and Shiga toxin A subunit (STxA-deficient EHEC O157:H7 bacteria with or without an additional waaJ mutation. BALB/c mice were immunized by eyedrop mOMVs, waaJ-mOMVs, and mOMVs plus polymyxin B (PMB. Mice were boosted at 2 weeks, and challenged peritoneally with wild-type OMVs (wtOMVs at 4 weeks. As parameters for evaluation of the OMV-mediated immune protection, serum and mucosal immunoglobulins, body weight change and blood urea nitrogen (BUN/Creatinin (Cr were tested, as well as histopathology of renal tissue. In order to confirm the safety of mOMVs for eyedrop use, body weight and ocular histopathological changes were monitored in mice. Modified OMVs having penta-acylated lipid A moiety did not contain STxA subunit proteins but retained non-toxic Shiga toxin B (STxB subunit. Removal of the polymeric O-antigen of O157 LPS was confirmed in waaJ-mOMVs. The mice group vaccinated with mOMVs elicited greater humoral and mucosal immune responses than did the waaJ-mOMVs and PBS-treated groups. Eyedrop vaccination of mOMVs plus PMB reduced the level of humoral and mucosal immune responses, suggesting that intact O157 LPS antigen can be a critical component for enhancing the immunogenicity of the mOMVs. After challenge, mice vaccinated with mOMVs were protected from a lethal dose of wtOMVs administered intraperitoneally, conversely mice in the PBS control group were not. Collectively, for the first time, EHEC O157-derived mOMV eyedrop vaccine was experimentally evaluated as an efficient and safe means of vaccine development against EHEC O157:H7 infection-associated HUS.

  14. Realizing a variable isotropic depolarizer.

    Science.gov (United States)

    Shaham, Assaf; Eisenberg, Hagai S

    2012-07-01

    We demonstrate an isotropic depolarizing channel with a controllable degree of depolarization. The depolarizer is composed of four birefringent crystals and half-wave plates. Quantum process tomography results of the depolarization effect on single photons agree well with the theoretical prediction. This depolarizer can be used to test quantum communication protocols with photons.

  15. Cysteine depletion causes oxidative stress and triggers outer membrane vesicle release by Neisseria meningitidis; implications for vaccine development.

    Directory of Open Access Journals (Sweden)

    Bas van de Waterbeemd

    Full Text Available Outer membrane vesicles (OMV contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV, which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (±1500 human doses per liter cultivation. Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteine-dependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis.

  16. On-Demand Formation of Supported Lipid Membrane Arrays by Trehalose-Assisted Vesicle Delivery for SPR Imaging.

    Science.gov (United States)

    Hinman, Samuel S; Ruiz, Charles J; Drakakaki, Georgia; Wilkop, Thomas E; Cheng, Quan

    2015-08-12

    The fabrication of large-scale, solid-supported lipid bilayer (SLB) arrays has traditionally been an arduous and complex task, primarily due to the need to maintain SLBs within an aqueous environment. In this work, we demonstrate the use of trehalose vitrified phospholipid vesicles that facilitate on-demand generation of microarrays, allowing each element a unique composition, for the label-free and high-throughput analysis of biomolecular interactions by SPR imaging (SPRi). Small, unilamellar vesicles (SUVs) are suspended in trehalose, deposited in a spatially defined manner, with the trehalose vitrifying on either hydrophilic or hydrophobic SPR substrates. SLBs are subsequently spontaneously formed on-demand simply by in situ hydration of the array in the SPR instrument flow cell. The resulting SLBs exhibit high lateral mobility, characteristic of fluidic cellular lipid membranes, and preserve the biological function of embedded cell membrane receptors, as indicated by SPR affinity measurements. Independent fluorescence and SPR imaging studies show that the individual SLBs stay localized at the area of deposition, without any encapsulating matrix, confining coral, or boundaries. The introduced methodology allows individually addressable SLB arrays to be analyzed with excellent label-free sensitivity in a real-time, high-throughput manner. Various protein-ganglioside interactions have been selected as a model system to illustrate discrimination of strong and weak binding responses in SPRi sensorgrams. This methodology has been applied toward generating hybrid bilayer membranes on hydrophobic SPR substrates, demonstrating its versatility toward a range of surfaces and membrane geometries. The stability of the fabricated arrays, over medium to long storage periods, was evaluated and found to be good. The highly efficient and easily scalable nature of the method has the potential to be applied to a variety of label-free sensing platforms requiring lipid membranes for

  17. Comparative analysis of the uropathogenic Escherichia coli surface proteome by tandem mass-spectrometry of artificially induced outer membrane vesicles.

    Science.gov (United States)

    Wurpel, Daniël J; Moriel, Danilo G; Totsika, Makrina; Easton, Donna M; Schembri, Mark A

    2015-02-06

    Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections. For successful colonisation of the urinary tract, UPEC employ multiple surface-exposed or secreted virulence factors, including adhesins and iron uptake systems. Whilst individual UPEC strains and their virulence factors have been the focus of extensive research, there have been no outer membrane (OM) proteomic studies based on large clinical UPEC collections, primarily due to limitations of traditional methods. In this study, a high-throughput method based on tandem mass-spectrometry of EDTA heat-induced outer membrane vesicles (OMVs) was developed for the characterisation of the UPEC surface-associated proteome. The method was applied to compare the OM proteome of fifty-four UPEC isolates, resulting in the identification of 8789 proteins, consisting of 619 unique proteins, which were subsequently interrogated for their subcellular origin, prevalence and homology to characterised virulence factors. Multiple distinct virulence-associated proteins were identified, including two novel putative iron uptake proteins, an uncharacterised type of chaperone-usher fimbriae and various highly prevalent hypothetical proteins. Our results give fundamental insight into the physiology of UPEC and provide a framework for understanding the composition of the UPEC OM proteome. In this study a high-throughput method based on tandem mass-spectrometry of EDTA heat-induced outer membrane vesicles was used to define the outer membrane proteome of a large uropathogenic E. coli (UPEC) collection. Our results provide an inventory of proteins expressed on the surface of UPEC, and provide a framework for understanding the composition of the UPEC OM proteome. The method enables the rapid characterisation of the E. coli surface proteome and could easily be applied to the large-scale outer membrane protein profiling of other Gram-negative bacteria. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Elastic Torques about Membrane Edges: A Study of Pierced Egg Lecithin Vesicles.

    Science.gov (United States)

    Lorenzen, S; Servuss, R M; Helfrich, W

    1986-10-01

    The shape of mechanically pierced giant vesicles is studied to obtain the elastic modulus of Gaussian curvature of egg lecithin bilayers. It is argued that such experiments are governed by an apparent modulus, kappa(app), not the true modulus of Gaussian curvature, kappa. A theory of kappa(app) is proposed, regarding the pierced bilayer vesicle as a closed monolayer vesicle. The quantity measured, i.e. kappa(app)/kappa, where kappa is the rigidity, agrees satisfactorily with the theory. We find kappa(app) = -(1.9 +/- 0.3) . 10(-12) erg (on the basis of kappa = (2.3 +/- 0.3) . 10(-12) erg). The result may have implications for bilayer fusion.

  19. Spontaneous Vesicle Self-Assembly: A Mesoscopic View of Membrane Dynamics

    DEFF Research Database (Denmark)

    Shillcock, J. C.

    2012-01-01

    Amphiphilic vesicles are ubiquitous in living cells and industrially interesting as drug delivery vehicles. Vesicle self-assembly proceeds rapidly from nanometer to micrometer length scales and is too fast to image experimentally but too slow for molecular dynamics simulations. Here, we use...... parallel dissipative particle dynamics (DPD) to follow spontaneous vesicle self-assembly for up to 445 mu s with near-molecular resolution. The mean mass and radius of gyration of growing amphiphilic clusters obey power laws with exponents of 0.85 +/- 0.03 and 0.41 +/- 0.02, respectively. We show that DPD...... provides a computational window onto fluid dynamics on scales unreachable by other explicit-solvent simulations....

  20. Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca²⁺, CaM and Depolarization of Plasma Membrane Potential.

    Science.gov (United States)

    Zhang, Xian-Chen; Gao, Hong-Jian; Yang, Tian-Yuan; Wu, Hong-Hong; Wang, Yu-Mei; Zhang, Zheng-Zhu; Wan, Xiao-Chun

    2016-01-05

    Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca(2+) efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca(2+) chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca(2+) fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca(2+) chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca(2+) and H⁺ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca(2+)-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants.

  1. Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca2+, CaM and Depolarization of Plasma Membrane Potential

    Science.gov (United States)

    Zhang, Xian-Chen; Gao, Hong-Jian; Yang, Tian-Yuan; Wu, Hong-Hong; Wang, Yu-Mei; Zhang, Zheng-Zhu; Wan, Xiao-Chun

    2016-01-01

    Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca2+ efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca2+ chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca2+ fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca2+ chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca2+ and H+ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca2+-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants. PMID:26742036

  2. Cephalosporinases associated with outer membrane vesicles released by Bacteroides spp. protect gut pathogens and commensals against β-lactam antibiotics.

    Science.gov (United States)

    Stentz, Régis; Horn, Nikki; Cross, Kathryn; Salt, Louise; Brearley, Charles; Livermore, David M; Carding, Simon R

    2015-03-01

    To identify β-lactamase genes in gut commensal Bacteroides species and to assess the impact of these enzymes, when carried by outer membrane vesicles (OMVs), in protecting enteric pathogens and commensals. A deletion mutant of the putative class A β-lactamase gene (locus tag BT_4507) found in the genome of the human commensal Bacteroides thetaiotaomicron was constructed and a phenotypic analysis performed. A phylogenetic tree was built from an alignment of nine Bacteroides cephalosporinase protein sequences, using the maximum likelihood method. The rate of cefotaxime degradation after incubation with OMVs produced by different Bacteroides species was quantified using a disc susceptibility test. The resistance of Salmonella Typhimurium and Bifidobacterium breve to cefotaxime in liquid culture in the presence of B. thetaiotaomicron OMVs was evaluated by measuring bacterial growth. The B. thetaiotaomicron BT_4507 gene encodes a β-lactamase related to the CepA cephalosporinase of Bacteroides fragilis. OMVs produced by B. thetaiotaomicron and several other Bacteroides species, except Bacteroides ovatus, carried surface-associated β-lactamases that could degrade cefotaxime. β-Lactamase-harbouring OMVs from B. thetaiotaomicron protected Salmonella Typhimurium and B. breve from an otherwise lethal dose of cefotaxime. The production of membrane vesicles carrying surface-associated β-lactamases by Bacteroides species, which constitute a major part of the human colonic microbiota, may protect commensal bacteria and enteric pathogens, such as Salmonella Typhimurium, against β-lactam antibiotics. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  3. K+-dependent paradoxical membrane depolarization and Na+ overload, major and reversible contributors to weakness by ion channel leaks

    DEFF Research Database (Denmark)

    Jurkat-Rott, Karin; Weber, Marc-André; Fauler, Michael

    2009-01-01

    Normal resting potential (P1) of myofibers follows the Nernst equation, exhibiting about -85 mV at a normal extracellular K(+) concentration ([K(+)](o)) of 4 mM. Hyperpolarization occurs with decreased [K(+)](o), although at [K(+)](o) < 1.0 mM, myofibers paradoxically depolarize to a second stabl...

  4. K⁺-dependent ³H-D-glucose transport by hepatopancreatic brush border membrane vesicles of a marine shrimp.

    Science.gov (United States)

    Obi, Ijeoma E; Sterling, Kenneth M; Ahearn, Gregory A

    2013-01-01

    The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-D-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl₂/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-D-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-D-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J(max) and lowered K(m) in both salts. ³H-D-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis-Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-D-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (D-galactose, α-methyl-D-gluco-pyranoside, unlabeled D-glucose, D-fructose, and D-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-D-glucose influx. Only unlabeled D-glucose, D-fructose, and D-mannose significantly (p glucose transport. An additional experiment using increasing concentrations of D-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-D-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-D-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, D-fructose, and D-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter, suggesting that an

  5. Membrane tubulation in lipid vesicles triggered by the local application of calcium ions

    DEFF Research Database (Denmark)

    Ali Doosti, Baharan; Pezeshkian, Weria; Bruhn, Dennis Skjøth

    2017-01-01

    Experimental and theoretical studies on ion-lipid interactions, predict that binding of calcium ions to cell membranes leads to macroscopic mechanical effects and membrane remodeling. Herein, we provide experimental evidence that a point-source of Ca2+ acting upon a negatively charged membrane, g...

  6. The ultrastructure of Ignicoccus: Evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon

    Directory of Open Access Journals (Sweden)

    Reinhard Rachel

    2002-01-01

    Full Text Available A novel genus of hyperthermophilic, strictly chemolithotrophic archaea, Ignicoccus, has been described recently, with (so far three isolates in pure culture. Cells were prepared for ultrastructural investigation by cultivation in cellulose capillaries and processing by high-pressure freezing, freeze-substitution and embedding in Epon. Cells prepared in accordance with this protocol consistently showed a novel cell envelope structure previously unknown among the Archaea: a cytoplasmic membrane; a periplasmic space with a variable width of 20 to 400 nm, containing membrane-bound vesicles; and an outer sheath, approximately 10 nm wide, resembling the outer membrane of gram-negative bacteria. This sheath contained three types of particles: numerous tightly, irregularly packed single particles, about 8 nm in diameter; pores with a diameter of 24 nm, surrounded by tiny particles, arranged in a ring with a diameter of 130 nm; and clusters of up to eight particles, each particle 12 nm in diameter. Freeze-etched cells exhibited a smooth surface, without a regular pattern, with frequent fracture planes through the outer sheath, indicating the presence of an outer membrane and the absence of an S-layer. The study illustrates the novel complex architecture of the cell envelope of Ignicoccus as well as the importance of elaborate preparation procedures for ultrastructural investigations.

  7. Segregative clustering of Lo and Ld membrane microdomains induced by local pH gradients in GM1-containing giant vesicles: a lipid model for cellular polarization.

    Science.gov (United States)

    Staneva, Galya; Puff, Nicolas; Seigneuret, Michel; Conjeaud, Hélène; Angelova, Miglena I

    2012-11-27

    Several cell polarization processes are coupled to local pH gradients at the membrane surface. We have investigated the involvement of a lipid-mediated effect in such coupling. The influence of lateral pH gradients along the membrane surface on lipid microdomain dynamics in giant unilamellar vesicles containing phosphatidylcholine, sphingomyelin, cholesterol, and the ganglioside GM1 was studied. Lo/Ld phase separation was generated by photosensitization. A lateral pH gradient was established along the external membrane surface by acid local microinjection. The gradient promotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, whereas Ld domains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo and Ld phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processes occurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup. LAURDAN fluorescence experiments on large unilamellar vesicles indicated that, with GM1, an increase in lipid packing occurs with decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ld phase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering and vesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. Such pH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients are known to be important, such as migrating cells and epithelial cells.

  8. Involvement of a cathepsin B-like cysteine proteinase in platelet aggregation induced by tumor cells and their shed membrane vesicles.

    Science.gov (United States)

    Cavanaugh, P G; Sloane, B F; Bajkowski, A S; Gasic, G J; Gasic, T B; Honn, K V

    1983-01-01

    Murine 15091A mammary adenocarcinoma cells and membrane vesicles spontaneously shed from these tumor cells in culture can induce aggregation of washed human platelets. A spectrum of proteinase inhibitors was tested for their ability to inhibit 15091A induced platelet aggregation. Of the inhibitors tested the most effective were those selective for cysteine proteinases. The effect of the spectrum of proteinase inhibitors on 15091A induced platelet aggregation was compared to the effect on cathepsin B-like cysteine proteinase activity in homogenates of 15091A tumor cells and their spontaneously shed vesicles. The results suggest that there is a correlation between activity of a cathepsin B-like proteinase in 15091A cells and vesicles and the ability of these cells and vesicles to induce aggregation of washed human platelets.

  9. Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria.

    Science.gov (United States)

    Lin, Hao-Peng; Zheng, De-Jin; Li, Yun-Pan; Wang, Na; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

    2016-06-01

    The objective of this study was to determine if plasma membrane vesicles (PMVs) could be exploited for efficient transfer of macro-biomolecules and mitochondria. PMVs were derived from mechanical extrusion, and made fusogenic (fPMVs) by incorporating the glycoprotein G of vesicular stomatitis virus (VSV-G). Confocal microscopy examination revealed that cytoplasmic proteins and mitochondria were enclosed in PMVs as evidenced by tracing with cytoplasmically localized and mitochondria-targeted EGFP, respectively. However, no fluorescence signal was detected in PMVs from cells whose nucleus was labeled with an EGFP-tagged histone H2B. Consistently, qRT-PCR measurement showed that mRNA, miRNA and mitochondrial DNA decreased slightly; while nuclear DNA was not measureable. Further, Western blot analysis revealed that cytoplasmic and membrane-bound proteins fell inconspicuously while nuclear proteins were barely detecsle. In addition, fPMVs carrying cytoplamic DsRed proteins transduced about ~40 % of recipient cells. The transfer of protein was further confirmed by using the inducible Cre/loxP system. Mitochondria transfer was found in about 20 % recipient cells after incubation with fPMVs for 5 h. To verify the functionalities of transferred mitochondria, mitochodria-deficient HeLa cells (Rho0) were generated and cultivated with fPMVs. Cell enumeration demonstrated that adding fPMVs into culture media stimulated Rho0 cell growth by 100 % as compared to the control. Lastly, MitoTracker and JC-1 staining showed that transferred mitochondria maintained normal shape and membrane potential in Rho0 cells. This study established a time-saving and efficient approach to delivering proteins and mitochondria by using fPMVs, which would be helpful for finding a cure to mitochondria-associated diseases. Graphical abstract Schematic of the delivery of macro-biomolecules and organelles by fPMVs. VSV-G-expressing cells were extruded through a 3 μm polycarbonate membrane filter to

  10. Xanthomonas campestris pv. vesicatoria Secretes Proteases and Xylanases via the Xps Type II Secretion System and Outer Membrane Vesicles

    Science.gov (United States)

    Solé, Magali; Scheibner, Felix; Hoffmeister, Anne-Katrin; Hartmann, Nadine; Hause, Gerd; Rother, Annekatrin; Jordan, Michael; Lautier, Martine; Arlat, Matthieu

    2015-01-01

    ABSTRACT Many plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system from Xanthomonas campestris pv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates from X. campestris pv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins from X. campestris pv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor of X. campestris pv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence and in planta growth of X. campestris pv. vesicatoria. When analyzed in the related pathogen X. campestris pv. campestris, several T2S substrates from X. campestris pv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, in X. campestris pv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed for X. campestris pv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes from X. campestris pv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles. IMPORTANCE The virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S

  11. S-Layered Aneurinibacillus and Bacillus spp. Are Susceptible to the Lytic Action of Pseudomonas aeruginosa Membrane Vesicles

    Science.gov (United States)

    Kadurugamuwa, J. L.; Mayer, A.; Messner, P.; Sára, M.; Sleytr, U. B.; Beveridge, T. J.

    1998-01-01

    When S-layered strains of Bacillus stearothermophilus and Aneurinibacillus thermoaerophilus, possessing S-layers of different lattice type and lattice constant as well as S-(glyco)protein chemistry, and isogenic S-layerless variants were subjected to membrane vesicles (MVs) from P. aeruginosa during plaque assays on plates or CFU measurements on cell suspensions, all bacterial types lysed. Electron microscopy of negative stains, thin sections, and immunogold-labelled MV preparations revealed that the vesicles adhered to all bacterial surfaces, broke open, and digested the underlying peptidoglycan-containing cell wall of all cell types. Reassembled S-layer did not appear to be affected by MVs, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the S-(glyco)proteins remained intact. meso-Diaminopimelic acid, as a peptidoglycan breakdown product, was found in all culture supernatants after MV attack. These results suggest that even though MVs are much larger than the channels which penetrate these proteinaceous arrays, S-layers on gram-positive bacteria do not form a defensive barrier against the lytic action of MVs. The primary mode of attack is by the liberation from the MVs of a peptidoglycan hydrolase, which penetrates through the S-layer to digest the underlying peptidoglycan-containing cell wall. The S-layer is not affected by MV protease. PMID:9573179

  12. Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

    DEFF Research Database (Denmark)

    Pszon-Bartosz, Kamila Justyna; Hansen, Jesper S.; Stibius, Karin B.

    2011-01-01

    establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein...... in a bilayer array with a total membrane area of 2mm2 within 20min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications....

  13. Preparation of large monodisperse vesicles.

    Directory of Open Access Journals (Sweden)

    Ting F Zhu

    Full Text Available Preparation of monodisperse vesicles is important both for research purposes and for practical applications. While the extrusion of vesicles through small pores (approximately 100 nm in diameter results in relatively uniform populations of vesicles, extrusion to larger sizes results in very heterogeneous populations of vesicles. Here we report a simple method for preparing large monodisperse multilamellar vesicles through a combination of extrusion and large-pore dialysis. For example, extrusion of polydisperse vesicles through 5-microm-diameter pores eliminates vesicles larger than 5 microm in diameter. Dialysis of extruded vesicles against 3-microm-pore-size polycarbonate membranes eliminates vesicles smaller than 3 microm in diameter, leaving behind a population of monodisperse vesicles with a mean diameter of approximately 4 microm. The simplicity of this method makes it an effective tool for laboratory vesicle preparation with potential applications in preparing large monodisperse liposomes for drug delivery.

  14. Colitogenic Bacteroides thetaiotaomicron Antigens Access Host Immune Cells in a Sulfatase-Dependent Manner via Outer Membrane Vesicles.

    Science.gov (United States)

    Hickey, Christina A; Kuhn, Kristine A; Donermeyer, David L; Porter, Nathan T; Jin, Chunsheng; Cameron, Elizabeth A; Jung, Haerin; Kaiko, Gerard E; Wegorzewska, Marta; Malvin, Nicole P; Glowacki, Robert W P; Hansson, Gunnar C; Allen, Paul M; Martens, Eric C; Stappenbeck, Thaddeus S

    2015-05-13

    Microbes interact with the host immune system via several potential mechanisms. One essential step for each mechanism is the method by which intestinal microbes or their antigens access specific host immune cells. Using genetically susceptible mice (dnKO) that develop spontaneous, fulminant colitis, triggered by Bacteroides thetaiotaomicron (B. theta), we investigated the mechanism of intestinal microbial access under conditions that stimulate colonic inflammation. B. theta antigens localized to host immune cells through outer membrane vesicles (OMVs) that harbor bacterial sulfatase activity. We deleted the anaerobic sulfatase maturating enzyme (anSME) from B. theta, which is required for post-translational activation of all B. theta sulfatase enzymes. This bacterial mutant strain did not stimulate colitis in dnKO mice. Lastly, access of B. theta OMVs to host immune cells was sulfatase dependent. These data demonstrate that bacterial OMVs and associated enzymes promote inflammatory immune stimulation in genetically susceptible hosts. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Interaction of n-octyl β,D-glucopyranoside with giant magnetic-fluid-loaded phosphatidylcholine vesicles: direct visualization of membrane curvature fluctuations as a function of surfactant partitioning between water and lipid bilayer.

    Science.gov (United States)

    Ménager, Christine; Guemghar, Dihya; Cabuil, Valérie; Lesieur, Sylviane

    2010-10-05

    The present study deals with the morphological modifications of giant dioleoyl phosphatidylcholine vesicles (DOPC GUVs) induced by the nonionic surfactant n-octyl β,D-glucopyranoside at sublytic levels, i.e., in the first steps of the vesicle-to-micelle transition process, when surfactant inserts into the vesicle bilayer without disruption. Experimental conditions were perfected to exactly control the surfactant bilayer composition of the vesicles, in line with former work focused on the mechanical properties of the membrane of magnetic-fluid-loaded DOPC GUVs submitted to a magnetic field. The purpose here was to systematically examine, in the absence of any external mechanical constraint, the dynamics of giant vesicle shape and membrane deformations as a function of surfactant partitioning between the aqueous phase and the lipid membrane, beforehand established by turbidity measurements from small unilamellar vesicles.

  16. Effects of fatty acid sucrose esters on ceftibuten transport by rat intestinal brush-border membrane vesicles.

    Science.gov (United States)

    Koga, K; Murakami, M; Kawashima, S

    1998-07-01

    The effects of fatty acid sucrose esters on membrane lipid dynamics and ceftibuten transport by rat intestinal brush-border membrane vesicles (BBMV) were examined to clarify the differences in the action of mono- and poly-acyl sucrose esters on the drug transport. Fatty acid sucrose mono-acyl ester (SS) inhibited ceftibuten transport by BBMV similar to the action of polyoxyethylene sorbitans (Tweens), while fatty acid sucrose polyacyl ester mixtures (F-160 and F-140) did not affect the drug transport by BBMV. SS but not F-160 and F-140 caused an increase in the anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH)- and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene iodide (TMA-DPH)-labeled BBMV in a concentration-dependent manner. Thus, the uptake of ceftibuten by BBMV was strongly correlated with the lipid fluidity of BBMV, in the outer layer and in the inner hydrophobic regions; however, there was no strong correlation between the membrane lipid fluidity and the drug uptake by BBMV. The micelle size and the size distribution of F-160 and F-140 were larger and more widely dispersed, respectively, compared to those of SS and Tweens. These results suggest that the effects of fatty acid sucrose esters on ceftibuten transport by BBMV are related to the dispersion parameter of these pharmaceutical adjuvants.

  17. Host cell interactions of outer membrane vesicle-associated virulence factors of Enterohemorrhagic Escherichia coli O157: intracellular delivery, trafficking and mechanisms of cell injury

    Science.gov (United States)

    Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, confocal laser...

  18. Health economics of a hexavalent meningococcal outer-membrane vesicle vaccine in children : potential impact of introduction in the Dutch vaccination program

    NARCIS (Netherlands)

    Bos, JM; Rumke, HC; Welte, R; Postma, MJ; Jager, JC

    2001-01-01

    The cost-effectiveness of universal vaccination of infants with a new hexavalent meningococcal B outer-membrane vesicle vaccine is projected for The Netherlands by applying decision analysis. The societal perspective is taken and direct and productivity costs (friction costs method) are considered.

  19. Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates

    NARCIS (Netherlands)

    Teuling, Eva; Ahmed, Suaad; Haasdijk, Elize; Demmers, Jeroen; Steinmetz, Michel O; Akhmanova, Anna; Jaarsma, Dick; Hoogenraad, Casper C

    2007-01-01

    The vesicle-associated membrane protein-associated proteins (VAPs) VAPA and VAPB interact with lipid-binding proteins carrying a short motif containing two phenylalanines in an acidic tract (FFAT motif) and targets them to the cytosolic surface of the endoplasmic reticulum (ER). A genetic mutation

  20. Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles

    DEFF Research Database (Denmark)

    Hummel, R; Nørgaard, P; Andreasen, P H

    1992-01-01

    of the Golgi apparatus and transport vesicles pointing to a role of TF-ag in membrane trafficking. Transcription of the TF-ag gene, as determined by run-on analyses, was only detectable in growing cells, and following transfer to starvation condition pre-existing TF-ag mRNA was rapidly degraded. The abundance...

  1. Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli.

    Science.gov (United States)

    Sedgwick, E G; Bragg, P D

    1987-12-17

    The fluorescence of the lipophilic probe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, D-lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.

  2. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules.

    Science.gov (United States)

    Heijnen, H F; Schiel, A E; Fijnheer, R; Geuze, H J; Sixma, J J

    1999-12-01

    Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and alpha-granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or alpha-thrombin: microvesicles of 100 nm to 1 microm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and alpha-granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains alpha(IIb)-beta(3) and beta(1), GPIbalpha, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63(+) internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

  3. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane

    DEFF Research Database (Denmark)

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels Henrik Helweg

    2013-01-01

    granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in...... subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes....

  4. Lack of Outer Membrane Protein A Enhances the Release of Outer Membrane Vesicles and Survival of Vibrio cholerae and Suppresses Viability of Acanthamoeba castellanii

    Directory of Open Access Journals (Sweden)

    Soni Priya Valeru

    2014-01-01

    Full Text Available Vibrio cholerae, the causative agent of the diarrhoeal disease cholera, survives in aquatic environments. The bacterium has developed a survival strategy to grow and survive inside Acanthamoeba castellanii. It has been shown that V. cholerae expresses outer membrane proteins as virulence factors playing a role in the adherence to interacted host cells. This study examined the role of outer membrane protein A (OmpA and outer membrane vesicles (OMVs in survival of V. cholerae alone and during its interaction with A. castellanii. The results showed that an OmpA mutant of V. cholerae survived longer than wild-type V. cholerae when cultivated alone. Cocultivation with A. castellanii enhanced the survival of both bacterial strains and OmpA protein exhibited no effect on attachment, engulfment, and survival inside the amoebae. However, cocultivation of the OmpA mutant of V. cholerae decreased the viability of A. castellanii and this bacterial strain released more OMVs than wild-type V. cholerae. Surprisingly, treatment of amoeba cells with OMVs isolated from the OmpA mutant significantly decreased viable counts of the amoeba cells. In conclusion, the results might highlight a regulating rule for OmpA in survival of V. cholerae and OMVs as a potent virulence factor for this bacterium towards eukaryotes in the environment.

  5. Visualization of lipid domains of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone

    DEFF Research Database (Denmark)

    Klymchenko, Andrey; Oncul, Sule; Didier, Pascal

    2009-01-01

    vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization...... of the photoselection with the Ld phase indicated no predominant orientation of the probe in the Ld phase. Comparison of the present results with those reported previously for F2N12S in living cells suggests a high content of the Lo phase in the outer leaflet of the cell plasma membranes. Taking into account the high...

  6. Overexpression of MicA induces production of OmpC-enriched outer membrane vesicles that protect against Salmonella challenge.

    Science.gov (United States)

    Choi, Hyun-Il; Kim, Moonjeong; Jeon, Jinseong; Han, Jin Kwan; Kim, Kwang-Sun

    2017-08-26

    Outer membrane vesicles (OMVs) derived from bacteria are promising candidates for subunit vaccines. Stresses that modulate the composition of outer membrane proteins (OMPs) are important for OMV synthesis. Small RNAs (sRNAs) expressed in response to stress regulate OMPs, although the mechanism underlying sRNA-mediated OMV biogenesis and its utility for developing vaccine platforms remains to be elucidated. Here, we characterized the role of a sRNA, MicA, which regulates OmpA, a major OMP involved in both production of OMVs and reactive immunity against Salmonella challenge. A Salmonella strain overexpressing MicA generated more OMVs than a control strain. In addition, OmpC was the major component of MicA-derived OMV proteins. MicA-derived OMVs induced Th1- and Th17-type immune responses in vitro and reduced Salmonella-mediated lethality in a mouse model. Thus, OmpA-regulatory sRNA-derived OMVs may facilitate production of Salmonella-protective vaccines. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins

    Directory of Open Access Journals (Sweden)

    Cristian Oliver

    2017-09-01

    Full Text Available Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

  8. Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection

    Science.gov (United States)

    Huang, Weiwei; Wang, Shijie; Yao, Yufeng; Xia, Ye; Yang, Xu; Li, Kui; Sun, Pengyan; Liu, Cunbao; Sun, Wenjia; Bai, Hongmei; Chu, Xiaojie; Li, Yang; Ma, Yanbing

    2016-11-01

    Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

  9. A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus

    Directory of Open Access Journals (Sweden)

    Ulrike Resch

    2016-11-01

    Full Text Available Export of macromolecules via extracellular membrane-derived vesicles (MVs plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS, the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to “anchorless surface proteins.” Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response.

  10. Neutron scattering to study membrane systems: from lipid vesicles to living cells.

    Energy Technology Data Exchange (ETDEWEB)

    Myles, Dean A A [ORNL; Standaert, Robert F. [ORNL; Stanley, Christopher B. [ORNL; Cheng, Xiaolin [ORNL; Elkins, James G. [ORNL; Katsaras, John [ORNL; Qian, Shuo [ORNL; Nickels, Jonathan D. [ORNL; Chatterjee, Sneha [ORNL

    2017-03-01

    The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent work using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.

  11. The cortical acto-myosin network: from diffusion barrier to functional gateway in the transport of neurosecretory vesicles to the plasma membrane

    Directory of Open Access Journals (Sweden)

    Andreas ePapadopulos

    2013-10-01

    Full Text Available Dysregulation of regulated exocytosis is linked to an array of pathological conditions, including neurodegenerative disorders, asthma and diabetes. Understanding the molecular mechanisms underpinning neuroexocytosis including the processes that allow neurosecretory vesicles to access and fuse with the plasma membrane and to recycle post-fusion, is therefore critical to the design of future therapeutic drugs that will efficiently tackle these diseases. Despite considerable efforts to determine the principles of vesicular fusion, the mechanisms controlling the approach of vesicles to the plasma membrane in order to undergo tethering, docking, priming, and fusion remain poorly understood. All these steps involve the cortical actin network, a dense mesh of actin filaments localized beneath the plasma membrane. Recent work overturned the long-held belief that the cortical actin network only plays a passive constraining role in neuroexocytosis functioning as a physical barrier that partly breaks down upon entry of Ca2+ to allow secretory vesicles to reach the plasma membrane. A multitude of new roles for the cortical actin network in regulated exocytosis have now emerged and point to highly dynamic novel functions of key myosin molecular motors. Myosins are not only believed to help bring about dynamic changes in the actin cytoskeleton, tethering and guiding vesicles to their fusion sites, but they also regulate the size and duration of the fusion pore, thereby directly contributing to the release of neurotransmitters and hormones.Here we discuss the functions of the cortical actin network, myosins and their effectors in controlling the processes that lead to tethering, directed transport, docking, and fusion of exocytotic vesicles in regulated exocytosis.

  12. Vesicle-associated Membrane Protein-2 (VAMP2) Mediates cAMP-stimulated Renin Release in Mouse Juxtaglomerular Cells*

    Science.gov (United States)

    Mendez, Mariela; Gross, Kenneth W.; Glenn, Sean T.; Garvin, Jeffrey L.; Carretero, Oscar A.

    2011-01-01

    Renin is essential for blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells, located in the pole of the renal afferent arterioles. The second messenger cAMP stimulates renin release. However, it is unclear whether fusion and exocytosis of renin-containing granules is involved. In addition, the role of the fusion proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins), in renin release from JG cells has not been studied. The vesicle SNARE proteins VAMP2 (vesicle associated membrane protein 2) and VAMP3 mediate cAMP-stimulated exocytosis in other endocrine cells. Thus, we hypothesized that VAMP2 and/or -3 mediate cAMP-stimulated renin release from JG cells. By fluorescence-activated cell sorting, we isolated JG cells expressing green fluorescent protein and compared the relative abundance of VAMP2/3 in JG cells versus total mouse kidney mRNA by quantitative PCR. We found that VAMP2 and VAMP3 mRNA are expressed and enriched in JG cells. Confocal imaging of primary cultures of JG cells showed that VAMP2 (but not VAMP3) co-localized with renin-containing granules. Cleavage of VAMP2 and VAMP3 with tetanus toxin blocked cAMP-stimulated renin release from JG cells by ∼50% and impaired cAMP-stimulated exocytosis by ∼50%, as monitored with FM1–43. Then we specifically knocked down VAMP2 or VAMP3 by adenoviral-mediated delivery of short hairpin silencing RNA. We found that silencing VAMP2 blocked cAMP-induced renin release by ∼50%. In contrast, silencing VAMP3 had no effect on basal or cAMP-stimulated renin release. We conclude that VAMP2 and VAMP3 are expressed in JG cells, but only VAMP2 is targeted to renin-containing granules and mediates the stimulatory effect of cAMP on renin exocytosis. PMID:21708949

  13. Free-flow electrophoresis of plasma membrane vesicles enriched by two-phase partitioning enhances the quality of the proteome from Arabidopsis seedlings

    DEFF Research Database (Denmark)

    de Michele, Roberto; McFarlane, Heather E; Parsons, Harriet Tempé

    2016-01-01

    The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane...... using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane...... isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including...

  14. Membrane-coating lattice scaffolds in the nuclear pore and vesicle coats

    Science.gov (United States)

    Leksa, Nina C

    2010-01-01

    The nuclear pore complex (NPC) regulates all traffic between the cytoplasm and the nucleus. It is a large protein assembly composed of multiple copies of ∼30 nucleoporins (nups). Structural studies of the NPC have been limited by its considerable size and complexity. Progress toward understanding the structure of this nanomachine has benefited from its modular nature, which allows for this 40–60 MDa assembly to be broken down into subcomplexes that can be studied individually. While recent work by both crystallographers and electron microscopists has greatly enhanced our model of the NPC, the resolution gap between crystal and EM structures remains too large to confidently place individual proteins within the context of the fully assembled NPC. In an effort to arrive at a veritable model of the NPC, we solved the structure of several scaffold nups and defined the ancestral coatomer element (ACE1) common to a set of nucleoporins and COPII vesicle coat proteins. Subsequently, we proposed a lattice-like model of the NPC, analogous to the COPII lattice, in which ACE1 proteins form the edge elements and β-propellers form the vertex elements. Here, we review our recent studies, speculate on how interactions between subcomplexes of the NPC are mediated, and outline the steps and challenges that lay ahead on the path to understanding this enormous assembly in molecular detail. PMID:21327078

  15. Novel elastic membrane vesicles (EMVs) and ethosomes-mediated effective topical delivery of aceclofenac: a new therapeutic approach for pain and inflammation.

    Science.gov (United States)

    Sharma, Gajanand; Goyal, Himanshu; Thakur, Kanika; Raza, Kaisar; Katare, O P

    2016-10-01

    Aceclofenac (ACE) is a systematically designed drug, developed to circumvent the concerns associated with diclofenac. But ACE is also associated with non-steroidal anti-inflammatory drug (NSAIDs)-tagged side effects, although of decreased amplitude. The present study aims to develop phospholipid-based vesicles (elastic membrane vesicles; EMV and ethosomes) loaded with ACE and explore their potential in topical delivery. Elastic membrane vesicles (EMVs) were prepared by thin-film hydration method and ethosomes by cold method. The composition of both the vesicular systems was selected on the basis of vesicle density and drug entrapment. The developed systems were characterized for micromeritics, surface charge, drug entrapment, and morphology. Ex vivo permeation and retention studies on Laca mice skin were performed. In vivo pharmacodynamic evaluation was performed by tail-flick method and carrageenan-induced rat paw-edema model. During stability studies, percent drug leakage was studied. The selected ratios of phospholpid:drug:stearylamine for EMVs and ethosomes were 8:1:1 and 3:1:1, respectively. The ethosomes were found to offer more vesicle density, drug loading, and deformability index as compared with that of EMVs. The drug permeation and the retention offered by both the vesicular systems were superior to that of the conventional cream; however, performance of ethosomes superseded that of EMVs. The phospholipid-based vesicles were found to be well tolerated on mice skin. Although, the in vivo performance of ethosomes was found to be better than that of EMVs in both the studied models. The phospholipid-based vesicular systems, especially, ethosomes can be a promising tool to enhance the delivery and safety of ACE by topical route.

  16. Immobilized membrane vesicle or proteoliposome affinity chromatography. Frontal analysis of interactions of cytochalasin B and D-glucose with the human red cell glucose transporter.

    Science.gov (United States)

    Brekkan, E; Lundqvist, A; Lundahl, P

    1996-09-17

    Human red cell membrane vesicles stripped of peripheral proteins and proteoliposomes with reconstituted red cell glucose transporter (Glut1) were sterically immobilized in gel beads by freezethawing. The specific interactions between the transport inhibitor cytochalasin B (CB), D-glucose, and Glut1 were analyzed by quantitative frontal affinity chromatography. The dissociation constants, Kd(CB), for the interaction between CB and Glut1 in vesicles and proteoliposomes were similar, the average value being 92 +/- 5 nM at an ionic strength I of 0.05. Kd(CB) for Glut1 in vesicles decreased with increasing ionic strength to become 46 nM at I = 0.5. The affinity of glucose was significantly higher for Glut1 in vesicles (Kd = 24 +/- 2 mM) than for reconstituted Glut1 (Kd = 37 +/- 2 mM). The frontal analysis allowed determination of the amount of CB binding sites, which was found to be 0.33 +/- 0.06 mol per mole of Glut1 monomer (Mr = 54 000). The CB binding capacity of Glut1 in the vesicles and the proteoliposomes was stable in the presence of dithioerythritol over periods of several weeks at room temperature.

  17. Depolarization by K*O+ and glutamate activates different neurotransmitter release mechanisms in gabaergic neurons: vesicular versus non-vesicular release of gaba

    DEFF Research Database (Denmark)

    Belhage, Bo; Hansen, G.H.; Schousboe, Arne

    1993-01-01

    Neurotransmitter release, gaba release, membrane transporter, vesicles, intracellular CA*OH, neuron cultures......Neurotransmitter release, gaba release, membrane transporter, vesicles, intracellular CA*OH, neuron cultures...

  18. Synaptic Vesicle Endocytosis

    Science.gov (United States)

    Saheki, Yasunori; De Camilli, Pietro

    2012-01-01

    Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property relies on a highly efficient local endocytic recycling of synaptic vesicle membranes, which can be reused for hundreds, possibly thousands, of exo-endocytic cycles. Morphological, physiological, molecular, and genetic studies over the last four decades have provided insight into the membrane traffic reactions that govern this recycling and its regulation. These studies have shown that synaptic vesicle endocytosis capitalizes on fundamental and general endocytic mechanisms but also involves neuron-specific adaptations of such mechanisms. Thus, investigations of these processes have advanced not only the field of synaptic transmission but also, more generally, the field of endocytosis. This article summarizes current information on synaptic vesicle endocytosis with an emphasis on the underlying molecular mechanisms and with a special focus on clathrin-mediated endocytosis, the predominant pathway of synaptic vesicle protein internalization. PMID:22763746

  19. Effect of Porphyromonas gingivalis outer membrane vesicles on gingipain-mediated detachment of cultured oral epithelial cells and immune responses.

    Science.gov (United States)

    Nakao, Ryoma; Takashiba, Shogo; Kosono, Saori; Yoshida, Minoru; Watanabe, Haruo; Ohnishi, Makoto; Senpuku, Hidenobu

    2014-01-01

    Porphyromonas gingivalis is a major etiological agent of periodontal diseases and the outer membrane vesicles (OMVs) contain virulence factors such as LPS and gingipains. In this study, we investigated the potential role of the OMVs in host immune response and tissue destruction during P. gingivalis infection. Firstly, we found that sera from periodontitis patients had significantly stronger reactivity against an OMV-producing wild type strain than the isogenic OMV-depleted strain. OMVs were found to be highly antigenic, as absorption of patient sera with OMVs greatly reduced reactivity with whole cells of P. gingivalis. LC-MS/MS analysis of OMVs revealed multiple forms of gingipains and several gingipain-related proteins. Western blots of OMVs using patient sera revealed a conserved immunoreactive antigen profile resembling the profile of OMV antigens that were recognized by gingipain antiserum, suggesting a potential role of OMV-associated gingipains in triggering antibody-mediated immune responses to P. gingivalis infection. When OMVs were added to a monolayer of an oral squamous epithelial cell line, OMVs caused cell detachment, which was inhibited by preincubating OMVs with anti-gingipain antiserum. These data suggest that gingipain-laden OMVs may contribute to tissue destruction in periodontal diseases by serving as a vehicle for the antigens and active proteases. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  20. The inhibitory effects of cephalosporin and dipeptide on ceftibuten uptake by human and rat intestinal brush-border membrane vesicles.

    Science.gov (United States)

    Sugawara, M; Toda, T; Kobayashi, M; Iseki, K; Miyazaki, K; Shiroto, H; Uchino, J; Kondo, Y

    1994-08-01

    The types of inhibitory effects caused by compound V (an analogue of ceftibuten) and alanylproline (dipeptide) on the uptake of ceftibuten by brush-border membrane vesicles (BBMV) prepared from human and rat small intestine were analysed. In the presence of an inward H(+)-gradient, the initial uptake rate of ceftibuten by both human and rat intestinal BBMV was concentration-dependent with apparent Km and Vmax values of 0.35 mM and 2.052 nmol (mg protein)-1 min-1 for human BBMV, and 0.50 mM and 3.056 nmol (mg protein)-1 min-1 for rat BBMV, respectively. For both human and rat BBMV, kinetic analysis by Dixon and Lineweaver-Burk plots demonstrated that the uptake of ceftibuten was competitively inhibited by compound V, whereas inhibition by alanylproline was noncompetitive or partially competitive. These results suggest that there is a stereospecific transport system which is common to ceftibuten and compound V, and that this system is not identical to the carrier system for the dipeptide, alanylproline.

  1. Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens.

    Science.gov (United States)

    Volgers, Charlotte; Benedikter, Birke J; Grauls, Gert E; Hellebrand, Pauline H M; Savelkoul, Paul H M; Stassen, Frank R M

    2017-05-01

    Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

    Science.gov (United States)

    Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.

  3. Exploring the midgut transcriptome and brush border membrane vesicle proteome of the rice stem borer, Chilo suppressalis (Walker).

    Science.gov (United States)

    Ma, Weihua; Zhang, Zan; Peng, Chuanhua; Wang, Xiaoping; Li, Fei; Lin, Yongjun

    2012-01-01

    The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt) toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV). Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac.

  4. Exploring the midgut transcriptome and brush border membrane vesicle proteome of the rice stem borer, Chilo suppressalis (Walker.

    Directory of Open Access Journals (Sweden)

    Weihua Ma

    Full Text Available The rice stem borer, Chilo suppressalis (Walker (Lepidoptera: Pyralidae, is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV. Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac.

  5. Recombinant outer membrane vesicles carrying Chlamydia muridarum HtrA induce antibodies that neutralize chlamydial infection in vitro

    Science.gov (United States)

    Bartolini, Erika; Ianni, Elvira; Frigimelica, Elisabetta; Petracca, Roberto; Galli, Giuliano; Berlanda Scorza, Francesco; Norais, Nathalie; Laera, Donatello; Giusti, Fabiola; Pierleoni, Andrea; Donati, Manuela; Cevenini, Roberto; Finco, Oretta; Grandi, Guido; Grifantini, Renata

    2013-01-01

    Background Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development. Methods Chlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro. Results CM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection. Conclusions When expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform. PMID:24009891

  6. Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

    Science.gov (United States)

    Kunsmann, Lisa; Greune, Lilo; Bauwens, Andreas; Zhang, Wenlan; Kuczius, Thorsten; Kim, Kwang Sik; Mellmann, Alexander; Schmidt, M. Alexander; Karch, Helge

    2013-01-01

    Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in

  7. Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

    Science.gov (United States)

    Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

    2016-07-01

    At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as

  8. Kemp elimination in membrane mimetic reaction media : Probing catalytic properties of catanionic vesicles formed from double-tailed amphiphiles

    NARCIS (Netherlands)

    Klijn, J.E.; Engberts, J.B.F.N.

    2003-01-01

    The rate-determining deprotonation of 5-nitrobenzisoxazole (Kemp elimination) by hydroxide is efficiently catalyzed by vesicles formed from dimethyldioctadecylammonium chloride (C18C18+). Gradual addition of sodium didecyl phosphate (C10C10-) leads to the formation of catanionic vesicles, which were

  9. A novel approach for purification and selective capture of membrane vesicles of the periodontopathic bacterium, Porphyromonas gingivalis: membrane vesicles bind to magnetic beads coated with epoxy groups in a noncovalent, species-specific manner.

    Directory of Open Access Journals (Sweden)

    Ryoma Nakao

    Full Text Available Membrane vesicles (MVs of Porphyromonas gingivalis are regarded as an offensive weapon of the bacterium, leading to tissue deterioration in periodontal disease. Therefore, isolation of highly purified MVs is indispensable to better understand the pathophysiological role of MVs in the progression of periodontitis. MVs are generally isolated by a conventional method based on ultracentrifugation of the bacterial culture supernatant. However, the resulting MVs are often contaminated with co-precipitating bacterial appendages sheared from the live bacteria. Here, we report an intriguing property of P. gingivalis MVs--their ability to bind superparamagnetic beads coated with epoxy groups (SB-Epoxy. Analysis of fractions collected during the purification revealed that all MVs of five tested P. gingivalis stains bound to SB-Epoxy. In contrast, free fimbriae in the crude MV preparation did not bind to the SB-Epoxy. The SB-Epoxy-bound MVs were easily dissociated from the SB-Epoxy using a mild denaturation buffer. These results suggest that the surface chemistry conferred by epoxy on the beads is responsible for the binding, which is mediated by noncovalent bonds. Both the structural integrity and purity of the isolated MVs were confirmed by electron microscopy. The isolated MVs also caused cell detachment from culture dishes at a physiologically relevant concentration. Assays of competitive binding between the SB-Epoxy and mixtures of MVs from five bacterial species demonstrated that only P. gingivalis MVs could be selectively eliminated from the mixtures. We suggest that this novel approach enables efficient purification and selective elimination of P. gingivalis MVs.

  10. A novel approach for purification and selective capture of membrane vesicles of the periodontopathic bacterium, Porphyromonas gingivalis: membrane vesicles bind to magnetic beads coated with epoxy groups in a noncovalent, species-specific manner.

    Science.gov (United States)

    Nakao, Ryoma; Kikushima, Kenji; Higuchi, Hideo; Obana, Nozomu; Nomura, Nobuhiko; Bai, Dongying; Ohnishi, Makoto; Senpuku, Hidenobu

    2014-01-01

    Membrane vesicles (MVs) of Porphyromonas gingivalis are regarded as an offensive weapon of the bacterium, leading to tissue deterioration in periodontal disease. Therefore, isolation of highly purified MVs is indispensable to better understand the pathophysiological role of MVs in the progression of periodontitis. MVs are generally isolated by a conventional method based on ultracentrifugation of the bacterial culture supernatant. However, the resulting MVs are often contaminated with co-precipitating bacterial appendages sheared from the live bacteria. Here, we report an intriguing property of P. gingivalis MVs--their ability to bind superparamagnetic beads coated with epoxy groups (SB-Epoxy). Analysis of fractions collected during the purification revealed that all MVs of five tested P. gingivalis stains bound to SB-Epoxy. In contrast, free fimbriae in the crude MV preparation did not bind to the SB-Epoxy. The SB-Epoxy-bound MVs were easily dissociated from the SB-Epoxy using a mild denaturation buffer. These results suggest that the surface chemistry conferred by epoxy on the beads is responsible for the binding, which is mediated by noncovalent bonds. Both the structural integrity and purity of the isolated MVs were confirmed by electron microscopy. The isolated MVs also caused cell detachment from culture dishes at a physiologically relevant concentration. Assays of competitive binding between the SB-Epoxy and mixtures of MVs from five bacterial species demonstrated that only P. gingivalis MVs could be selectively eliminated from the mixtures. We suggest that this novel approach enables efficient purification and selective elimination of P. gingivalis MVs.

  11. In Silico Analysis of the Small Molecule Content of Outer Membrane Vesicles Produced by Bacteroides thetaiotaomicron Indicates an Extensive Metabolic Link between Microbe and Host

    Directory of Open Access Journals (Sweden)

    William A. Bryant

    2017-12-01

    Full Text Available The interactions between the gut microbiota and its host are of central importance to the health of the host. Outer membrane vesicles (OMVs are produced ubiquitously by Gram-negative bacteria including the gut commensal Bacteroides thetaiotaomicron. These vesicles can interact with the host in various ways but until now their complement of small molecules has not been investigated in this context. Using an untargeted high-coverage metabolomic approach we have measured the small molecule content of these vesicles in contrasting in vitro conditions to establish what role these metabolites could perform when packed into these vesicles. B. thetaiotaomicron packs OMVs with a highly conserved core set of small molecules which are strikingly enriched with mouse-digestible metabolites and with metabolites previously shown to be associated with colonization of the murine GIT. By use of an expanded genome-scale metabolic model of B. thetaiotaomicron and a potential host (the mouse we have established many possible metabolic pathways between the two organisms that were previously unknown, and have found several putative novel metabolic functions for mouse that are supported by gene annotations, but that do not currently appear in existing mouse metabolic networks. The lipidome of these OMVs bears no relation to the mouse lipidome, so the purpose of this particular composition of lipids remains unclear. We conclude from this analysis that through intimate symbiotic evolution OMVs produced by B. thetaiotaomicron are likely to have been adopted as a conduit for small molecules bound for the mammalian host in vivo.

  12. Characterization of Innate Immune Responses of Human Endothelial Cells Induced by Porphyromonas gingivalis and Their Derived Outer Membrane Vesicles.

    Science.gov (United States)

    Ho, Meng-Hsuan; Guo, Zhong-Mao; Chunga, Julio; Goodwin, J Shawn; Xie, Hua

    2016-01-01

    Atherosclerosis, a chronic inflammatory disease of the blood vessels, is one of the most common causes of morbidity and mortality world-wide. Involvement of Porphyromonas gingivalis in atherosclerosis is supported by observations from epidemiological, clinical, immunological, and molecular studies. Previously we reported that P. gingivalis vesicles have a much higher invasive efficiency than their originating cells. Here, we further compare the role of P. gingivalis cells and their vesicles in expression of chemoattractant proteins including CXCL1, CXCL2, and CXCL8, and adhesive molecules such as E-selectin in human umbilical vein endothelial cells (HUVECs). Both P. gingivalis 33277 cells and vesicles were able to up-regulate expression of these molecules, while the vesicles acted as more potent inducers of the inflammatory response associated with the development of atherosclerosis, consequently resulting in significant monocyte adhesion to a monolayer of HUVECs. Interestingly, we found that elevated expression of CXCL8 and E-selectin in endothelial cells induced by P. gingivalis correlated with the invasive ability of P. gingivalis cells and vesicles. Non-invasive bacterial cells and vesicles had no effect on expression of these genes. This study highlights the potential risk of P. gingivalis cells and vesicles in initiation of atherosclerosis and provides a potential target for the development of novel therapeutics against bacteria-associated atherosclerosis.

  13. Metabolic Remodeling, Inflammasome Activation, and Pyroptosis in Macrophages Stimulated by Porphyromonas gingivalis and Its Outer Membrane Vesicles

    Science.gov (United States)

    Fleetwood, Andrew J.; Lee, Man K.S.; Singleton, William; Achuthan, Adrian; Lee, Ming-Chin; O'Brien-Simpson, Neil M.; Cook, Andrew D.; Murphy, Andrew J.; Dashper, Stuart G.; Reynolds, Eric C.; Hamilton, John A.

    2017-01-01

    Porphyromonas gingivalis is one of the bacterial species most closely associated with periodontitis and can shed large numbers of outer membrane vesicles (OMVs), which are increasingly thought to play a significant role in bacterial virulence and pathogenicity. Macrophages are amongst the first immune cells to respond to bacteria and their products, so we sought to directly compare the response of macrophages to P. gingivalis or its purified OMVs. Macrophages stimulated with OMVs produced large amounts of TNFα, IL-12p70, IL-6, IL-10, IFNβ, and nitric oxide compared to cells infected with P. gingivalis, which produced very low levels of these mediators. Both P. gingivalis and OMVs induced a shift in macrophage metabolism from oxidative phosphorylation (OXPHOS) to glycolysis, which was supported by enhanced lactate release, decreased mitochondrial oxygen consumption with reduced spare respiratory capacity, as well as increased mitochondrial reactive oxygen species (ROS) production. Corresponding to this metabolic shift, gene expression analysis of macrophages infected with P. gingivalis or stimulated with OMVs revealed a broad transcriptional upregulation of genes critical to glycolysis and a downregulation of genes associated with the TCA cycle. Upon examination of inflammasome signaling and pyroptosis it was found that P. gingivalis did not activate the inflammasome in macrophages as the mature forms of caspase-1, IL-1β, and IL-18 were not detected and there was no extracellular release of lactate dehydrogenase (LDH) or 7-AAD staining. In comparison, macrophages stimulated with OMVs potently activated caspase-1, produced large amounts of IL-1β, IL-18, released LDH, and were positive for 7-AAD indicative of pyroptotic cell death. These data directly quantitate the distinct effects of P. gingivalis and its OMVs on macrophage inflammatory phenotype, mitochondrial function, inflammasome activation, and pyroptotic cell death that may have potential implications for

  14. The t-SNAREs syntaxin4 and SNAP23 but not v-SNARE VAMP2 are indispensable to tether GLUT4 vesicles at the plasma membrane in adipocyte

    Energy Technology Data Exchange (ETDEWEB)

    Kawaguchi, Takayuki [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine (Japan); Tamori, Yoshikazu, E-mail: tamori@med.kobe-u.ac.jp [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine (Japan); Kanda, Hajime; Yoshikawa, Mari; Tateya, Sanshiro; Nishino, Naonobu [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine (Japan); Kasuga, Masato [Research Institute, International Medical Center of Japan (Japan)

    2010-01-15

    SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.

  15. Lamin B receptor plays a role in stimulating nuclear envelope production and targeting membrane vesicles to chromatin during nuclear envelope assembly through direct interaction with importin beta.

    Science.gov (United States)

    Ma, Yan; Cai, Shang; Lv, Quanlong; Jiang, Qing; Zhang, Quan; Sodmergen; Zhai, Zhonghe; Zhang, Chuanmao

    2007-02-01

    Lamin B receptor (LBR), a chromatin and lamin binding protein in the inner nuclear membrane, has been proposed to play a vital role in nuclear envelope (NE) assembly. But the specific role for LBR in NE assembly remains unknown. In the present study, we show that overexpression of LBR causes membrane overproduction, inducing NE invagination and membrane stack formation, and that these processes require the transmembrane domain of LBR. Biochemical analysis shows that the N-terminal domain of LBR directly interacts with importin beta in a Ran sensitive and importin alpha independent manner. Using an in vitro NE assembly assay, we also demonstrate that blocking full length LBR binding sites on importin beta, by the addition of the LBR N-terminal domain inhibits the recruitment of LBR-containing vesicles to importin beta- or Ran-coated beads to form NE structure. Our results suggest that LBR is recruited to chromatin through direct interaction with importin beta to contribute to the fusion of membrane vesicles and formation of the NE.

  16. A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane*

    Science.gov (United States)

    Verma, Richa; Malik, Chetan; Azmi, Sarfuddin; Srivastava, Saurabh; Ghosh, Subhendu; Ghosh, Jimut Kanti

    2011-01-01

    KvAP is a voltage-gated tetrameric K+ channel with six transmembrane (S1–S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218–239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel. PMID:21592970

  17. Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

    Science.gov (United States)

    Liao, Sumei; Klein, Marlise I.; Heim, Kyle P.; Fan, Yuwei; Bitoun, Jacob P.; Ahn, San-Joon; Burne, Robert A.; Koo, Hyun; Brady, L. Jeannine

    2014-01-01

    Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA. PMID:24748612

  18. The Kemp elimination in membrane mimetic reaction media. Probing catalytic properties of cationic vesicles formed from a double-tailed amphiphile and linear long-tailed alcohols or alkyl pyranosides

    NARCIS (Netherlands)

    Klijn, JE; Engberts, JBFN

    2004-01-01

    Vesicles formed from synthetic, double-tailed amphiphiles are often used as mimics for biological membranes. However, biological membranes are a complex mixture of various compounds. In the present paper we describe a first attempt to study the importance of additives on vesicular catalysis. The

  19. The Antitoxin Protein of a Toxin-Antitoxin System fromXylella fastidiosaIs Secreted via Outer Membrane Vesicles.

    Science.gov (United States)

    Santiago, André da Silva; Mendes, Juliano S; Dos Santos, Clelton A; de Toledo, Marcelo A S; Beloti, Lilian L; Crucello, Aline; Horta, Maria A C; Favaro, Marianna T de Pinho; Munar, Duber M M; de Souza, Alessandra A; Cotta, Mônica A; de Souza, Anete P

    2016-01-01

    The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli . The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible

  20. The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    André Santiago

    2016-12-01

    Full Text Available The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp pauca strain 9a5c. These proteins display a high similarity to their homologues in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC, size exclusion chromatography, isothermal titration calorimetry and western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss

  1. trans-10,cis-12 Conjugated linoleic acid induces depolarization of mitochondrial membranes in HT-29 human colon cancer cells: a possible mechanism for induction of apoptosis.

    Science.gov (United States)

    Cho, Han Jin; Kwon, Gyoo Taik; Park, Jung Han Yoon

    2009-10-01

    Conjugated linoleic acid (CLA), which is naturally present in a variety of foods such as milk fat and the meat of ruminant animals, has been demonstrated to exert chemoprotective effects in several tissues in experimental animals. CLA is a collective term, which denotes one or more positional and geometric isomers of octadecadienoic acid, with cis-9,trans-11 (c9t11) and trans-10,cis-12 CLA (t10c12) being the principal isomers in commercial preparations. We observed previously that physiological levels of CLA inhibited HT-29 cell growth, and the growth inhibitory effects of CLA were attributed to the effect of t10c12, but not c9t11. In the present study, we assessed the mechanisms by which physiological levels of CLA and t10c12 induce apoptosis in HT-29 cells. HT-29 cells were cultured for 3 days in serum-free medium in the presence of various concentrations of CLA (0-20 micromol/L) or t10c12 (0-4 micromol/L). Addition of CLA or t10c12 to culture medium resulted in a dose-dependent increase in the numbers of apoptotic cells. The results of western blot analysis of total cell lysates showed that CLA and t10c12 increased the levels of cleaved caspase-9, caspase-3, and poly(ADP-ribose) polymerase but did not alter the levels of Bcl-2 family member proteins. However, these fatty acids were shown to increase the translocation of Bad and Bax to the mitochondria, increase mitochondrial membrane permeability, and induce the release of cytochrome c and Smac/Diablo from the mitochondria. In addition, CLA and t10c12 diminished Akt content and Akt phosphorylation. These findings indicate that physiological levels of t10c12 induce apoptosis in HT-29 colon cancer cells, which is mediated via mitochondrion-mediated events associated with a decline in Akt activity, an increase in the translocation of the pro-apototic Bad and Bax to the mitochondria, and the subsequent disruption of normal mitochondrial membrane potential.

  2. Size and CT density of iodine-containing ethosomal vesicles obtained by membrane extrusion: potential for use as CT contrast agents.

    Science.gov (United States)

    Na, Bomin; Choi, Byoung Wook; Kim, Bumsang

    2013-11-01

    Computed tomography (CT) is the primary non-invasive imaging technique used for most patients with suspected liver disease. In order to improve liver-specific imaging properties and prevent toxic effects in patients with compromised renal function, we investigated the encapsulation of iodine within ethosomal vesicles. As a first step in the development of novel contrast agents using ethosomes for CT imaging applications, iodine was entrapped within ethosomes and iodine-containing ethosomes of the desired size were obtained by extrusion using a polycarbonate membrane with a defined pore size. Ethosomes containing iodine showed a relatively high CT density, which decreased when they were extruded, due to the rupture and re-formation of the lipid bilayer of the ethosome. However, when a solution with a high iodine concentration was used as a dispersion media during the extrusion process, the decrease in CT density could be prevented. In addition, ethosomes containing iodine were taken up efficiently by macrophages, which are abundant in the liver, and these ethosomes exhibited no cellular toxicity. These results demonstrate that iodine could be entrapped within ethosomal vesicles, giving the ethosomes a relatively high CT density, and that the extrusion technique used in this study could conveniently and reproducibly produce ethosomal vesicles with a desired size. Therefore, ethosomes containing iodine, as prepared in this study, have potential as contrast agents with applications in CT imaging. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Membrane Vesicles Released by a hypervesiculating Escherichia coli Nissle 1917 tolR Mutant Are Highly Heterogeneous and Show Reduced Capacity for Epithelial Cell Interaction and Entry.

    Directory of Open Access Journals (Sweden)

    Carla Pérez-Cruz

    Full Text Available Membrane vesicles (MVs produced by Gram-negative bacteria are being explored for novel clinical applications due to their ability to deliver active molecules to distant host cells, where they can exert immunomodulatory properties. MVs released by the probiotic Escherichia coli Nissle 1917 (EcN are good candidates for testing such applications. However, a drawback for such studies is the low level of MV isolation from in vitro culture supernatants, which may be overcome by the use of mutants in cell envelope proteins that yield a hypervesiculation phenotype. Here, we confirm that a tolR mutation in EcN increases MV production, as determined by protein, LPS and fluorescent lipid measurements. Transmission electron microscopy (TEM of negatively stained MVs did not reveal significant differences with wild type EcN MVs. Conversely, TEM observation after high-pressure freezing followed by freeze substitution of bacterial samples, together with cryo-TEM observation of plunge-frozen hydrated isolated MVs showed considerable structural heterogeneity in the EcN tolR samples. In addition to common one-bilayer vesicles (OMVs and the recently described double-bilayer vesicles (O-IMVs, other types of MVs were observed. Time-course experiments of MV uptake in Caco-2 cells using rhodamine- and DiO-labelled MVs evidenced that EcN tolR MVs displayed reduced internalization levels compared to the wild-type MVs. The low number of intracellular MVs was due to a lower cell binding capacity of the tolR-derived MVs, rather than a different entry pathway or mechanism. These findings indicate that heterogeneity of MVs from tolR mutants may have a major impact on vesicle functionality, and point to the need for conducting a detailed structural analysis when MVs from hypervesiculating mutants are to be used for biotechnological applications.

  4. Extracellular vesicle-mediated transfer of membranous components from the highly malignant T24 urinary carcinoma cell line to the non-malignant RT4 urinary papilloma cell line.

    Science.gov (United States)

    Ogorevc, Eva; Hudoklin, Samo; Veranič, Peter; Kralj-Iglič, Veronika

    2014-05-01

    This communication reports the first experimental evidence that in the bladder cancer model, membranous components labelled with the DiO dye and the cholera toxin subunit B can be transported from highly malignant (T24) to non-malignant (RT4) cells by extracellular vesicles. Taking into account the presence of stable membranous nanostructures found by scanning electron microscopy, we suggest a possible uptake mechanism in recipient cells through fusion with highly curved membranous regions.

  5. A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

    Science.gov (United States)

    Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

    2013-03-01

    Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Proteomic study via a non-gel based approach of meningococcal outer membrane vesicle vaccine obtained from strain CU385: a road map for discovering new antigens.

    Science.gov (United States)

    Gil, Jeovanis; Betancourt, L Zaro H; Sardiñas, Gretel; Yero, Daniel; Niebla, Olivia; Delgado, Maité; García, Darien; Pajón, Rolando; Sánchez, Aniel; González, Luis J; Padrón, Gabriel; Campa, Concepción; Sotolongo, Franklin; Barberó, Ramón; Guillén, Gerardo; Herrera, Luis; Besada, Vladimir

    2009-05-01

    This work presents the results from a study of the protein composition of outer membrane vesicles from VA-MENGOC-BC (Finlay Institute, Cuba), an available vaccine against serogroup B Neisseria meningitidis. Proteins were identified by means of SCAPE, a 2DE-free method for proteome studies. More than one hundred proteins were detected by tandem liquid chromatographymass spectrometry analysis of fractions enriched in peptides devoid of histidine or arginine residues, providing a detailed description of the vaccine. A bioinformatic analysis of the identified components resulted in the identification of 31 outer membrane proteins and three conserved hypothetical proteins, allowing the cloning, expression, purification and immunological study of two of them (NMB0088 and NMB1796) as new antigens.

  7. Comparison of three tetramic acids and their ability to alter membrane function in cultured skeletal muscle cells and sarcoplasmic reticulum vesicles.

    Science.gov (United States)

    Riley, R T; Goeger, D E; Yoo, H; Showker, J L

    1992-06-01

    Cyclopiazonic acid is a potent inhibitor of calcium uptake and Ca(2+)-ATPase activity in sarcoplasmic and endoplasmic reticulum. In L6 muscle myoblasts, cyclopiazonic acid stimulates the uptake of tetraphenylphosphonium, a lipophilic membrane potential probe, and has antioxidant properties. The purpose of the present study was to investigate the structural requirements necessary for causing the surface charge alterations, and the antioxidant activity in L6 skeletal muscle myoblasts, and for inhibition of calcium transport by rat skeletal muscle sarcoplasmic reticulum vesicles. This was accomplished by comparing the effects of two structurally related tetramic acids, cyclopiazonic acid imine and tenuazonic acid, with cyclopiazonic acid. Cyclopiazonic acid imine inhibited oxalate-assisted 45Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles and stimulated tetraphenylphosphonium accumulation by L6 muscle myoblasts. However, these effects required an approximately fourfold higher concentration than that of cyclopiazonic acid. Tenuazonic acid, up to 1 mM, had no effect on oxalate-assisted 45Ca2+ uptake or Ca(2+)-ATPase activity in sarcoplasmic reticulum vesicles and did not stimulate tetraphenylphosphonium accumulation by L6 muscle myoblasts. Cyclopiazonic acid was only slightly more effective than cyclopiazonic acid imine at preventing the patulin-induced increase in thiobarbituric acid positive substance (used to estimate lipid peroxidation); tenuazonic acid was totally ineffective. Previously, it was shown that cyclopiazonic acid was twice as effective as cyclopiazonic acid imine at preventing increases in thiobarbituric acid positive substance in cultured renal cells, LLC-PK1. Thus, the indole nucleus of cyclopiazonic acid is essential for the membrane-associated biological activity; however, modification of the acetyl group reduces the potency of the activity.

  8. Membrane vesicle protein PagC as a novel biomarker for detecting pathogenic Salmonella in the viable but not culturable state.

    Science.gov (United States)

    Xu, Jun; Suita, Kazuasa; Okuno, Katsuya; Takaya, Akiko; Yamamoto, Tomoko; Isogai, Emiko

    2017-12-04

    The viable but non-culturable (VBNC) state is a remarkable survival mechanism in which cells exist in a physiologically inactive state. Bacteria in the VBNC state do not form colonies, and thus, are difficult to detect using colony-based methods. As a result, VBNC bacteria are potentially virulent and can cause widespread contamination during food production. In the present study, we reported a novel biomarker, the membrane vesicle protein PagC, for the detection of VBNC Salmonella. Salmonella cells were chemically induced into the VBNC state by H2O2 treatment. The bacterial cells retained their shapes but were observed to release numerous membrane vesicles, which were accompanied by a transient PagC overexpression. Immunoblotting was performed to detect PagC in pathogenic strains, including Salmonella Enteritidis and S. Typhimurium, which are harmful and known to cause food-borne gastroenteritis in humans and other animals. Therefore, our findings demonstrated the potential use of PagC as a biomarker for the detection of VBNC Salmonella in food production.

  9. Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Le Thi Kim, E-mail: ngocanh@nutr.med.tokushima-u.ac.jp [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Hosaka, Toshio [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan); Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Teshigawara, Kiyoshi [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan); Sakai, Tohru [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan); Nakaya, Yutaka [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Funaki, Makoto, E-mail: m-funaki@clin.med.tokushima-u.ac.jp [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)

    2010-01-01

    In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

  10. The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells

    Directory of Open Access Journals (Sweden)

    Andres Marilou A

    2007-09-01

    Full Text Available Abstract Background The cyclic nucleotide-gated ion channels (CNGCs maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots. Results AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50–150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents. Conclusion AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported

  11. Intestinal Anti-inflammatory Effects of Outer Membrane Vesicles from Escherichia coli Nissle 1917 in DSS-Experimental Colitis in Mice

    Directory of Open Access Journals (Sweden)

    María-José Fábrega

    2017-07-01

    Full Text Available Escherichia coli Nissle 1917 (EcN is a probiotic strain with proven efficacy in inducing and maintaining remission of ulcerative colitis. However, the microbial factors that mediate these beneficial effects are not fully known. Gram-negative bacteria release outer membrane vesicles (OMVs as a direct pathway for delivering selected bacterial proteins and active compounds to the host. In fact, vesicles released by gut microbiota are emerging as key players in signaling processes in the intestinal mucosa. In the present study, the dextran sodium sulfate (DSS-induced colitis mouse model was used to investigate the potential of EcN OMVs to ameliorate mucosal injury and inflammation in the gut. The experimental protocol involved pre-treatment with OMVs for 10 days before DSS intake, and a 5-day recovery period. Oral administration of purified EcN OMVs (5 μg/day significantly reduced DSS-induced weight loss and ameliorated clinical symptoms and histological scores. OMVs treatment counteracted altered expression of cytokines and markers of intestinal barrier function. This study shows for the first time that EcN OMVs can mediate the anti-inflammatory and barrier protection effects previously reported for this probiotic in experimental colitis. Remarkably, translation of probiotics to human healthcare requires knowledge of the molecular mechanisms involved in probiotic–host interactions. Thus, OMVs, as a non-replicative bacterial form, could be explored as a new probiotic-derived therapeutic approach, with even lower risk of adverse events than probiotic administration.

  12. Phosphatidylethanolamine: ceramide-ethanolaminephosphotransferase activity in synaptic plasma membrane vesicles. Influence of some cations and phospholipid environment on transferase activity. Further proof of its location.

    Science.gov (United States)

    Maurice, A; Malgat, M

    1993-08-01

    1. Synaptic plasma membrane vesicles (SPMV) from rat brain synthesized ceramide-phosphoethanolamine (SpE), an analogue of sphingomyelin (SpC) from phosphatidylethanolamine (PE) and ceramide. 2. This reaction was catalyzed by PE: ceramide-phosphotransferase. 3. The presence of PC did not modify the SpE synthesis and PI and PS at twice PE concentration seemed to be activators; only PG was an inhibitor at all concentrations. 4. Some cations (Mg2+, Mn2+) were without effect, while Ca2+ increased transferase activity, so was interesting to study. 5. Transferase was compared with sialidase (external enzyme). 6. Kinetics other than those already performed by us were undertaken in order to confirm its location.

  13. Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies

    Directory of Open Access Journals (Sweden)

    Masaaki Nameta

    2016-09-01

    Full Text Available Aquaporin-2 (AQP2 is present in urine extracellular vesicles (EVs and is a useful biomarker for water balance disorders. We previously found that pre-treatment of urine with alkali/detergent or storage at −25 °C is required for enzyme-linked immunosorbent assay (ELISA measurement. We speculated that disruptions of EVs membranes are necessary to allow for the direct contact of antibodies with their epitopes. Human urine EVs were prepared using an ultracentrifugation method. Urine EV samples were stored at different temperatures for a week. Electron microscopy showed abundant EVs with diameters of 20–100 nm, consistent with those of exosomes, in normal urine, whereas samples from alkali/detergent pre-treated urine showed fewer EVs with large swollen shapes and frequent membrane disruptions. The abundance and structures of EVs were maintained during storage at −80 °C, but were severely damaged at −25 °C. Binding and competitive inhibition assays showed that epitopes of monoclonal antibody and polyclonal antibody were the hydrophilic Loop D and C-terminus of AQP2, respectively, both of which are present on the inner surface of EVs. Thus, urine storage at −25 °C or pre-treatment with alkali/detergent disrupt EVs membranes and allow AQP2 antibodies to bind to their epitopes located inside EVs.

  14. Transcription factor σB plays an important role in the production of extracellular membrane-derived vesicles in Listeria monocytogenes.

    Science.gov (United States)

    Lee, Jung Hwa; Choi, Chi-Won; Lee, Taewon; Kim, Seung Il; Lee, Je-Chul; Shin, Ji-Hyun

    2013-01-01

    Gram-negative bacteria produce extracellular outer membrane vesicles (OMVs) that interact with host cells. Unlike Gram-negative bacteria, less is known about the production and role of extracellular membrane vesicles (MVs) in Gram-positive bacteria. The food-borne pathogen Listeria monocytogenes can survive under extreme environmental and energy stress conditions and the transcription factor σ(B) is involved in this survival ability. Here, we first determined the production of MVs from L. monocytogenes and evaluated whether general stress transcription factor σ(B) affected production of MVs in L. monocytogenes. L. monocytogenes secreted MVs during in vitro broth culture. The wild-type strain actively produced MVs approximately nine times more and also produced more intact shapes of MVs than those of the isogenic ΔsigB mutant. A proteomic analysis showed that 130 and 89 MV proteins were identified in the wild-type and ΔsigB mutant strains, respectively. Wild-type strain-derived MVs contained proteins regulated by σ(B) such as transporters (OpuCA and OpuCC), stress response (Kat), metabolism (LacD), translation (InfC), and cell division protein (FtsZ). Gene Ontology (GO) enrichment analysis showed that wild-type-derived MV proteins corresponded to several GO terms, including response to stress (heat, acid, and bile resistance) and extracellular polysaccharide biosynthetic process, but not the ΔsigB mutant. Internalin B (InlB) was almost three times more contained in MVs derived from the wild-type strain than in MVs derived from the ΔsigB mutant. Taken together, these results suggest that σ(B) plays a pivotal role in the production of MVs and protein profiles contained in MVs. L. monocytogenes MVs may contribute to host infection and survival ability under various stressful conditions.

  15. Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms.

    Science.gov (United States)

    Sedlik, Christine; Vigneron, James; Torrieri-Dramard, Lea; Pitoiset, Fabien; Denizeau, Jordan; Chesneau, Caroline; de la Rochere, Philippe; Lantz, Olivier; Thery, Clotilde; Bellier, Bertrand

    2014-01-01

    The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs) to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccines encoding the non-EV-associated antigen. Here, we compared our two approaches to associate the ovalbumin (OVA) antigen to EVs: (a) by fusion to the lipid-binding domain C1C2 of MFGE8(=lactadherin), which is exposed on the surface of secreted membrane vesicles; and (b) by fusion to retroviral Gag capsid protein, which is incorporated inside membrane-enclosed virus-like particles. Plasmids encoding either form of modified OVA were used as DNA-based vaccines (i.e. injected into mice to allow in vivo expression of the antigen associated to EVs). We show that both DNA vaccines induced, with similar efficiency, OVA-specific CD8(+) T cells and total IgG antibodies. By contrast, each vaccine preferentially stimulated different isotypes of immunoglobulins, and the OVA-C1C2-encoding vaccine favoured antigen-specific CD4(+) T lymphocyte induction as compared to the Gag-OVA vaccine. Nevertheless, both OVA-C1C2 and Gag-OVA vaccines efficiently prevented in vivo outgrowth of OVA-expressing tumours and reduced tumour progression when administered to tumour-bearing mice, although with variable efficacies depending on the tumour models. DNA vaccines encoding EV-associated antigens are thus promising immunotherapy tools in cancer but also potentially other diseases.

  16. Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms

    Science.gov (United States)

    Sedlik, Christine; Vigneron, James; Torrieri-Dramard, Lea; Pitoiset, Fabien; Denizeau, Jordan; Chesneau, Caroline; de la Rochere, Philippe; Lantz, Olivier; Thery, Clotilde; Bellier, Bertrand

    2014-01-01

    The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs) to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccines encoding the non-EV-associated antigen. Here, we compared our two approaches to associate the ovalbumin (OVA) antigen to EVs: (a) by fusion to the lipid-binding domain C1C2 of MFGE8(=lactadherin), which is exposed on the surface of secreted membrane vesicles; and (b) by fusion to retroviral Gag capsid protein, which is incorporated inside membrane-enclosed virus-like particles. Plasmids encoding either form of modified OVA were used as DNA-based vaccines (i.e. injected into mice to allow in vivo expression of the antigen associated to EVs). We show that both DNA vaccines induced, with similar efficiency, OVA-specific CD8+ T cells and total IgG antibodies. By contrast, each vaccine preferentially stimulated different isotypes of immunoglobulins, and the OVA-C1C2-encoding vaccine favoured antigen-specific CD4+ T lymphocyte induction as compared to the Gag-OVA vaccine. Nevertheless, both OVA-C1C2 and Gag-OVA vaccines efficiently prevented in vivo outgrowth of OVA-expressing tumours and reduced tumour progression when administered to tumour-bearing mice, although with variable efficacies depending on the tumour models. DNA vaccines encoding EV-associated antigens are thus promising immunotherapy tools in cancer but also potentially other diseases. PMID:25206960

  17. Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms

    Directory of Open Access Journals (Sweden)

    Christine Sedlik

    2014-08-01

    Full Text Available The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccines encoding the non-EV-associated antigen. Here, we compared our two approaches to associate the ovalbumin (OVA antigen to EVs: (a by fusion to the lipid-binding domain C1C2 of MFGE8(=lactadherin, which is exposed on the surface of secreted membrane vesicles; and (b by fusion to retroviral Gag capsid protein, which is incorporated inside membrane-enclosed virus-like particles. Plasmids encoding either form of modified OVA were used as DNA-based vaccines (i.e. injected into mice to allow in vivo expression of the antigen associated to EVs. We show that both DNA vaccines induced, with similar efficiency, OVA-specific CD8+ T cells and total IgG antibodies. By contrast, each vaccine preferentially stimulated different isotypes of immunoglobulins, and the OVA-C1C2-encoding vaccine favoured antigen-specific CD4+ T lymphocyte induction as compared to the Gag-OVA vaccine. Nevertheless, both OVA-C1C2 and Gag-OVA vaccines efficiently prevented in vivo outgrowth of OVA-expressing tumours and reduced tumour progression when administered to tumour-bearing mice, although with variable efficacies depending on the tumour models. DNA vaccines encoding EV-associated antigens are thus promising immunotherapy tools in cancer but also potentially other diseases.

  18. Gramicidin-perforated patch revealed depolarizing effect of GABA in cultured frog melanotrophs

    National Research Council Canada - National Science Library

    Frank Le Foll; Hélène Castel; Olivier Soriani; Hubert Vaudry; Lionel Cazin

    1998-01-01

    ...ˆ’ ] i ) in cultured frog melanotrophs. In the gramicidin-perforated patch configuration, 1 μM GABA caused a depolarization associated with an action potential discharge and a slight fall of membrane resistance...

  19. Exosomes: secreted vesicles and intercellular communications

    OpenAIRE

    Théry, Clotilde

    2011-01-01

    Exosomes are small membrane vesicles of endocytic origin secreted by most cell types, and are thought to play important roles in intercellular communications. Although exosomes were originally described in 1983, interest in these vesicles has really increased dramatically in the last 3 years, after the finding that they contain mRNA and microRNA. This discovery sparked renewed interest for the general field of membrane vesicles involved in intercellular communications, and research on these s...

  20. Outer membrane vesicles harboring modified lipid A moiety augment the efficacy of an influenza vaccine exhibiting reduced endotoxicity in a mouse model.

    Science.gov (United States)

    Lee, Tae-Young; Kim, Chang-Ung; Bae, Eun-Hye; Seo, Sang-Hwan; Jeong, Dae Gwin; Yoon, Sun-Woo; Chang, Kyu-Tae; Kim, Young Sang; Kim, Sang-Hyun; Kim, Doo-Jin

    2017-01-23

    Influenza is an acute respiratory disease and a major health problem worldwide. Since mucosal immunity plays a critical role in protection against influenza virus infection, mucosal immunization is considered a promising vaccination route. However, except for live-attenuated vaccines, there are no effective killed or recombinant mucosal influenza vaccines to date. Outer membrane vesicles (OMVs) are nano-sized vesicles produced by gram-negative bacteria, and contain various bacterial components capable of stimulating the immune system of the host. We generated an OMV with low endotoxicity (fmOMV) by modifying the structure of the lipid A moiety of lipopolysaccharide and investigated its effect as an intranasal vaccine adjuvant in an influenza vaccine model. In this model, fmOMV exhibited reduced toll-like receptor 4-stimulating activity and attenuated endotoxicity compared to that of native OMV. Intranasal injection of the vaccine antigen with fmOMV significantly increased systemic antibody and T cell responses, mucosal IgA levels, and the frequency of lung-resident influenza-specific T cells. In addition, the number of antigen-bearing CD103+ dendritic cells in the mediastinal lymph nodes was significantly increased after fmOMV co-administration. Notably, the mice co-immunized with fmOMV showed a significantly higher protection rate against challenge with a lethal dose of homologous or heterologous influenza viruses without adverse effects. These results show the potential of fmOMV as an effective mucosal adjuvant for intranasal vaccines. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning

    Directory of Open Access Journals (Sweden)

    Lotta Kilpinen

    2013-12-01

    Full Text Available Background: Mesenchymal stromal cells (MSC are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs from human umbilical cord blood derived MSCs (hUCBMSC in the presence (MVstim or absence (MVctrl of inflammatory stimulus. Methods: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays. Results: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5 and lipid binding proteins (i.e apolipoproteins were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81 and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations. Conclusions: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs

  2. Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Rama Garimella

    2017-03-01

    Full Text Available Osteosarcoma (OS is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a inflammation and immunity; (b formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium, quantity of gap junctions and skeletogenesis; (c bone mineral density; and (d cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12, bone morphogenetic factor-1 (BMP1, SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4, Matrix extracellular phosphoglycoprotein (MEPE, Integrin, β4 (ITGBP4, Matrix Metalloproteinase -1, -28 (MMP1 and MMP28, and signal transducer and activator of transcription-4 (STAT4 in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7, but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

  3. A GALA lipopeptide mediates pH- and membrane charge dependent fusion with stable giant unilamellar vesicles

    DEFF Research Database (Denmark)

    Etzerodt, Thomas P.; Trier, Sofie; Henriksen, Jonas R.

    2012-01-01

    Peptides capable of mediating fusion between lipid membranes are widely observed in nature, and have attracted considerable attention in the liposome drug delivery field. However, studies that are proving the benefit of small synthetic fusion peptides as components in drug delivery systems remain...

  4. Tooth enamel protein amelogenin binds to ameloblast cell membrane-mimicking vesicles via its N-terminus.

    Science.gov (United States)

    Lokappa, Sowmya Bekshe; Chandrababu, Karthik Balakrishna; Moradian-Oldak, Janet

    2015-08-28

    We have recently reported that the extracellular enamel protein amelogenin has affinity to interact with phospholipids and proposed that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. Here, in order to identify the liposome-interacting domains of amelogenin we designed four different amelogenin mutants containing only a single tryptophan at positions 25, 45, 112 and 161. Circular dichroism studies of the mutants confirmed that they are structurally similar to the wild-type amelogenin. Utilizing the intrinsic fluorescence of single tryptophan residue and fluorescence resonance energy transfer [FRET], we analyzed the accessibility and strength of their binding with an ameloblast cell membrane-mimicking model membrane (ACML) and a negatively charged liposome used as a membrane model. We found that amelogenin has membrane-binding ability mainly via its N-terminal, close to residues W25 and W45. Significant blue shift was also observed in the fluorescence of a N-terminal peptide following addition of liposomes. We suggest that, among other mechanisms, enamel malformation in cases of Amelogenesis Imperfecta (AI) with mutations at the N-terminal may be the result of defective amelogenin-cell interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors.

    Science.gov (United States)

    Ojeda Naharros, Irene; Gesemann, Matthias; Mateos, José M; Barmettler, Gery; Forbes, Austin; Ziegler, Urs; Neuhauss, Stephan C F; Bachmann-Gagescu, Ruxandra

    2017-12-01

    Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming

  6. Extracellular Vesicles in Cardiovascular Theranostics

    OpenAIRE

    Bei, Yihua; Das, Saumya; Rodosthenous, Rodosthenis S.; Holvoet, Paul; Vanhaverbeke, Maarten; Monteiro,Marta Chagas; Monteiro, Valter Vinicius Silva; Radosinska, Jana; Bartekova, Monika; Jansen, Felix; Li, Qian; Rajasingh, Johnson; Xiao, Junjie

    2017-01-01

    Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells pla...

  7. Immunotherapeutic Potential of Extracellular Vesicles

    OpenAIRE

    Zhang, Bin; Yin, Yijun; Lai, Ruenn Chai; Lim, Sai Kiang

    2014-01-01

    Extracellular vesicle or EV is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes, the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized...

  8. Reexamination of depolarization in lidar measurements.

    Science.gov (United States)

    Gimmestad, Gary G

    2008-07-20

    Almost all of the depolarization papers in the lidar literature employ a physically inappropriate notation and they use a definition of the depolarization ratio that is not linear in the quantity of interest. This depolarization lidar legacy is misleading and confusing. In particular, subscripts meaning parallel and perpendicular do not apply to atmospheric parameters, such as the volume backscatter coefficient, because (for linear polarization) the two components of the backscattered light are polarized in the transmitted sense and completely unpolarized; the unpolarized component is not "perpendicular." An analysis of lidar depolarization measurements with a particle scattering matrix recently provided in the literature yields algorithms for retrieving the depolarization parameter from either linear or circular depolarization lidar measurements. The analysis, notation, and definitions recommended here harmonize lidar depolarization analysis with radiative transfer theory, particle scattering theory, and standard polarization measurement techniques.

  9. Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.

    Directory of Open Access Journals (Sweden)

    Martina Bielaszewska

    2017-02-01

    Full Text Available Outer membrane vesicles (OMVs are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a, cytolethal distending toxin V (CdtV, EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV

  10. Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury

    Science.gov (United States)

    Greune, Lilo; Jarosch, Kevin-André; Steil, Daniel; Zhang, Wenlan; He, Xiaohua; Lloubes, Roland; Fruth, Angelika; Kim, Kwang Sik; Schmidt, M. Alexander; Dobrindt, Ulrich; Mellmann, Alexander; Karch, Helge

    2017-01-01

    Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors

  11. Effects of extracellular calcium and sodium on depolarization-induced automaticity in guinea pig papillary muscle.

    Science.gov (United States)

    Katzung, B G

    1975-07-01

    Regenerative discharge of action potentials is induced in mammalian papillary muscles by passage of small depolarizing currents. In this paper, the effects of various extracellular calcium and sodium concentrations and of tetrodotoxin on this phenomenon were studied in guinea pig papillary muscles in a sucrose gap chamber. Phase 4 diastolic depolarization was found to be associated with an increase in membrane resistance. The slope of phase 4 depolarization was decreased by reductions in extracellular calcium or sodium concentration. The range of maximum diastolic potentials and the thresholds from which regenerative potentials arose were reduced, especially at the positive limit of potentials, by a reduction in either ion. It was concluded that both calcium and sodium influence diastolic depolarization and participate in the regenerative action potentials of depolarization-induced ventricular automaticity.

  12. Assessment of different fixation protocols on the presence of membrane-bound vesicles in Caco-2 cells: a multidimensional view by means of correlative light and 3-D transmission electron microscopy.

    Science.gov (United States)

    Shami, Gerald; Cheng, Delfine; Henriquez, Jeffrey; Braet, Filip

    2014-12-01

    Herein, we present a comparative analysis of a variety of chemical and physical fixation protocols for the specific visualisation of the membrane-bound vesicles (MBVs) in the Caco-2 colorectal cancer (CRC) cell line. In so doing, we validated the applicability of specific specimen preparation protocols for the preservation and contrasting of membrane-associated vesicles. Next, by employing the best respective chemical (GOT) and physical (SHPF) fixation methods for the application of transmission electron tomography and modelling we were able to characterise MBVs in three-dimensions and at the nanometer scale. In the second part of this study, we employ a correlative light and electron microscopy (CLEM) approach in order to determine which vesicular compartments are implicated in the uptake of FITC-BSA as a model protein drug. In so doing, we provide a solid foundation for future studies investigating chemotherapeutic drug uptake, transport and fate in cancer cell lines. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Lack of intestinal transport of [3H]-demethylphalloin: comparative studies with phallotoxins and bile acids on isolated small intestinal cells and ileal brush border membrane vesicles.

    Science.gov (United States)

    Petzinger, E; Burckhardt, G; Schwenk, M; Faulstich, H

    1982-08-01

    Several earlier studies suggested that the uptake of phallotoxins by liver cells is a carrier mediated process using a transport system normally handling bile acids (see Frimmer 1982). In this study we have shown whether ileal cells, well known to transport bile acids too, are able to take up phallotoxins. Isolated epithelial cells prepared from guinea pig ileum accumulated [14C]-cholate, whereas [3H]-demethylphalloin ([3H]-DMP) was not taken up. The same observation was made with isolated jejunal cells but the uptake of [14C]-cholate was much slower. [3H]-DMP, however, was partly bound to intestinal cells. This process was not inhibited by cholate, iodipamide, oligomycin and carbonylcyano-chlorophenylhydrazone (CCCP), compounds known to decrease the uptake of phallotoxins into liver cells. Substituting Na+ for choline+ and also Cl- for SCN- did not influence the binding of [3H]-DMP. Frozen intestinal cells from the guinea pig bound two time more [3H]-DMP after thawing compared with intact cells. Supplementary uptake experiments on isolated brush border membrane vesicles from rat ileum revealed that phalloidin does not inhibit taurocholate uptake and that taurocholate does not interfere with [3H]-DMP binding. The results suggest that [3H]-demethylphalloin is not recognized by the bile acid carrier of the guinea pig and the rat ileum. It is concluded that the transport system for bile acids present in ileal cell is different from that of liver cells.

  14. Preparation and evaluation of antifungal efficacy of griseofulvin loaded deformable membrane vesicles in optimized guinea pig model of Microsporum canis--dermatophytosis.

    Science.gov (United States)

    Aggarwal, Nidhi; Goindi, Shishu

    2012-11-01

    The present study is aimed at the encapsulation of griseofulvin in the deformable membrane vesicles (DMVs) for dermal delivery. Presently, griseofulvin is available only in conventional oral dosage forms that suffer from the issues of poor and highly variable bioavailability, numerous systemic side effects and long duration of treatment. Multi-lamellar drug-loaded DMVs of griseofulvin (Indian Patent Application 208/DEL/2009) were prepared by thin-film hydration method and were optimized for type and concentration of edge activator (EA). The optimized formulation was evaluated for vesicular shape, size, drug entrapment efficiency, drug content, pH, stability, spreadability, ex vivo skin permeation, dermatokinetics, skin sensitivity, in vitro antifungal assay and in vivo antifungal activity against Microsporum canis using guinea pig model for dermatophytosis. The optimized DMVs illustrated remarkably higher drug permeation and skin retention when compared with liposomes. A complete clinical and mycological cure was observed in animals treated with topical griseofulvin formulation in 10 days. The formulation was observed to be non-sensitizing, histopathologically safe, and stable at 5±3 °C, 25±2 °C and 40±2 °C for a period of six months. The results indicated that the topical formulation of DMVs of griseofulvin could be utilized as an alternative to reduce the encumbrance of conventional oral formulations. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Moraxella catarrhalis Outer Membrane Vesicles Carry β-Lactamase and Promote Survival of Streptococcus pneumoniae and Haemophilus influenzae by Inactivating Amoxicillin▿

    Science.gov (United States)

    Schaar, Viveka; Nordström, Therése; Mörgelin, Matthias; Riesbeck, Kristian

    2011-01-01

    Moraxella catarrhalis is a common pathogen found in children with upper respiratory tract infections and in patients with chronic obstructive pulmonary disease during exacerbations. The bacterial species is often isolated together with Streptococcus pneumoniae and Haemophilus influenzae. Outer membrane vesicles (OMVs) are released by M. catarrhalis and contain phospholipids, adhesins, and immunomodulatory compounds such as lipooligosaccharide. We have recently shown that M. catarrhalis OMVs exist in patients upon nasopharyngeal colonization. As virtually all M. catarrhalis isolates are β-lactamase positive, the goal of this study was to investigate whether M. catarrhalis OMVs carry β-lactamase and to analyze if OMV consequently can prevent amoxicillin-induced killing. Recombinant β-lactamase was produced and antibodies were raised in rabbits. Transmission electron microscopy, flow cytometry, and Western blotting verified that OMVs carried β-lactamase. Moreover, enzyme assays revealed that M. catarrhalis OMVs contained active β-lactamase. OMVs (25 μg/ml) incubated with amoxicillin for 1 h completely hydrolyzed amoxicillin at concentrations up to 2.5 μg/ml. In functional experiments, preincubation of amoxicillin (10× MIC) with M. catarrhalis OMVs fully rescued amoxicillin-susceptible M. catarrhalis, S. pneumoniae, and type b or nontypeable H. influenzae from β-lactam-induced killing. Our results suggest that the presence of amoxicillin-resistant M. catarrhalis originating from β-lactamase-containing OMVs may pave the way for respiratory pathogens that by definition are susceptible to β-lactam antibiotics. PMID:21576428

  16. Comparative Proteomic Analysis of the Effect of Periplocoside P from Periploca sepium on Brush Border Membrane Vesicles in Midgut Epithelium of Mythimna separata Larvae

    Directory of Open Access Journals (Sweden)

    Mingxing Feng

    2017-12-01

    Full Text Available Periplocoside P (PSP, a novel compound isolated from Periploca sepium Bunge, possesses insecticidal activity against some lepidopterans, such as Mythimna separata. In M. separata, the brush border membrane vesicles of the midgut epithelium are the initial site of action of periplocosides. We conducted two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry analysis to analyze differentially expressed proteins (DEPs from periplocoside P (PSP-treated M. separata. We successfully isolated seven up-regulated and three down-regulated DEPs that have been previously identified, as well as a novel DEP. The DEPs are implicated in protein degradation, transporter, folding, and synthesis, and in juvenile hormone biosynthesis. DEPs involved in the oxidative phosphorylation energy metabolism pathway are enriched. Through real-time polymerase chain reaction assay, we confirmed that vma1 expression is significantly up-regulated expression levels in PSP-treated M. separata larvae. Enzymology validation further indicated that PSP can significantly inhibit V-type ATPase activity in a concentration-dependent manner. Given these results, we speculate that in M. separata, the V-type ATPase A subunit in the midgut epithelium is the putative target binding site of periplocosides. This finding provides preliminary evidence for the mode of action of periplocosides.

  17. Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

    LENUS (Irish Health Repository)

    Tudor, E L

    2010-05-19

    Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

  18. The activation of membrane targeted CaMK-II in the zebrafish Kupffer's vesicle is required for left-right asymmetry.

    Science.gov (United States)

    Francescatto, Ludmila; Rothschild, Sarah C; Myers, Alexandra L; Tombes, Robert M

    2010-08-01

    Intracellular calcium ion (Ca(2+)) elevation on the left side of the mouse embryonic node or zebrafish Kupffer's vesicle (KV) is the earliest asymmetric molecular event that is functionally linked to lateral organ placement in these species. In this study, Ca(2+)/CaM-dependent protein kinase (CaMK-II) is identified as a necessary target of this Ca(2+) elevation in zebrafish embryos. CaMK-II is transiently activated in approximately four interconnected cells along the anterior left wall of the KV between the six- and 12-somite stages, which is coincident with known left-sided Ca(2+) elevations. Within these cells, activated CaMK-II is observed at the surface and in clusters, which appear at the base of some KV cilia. Although seven genes encode catalytically active CaMK-II in early zebrafish embryos, one of these genes also encodes a truncated inactive variant (alphaKAP) that can hetero-oligomerize with and target active enzyme to membranes. alphaKAP, beta2 CaMK-II and gamma1 CaMK-II antisense morpholino oligonucleotides, as well as KV-targeted dominant negative CaMK-II, randomize organ laterality and southpaw (spaw) expression in lateral plate mesoderm (LPM). Left-sided CaMK-II activation was most dependent on an intact KV, the PKD2 Ca(2+) channel and gamma1 CaMK-II; however, alphaKAP, beta2 CaMK-II and the RyR3 ryanodine receptor were also necessary for full CaMK-II activation. This is the first report to identify a direct Ca(2+)-sensitive target in left-right asymmetry and supports a model in which membrane targeted CaMK-II hetero-oligomers in nodal cells transduce the left-sided PKD2-dependent Ca(2+) signals to the LPM.

  19. Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking.

    Science.gov (United States)

    Wang, Shan Shan H; Held, Richard G; Wong, Man Yan; Liu, Changliang; Karakhanyan, Aziz; Kaeser, Pascal S

    2016-08-17

    In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near-complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. C11ORF24 is a novel type I membrane protein that cycles between the Golgi apparatus and the plasma membrane in Rab6-positive vesicles.

    Science.gov (United States)

    Fraisier, Vincent; Kasri, Amal; Miserey-Lenkei, Stéphanie; Sibarita, Jean-Baptiste; Nair, Deepak; Mayeux, Adeline; Bardin, Sabine; Toyoda, Yusuke; Poser, Ina; Poznyakovskiy, Andrei; Goud, Bruno; Hyman, Anthony A; Dimitrov, Ariane

    2013-01-01

    The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.

  1. Extracellular Vesicles: Evolving Contributors in Autoimmunity

    OpenAIRE

    Katsiougiannis, Stergios

    2015-01-01

    Extracellular vesicles, including microvesicles, exosomes and apoptotic bodies are recognized as carriers of pathogen-associated molecules with direct involvement in immune signaling and inflammation. Those observations have enforced the way these membranous vesicles are being considered as promising immunotherapeutic targets. In this review, we discuss the emerging roles of extracellular vesicles in autoimmunity and highlights their potential use as disease biomarkers as well as targets for ...

  2. Water and membrane dynamics in suspensions of lipid vesicles functionalized with poly(ethylene glycol)s.

    Science.gov (United States)

    Clop, Eduardo M; Chattah, Ana K; Perillo, María A

    2014-06-12

    The present work was aimed at studying the molecular dynamics at different levels of model membranes having a simulated glycoclix, with focus on the molecular crowding conditions at the lipid-water interfacial region. Thus, binary mixtures of dipalmitoylphosphatidylcholine (dpPC) and a poly(ethylene glycol) (PEG(n)) derivative of dipalmitoylphosphatidylethanolamine (PE) (where n = 350, 1000, and 5000, respectively, refer to PEG molecular masses) were submitted to (1)H spin-lattice relaxation time (T1) and (31)P NMR spectra analysis. (1)H NMR relaxation times revealed two contributing components in each proton system (PEG, phospholipids, and water), for all the mixtures studied, exhibiting values of T1 with very different orders of magnitude. This allowed identifying confined and bulk water populations as well as PEG moieties becoming more disordered and independent from the phospholipid moiety as n increased. (31)P spectra showed a typical broad bilayer signal for n = 350 and 1000, and an isotropic signal characteristic of micelles for n = 5000. Surface pressure (π)-molecular area isotherms and compressional modulus measurements provided further structural information. Moreover, epifluorescence microscopy data from monolayers at π ∼ 30 mN/m, the expected equilibrium π in lipid bilayers, allowed us to postulate that both (1)H populations resolved through NMR in phospholipids and lipopolymers corresponded to different phase domains.

  3. Vesicle-cholesterol interactions : Effects of added cholesterol on gel-to-liquid crystal transitions in a phospholipid membrane and five dialkyl-based vesicles as monitored using DSC

    NARCIS (Netherlands)

    Blandamer, Michael J.; Briggs, B; Cullis, PM; Rawlings, BJ; Engberts, Jan B. F. N.; Cullis, Paul M.; Rawlings, Bernard J.

    2003-01-01

    Differential scanning calorimetry (DSC) results are reported characterising the effects of added cholesterol (CHOL) on vesicle bilayer systems formed in aqueous systems by three sodium dialkylphosphates, (RO)(2)PO2--Na+ where R = dodecyl (DDP), tetradecyl (DTP) and octadecyl (DOP), (ii) two

  4. Interaction of insulin with SDS/CTAB catanionic Vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Tah, Bidisha; Pal, Prabir; Talapatra, G.B., E-mail: spgbt@iacs.res.in

    2014-01-15

    In the present study, a novel method was used for entrapping the protein, insulin into the catanionic SDS/CTAB vesicle membrane. The anionic SDS and cationic CTAB formed catanionic vesicles at particular concentration (35:65 by volume). In this study, vesicle membrane can be considered as model membrane. The vesicle formation and entrapment efficiency depend on the pH of the aqueous solution. The insulin molecules have attached with the vesicular membrane at pH 7.0. However, at acidic pH, the vesicles were ruptured and the insulin did not entrap into the vesicle membrane, whereas at alkaline pH insulin became fibriller. The scanning electron microscope (SEM), Dynamic light scattering (DLS), and Zeta potential studies established the self-assembled structure formation of insulin and catanionic vesicles. To know the protein confirmations, Circular dichroism (CD) was also employed. The temperature dependent steady state and time resolved emission spectroscopy show that at room temperature (25 °C), apart from the 305 nm tyrosine fluorescence, a new emission peak at 450 nm was observed only in case of insulin-vesicle system, and was assigned as the tyrosine phosphorescence. This phosphorescence peak is the signature of the entrapment of insulin into the vesicle membrane. Highlights: • SDS-CTAB based catanionic vesicle has been fabricated. • Insulin has been successfully immobilized on these vesicles. • Immobilized insulin shows room temperature phosphorescence.

  5. Vesicles and vesicle fusion: coarse-grained simulations

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2010-01-01

    Biological cells are highly dynamic, and continually move material around their own volume and between their interior and exterior. Much of this transport encapsulates the material inside phospholipid vesicles that shuttle to and fro, fusing with, and budding from, other membranes. A feature of v...

  6. Functional and specific antibody responses in adult volunteers in new zealand who were given one of two different meningococcal serogroup B outer membrane vesicle vaccines.

    Science.gov (United States)

    Wedege, E; Bolstad, K; Aase, A; Herstad, T K; McCallum, L; Rosenqvist, E; Oster, P; Martin, D

    2007-07-01

    This study presents detailed analyses of total and specific serum antibody levels among 26 and 24 adult volunteers before vaccination and after the third dose of the meningococcal serogroup B outer membrane vesicle (OMV) vaccines MeNZB and MenBvac, respectively, in a clinical trial in New Zealand (V. Thornton, D. Lennon, K. Rasanathan, J. O'Hallahan, P. Oster, J. Stewart, S. Tilman, I. Aaberge, B. Feiring, H. Nokleby, E. Rosenqvist, K. White, S. Reid, K. Mulholland, M. J. Wakefield, and D. Martin, Vaccine 24:1395-1400, 2006). With the homologous vaccine strains as targets, both vaccines induced significant increases in serum bactericidal and opsonophagocytic activities and in the levels of immunoglobulin G (IgG) to OMV antigens in an enzyme-linked immunosorbent assay (ELISA) and to live meningococci by flow cytometry. They also induced high levels of activity against the heterologous strains, particularly in terms of opsonophagocytic activity and IgG binding to live bacteria. The antibody levels with the homologous and heterologous strains in the four assays showed high and significant positive correlations. Specific IgG binding to 10 major OMV antigens in each vaccine was measured by scanning of immunoblots; ELISAs for two antigens, lipopolysaccharide and Neisseria surface protein A (NspA), were also performed. Both vaccines elicited significant increases in IgG binding to all homologous and heterologous OMV antigens except NspA. The total IgG band intensity on the blots correlated significantly with the IgG levels determined by the OMV ELISA and flow cytometry. In conclusion, the results of the various immunological assays showed that both OMV vaccines gave rise to high levels of specific and cross-reacting antibodies.

  7. Local and Systemic Antibody Responses in Mice Immunized Intranasally with Native and Detergent-Extracted Outer Membrane Vesicles from Neisseria meningitidis

    Science.gov (United States)

    Guthrie, Terry; Wong, Simon Y. C.; Liang, Bin; Hyland, Lisa; Hou, Sam; Høiby, E. Arne; Andersen, Svein Rune

    2004-01-01

    The mouse humoral immune response toward native or detergent-extracted outer membrane vesicles (NOMVs and DOMVs, respectively) from Neisseria meningitidis was determined after intranasal immunization. Both preparations elicited high frequencies of NOMV-specific antibody-forming cells (AFCs) locally in the nasal associated lymphoid tissue (NALT) after three or four weekly doses. The diffuse NALT (D-NALT) contained ca. 10-fold more NOMV-specific AFCs than those observed in the mediastinal lymph node, spleen, and bone marrow. AFCs observed in the D-NALT were primarily immunoglobulin A positive (IgA+) and were maintained for at least 1 month. In contrast, the organized NALT (O-NALT) contained low numbers of AFCs, and the response was relatively short-lived. In other lymphoid tissues, AFCs producing various IgG subclasses and IgM were present with IgG2b-producing AFCs being dominant or codominant with IgA or IgG2a. In serum and in all of the tissues examined, with the exception of the NALT, NOMVs clearly induced a stronger antibody response and a broader range of antibody isotypes than DOMVs. The development of NOMV-specific AFCs in spleen and bone marrow after intranasal immunization was slow compared to intravenous immunization but, once established, the intranasally elicited responses increased steadily for at least 75 days. NOMV-specific antibodies induced via several routes of immunization had high bactericidal activities in serum. Our results indicated that intranasally administered OMVs induced strong local and systemic antibody responses in mice that were relatively long-lived. PMID:15102760

  8. Sensor kinase RscS induces the production of antigenically distinct outer membrane vesicles that depend on the symbiosis polysaccharide locus in Vibrio fischeri.

    Science.gov (United States)

    Shibata, Satoshi; Visick, Karen L

    2012-01-01

    Robust biofilm formation by Vibrio fischeri depends upon activation of the symbiosis polysaccharide (syp) locus, which is achieved by overexpressing the RscS sensor kinase (RscS(+)). Other than the Syp polysaccharide, however, little is known about V. fischeri biofilm matrix components. In other bacteria, biofilms contain polysaccharides, secreted proteins, and outer membrane vesicles (OMVs). Here, we asked whether OMVs are part of V. fischeri biofilms. Transmission electron microscopy revealed OMV-like particles between cells within colonies. In addition, OMVs could be purified from culture supernatants of both RscS(+) and control cells, with the former releasing 2- to 3-fold more OMVs. The increase depended upon the presence of an intact syp locus, as an RscS(+) strain deleted for sypK, which encodes a putative oligosaccharide translocase, exhibited reduced production of OMVs; it also showed a severe defect in biofilm formation. Western immunoblot analyses revealed that the RscS(+) strain, but not the control strain or the RscS(+) sypK mutant, produced a distinct set of nonproteinaceous molecules that could be detected in whole-cell extracts, OMV preparations, and lipopolysaccharide (LPS) extracts. Finally, deletion of degP, which in other bacteria influences OMV production, decreased OMV production and reduced the ability of the cells to form biofilms. We conclude that overexpression of RscS induces OMV production in a manner that depends on the presence of the syp locus and that OMVs produced under these conditions contain antigenically distinct molecules, possibly representing a modified form of lipopolysaccharide (LPS). Finally, our data indicate a correlation between OMV production and biofilm formation by V. fischeri.

  9. Activation of immune and defense responses in the intestinal mucosa by outer membrane vesicles of commensal and probiotic Escherichia coli strains

    Directory of Open Access Journals (Sweden)

    Maria José eFábrega

    2016-05-01

    Full Text Available The influence of microbiota in human health is well known. Imbalances in microbiome structure have been linked to several diseases. Modulation of microbiota composition through probiotic therapy is an attempt to harness the beneficial effects of commensal microbiota. Although there is wide knowledge of the responses induced by gut microbiota, the microbial factors that mediate these effects are not well known. Gram-negative bacteria release outer membrane vesicles (OMVs as a secretion mechanism of microbial factors, which have an important role in intercellular communication. Here, we investigated whether OMVs from the probiotic Escherichia coli strain Nissle 1917 or the commensal E. coli strain ECOR12 trigger immune responses in various cellular models: (i peripheral blood mononuclear cells (PBMCs as a model of intestinal barrier disruption, (ii apical stimulation of Caco-2/PMBCs co-culture as a model of intact intestinal mucosa, and (iii colonic mucosa explants as an ex vivo model. Stimulations with bacterial lysates were also performed. Whereas both OMVs and lysates activated expression and secretion of several cytokines and chemokines in PBMCs, only OMVs induced basolateral secretion and mRNA upregulation of these mediators in the co-culture model. We provide evidence that OMVs are internalized in polarized Caco-2 cells. The activated epithelial cells elicit a response in the underlying immunocompetent cells. The OMVs effects were corroborated in the ex vivo model. This experimental study shows that OMVs are an effective strategy used by beneficial gut bacteria to communicate with and modulate host responses, activating signaling events through the intestinal epithelial barrier.

  10. Isomerization and fluorescence depolarization of merocyanine 540 ...

    Indian Academy of Sciences (India)

    It is observed that the dynamics of isomerization and depolarization are sensitive enough to monitor the uncoiling of PAA at high H (> 6). At low H (< 3), when the polymer remains in a hypercoiled form, polymer bound MC540 experiences very high microscopic friction and, hence, the isomerization and depolarization ...

  11. Kinetic regulation of coated vesicle secretion

    CERN Document Server

    Foret, Lionel

    2008-01-01

    The secretion of vesicles for intracellular transport often rely on the aggregation of specialized membrane-bound proteins into a coat able to curve cell membranes. The nucleation and growth of a protein coat is a kinetic process that competes with the energy-consuming turnover of coat components between the membrane and the cytosol. We propose a generic kinetic description of coat assembly and the formation of coated vesicles, and discuss its implication to the dynamics of COP vesicles that traffic within the Golgi and with the Endoplasmic Reticulum. We show that stationary coats of fixed area emerge from the competition between coat growth and the recycling of coat components, in a fashion resembling the treadmilling of cytoskeletal filaments. We further show that the turnover of coat components allows for a highly sensitive switching mechanism between a quiescent and a vesicle producing membrane, upon a slowing down of the exchange kinetics. We claim that the existence of this switching behaviour, also tri...

  12. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  13. The fluorescence intensity of the lipophilic probe N-phenyl-1-naphthylamine responds to the oxidation-reduction state of the respiratory chain in everted membrane vesicles of Escherichia coli.

    Science.gov (United States)

    Sedgwick, E G; Bragg, P D

    1987-06-22

    N-Phenyl-1-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to metabolic changes in everted inner membrane vesicles from this organism. NPN was bound to the vesicles to produce a steady-state level of fluorescence intensity. Addition of substrate or ATP did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of substrate through the respiratory chain, there was an increase in the fluorescence of NPN. Reoxidation of the components of the respiratory chain by the addition of oxygen, ferricyanide, fumarate or nitrate decreased fluorescence to the steady-state level until the oxidant had been completely reduced. The fluorescence changes were insensitive to the state of energization of the membrane. It is proposed that NPN responds to the state of reduction of components of the respiratory chain either directly by reacting with a component of the chain or indirectly through an effect transmitted to the membrane by a change in the conformation of respiratory chain components.

  14. Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p ▿

    Science.gov (United States)

    Basso, Luiz R.; Gast, Charles E.; Mao, Yuxin; Wong, Brian

    2010-01-01

    A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1- and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [3H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1-regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1-, CDR2-, or MDR1-overexpressing cells accumulated much more [3H]fluconazole than did PGVs from empty-vector controls. The Kms (expressed in micromolar concentrations) and Vmaxs (expressed in picomoles per milligram of protein per minute), respectively, for [3H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [3H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [3H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [3H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC50s; expressed as proportions of the [3H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi. PMID:20348384

  15. Trafficking of astrocytic vesicles in hippocampal slices

    Energy Technology Data Exchange (ETDEWEB)

    Potokar, Maja; Kreft, Marko [Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana (Slovenia); Celica Biomedical Center, Technology Park 24, 1000 Ljubljana (Slovenia); Lee, So-Young; Takano, Hajime; Haydon, Philip G. [Department of Neuroscience, Room 215, Stemmler Hall, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104 (United States); Zorec, Robert, E-mail: Robert.Zorec@mf.uni-lj.si [Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana (Slovenia); Celica Biomedical Center, Technology Park 24, 1000 Ljubljana (Slovenia)

    2009-12-25

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  16. Extracellular Vesicles in Lung Disease.

    Science.gov (United States)

    Kubo, Hiroshi

    2018-01-01

    Accumulating evidence suggests that extracellular vesicles (EVs) play a role in the pathogenesis of lung diseases. These vesicles include exosomes, ectosomes (ie, microparticles, extracellular vesicles, microvesicles, and shedding vesicles), and apoptotic bodies. Exosomes are generated by inward budding of the membrane (endocytosis), subsequent forming of multivesicular bodies, and release by exocytosis. Ectosomes are formed by outward blebbing from the plasma membrane and are then released by proteolytic cleavage from the cell surface. Apoptotic bodies are generated on apoptotic cell shrinkage and death. Extracellular vesicles are released when the cells are activated or undergo apoptosis under inflammatory conditions. The number and types of released EVs are different according to the pathophysiological status of the disease. Therefore, EVs can be novel biomarkers for various lung diseases. EVs contain several molecules, including proteins, mRNA, microRNA, and DNA; they transfer these molecules to distant recipient cells. Circulating EVs modify the targeted cells and influence the microenvironment of the lungs. For this unique capability, EVs are expected to be a new drug delivery system and a novel therapeutic target. Copyright © 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  17. SMALL VESICLES, BIG VEHICLES: EXOSOMES.

    Directory of Open Access Journals (Sweden)

    Saiz-Lopez P

    2016-09-01

    Full Text Available Exosomes are small membranous vesicles released by different cell types. Since their discovery, they have evolved from being considered simple vehicles for the liberation of cellular wastes, to become one of the most promising fields in the area of biomedical research, and more specifically in oncology, since the different malignant tumors release exosomes to all biological fluids, being involved in various functions of the neoplastic process. At present, it is possible to study these vesicles by minimally invasive techniques in patients, which approach us to obtain a more detailed diagnosis and prognosis, as well as to the discovery of new antitumoral therapies

  18. Stability of Spherical Vesicles in Electric Fields

    Science.gov (United States)

    2010-01-01

    The stability of spherical vesicles in alternating (ac) electric fields is studied theoretically for asymmetric conductivity conditions across their membranes. The vesicle deformation is obtained from a balance between the curvature elastic energies and the work done by the Maxwell stresses. The present theory describes and clarifies the mechanisms for the four types of morphological transitions observed experimentally on vesicles exposed to ac fields in the frequency range from 500 to 2 × 107 Hz. The displacement currents across the membranes redirect the electric fields toward the membrane normal to accumulate electric charges by the Maxwell−Wagner mechanism. These accumulated electric charges provide the underlying molecular mechanism for the morphological transitions of vesicles as observed on the micrometer scale. PMID:20575588

  19. An Active Form of Sphingosine Kinase-1 Is Released in the Extracellular Medium as Component of Membrane Vesicles Shed by Two Human Tumor Cell Lines

    Directory of Open Access Journals (Sweden)

    Salvatrice Rigogliuso

    2010-01-01

    Full Text Available Expression of sphingosine kinase-1 (SphK-1 correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P, a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis.

  20. Mechanics of post-fusion exocytotic vesicle.

    Science.gov (United States)

    Stephens, Thomas; Wu, Zhanghan; Liu, Jian

    2017-05-23

    Exocytosis is an important cellular process controlled by metabolic signaling. It involves vesicle fusion to the plasma membrane, followed by the opening of a fusion pore, and the subsequent release of the vesicular lumen content into the extracellular space. While most modeling efforts focus on the events leading to membrane fusion, how the vesicular membrane remodels after fusing to plasma membrane remains unclear. This latter event dictates the nature and the efficiency of exocytotic vesicular secretions, and is thus critical for exocytotic function. We provide a generic membrane mechanical model to systematically study the fate of post-fusion vesicles. We show that while membrane stiffness favors full-collapse vesicle fusion into the plasma membrane, the intravesicular pressure swells the vesicle and causes the fusion pore to shrink. Dimensions of the vesicle and its associated fusion pore further modulate this mechanical antagonism. We systematically define the mechanical conditions that account for the full spectrum of the observed vesicular secretion modes. Our model therefore can serve as a unified theoretical framework that sheds light on the elaborate control mechanism of exocytosis.

  1. Mechanics of post-fusion exocytotic vesicle

    Science.gov (United States)

    Stephens, Thomas; Wu, Zhanghan; Liu, Jian

    2017-06-01

    Exocytosis is an important cellular process controlled by metabolic signaling. It involves vesicle fusion to the plasma membrane, followed by the opening of a fusion pore, and the subsequent release of the vesicular lumen content into the extracellular space. While most modeling efforts focus on the events leading to membrane fusion, how the vesicular membrane remodels after fusing to plasma membrane remains unclear. This latter event dictates the nature and the efficiency of exocytotic vesicular secretions, and is thus critical for exocytotic function. We provide a generic membrane mechanical model to systematically study the fate of post-fusion vesicles. We show that while membrane stiffness favors full-collapse vesicle fusion into the plasma membrane, the intravesicular pressure swells the vesicle and causes the fusion pore to shrink. Dimensions of the vesicle and its associated fusion pore further modulate this mechanical antagonism. We systematically define the mechanical conditions that account for the full spectrum of the observed vesicular secretion modes. Our model therefore can serve as a unified theoretical framework that sheds light on the elaborate control mechanism of exocytosis.

  2. Minimal experimental requirements for definition of extracellular vesicles and their functions : a position statement from the International Society for Extracellular Vesicles

    NARCIS (Netherlands)

    Lötvall, Jan; Hill, Andrew F; Hochberg, Fred; Buzás, Edit I; Di Vizio, Dolores; Gardiner, Christopher; Gho, Yong Song; Kurochkin, Igor V; Mathivanan, Suresh; Quesenberry, Peter; Sahoo, Susmita; Tahara, Hidetoshi; Wauben, Marca H|info:eu-repo/dai/nl/112675735; Witwer, Kenneth W; Théry, Clotilde

    2014-01-01

    Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently

  3. Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake.

    Science.gov (United States)

    Abels, Erik R; Breakefield, Xandra O

    2016-04-01

    Extracellular vesicles are a heterogeneous group of membrane-limited vesicles loaded with various proteins, lipids, and nucleic acids. Release of extracellular vesicles from its cell of origin occurs either through the outward budding of the plasma membrane or through the inward budding of the endosomal membrane, resulting in the formation of multivesicular bodies, which release vesicles upon fusion with the plasma membrane. The release of vesicles can facilitate intercellular communication by contact with or by internalization of contents, either by fusion with the plasma membrane or by endocytosis into "recipient" cells. Although the interest in extracellular vesicle research is increasing, there are still no real standards in place to separate or classify the different types of vesicles. This review provides an introduction into this expanding and complex field of research focusing on the biogenesis, nucleic acid cargo loading, content, release, and uptake of extracellular vesicles.

  4. Hierarchical unilamellar vesicles of controlled compositional heterogeneity.

    Directory of Open Access Journals (Sweden)

    Maik Hadorn

    Full Text Available Eukaryotic life contains hierarchical vesicular architectures (i.e. organelles that are crucial for material production and trafficking, information storage and access, as well as energy production. In order to perform specific tasks, these compartments differ among each other in their membrane composition and their internal cargo and also differ from the cell membrane and the cytosol. Man-made structures that reproduce this nested architecture not only offer a deeper understanding of the functionalities and evolution of organelle-bearing eukaryotic life but also allow the engineering of novel biomimetic technologies. Here, we show the newly developed vesicle-in-water-in-oil emulsion transfer preparation technique to result in giant unilamellar vesicles internally compartmentalized by unilamellar vesicles of different membrane composition and internal cargo, i.e. hierarchical unilamellar vesicles of controlled compositional heterogeneity. The compartmentalized giant unilamellar vesicles were subsequently isolated by a separation step exploiting the heterogeneity of the membrane composition and the encapsulated cargo. Due to the controlled, efficient, and technically straightforward character of the new preparation technique, this study allows the hierarchical fabrication of compartmentalized giant unilamellar vesicles of controlled compositional heterogeneity and will ease the development of eukaryotic cell mimics that resemble their natural templates as well as the fabrication of novel multi-agent drug delivery systems for combination therapies and complex artificial microreactors.

  5. Morphology transition of raft-model membrane induced by osmotic pressure: Formation of double-layered vesicle similar to an endo- and/or exocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Onai, Teruaki; Hirai, Mitsuhiro, E-mail: mhirai@fs.aramaki.gunma-u.ac.j [Department of Physics, Gunma University, Maebashi 371-8510 (Japan)

    2010-10-01

    The effect of osmotic pressure on the structure of large uni-lamellar vesicle (LUV) of the lipid mixtures of monosialoganglioside (G{sub M1})-cholesterol-dioleoyl-phosphatidylcholine (DOPC) was studies by using wide-angle X-ray scattering (WAXS) method. The molar ratios of the mixtures were 0.1/0.1/1, 0/0.1/1, and 0/0/1. The ternary lipid mixture is a model of lipid rafts. The value of osmotic pressure was varied from 0 to 4.16x10{sup 5} N/m{sup 2} by adding the polyvinylpyrrolidone (PVP) in the range from 0 to 25 % w/v. In the case of the mixtures without G{sub M1}, the rise of the osmotic pressure just enhances the multi-lamellar stacking with deceasing the inter-lamellar spacing. On the other hand, the mixture containing G{sub M1} shows the structural transition from a uni-lamellar vesicle to a double-layered vesicle (a liposome including a smaller one inside) by the rise of osmotic pressure. In this morphology transition the total surface area of the double-layered vesicle is mostly as same as that of the LUV at the initial state. The polar head region of G{sub M1} is bulky and highly hydrophilic due to the oligosaccharide chain containing a sialic acid residue. Then, the present results suggest that the existence of G{sub M1} in the outer-leaflet of the LUV is essentially important for such a double-layered vesicle formation. Alternatively, a phenomenon similar to an endo- and/or exocytosis in cells can be caused simply by a variation of osmotic pressure.

  6. Pushing synaptic vesicles over the RIM.

    Science.gov (United States)

    Kaeser, Pascal S

    2011-05-01

    In a presynaptic nerve terminal, neurotransmitter release is largely restricted to specialized sites called active zones. Active zones consist of a complex protein network, and they organize fusion of synaptic vesicles with the presynaptic plasma membrane in response to action potentials. Rab3-interacting molecules (RIMs) are central components of active zones. In a recent series of experiments, we have systematically dissected the molecular mechanisms by which RIMs operate in synaptic vesicle release. We found that RIMs execute two critical functions of active zones by virtue of independent protein domains. They tether presyanptic Ca(2+) channels to the active zone, and they activate priming of synaptic vesicles by monomerizing homodimeric, constitutively inactive Munc13. These data indicate that RIMs orchestrate synaptic vesicle release into a coherent process. In conjunction with previous studies, they suggest that RIMs form a molecular platform on which plasticity of synaptic vesicle release can operate.

  7. Apoptotic Bodies: Selective Detection in Extracellular Vesicles.

    Science.gov (United States)

    Hauser, Paul; Wang, Sha; Didenko, Vladimir V

    2017-01-01

    Normal and dying cells release various types of membrane-bound vesicles including microvesicles, exosomes, and apoptotic bodies. These vesicles play important roles in intercellular communication and signal transduction. However, their diverse forms and subtypes fluctuate in size and other properties. In result current purification approaches do not fully discriminate between different categories of extracellular vesicles. Here, we present a fluorescence technique that specifically identifies apoptotic bodies in preparations of microvesicles, exosomes, and other extracellular vesicles.The approach exclusively labels the vesicles that contain DNA with 5'PO 4 blunt-ended DNA breaks, such as those produced by the apoptotic CAD nuclease during apoptotic DNA degradation. The technique can be useful in studies of apoptosis involving microvesicles and exosomes.

  8. Weaver mutant mouse cerebellar granule cells respond normally to chronic depolarization

    DEFF Research Database (Denmark)

    Bjerregaard, Annette; Mogensen, Helle Smidt; Hack, N

    1997-01-01

    We studied the effects of chronic K(+)-induced membrane depolarization and treatment with N-methyl-D-aspartate (NMDA) on cerebellar granule cells (CGCs) from weaver mutant mice and non-weaver litter-mates. The weaver mutation is a Gly-to-Ser substitution in a conserved region of the Girk2 G prote...

  9. Effects of D-600 on intramembrane charge movement of polarized and depolarized frog muscle fibers.

    Science.gov (United States)

    Caputo, C; Bolaños, P

    1989-07-01

    Intramembrane charge movement has been measured in frog cut skeletal muscle fibers using the triple vaseline gap voltage-clamp technique. Ionic currents were reduced using an external solution prepared with tetraethylammonium to block potassium currents, and O sodium + tetrodotoxin to abolish sodium currents. The internal solution contained 10 mM EGTA to prevent contractions. Both the internal and external solutions were prepared with impermeant anions. Linear capacitive currents were subtracted using the P-P/4 procedure, with the control pulses being subtracted either at very negative potentials, for the case of polarized fibers, or at positive potentials, for the case of depolarized fibers. In 63 polarized fibers dissected from Rana pipiens or Leptodactylus insularis frogs the following values were obtained for charge movement parameters: Qmax = 39 nC/microF, V = 36 mV, k = 18.5 mV. After depolarization we found that the total amount of movable charge was not appreciably reduced, while the voltage sensitivity was much changed. For 10 fibers, in which charge movement was measured at -100 and at 0 mV, Qmax changed from 46 to 41 nC/microF, while V changed from -41 to -103 mV and k changed from 20.5 to 30 mV. Thus membrane depolarization to 0 mV produces a shift of greater than 50 mV in the Q-V relationship and a decrease of the slope. Membrane depolarization to -20 and -30 mV, caused a smaller shift of the Q-V relationship. In normally polarized fibers addition of D-600 at concentrations of 50-100 microM, does not cause important changes in charge movement parameters. However, the drug appears to have a use-dependent effect after depolarization. Thus in depolarized fibers, total charge is reduced by approximately 20%. D-600 causes no further changes in the voltage sensitivity of charge movement in fibers depolarized to 0 mV, while in fibers depolarized to -20 and -30 mV it causes the same effects as that obtained with depolarization to 0 mV. These results are

  10. Dynamics of endocytic vesicle creation.

    Science.gov (United States)

    Perrais, David; Merrifield, Christien J

    2005-11-01

    Clathrin-mediated endocytosis is the main path for receptor internalization in metazoans and is essential for controlling cell integrity and signaling. It is driven by a large array of protein and lipid interactions that have been deciphered mainly by biochemical and genetic means. To place these interactions into context, and ultimately build a fully operative model of endocytosis at the molecular level, it is necessary to know the kinetic details of the role of each protein in this process. In this review, we describe the recent efforts made, by using live cell imaging, to define clear steps in the formation of endocytic vesicles and to observe the recruitment of key proteins during membrane invagination, the scission of a newly formed vesicle, and its movement away from the plasma membrane.

  11. Extracellular vesicles and blood diseases.

    Science.gov (United States)

    Nomura, Shosaku

    2017-04-01

    Extracellular vesicles (EVs) are small membrane vesicles released from many different cell types by the exocytic budding of the plasma membrane in response to cellular activation or apoptosis. EVs disseminate various bioactive effectors originating from the parent cells and transfer functional RNA and protein between cells, enabling them to alter vascular function and induce biological responses involved in vascular homeostasis. Although most EVs in human blood originate from platelets, EVs are also released from leukocytes, erythrocytes, endothelial cells, smooth muscle cells, and cancer cells. EVs were initially thought to be small particles with procoagulant activity; however, they can also evoke cellular responses in the immediate microenvironments and transport microRNAs (miRNA) into target cells. In this review, we summarize the recent literature relevant to EVs, including a growing list of clinical disorders that are associated with elevated EV levels. These studies suggest that EVs play roles in various blood diseases.

  12. Coated vesicles as protein release mechanism in myeloma cells.

    Science.gov (United States)

    Trombetta, L D; Lazarus, S S

    An electron microscopic study was undertaken of the protein release mechanism within myeloma cells showing a very high degree of protein production. Smooth surfaced vesicles (50 millimicrons) were seen to originate from the outer margin of the perinuclear cistern. Similar vesicles were also associated with distended Golgi sacs. Possible function of these vesicles could not be determined. Coated vesicles (60 millimicrons) originated as evaginations from endoplasmic reticulum in the transitional region. They were present throughout the cytoplasm and were seen to fuse with the cell membrane discharging an electron dense material. These vesicles are, therefore, thought to transport protein from the rough endoplasmic reticulum and discharge it at the cell surface.

  13. Electrohydrodynamics of a compound vesicle under an AC electric field.

    Science.gov (United States)

    Sinha, Kumari Priti; Thaokar, Rochish M

    2017-07-12

    Compound vesicles are relevant as simplified models for biological cells as well as in technological applications such as drug delivery. Characterization of these compound vesicles, especially the inner vesicle, remains a challenge. Similarly their response to electric field assumes importance in light of biomedical applications such as electroporation. Fields lower than that required for electroporation cause electrodeformation in vesicles and can be used to characterize their mechanical and electrical properties. A theoretical analysis of the electrohydrodynamics of a compound vesicle with outer vesicle of radius R o and an inner vesicle of radius [Formula: see text], is presented. A phase diagram for the compound vesicle is presented and elucidated using detailed plots of electric fields, free charges and electric stresses. The electrohydrodynamics of the outer vesicle in a compound vesicle shows a prolate-sphere and prolate-oblate-sphere shape transitions when the conductivity of the annular fluid is greater than the outer fluid, and vice-versa respectively, akin to single vesicle electrohydrodynamics reported in the literature. The inner vesicle in contrast shows sphere-prolate-sphere and sphere-prolate-oblate-sphere transitions when the inner fluid conductivity is greater and smaller than the annular fluid, respectively. Equations and methodology are provided to determine the bending modulus and capacitance of the outer as well as the inner membrane, thereby providing an easy way to characterize compound vesicles and possibly biological cells.

  14. Electrohydrodynamics of a compound vesicle under an AC electric field

    Science.gov (United States)

    Priti Sinha, Kumari; Thaokar, Rochish M.

    2017-07-01

    Compound vesicles are relevant as simplified models for biological cells as well as in technological applications such as drug delivery. Characterization of these compound vesicles, especially the inner vesicle, remains a challenge. Similarly their response to electric field assumes importance in light of biomedical applications such as electroporation. Fields lower than that required for electroporation cause electrodeformation in vesicles and can be used to characterize their mechanical and electrical properties. A theoretical analysis of the electrohydrodynamics of a compound vesicle with outer vesicle of radius R o and an inner vesicle of radius λ {{R}o} , is presented. A phase diagram for the compound vesicle is presented and elucidated using detailed plots of electric fields, free charges and electric stresses. The electrohydrodynamics of the outer vesicle in a compound vesicle shows a prolate-sphere and prolate-oblate-sphere shape transitions when the conductivity of the annular fluid is greater than the outer fluid, and vice-versa respectively, akin to single vesicle electrohydrodynamics reported in the literature. The inner vesicle in contrast shows sphere-prolate-sphere and sphere-prolate-oblate-sphere transitions when the inner fluid conductivity is greater and smaller than the annular fluid, respectively. Equations and methodology are provided to determine the bending modulus and capacitance of the outer as well as the inner membrane, thereby providing an easy way to characterize compound vesicles and possibly biological cells.

  15. Preeclampsia and Extracellular Vesicles.

    Science.gov (United States)

    Gilani, Sarwat I; Weissgerber, Tracey L; Garovic, Vesna D; Jayachandran, Muthuvel

    2016-09-01

    Preeclampsia is a hypertensive pregnancy disorder characterized by development of hypertension and proteinuria after 20 weeks of gestation that remains a leading cause of maternal and neonatal morbidity and mortality. While preeclampsia is believed to result from complex interactions between maternal and placental factors, the proximate pathophysiology of this syndrome remains elusive. Cell-to-cell communication is a critical signaling mechanism for feto-placental development in normal pregnancies. One mechanism of cellular communication relates to activated cell-derived sealed membrane vesicles called extracellular vesicles (EVs). The concentrations and contents of EVs in biological fluids depend upon their cells of origin and the stimuli which trigger their production. Research on EVs in preeclampsia has focused on EVs derived from the maternal vasculature (endothelium, vascular smooth muscle) and blood (erythrocytes, leukocytes, and platelets), as well as placental syncytiotrophoblasts. Changes in the concentrations and contents of these EVs may contribute to the pathophysiology of preeclampsia by accentuating the pro-inflammatory and pro-coagulatory states of pregnancy. This review focuses on possible interactions among placental- and maternal-derived EVs and their contents in the initiation and progression of the pathogenesis of preeclampsia. Understanding the contributions of EVs in the pathogenesis of preeclampsia may facilitate their use as diagnostic and prognostic biomarkers.

  16. Ultracold neutron depolarization in magnetic bottles

    CERN Document Server

    Steyerl, A; Müller, G; Malik, S S; Desai, A M

    2013-01-01

    We analyze the depolarization of ultracold neutrons confined in a magnetic field configuration similar to those used in existing or proposed magneto-gravitational storage experiments aiming at a precise measurement of the neutron lifetime. We use an extension of the semi-classical Majorana approach as well as an approximate quantum mechanical analysis, both pioneered by Walstrom et al. [Nucl. Instr. Meth. Phys. Res. A 599, 82 (2009)]. In contrast with this previous work we do not restrict the analysis to purely vertical modes of neutron motion. The lateral motion is shown to cause the predominant depolarization loss in a magnetic storage trap. The system studied also allowed us to estimate the depolarization loss suffered by ultracold neutrons totally reflected on a non-magnetic mirror immersed in a magnetic field. This problem is of preeminent importance in polarized neutron decay studies such as the measurement of the asymmetry parameter A using ultracold neutrons, and it may limit the efficiency of ultraco...

  17. Zinc depolarized electrochemical CO2 concentration

    Science.gov (United States)

    Woods, R. R.; Marshall, R. D.; Schubert, F. H.

    1975-01-01

    Two zinc depolarized electrochemical carbon dioxide concentrator concepts were analytically and experimentally evaluated for portable life support system carbon dioxide (CO2) removal application. The first concept, referred to as the zinc hydrogen generator electrochemical depolarized CO2 concentrator, uses a ZHG to generate hydrogen for direct use in an EDC. The second concept, referred to as the zinc/electrochemical depolarized concentrator, uses a standard EDC cell construction modified for use with the Zn anode. The Zn anode is consumed and subsequently regenerated, thereby eliminating the need to supply H2 to the EDC for the CO2 removal process. The evaluation was based primarily on an analytical evaluation of the two ZnDCs at projected end item performance and hardware design levels. Both ZnDC concepts for PLSS CO2 removal application were found to be noncompetitive in both total equivalent launch weight and individual extravehicular activity mission volume when compared to other candidate regenerable PLSS CO2 scrubbers.

  18. Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms

    OpenAIRE

    Bellier, Bertrand; Sedlik, Christine; Vigneron, James; Torrieri-Dramard, Lea; Pitoiset, Fabien; Denizeau, Jordan; Chesneau, Caroline; de la Rochere, Philippe; Lantz, Olivier; Thery, Clotilde

    2014-01-01

    The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs) to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccine...

  19. Extracellular vesicles in coronary artery disease.

    Science.gov (United States)

    Boulanger, Chantal M; Loyer, Xavier; Rautou, Pierre-Emmanuel; Amabile, Nicolas

    2017-05-01

    Membrane vesicles released in the extracellular space are composed of a lipid bilayer enclosing soluble cytosolic material and nuclear components. Extracellular vesicles include apoptotic bodies, exosomes, and microvesicles (also known previously as microparticles). Originating from different subcellular compartments, the role of extracellular vesicles as regulators of transfer of biological information, acting locally and remotely, is now acknowledged. Circulating vesicles released from platelets, erythrocytes, leukocytes, and endothelial cells contain potential valuable biological information for biomarker discovery in primary and secondary prevention of coronary artery disease. Extracellular vesicles also accumulate in human atherosclerotic plaques, where they affect major biological pathways, including inflammation, proliferation, thrombosis, calcification, and vasoactive responses. Extracellular vesicles also recapitulate the beneficial effect of stem cells to treat cardiac consequences of acute myocardial infarction, and now emerge as an attractive alternative to cell therapy, opening new avenues to vectorize biological information to target tissues. Although interest in microvesicles in the cardiovascular field emerged about 2 decades ago, that for extracellular vesicles, in particular exosomes, started to unfold a decade ago, opening new research and therapeutic avenues. This Review summarizes current knowledge on the role of extracellular vesicles in coronary artery disease, and their emerging potential as biomarkers and therapeutic agents.

  20. Luminescent functionalized vesicles: synthesis, characterization and analytical applications

    OpenAIRE

    Balk, Stefan

    2014-01-01

    This work describes the membrane functionalization of small unilamellar phospholipid vesicles by incorporation of artificial amphiphiles. The presented investigations demonstrate a fast and simple approach for sensing molecular recognition events at the membrane-water interface. Chapter 1 describes the dynamic recognition of multivalent ligands by receptor recruiting in fluid vesicle membranes. Two amphiphilic metal-complexes with attached FRET-pair labels were prepared and embedded into D...

  1. Functionalization of Block Copolymer Vesicle Surfaces

    Directory of Open Access Journals (Sweden)

    Wolfgang Meier

    2011-01-01

    Full Text Available In dilute aqueous solutions certain amphiphilic block copolymers self-assemble into vesicles that enclose a small pool of water with a membrane. Such polymersomes have promising applications ranging from targeted drug-delivery devices, to biosensors, and nanoreactors. Interactions between block copolymer membranes and their surroundings are important factors that determine their potential biomedical applications. Such interactions are influenced predominantly by the membrane surface. We review methods to functionalize block copolymer vesicle surfaces by chemical means with ligands such as antibodies, adhesion moieties, enzymes, carbohydrates and fluorophores. Furthermore, surface-functionalization can be achieved by self-assembly of polymers that carry ligands at their chain ends or in their hydrophilic blocks. While this review focuses on the strategies to functionalize vesicle surfaces, the applications realized by, and envisioned for, such functional polymersomes are also highlighted.

  2. Modeling of thermally stimulated depolarization current (TSDC ...

    Indian Academy of Sciences (India)

    Keywords. Dipole–dipole interaction; relaxation; modeling; TSDC; activation energy; PVC; ABS. Abstract. The study of thermally stimulated depolarization current (TSDC) using the dipole–dipole interaction model is described in this work. The dipole–dipole interactionmodel (DDIM) determines the TSDC peak successfully ...

  3. Slow Sedimentation and Deformability of Charged Lipid Vesicles

    Science.gov (United States)

    Rey Suárez, Iván; Leidy, Chad; Téllez, Gabriel; Gay, Guillaume; Gonzalez-Mancera, Andres

    2013-01-01

    The study of vesicles in suspension is important to understand the complicated dynamics exhibited by cells in in vivo and in vitro. We developed a computer simulation based on the boundary-integral method to model the three dimensional gravity-driven sedimentation of charged vesicles towards a flat surface. The membrane mechanical behavior was modeled using the Helfrich Hamiltonian and near incompressibility of the membrane was enforced via a model which accounts for the thermal fluctuations of the membrane. The simulations were verified and compared to experimental data obtained using suspended vesicles labelled with a fluorescent probe, which allows visualization using fluorescence microscopy and confers the membrane with a negative surface charge. The electrostatic interaction between the vesicle and the surface was modeled using the linear Derjaguin approximation for a low ionic concentration solution. The sedimentation rate as a function of the distance of the vesicle to the surface was determined both experimentally and from the computer simulations. The gap between the vesicle and the surface, as well as the shape of the vesicle at equilibrium were also studied. It was determined that inclusion of the electrostatic interaction is fundamental to accurately predict the sedimentation rate as the vesicle approaches the surface and the size of the gap at equilibrium, we also observed that the presence of charge in the membrane increases its rigidity. PMID:23874582

  4. Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis.

    Science.gov (United States)

    Lee, Jaewook; Kim, Si-Hyun; Choi, Dong-Sic; Lee, Jong Seok; Kim, Dae-Kyum; Go, Gyeongyun; Park, Seon-Min; Kim, Si Hyun; Shin, Jeong Hwan; Chang, Chulhun L; Gho, Yong Song

    2015-10-01

    The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC-MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 (http://proteomecentral.proteomexchange.org/dataset/PXD001160). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Exocytosis at the ribbon synapse of retinal bipolar cells studied in patches of presynaptic membrane.

    Science.gov (United States)

    Llobet, Artur; Cooke, Anne; Lagnado, Leon

    2003-04-01

    The distribution of exocytic sites and ion channels in the synaptic terminal of retinal bipolar cells was investigated by measuring capacitance and conductance changes in cell-attached patches of presynaptic membrane. Patch depolarization evoked capacitance and conductance increases that were inhibited by blocking Ca(2+) influx or loading the terminal with EGTA. The increase in capacitance declined as the depolarization approached the reversal potential for Ca(2+), indicating that it was a result of Ca(2+)-dependent exocytosis. The conductance increase was caused by K(Ca) channels that were also activated by Ca(2+) influx. Two observations indicated that sites of exocytosis and endocytosis colocalized with clusters of Ca(2+) channels and K(Ca) channels; the initial rate of exocytosis was correlated with the activation of K(Ca) channels, and exocytosis did not occur in the 41% of patches lacking this conductance. Electron microscopy demonstrated that there were approximately 16 vesicles docked to the plasma membrane at each active zone marked by a ribbon, but vesicles were also attached to the rest of the membrane at a density of 1.5/microm(2). The density of ribbons was 0.10 +/- 0.02/microm(2), predicting that approximately 43% of cell-attached patches would lack an active zone. The density of Ca(2+) channel clusters assayed by capacitance and conductance responses was therefore similar to the density of ribbons. These results are consistent with the idea that Ca(2+) channel clusters were colocalized with ribbons but do not exclude the possibility that calcium channels also occurred at other sites. The wide distribution of vesicles docked to the plasma membrane suggests that exocytosis might also be triggered by the spread of Ca(2+) from Ca(2+) channel clusters.

  6. Outer Membrane Vesicles from the Probiotic Escherichia coli Nissle 1917 and the Commensal ECOR12 Enter Intestinal Epithelial Cells via Clathrin-Dependent Endocytosis and Elicit Differential Effects on DNA Damage.

    Directory of Open Access Journals (Sweden)

    María-Alexandra Cañas

    Full Text Available Interactions between intestinal microbiota and the human host are complex. The gut mucosal surface is covered by a mucin layer that prevents bacteria from accessing the epithelial cells. Thus, the crosstalk between microbiota and the host mainly rely on secreted factors that can go through the mucus layer and reach the epithelium. In this context, vesicles released by commensal strains are seen as key players in signaling processes in the intestinal mucosa. Studies with Gram-negative pathogens showed that outer membrane vesicles (OMVs are internalized into the host cell by endocytosis, but the entry mechanism for microbiota-derived vesicles is unknown. Escherichia coli strains are found as part of normal human gut microbiota. In this work, we elucidate the pathway that mediate internalization of OMVs from the probiotic E.coli Nissle 1917 (EcN and the commensal ECOR12 strains in several human intestinal epithelial cell lines. Time course measurement of fluorescence and microscopy analysis performed with rhodamine B-R18-labeled OMVs in the presence of endocytosis inhibitors showed that OMVs from these strains enter epithelial cells via clathrin-mediated endocytosis. Vesicles use the same endocytosis pathway in polarized epithelial monolayers. Internalized OMVs are sorted to lysosomal compartments as shown by their colocalization with clathrin and specific markers of endosomes and lysosomes. OMVs from both strains did not affect cell viability, but reduce proliferation of HT-29 cells. Labeling of 8-oxo-dG adducts in DNA revealed that neither OMVs from EcN nor from ECOR12 promoted oxidative DNA damage. In contrast, flow cytometry analysis of phosphorylated γH2AX evidenced that OMVs from the probiotic EcN significantly produced more double strand breaks in DNA than ECOR12 OMVs. The EcN genotoxic effects have been attributed to the synthesis of colibactin. However, it is not known how colibactin is exported and delivered into host cells. Whether

  7. Soft vesicles in the synthesis of hard materials.

    Science.gov (United States)

    Dong, Renhao; Liu, Weimin; Hao, Jingcheng

    2012-04-17

    Vesicles of surfactants in aqueous solution have received considerable attention because of their use as simple model systems for biological membranes and their applications in various fields including colloids, pharmaceuticals, and materials. Because of their architecture, vesicles could prove useful as "soft" templates for the synthesis of "hard materials". The vesicle phase, however, has been challenging and difficult to work with in the construction of hard materials. In the solution-phase synthesis of various inorganic or macromolecular materials, templating methods provide a powerful strategy to control the size, morphology, and composition of the resulting micro- and nanostructures. In comparison with hard templates, soft templates are generally constructed using amphiphilic molecules, especially surfactants and amphiphilic polymers. These types of compounds offer advantages including the wide variety of available templates, simple fabrication processes under mild conditions, and easy removal of the templates with less damage to the final structures. Researchers have used many ordered molecular aggregates such as vesicles, micelles, liquid crystals, emulsion droplets, and lipid nanotubes as templates or structure-directing agents to control the synthesis or assembly hard micro- and nanomaterials composed from inorganic compounds or polymers. In addition to their range of sizes and morphologies, vesicles present unique structures that can simultaneously supply different microenvironments for the growth and assembly of hard materials: the inner chamber of vesicles, the outer surface of the vesicles, and the space between bilayers. Two main approaches for applying vesicles in the field of hard materials have been explored: (i) in situ synthesis of micro- or nanomaterials within a specific microenvironment by vesicle templating and (ii) the assembly or incorporation of guest materials during the formation of vesicles. This Account provides an in-depth look at

  8. MAA-1, a novel acyl-CoA-binding protein involved in endosomal vesicle transport in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Kobæk Larsen, Morten; Tuck, Simon; Færgeman, Nils J.

    2006-01-01

    The budding and fission of vesicles during membrane trafficking requires many proteins, including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted by the hydro...

  9. Optimal posttranslational translocation of the precursor of PhoE protein across Escherichia coli membrane vesicles requires both ATP and the protonmotive force

    NARCIS (Netherlands)

    Vrije, T. de; Tommassen, J.P.M.; Kruijff, B. de

    1987-01-01

    In order to reach their final destination, periplasmic and outer membrane proteins have to pass the cytoplasmic membrane of Escherichia coli cells. To study the transport of PhoE protein, we developed an in vitro transcription-translation and translocation system. In this in vitro system, the

  10. Recording, analysis, and interpretation of spreading depolarizations in neurointensive care

    DEFF Research Database (Denmark)

    Dreier, Jens P; Fabricius, Martin; Ayata, Cenk

    2017-01-01

    Spreading depolarizations (SD) are waves of abrupt, near-complete breakdown of neuronal transmembrane ion gradients, are the largest possible pathophysiologic disruption of viable cerebral gray matter, and are a crucial mechanism of lesion development. Spreading depolarizations are increasingly r...

  11. Extracellular vesicles as emerging intercellular communicasomes.

    Science.gov (United States)

    Yoon, Yae Jin; Kim, Oh Youn; Gho, Yong Song

    2014-10-01

    All living cells release extracellular vesicles having pleiotropic functions in intercellular communication. Mammalian extracellular vesicles, also known as exosomes and microvesicles, are spherical bilayered proteolipids composed of various bioactive molecules, including RNAs, DNAs, proteins, and lipids. Extracellular vesicles directly and indirectly control a diverse range of biological processes by transferring membrane proteins, signaling molecules, mRNAs, and miRNAs, and activating receptors of recipient cells. The active interaction of extracellular vesicles with other cells regulates various physiological and pathological conditions, including cancer, infectious diseases, and neurodegenerative disorders. Recent developments in high-throughput proteomics, transcriptomics, and lipidomics tools have provided ample data on the common and specific components of various types of extracellular vesicles. These studies may contribute to the understanding of the molecular mechanism involved in vesicular cargo sorting and the biogenesis of extracellular vesicles, and, further, to the identification of disease-specific biomarkers. This review focuses on the components, functions, and therapeutic and diagnostic potential of extracellular vesicles under various pathophysiological conditions.

  12. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles. Volume I

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR, HSR) were isolated from rabbit leg muscle. They were then diluted and washed with sucrose or KCl and referred to as sucrose or KCl washed vesicles. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes where as the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material. The LSR consists of predominantly Ca2+ + Mg2+ ATPase (80 to 90%), a small amount of the high affinity Ca binding protein (5%), and a 5000 dalton proteolipid. The sucrose HSR vesicles contain the Ca2+ + Mg2+ ATPase (50%), Calsequestrin (25%), high affinity Ca binding protein (5%), one extrinsic 34,000 dalton protein (3%), one intrinsic 30,000 dalton protein (3%), a 9000 dalton proteolipid, and a 5000 dalton proteolipid. The sucrose--washed HSR vesicles contain greater than three times the calcium content of the sucrose washed LSR vesicles where as the KCl--washed vesicles contain less than 15 nmoles Ca2+ mg of protein each. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP. Exchange of methanesulfonate for chloride resulted in the release of calcium from both the light and heavy SR vesicles. Sucrose causes a slight inhibition of chloride--induced calcium release from the heavy SR vesicles but it greatly reduces the release of calcium from the light SR vesicles. Sodium dantrolene (20 uM) has no effect on the release of calcium from the light SR vesicles but it inhibits the release of calcium from the heavy SR vesicles. The results indicate that the chloride--induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization.

  13. Hybrid, Nanoscale Phospholipid/Block Copolymer Vesicles

    Directory of Open Access Journals (Sweden)

    Bo Liedberg

    2013-09-01

    Full Text Available Hybrid phospholipid/block copolymer vesicles, in which the polymeric membrane is blended with phospholipids, display interesting self-assembly behavior, incorporating the robustness and chemical versatility of polymersomes with the softness and biocompatibility of liposomes. Such structures can be conveniently characterized by preparing giant unilamellar vesicles (GUVs via electroformation. Here, we are interested in exploring the self-assembly and properties of the analogous nanoscale hybrid vesicles (ca. 100 nm in diameter of the same composition prepared by film-hydration and extrusion. We show that the self-assembly and content-release behavior of nanoscale polybutadiene-b-poly(ethylene oxide (PB-PEO/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC hybrid phospholipid/block copolymer vesicles can be tuned by the mixing ratio of the amphiphiles. In brief, these hybrids may provide alternative tools for drug delivery purposes and molecular imaging/sensing applications and clearly open up new avenues for further investigation.

  14. IN-VITRO FUSION OF RETICULOCYTE ENDOCYTIC VESICLES WITH LIPOSOMES

    NARCIS (Netherlands)

    VIDAL, M; HOEKSTRA, D

    1995-01-01

    Since reticulocytes have a high demand for iron, which is required for heme biosynthesis, these cells are highly specialized in the endocytosis of the iron carrier transferrin (Tf). From the resulting endocytic vesicles (EVs), iron is released and the vesicles rapidly return to the cell membrane

  15. Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation

    NARCIS (Netherlands)

    Kalra, Hina; Simpson, Richard J.; Ji, Hong; Aikawa, Elena; Altevogt, Peter; Askenase, Philip; Bond, Vincent C.; Borràs, Francesc E.; Breakefield, Xandra; Budnik, Vivian; Buzas, Edit; Camussi, Giovanni; Clayton, Aled; Cocucci, Emanuele; Falcon-Perez, Juan M.; Gabrielsson, Susanne; Gho, Yong Song; Gupta, Dwijendra; Harsha, H. C.; Hendrix, An; Hill, Andrew F.; Inal, Jameel M.; Jenster, Guido; Krämer-Albers, Eva-Maria; Lim, Sai Kiang; Llorente, Alicia; Lötvall, Jan; Marcilla, Antonio; Mincheva-Nilsson, Lucia; Nazarenko, Irina; Nieuwland, Rienk; Nolte-'t Hoen, Esther N. M.; Pandey, Akhilesh; Patel, Tushar; Piper, Melissa G.; Pluchino, Stefano; Prasad, T. S. Keshava; Rajendran, Lawrence; Raposo, Graca; Record, Michel; Reid, Gavin E.; Sánchez-Madrid, Francisco; Schiffelers, Raymond M.; Siljander, Pia; Stensballe, Allan; Stoorvogel, Willem; Taylor, Douglas; Thery, Clotilde; Valadi, Hadi; van Balkom, Bas W. M.; Vázquez, Jesús; Vidal, Michel; Wauben, Marca H. M.; Yáñez-Mó, María; Zoeller, Margot; Mathivanan, Suresh

    2012-01-01

    Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These

  16. NK-3 receptor activation depolarizes and induces an after-depolarization in pyramidal neurons in gerbil cingulate cortex

    DEFF Research Database (Denmark)

    Rekling, Jens C

    2004-01-01

    M), a selective NK3 receptor agonist, induced a transient increase in spontaneous EPSPs in layer V pyramidal neurons, accompanied by a small depolarization ( approximately 4 mV). EPSPs during senktide had a larger amplitude and faster 10-90% rise time than during control. Senktide induced a transient...... depolarization in layer II/III pyramidal neurons, which often reached threshold for spikes. The depolarization ( approximately 6 mV) persisted in TTX, and was accompanied by an increase in input resistance. Senktide also transiently induced a slow after-depolarization, which appeared following a depolarizing...... pulse. The slow after-depolarization persisted in TTX. These data suggest that activation of NK3 receptors on layer II/III pyramidal neurons induce post-synaptic depolarization and an after-depolarization, which could be mediated by blockade of a leak potassium conductance and a non-selective cation...

  17. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  18. Exploring neuronal bistability at the depolarization block.

    Directory of Open Access Journals (Sweden)

    Andrey Dovzhenok

    Full Text Available Many neurons display bistability--coexistence of two firing modes such as bursting and tonic spiking or tonic spiking and silence. Bistability has been proposed to endow neurons with richer forms of information processing in general and to be involved in short-term memory in particular by allowing a brief signal to elicit long-lasting changes in firing. In this paper, we focus on bistability that allows for a choice between tonic spiking and depolarization block in a wide range of the depolarization levels. We consider the spike-producing currents in two neurons, models of which differ by the parameter values. Our dopaminergic neuron model displays bistability in a wide range of applied currents at the depolarization block. The Hodgkin-Huxley model of the squid giant axon shows no bistability. We varied parameter values for the model to analyze transitions between the two parameter sets. We show that bistability primarily characterizes the inactivation of the Na(+ current. Our study suggests a connection between the amount of the Na(+ window current and the length of the bistability range. For the dopaminergic neuron we hypothesize that bistability can be linked to a prolonged action of antipsychotic drugs.

  19. Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

    Directory of Open Access Journals (Sweden)

    R.M. Leão

    2009-01-01

    Full Text Available The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7 or older (p10-12 rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

  20. Effect of the achondroplasia mutation on FGFR3 dimerization and FGFR3 structural response to fgf1 and fgf2: A quantitative FRET study in osmotically derived plasma membrane vesicles.

    Science.gov (United States)

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-07-01

    The G380R mutation in the transmembrane domain of FGFR3 is a germline mutation responsible for most cases of Achondroplasia, a common form of human dwarfism. Here we use quantitative Fӧster Resonance Energy Transfer (FRET) and osmotically derived plasma membrane vesicles to study the effect of the achondroplasia mutation on the early stages of FGFR3 signaling in response to the ligands fgf1 and fgf2. Using a methodology that allows us to capture structural changes on the cytoplasmic side of the membrane in response to ligand binding to the extracellular domain of FGFR3, we observe no measurable effects of the G380R mutation on FGFR3 ligand-bound dimer configurations. Instead, the most notable effect of the achondroplasia mutation is increased propensity for FGFR3 dimerization in the absence of ligand. This work reveals new information about the molecular events that underlie the achondroplasia phenotype, and highlights differences in FGFR3 activation due to different single amino-acid pathogenic mutations. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Compartmentalization and Transport in Synthetic Vesicles

    Directory of Open Access Journals (Sweden)

    Christine eSchmitt

    2016-02-01

    Full Text Available Nano-scale vesicles have become a popular tool in life sciences. Besides liposomes that are generated from phospholipids of natural origin, polymersomes fabricated of synthetic block copolymers enjoy increasing popularity, as they represent more versatile membrane building blocks that can be selected based on their specific physicochemical properties, like permeability, stability or chemical reactivity.In this review, we focus on the application of simple and nested artificial vesicles in synthetic biology. First, we provide an introduction into the utilization of multi-compartmented vesosomes as compartmentalized nano-scale bioreactors. In the bottom-up development of protocells from vesicular nano-reactors, the specific exchange of pathway intermediates across compartment boundaries represents a bottleneck for future studies. To date, most compartmented bioreactors rely on unspecific exchange of substrates and products. This is either based on changes in permeability of the coblock polymer shell by physicochemical triggers or by the incorporation of unspecific porin proteins into the vesicle membrane. Since the incorporation of membrane transport proteins into simple and nested artificial vesicles offers the potential for specific exchange of substances between subcompartments, it opens new vistas in the design of protocells. Therefore we devote the main part of the review to summarize the technical advances in the use of phospholipids and block copolymers for the reconstitution of membrane proteins.

  2. Lipid Directed Intrinsic Membrane Protein Segregation

    DEFF Research Database (Denmark)

    Hansen, Jesper S.; Thompson, James R.; Helix Nielsen, Claus

    2013-01-01

    We demonstrate a new approach for direct reconstitution of membrane proteins during giant vesicle formation. We show that it is straightforward to create a tissue-like giant vesicle film swelled with membrane protein using aquaporin SoPIP2;1 as an illustration. These vesicles can also be easily h...

  3. Scolopendin 2, a cationic antimicrobial peptide from centipede, and its membrane-active mechanism.

    Science.gov (United States)

    Lee, Heejeong; Hwang, Jae-Sam; Lee, Jaeho; Kim, Jae Il; Lee, Dong Gun

    2015-02-01

    Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans. We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans, resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3'-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Polymer/TiO₂ hybrid vesicles for excellent UV screening and effective encapsulation of antioxidant agents.

    Science.gov (United States)

    Du, Jianzhong; Sun, Hui

    2014-08-27

    Presented in this paper is a hybrid polymer/titanium dioxide (TiO2) vesicle that has excellent UV-screening efficacy and strong capacity to encapsulate antioxidant agents. Poly(ethylene oxide)-block-poly(2-(dimethylamino)ethyl methacrylate)-block-polystyrene (PEO-b-PDMAEMA-b-PS) triblock terpolymer was synthesized by atom transfer radical polymerization (ATRP) and then self-assembled into vesicles. Those vesicles showed excellent UV-screening property due to the scattering by vesicles and the absorption by PS vesicle membrane. The selective deposition of solvophobic tetrabutyl titanate in the PDMAEMA shell and the PS membrane of the vesicles led to the formation of polymer/TiO2 hybrid vesicles, resulting in an enhanced UV-screening property by further reflecting and scattering UV radiation. The vesicles can effectively encapsulate antioxidant agents such as ferulic acid (up to 57%), showing a rapid antioxidant capability (within 1 min) and a long-lasting antioxidant effect.

  5. Role of extracellular vesicles in de novo mineralization: an additional novel mechanism of cardiovascular calcification.

    Science.gov (United States)

    New, Sophie E P; Aikawa, Elena

    2013-08-01

    Extracellular vesicles are membrane micro/nanovesicles secreted by many cell types into the circulation and the extracellular milieu in physiological and pathological conditions. Evidence suggests that extracellular vesicles, known as matrix vesicles, play a role in the mineralization of skeletal tissue, but emerging ultrastructural and in vitro studies have demonstrated their contribution to cardiovascular calcification as well. Cells involved in the progression of cardiovascular calcification release active vesicles capable of nucleating hydroxyapatite on their membranes. This review discusses the role of extracellular vesicles in cardiovascular calcification and elaborates on this additional mechanism of calcification as an alternative pathway to the currently accepted mechanism of biomineralization via osteogenic differentiation.

  6. CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

    NARCIS (Netherlands)

    S. Basu (Saonli); S. Sladecek (Stefan); I.M. De La Peña Y Valenzuela (Isabel Martinez); M. Akaaboune (Mohammed); I. Smal (Ihor); K. Martin (Katrin); N.J. Galjart (Niels); H.R. Brenner (Hans Rudolf)

    2015-01-01

    textabstractA hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to

  7. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.P.

    1978-01-01

    Light and heavy sarcoplasmic reticulum vesicles isolated from rabbit leg muscle have been used in a study of chloride-induced calcium release. The biochemical and morphological data indicate that light sarcoplasmic reticulum vesicles are derived from the longitudinal reticulum and heavy sarcoplasmic reticulum vesicles are derived from the terminal cisternae of the sarcoplasmic reticulum. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP to amounts greater than 100 nmoles Ca/sup + +/ per mg of protein in less than one minute. Light and heavy sarcoplasmic reticulum vesicles each had a biphasic time course of calcium uptake. The initial uptake was followed by a rapid release after approximately one minute, of 30 to 40% of the accumulated calcium, which was then followed by a slower phase of calcium accumulation. Results indicate that the chloride induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization. The release of calcium from the light SR vesicles is probably due to osmotic swelling and the release of calcium from the heavy SR vesicles is probably due to depolarization.

  8. Gram-negative and Gram-positive bacterial extracellular vesicles.

    Science.gov (United States)

    Kim, Ji Hyun; Lee, Jaewook; Park, Jaesung; Gho, Yong Song

    2015-04-01

    Like mammalian cells, Gram-negative and Gram-positive bacteria release nano-sized membrane vesicles into the extracellular environment either in a constitutive manner or in a regulated manner. These bacterial extracellular vesicles are spherical bilayered proteolipids enriched with bioactive proteins, lipids, nucleic acids, and virulence factors. Recent progress in this field supports the critical pathophysiological functions of these vesicles in both bacteria-bacteria and bacteria-host interactions. This review provides an overview of the current understanding on Gram-negative and Gram-positive bacterial extracellular vesicles, especially regarding the biogenesis, components, and functions in poly-species communities. We hope that this review will stimulate additional research in this emerging field of bacterial extracellular vesicles and contribute to the development of extracellular vesicle-based diagnostic tools and effective vaccines against pathogenic Gram-negative and Gram-positive bacteria. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Immunotherapeutic potential of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Bin eZhang

    2014-10-01

    Full Text Available Extracellular vesicles or EVs is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized endosome-derived vesicles secreted by many cell types and their immunomodulatory potential is independent of their cell source. Besides immune cells such as dendritic cells, macrophages and T cells, cancer and stem cells also secrete immunologically active exosomes that could influence both physiological and pathological processes. The immunological activities of exosomes affect both innate and adaptive immunity and include antigen presentation, T cell activation, T cell polarisation to Tregs, immune suppression and anti-inflammation. As such, exosomes carry much immunotherapeutic potential as a therapeutic agent and a therapeutic target.

  10. RNA in extracellular vesicles.

    Science.gov (United States)

    Kim, Kyoung Mi; Abdelmohsen, Kotb; Mustapic, Maja; Kapogiannis, Dimitrios; Gorospe, Myriam

    2017-07-01

    Cells release a range of membrane-enclosed extracellular vesicles (EVs) into the environment. Among them, exosomes and microvesicles (collectively measuring 40-1000 nm in diameter) carry proteins, signaling lipids, and nucleic acids from donor cells to recipient cells, and thus have been proposed to serve as intercellular mediators of communication. EVs transport cellular materials in many physiologic processes, including differentiation, stem cell homeostasis, immune responses, and neuronal signaling. EVs are also increasingly recognized as having a direct role in pathologies such as cancer and neurodegeneration. Accordingly, EVs have been the focus of intense investigation as biomarkers of disease, prognostic indicators, and even therapeutic tools. Here, we review the classes of RNAs present in EVs, both coding RNAs (messenger RNAs) and noncoding RNAs (long noncoding RNAs, microRNAs, and circular RNAs). The rising attention to EV-resident RNAs as biomarkers stems from the fact that RNAs can be detected at extremely low quantities using a number of methods. To illustrate the interest in EV biology, we discuss EV RNAs in cancer and neurodegeneration, two major age-associated disease processes. WIREs RNA 2017, 8:e1413. doi: 10.1002/wrna.1413 For further resources related to this article, please visit the WIREs website. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  11. Astrocytic Vesicle Mobility in Health and Disease

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    Robert Zorec

    2013-05-01

    Full Text Available Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide, (ii plasma membrane exchange of transporters and receptors (EAAT2, MHC-II, and (iii the involvement of vesicle mobility carrying aquaporins (AQP4 in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

  12. Formation of Giant Protein Vesicles by a Lipid Cosolvent Method

    DEFF Research Database (Denmark)

    Hansen, Jesper S.; Vararattanavech, Ardcharaporn; Vissing, Thomas

    2011-01-01

    This paper describes a method to create giant protein vesicles (GPVs) of ≥10 μm by solvent‐driven fusion of large vesicles (0.1–0.2 μm) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein‐reconstituted large unilamellar vesicles (LUVs)...... of spinach SoPIP2;1 and E. coli AqpZ aquaporins. Our findings show that hydrophobic interactions within the bilayer of formed GPVs are influenced not only by the solvent partitioning propensity, but also by lipid composition and membrane protein isoform....

  13. Cortical spreading depolarization: Pathophysiology, implications, and future directions.

    Science.gov (United States)

    Kramer, Daniel R; Fujii, Tatsuhiro; Ohiorhenuan, Ifije; Liu, Charles Y

    2016-02-01

    Cortical spreading depolarization (CSD) is a spreading loss of ion homeostasis, altered vascular response, change in synaptic architecture, and subsequent depression in electrical activity following an inciting neurological injury. First described by Leão in 1944, this disturbance in neuronal electrophysiology has since been demonstrated in a number of animal studies, and recently a few human studies that examine the occurrence of this depolarizing phenomenon in the setting of a variety of pathological states, including migraines, cerebrovascular accidents, epilepsy, intracranial hemorrhages, and traumatic brain injuries. The onset of CSD has been demonstrated experimentally following a disruption in the neuronal environment leading to glutamate-induced toxicity. This initial event leads to pathological changes in the activity of ion channels that maintain membrane potential. Recovery mechanisms such as sodium-potassium pumps that aim to restore homeostasis fail, leading to osmolar shifts of fluid, swelling of the neuron, and ultimately a measurable depression in cortical activity that spreads in the order of millimeters per minute. Equally important is the resulting change in vascular response. In healthy tissue, increased electrical activity is coupled with release of vasodilatory factors such as nitric oxide and arachidonic acid metabolites that increase local blood flow to meet increased energy expenditure. In damaged tissue, not only is the restorative vascular response lacking but a vasoconstrictive response is promoted and the ischemia that follows adds to the severity of the initial injury. Tissue threatened by this ischemic response is then at elevated risk for CSD propagation and falls into a vicious cycle of electrical and hemodynamic disturbance. Efforts have been made to halt this spreading cortical depression using N-methyl-D-aspartate receptor antagonists and other ion channel blockers to minimize the damaging effects of CSD that can persist long

  14. DNA-mediated self-assembly of artificial vesicles.

    Science.gov (United States)

    Hadorn, Maik; Eggenberger Hotz, Peter

    2010-03-26

    Although multicompartment systems made of single unilamellar vesicles offer the potential to outperform single compartment systems widely used in analytic, synthetic, and medical applications, their use has remained marginal to date. On the one hand, this can be attributed to the binary character of the majority of the current tethering protocols that impedes the implementation of real multicomponent or multifunctional systems. On the other hand, the few tethering protocols theoretically providing multicompartment systems composed of several distinct vesicle populations suffer from the readjustment of the vesicle formation procedure as well as from the loss of specificity of the linking mechanism over time. In previous studies, we presented implementations of multicompartment systems and resolved the readjustment of the vesicle formation procedure as well as the loss of specificity by using linkers consisting of biotinylated DNA single strands that were anchored to phospholipid-grafted biotinylated PEG tethers via streptavidin as a connector. The systematic analysis presented herein provides evidences for the incorporation of phospholipid-grafted biotinylated PEG tethers to the vesicle membrane during vesicle formation, providing specific anchoring sites for the streptavidin loading of the vesicle membrane. Furthermore, DNA-mediated vesicle-vesicle self-assembly was found to be sequence-dependent and to depend on the presence of monovalent salts. This study provides a solid basis for the implementation of multi-vesicle assemblies that may affect at least three distinct domains. (i) Analysis. Starting with a minimal system, the complexity of a bottom-up system is increased gradually facilitating the understanding of the components and their interaction. (ii) Synthesis. Consecutive reactions may be implemented in networks of vesicles that outperform current single compartment bioreactors in versatility and productivity. (iii) Personalized medicine. Transport and

  15. [The role of cholesterol in exo- and endocytosis of the synaptic vesicles at the frog motor nerve terminal].

    Science.gov (United States)

    Petrov, A M; kasimov, M R; Giniatullin, A R; Tarakanova, O I; Zefirov, A L

    2009-07-01

    In experiments on the frog neuro-muscular preparations using electrophysiological (two electrode fixing of potential) and optical (fluorescent endocytic dye FM1-43) methods, the value of surface cholestertol for exo-endocytic cycle of synaptic vesicles at the prolonged rhythmic activity (20 Hz--3 minutes) was investigated. It is shown that extraction of cholesterol from surface membranes by methyl-betta-cyclodextrin (1 mM MCD) leads to the expressed shifts in recycling of synaptic vesicles. Exocytosis of vesicles is decreased, and oppression of processes leading to restoration of the number of vesicles of ready releasable pool is observed. Cholesterol replacement from external membranes and membranes of recycling synaptic vesicles in addition to above described effects breaks processes of endocytosis and recycle of synaptic vesicles. Thus, in the processes of exocytosis, the key role is played by cholesterol of plasmatic membranes, and endocytosis critically depends on the amount of cholesterol in the membranes of synaptic vesicles.

  16. Substance P Depolarizes Lamprey Spinal Cord Neurons by Inhibiting Background Potassium Channels.

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    Carolina Thörn Pérez

    Full Text Available Substance P is endogenously released in the adult lamprey spinal cord and accelerates the burst frequency of fictive locomotion. This is achieved by multiple effects on interneurons and motoneurons, including an attenuation of calcium currents, potentiation of NMDA currents and reduction of the reciprocal inhibition. While substance P also depolarizes spinal cord neurons, the underlying mechanism has not been resolved. Here we show that effects of substance P on background K+ channels are the main source for this depolarization. Hyperpolarizing steps induced inward currents during whole-cell voltage clamp that were reduced by substance P. These background K+ channels are pH sensitive and are selectively blocked by anandamide and AVE1231. These blockers counteracted the effect of substance P on these channels and the resting membrane potential depolarization in spinal cord neurons. Thus, we have shown now that substance P inhibits background K+ channels that in turn induce depolarization, which is likely to contribute to the frequency increase observed with substance P during fictive locomotion.

  17. Theory of Disk-to-Vesicle Transformation

    Science.gov (United States)

    Li, Jianfeng; Shi, An-Chang

    2009-03-01

    Self-assembled membranes from amphiphilic molecules, such as lipids and block copolymers, can assume a variety of morphologies dictated by energy minimization of system. The membrane energy is characterized by a bending modulus (κ), a Gaussian modulus (κG), and the line tension (γ) of the edge. Two basic morphologies of membranes are flat disks that minimize the bending energy at the cost of the edge energy, and enclosed vesicles that minimize the edge energy at the cost of bending energy. In our work, the transition from disk to vesicle is studied theoretically using the string method, which is designed to find the minimum energy path (MEP) or the most probable transition path between two local minima of an energy landscape. Previous studies of disk-to-vesicle transition usually approximate the transitional states by a series of spherical cups, and found that the spherical cups do not correspond to stable or meta-stable states of the system. Our calculation demonstrates that the intermediate shapes along the MEP are very different from spherical cups. Furthermore, some of these transitional states can be meta-stable. The disk-to-vesicle transition pathways are governed by two scaled parameters, κG/κ and γR0/4κ, where R0 is the radius of the disk. In particular, a meta-stable intermediate state is predicted, which may correspond to the open morphologies observed in experiments and simulations.

  18. Docking of secretory vesicles is syntaxin dependent.

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    Heidi de Wit

    Full Text Available Secretory vesicles dock at the plasma membrane before they undergo fusion. Molecular docking mechanisms are poorly defined but believed to be independent of SNARE proteins. Here, we challenged this hypothesis by acute deletion of the target SNARE, syntaxin, in vertebrate neurons and neuroendocrine cells. Deletion resulted in fusion arrest in both systems. No docking defects were observed in synapses, in line with previous observations. However, a drastic reduction in morphologically docked secretory vesicles was observed in chromaffin cells. Syntaxin-deficient chromaffin cells showed a small reduction in total and plasma membrane staining for the docking factor Munc18-1, which appears insufficient to explain the drastic reduction in docking. The sub-membrane cortical actin network was unaffected by syntaxin deletion. These observations expose a docking role for syntaxin in the neuroendocrine system. Additional layers of regulation may have evolved to make syntaxin redundant for docking in highly specialized systems like synaptic active zones.

  19. Spreading depolarizations and late secondary insults after traumatic brain injury

    DEFF Research Database (Denmark)

    Hartings, Jed A; Strong, Anthony J; Fabricius, Martin

    2009-01-01

    Here we investigated the incidence of cortical spreading depolarizations (spreading depression and peri-infarct depolarization) after traumatic brain injury (TBI) and their relationship to systemic physiologic values during neurointensive care. Subdural electrode strips were placed on peri......-contusional cortex in 32 patients who underwent surgical treatment for TBI. Prospective electrocorticography was performed during neurointensive care with retrospective analysis of hourly nursing chart data. Recordings were 84 hr (median) per patient and 2,503 hr in total. In 17 patients (53%), 280 spreading...... depolarizations (spreading depressions and peri-infarct depolarizations) were observed. Depolarizations occurred in a bimodal pattern with peak incidence on days 1 and 7. The probability of a depolarization occurring increased significantly as a function of declining mean arterial pressure (MAP; R(2) = 0.78; p...

  20. Overcoming an intrinsic depolarizing resonance with a partial Siberian snake

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    H. Huang

    2004-07-01

    Full Text Available An 11.4% partial Siberian snake was used to successfully accelerate polarized protons through a strong intrinsic depolarizing spin resonance in the Alternating Gradient Synchrotron (AGS. No noticeable depolarization was observed. This opens up the possibility of using a 20% to 30% partial Siberian snake in the AGS or other medium energy proton synchrotrons to overcome all weak and strong depolarizing spin resonances.

  1. Comparison of Intranasal Outer Membrane Vesicles with Cholera Toxin and Injected MF59C.1 as Adjuvants for Malaria Transmission Blocking Antigens AnAPN1 and Pfs48/45

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    Michael Pritsch

    2016-01-01

    Full Text Available Purified protein vaccines often require adjuvants for efficient stimulation of immune responses. There is no licensed mucosal adjuvant on the market to adequately boost the immune response to purified antigens for intranasal applications in humans. Bacterial outer membrane vesicles (OMV are attractive candidates potentially combining antigenic and adjuvant properties in one substance. To more precisely characterize the potential of Escherichia coli OMV for intranasal vaccination with heterologous antigens, immune responses for AnAPN1 and Pfs48/45 as well as ovalbumin as a reference antigen were assessed in mice. The intranasal adjuvant cholera toxin (CT and parenteral adjuvant MF59C.1 were used in comparison. Vaccinations were administered intranasally or subcutaneously. Antibodies (total IgG and IgM as well as subclasses IgG1, IgG2a, IgG2b, and IgG3 were measured by ELISA. T cell responses (cytotoxic T cells, Th1, Th17, and regulatory T cells were determined by flow cytometry. When OMV were used as adjuvant for intranasal immunization, antibody and cellular responses against all three antigens could be induced, comparable to cholera toxin and MF59C.1. Antigen-specific IgG titres above 1 : 105 could be detected in all groups. This study provides the rationale for further development of OMV as a vaccination strategy in malaria and other diseases.

  2. Why the need and how to approach the functional diversity of extracellular vesicles

    OpenAIRE

    Tkach, Mercedes; Kowal, Joanna; Théry, Clotilde

    2017-01-01

    In the past decade, cell-to-cell communication mediated by exosomes has attracted growing attention from biomedical scientists and physicians, leading to several recent publications in top-tier journals. Exosomes are generally defined as secreted membrane vesicles, or extracellular vesicles (EVs), corresponding to the intraluminal vesicles of late endosomal compartments, which are secreted upon fusion of multi-vesicular endosomes with the cell's plasma membrane. Cells, however, were shown to ...

  3. Spreading depolarization monitoring in neurocritical care of acute brain injury.

    Science.gov (United States)

    Hartings, Jed A

    2017-04-01

    Spreading depolarizations are unique in being discrete pathologic entities that are well characterized experimentally and also occur commonly in patients with substantial acute brain injury. Here, we review essential concepts in depolarization monitoring, highlighting its clinical significance, interpretation, and future potential. Cortical lesion development in diverse animal models is mediated by tissue waves of mass spreading depolarization that cause the toxic loss of ion homeostasis and limit energy substrate supply through associated vasoconstriction. The signatures of such deterioration are observed in electrocorticographic recordings from perilesional cortex of patients with acute stroke or brain trauma. Experimental work suggests that depolarizations are triggered by energy supply-demand mismatch in focal hotspots of the injury penumbra, and depolarizations are usually observed clinically when other monitoring variables are within recommended ranges. These results suggest that depolarizations are a sensitive measure of relative ischemia and ongoing secondary injury, and may serve as a clinical guide for personalized, mechanistically targeted therapy. Both existing and future candidate therapies offer hope to limit depolarization recurrence. Electrocorticographic monitoring of spreading depolarizations in patients with acute brain injury provides a sensitive measure of relative energy shortage in focal, vulnerable brains regions and indicates ongoing secondary damage. Depolarization monitoring holds potential for targeted clinical trial design and implementation of precision medicine approaches to acute brain injury therapy.

  4. Optogenetic Analysis of Depolarization-Dependent Glucagonlike Peptide-1 Release.

    Science.gov (United States)

    Chimerel, Catalin; Riccio, Cristian; Murison, Keir; Gribble, Fiona M; Reimann, Frank

    2017-10-01

    Incretin hormones play an important role in the regulation of food intake and glucose homeostasis. Glucagonlike peptide-1 (GLP-1)-secreting cells have been demonstrated to be electrically excitable and to fire action potentials (APs) with increased frequency in response to nutrient exposure. However, nutrients can also be metabolized or activate G-protein-coupled receptors, thus potentially stimulating GLP-1 secretion independent of their effects on the plasma membrane potential. Here we used channelrhodopsins to manipulate the membrane potential of GLUTag cells, a well-established model of GLP-1-secreting enteroendocrine L cells. Using channelrhodopsins with fast or slow on/off kinetics (CheTA and SSFO, respectively), we found that trains of light pulses could trigger APs and calcium elevation in GLUTag cells stably expressing either CheTA or SSFO. Tetrodotoxin reduced light-triggered AP frequency but did not impair calcium responses, whereas further addition of the calcium-channel blockers nifedipine and ω-conotoxin GVIA abolished both APs and calcium transients. Light pulse trains did not trigger GLP-1 secretion from CheTA-expressing cells under basal conditions but were an effective stimulus when cyclic adenosine monophosphate (cAMP) concentrations were elevated by forskolin plus 3-isobutyl 1-methylxanthine. In SSFO-expressing cells, light-stimulated GLP-1 release was observed at resting and elevated cAMP concentrations and was blocked by nifedipine plus ω-conotoxin GVIA but not tetrodotoxin. We conclude that cAMP elevation or cumulative membrane depolarization triggered by SSFO enhances the efficiency of light-triggered action potential firing, voltage-gated calcium entry, and GLP-1 secretion.

  5. Prostasome-like vesicles stimulate acrosome reaction of pig spermatozoa

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    Marcianò Vito

    2008-01-01

    Full Text Available Abstract Background The presence of small membranous particles characterizes the male genital fluids of different mammalian species. The influence of semen vesicles, denominated prostasomes, on sperm functional properties has been well documented in humans, but their biological activity is scarcely known in other species. The present work investigated prostasome-like vesicles in pig semen for their ability to interact with spermatozoa and to affect acrosome reaction. Methods Prostasome-like vesicles have been isolated from pig seminal plasma by high-speed centrifugation and Sephadex G-200 gel chromatography. Morphology of purified vesicles has been checked by scanning electron microscopy while their protein pattern has been investigated by SDS-PAGE. Then prostasome- like vesicles have been incubated with pig spermatozoa and their ability to interact with sperm has been tested by the aminopeptidase assay. In addition, the efficiency of vesicles to influence the acrosome reaction has been investigated by assessing the sperm acrosomal status by the PI/FITC-PNA (propidium iodide/fluorescein isothiocyanate-labeled peanut agglutinin stainings. Results Purified vesicles revealed a complex protein pattern with the occurrence of bands in the high, medium and low molecular weight range. However, the two major bands were observed at ~90 kDa and ~60 kDa. A vesicle-mediated transfer of aminopeptidase to sperm cells has been also detected. Furthermore, a significant increase of acrosome reaction extent has been revealed in spermatozoa incubated with prostasome-like vesicles in comparison to control sperm. Conclusion This is the first report demonstrating that pig prostasome-like vesicles are able, in vitro, to interact with spermatozoa and to stimulate the acrosome reaction. These findings lead to hypothesize a transfer of molecules from vesicles to sperm membrane, thus sensitizing male gametes to undergo the acrosome reaction

  6. Strong Resilience of Topological Codes to Depolarization

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    H. Bombin

    2012-04-01

    Full Text Available The inevitable presence of decoherence effects in systems suitable for quantum computation necessitates effective error-correction schemes to protect information from noise. We compute the stability of the toric code to depolarization by mapping the quantum problem onto a classical disordered eight-vertex Ising model. By studying the stability of the related ferromagnetic phase via both large-scale Monte Carlo simulations and the duality method, we are able to demonstrate an increased error threshold of 18.9(3% when noise correlations are taken into account. Remarkably, this result agrees within error bars with the result for a different class of codes—topological color codes—where the mapping yields interesting new types of interacting eight-vertex models.

  7. The puzzle of chloroplast vesicle transport – involvement of GTPases

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    Sazzad eKarim

    2014-09-01

    Full Text Available In the cytosol of plant cells vesicle transport occurs via secretory pathways among the endoplasmic reticulum (ER network, Golgi bodies, secretory granules, endosome and plasma membrane. Three systems transfer lipids, proteins and other important molecules through aqueous spaces to membrane-enclosed compartments, via vesicles that bud from donor membranes, being coated and uncoated before tethered and fused with acceptor membranes. In addition, molecular, biochemical and ultrastructural evidence indicates presence of a vesicle transport system in chloroplasts. Little is known about the protein components of this system. However, as chloroplasts harbour the photosynthetic apparatus that ultimately supports most organisms on the planet, close attention to their pathways is warranted. This may also reveal novel diversification and/or distinct solutions to the problems posed by the targeted intra-cellular trafficking of important molecules. To date two homologues to well-known yeast cytosolic vesicle transport proteins, CPSAR1 and CPRabA5e, have been shown to have roles in chloroplast vesicle transport, both being GTPases. Bioinformatic data indicate that several homologues of cytosolic vesicle transport system components are putatively chloroplast-localized and in addition other proteins have been implicated to participate in chloroplast vesicle transport, including vesicle-inducing protein in plastids 1 (VIPP1, thylakoid formation 1 (THF1, snowy cotyledon 2/cotyledon chloroplast biogenesis factor (SCO2/CYO1, curvature thylakoid 1 (CURT1 proteins, and a dynamin like GTPase FZO-like (FZL protein. Several putative potential cargo proteins have also been identified, including building blocks of the photosynthetic apparatus. Here we discuss details of the largely unknown putative chloroplast vesicle transport system, focusing on GTPase-related components.

  8. Dynamic properties of the alkaline vesicle population at hippocampal synapses.

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    Mareike Röther

    Full Text Available In compensatory endocytosis, scission of vesicles from the plasma membrane to the cytoplasm is a prerequisite for intravesicular reacidification and accumulation of neurotransmitter molecules. Here, we provide time-resolved measurements of the dynamics of the alkaline vesicle population which appears upon endocytic retrieval. Using fast perfusion pH-cycling in live-cell microscopy, synapto-pHluorin expressing rat hippocampal neurons were electrically stimulated. We found that the relative size of the alkaline vesicle population depended significantly on the electrical stimulus size: With increasing number of action potentials the relative size of the alkaline vesicle population expanded. In contrast to that, increasing the stimulus frequency reduced the relative size of the population of alkaline vesicles. Measurement of the time constant for reacification and calculation of the time constant for endocytosis revealed that both time constants were variable with regard to the stimulus condition. Furthermore, we show that the dynamics of the alkaline vesicle population can be predicted by a simple mathematical model. In conclusion, here a novel methodical approach to analyze dynamic properties of alkaline vesicles is presented and validated as a convenient method for the detection of intracellular events. Using this method we show that the population of alkaline vesicles is highly dynamic and depends both on stimulus strength and frequency. Our results implicate that determination of the alkaline vesicle population size may provide new insights into the kinetics of endocytic retrieval.

  9. A Network of Three Types of Filaments Organizes Synaptic Vesicles for Storage, Mobilization, and Docking.

    Science.gov (United States)

    Cole, Andy A; Chen, Xiaobing; Reese, Thomas S

    2016-03-16

    Synaptic transmission between neurons requires precise management of synaptic vesicles. While individual molecular components of the presynaptic terminal are well known, exactly how the molecules are organized into a molecular machine serving the storage and mobilization of synaptic vesicles to the active zone remains unclear. Here we report three filament types associated with synaptic vesicles in glutamatergic synapses revealed by electron microscope tomography in unstimulated, dissociated rat hippocampal neurons. One filament type, likely corresponding to the SNAREpin complex, extends from the active zone membrane and surrounds docked vesicles. A second filament type contacts all vesicles throughout the active zone and pairs vesicles together. On the third filament type, vesicles attach to side branches extending from the long filament core and form vesicle clusters that are distributed throughout the vesicle cloud and along the active zone membrane. Detailed analysis of presynaptic structure reveals how each of the three filament types interacts with synaptic vesicles, providing a means to traffic reserved and recycled vesicles from the cloud of vesicles into the docking position at the active zone. The formation and release of synaptic vesicles has been extensively investigated. Explanations of the release of synaptic vesicles generally begin with the movement of vesicles from the cloud into the synaptic active zone. However, the presynaptic terminal is filled with filamentous material that would appear to limit vesicular diffusion. Here, we provide a systematic description of three filament types connecting synaptic vesicles. A picture emerges illustrating how the cooperative attachment and release of these three filament types facilitate the movement of vesicles to the active zone to become docked in preparation for release. Copyright © 2016 the authors 0270-6474/16/363222-09$15.00/0.

  10. Membrane potential responses of paramecium caudatum to external Na+.

    Science.gov (United States)

    Oami, Kazunori; Takahashi, Mihoko

    2004-11-01

    The membrane potential responses of Paramecium caudatum to Na+ ions were examined to understand the mechanisms underlying the sensation of external inorganic ions in the ciliate by comparing the responses of the wild type and the behavioral mutant. Wild-type cells exhibited initial continuous backward swimming followed by repeated transient backward swimming in the Na+-containing test solution. A wild-type cell impaled by a microelectrode produced initial action potentials and a sustained depolarization to an application of the test solution. The prolonged depolarization, the depolarizing afterpotential, took place subsequently after stimulation. The ciliary reversal of the cell was closely associated with the depolarizing responses. When the application of the test solution was prolonged, the wild-type cell produced sustained depolarization overlapped by repeated transient depolarization. A behavioral mutant defective in the Ca2+ channel, CNR (caudatum non reversal), produced a sustained depolarization but no action potential or depolarizing afterpotential. The mutant cell responded to prolonged stimulation with sustained depolarization overlapped by transient depolarization, although it did not show backward swimming. The results suggest that Paramecium shows at least two kinds of membrane potential responses to Na+ ions: a depolarizing afterpotential mediating initial backward swimming and repeated transient depolarization responsible for the repeated transient backward swimming.

  11. Fusion of Nonionic Vesicles

    DEFF Research Database (Denmark)

    Bulut, Sanja; Oskolkova, M. Z.; Schweins, R.

    2010-01-01

    We present an experimental study of vesicle fusion using light and neutron scattering to monitor fusion events. Vesicles are reproducibly formed with an extrusion procedure using an single amphiphile triethylene glycol mono-n-decyl ether in water. They show long-term stability for temperatures ar...... a barrier to fusion changing from 15 k(B)T at T = 26 degrees C to 10k(H) T at T = 35 degrees C. These results are compatible with the theoretical predictions using the stalk model of vesicle fusion....

  12. The effects of polyamine agonists and antagonists on N-methyl-D-aspartate-induced depolarizations of amphibian motoneurons in situ.

    Science.gov (United States)

    Hackman, John C; Holohean, Alice M

    2010-04-14

    Polyamines have been found to reduce proton inhibition of isolated N-methyl-D-aspartate (NMDA) channels recorded in vitro. This study examines the role of polyamine modulation of motoneuronal excitation in situ, with an emphasis on possible interactions with NMDA-mediated depolarization of motoneurons and receptor mediated modulation of NMDA receptors by L-glutamate and serotonin (5-HT). Motoneuron membrane potential changes were electrotonically recorded in situ from the ventral root of isolated, hemisected amphibian spinal cords using sucrose gap techniques. The methods provided highly stable recordings (polyamine antagonist arcaine or the allosteric modulator ifenprodil had no effect on NMDA-induced changes in motoneuron membrane potentials recorded in situ but blocked the effects of spermine. Synthalin did not block spermine enhancement of NMDA-induced depolarization of motoneurons but mimicked Mg(2+) block of the NMDA channel. The data provide evidence that the proton block of the NMDA receptor is maximized in frog motoneurons in situ and also for a spermine specific polyamine site on native NMDA receptors of motoneurons that can enhance NMDA-induced depolarization when activated. Polyamines do not appear to be constitutively active at the motoneurons recorded since polyamine antagonists had no effect on either membrane depolarization or modulation of NMDA receptors. Published by Elsevier B.V.

  13. Astrocyte VAMP3 vesicles undergo Ca2+-independent cycling and modulate glutamate transporter trafficking

    Science.gov (United States)

    Li, Dongdong; Hérault, Karine; Zylbersztejn, Kathleen; Lauterbach, Marcel A; Guillon, Marc; Oheim, Martin; Ropert, Nicole

    2015-01-01

    Key points Mouse cortical astrocytes express VAMP3 but not VAMP2. VAMP3 vesicles undergo Ca2+-independent exo- and endocytotic cycling at the plasma membrane. VAMP3 vesicle traffic regulates the recycling of plasma membrane glutamate transporters. cAMP modulates VAMP3 vesicle cycling and glutamate uptake. Abstract Previous studies suggest that small synaptic-like vesicles in astrocytes carry vesicle-associated vSNARE proteins, VAMP3 (cellubrevin) and VAMP2 (synaptobrevin 2), both contributing to the Ca2+-regulated exocytosis of gliotransmitters, thereby modulating brain information processing. Here, using cortical astrocytes taken from VAMP2 and VAMP3 knock-out mice, we find that astrocytes express only VAMP3. The morphology and function of VAMP3 vesicles were studied in cultured astrocytes at single vesicle level with stimulated emission depletion (STED) and total internal reflection fluorescence (TIRF) microscopies. We show that VAMP3 antibodies label small diameter (∼80 nm) vesicles and that VAMP3 vesicles undergo Ca2+-independent exo-endocytosis. We also show that this pathway modulates the surface expression of plasma membrane glutamate transporters and the glutamate uptake by astrocytes. Finally, using pharmacological and optogenetic tools, we provide evidence suggesting that the cytosolic cAMP level influences astrocytic VAMP3 vesicle trafficking and glutamate transport. Our results suggest a new role for VAMP3 vesicles in astrocytes. PMID:25864578

  14. Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles

    Science.gov (United States)

    Boguslavsky, Shlomit; Chiu, Tim; Foley, Kevin P.; Osorio-Fuentealba, Cesar; Antonescu, Costin N.; Bayer, K. Ulrich; Bilan, Philip J.; Klip, Amira

    2012-01-01

    GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding–deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells. PMID:22918957

  15. The Q-soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (Q-SNARE) SNAP-47 Regulates Trafficking of Selected Vesicle-associated Membrane Proteins (VAMPs)*

    Science.gov (United States)

    Kuster, Aurelia; Nola, Sebastien; Dingli, Florent; Vacca, Barbara; Gauchy, Christian; Beaujouan, Jean-Claude; Nunez, Marcela; Moncion, Thomas; Loew, Damarys; Formstecher, Etienne; Galli, Thierry; Proux-Gillardeaux, Veronique

    2015-01-01

    SNAREs constitute the core machinery of intracellular membrane fusion, but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here, we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum (ER), and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with the trans-Golgi network VAMP4 and post-Golgi VAMP7 and -8. SNAP-47 also interacted with ER and Golgi syntaxin 5 and with syntaxin 1 in the absence of Munc18a, when syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway. PMID:26359495

  16. The Q-soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (Q-SNARE) SNAP-47 Regulates Trafficking of Selected Vesicle-associated Membrane Proteins (VAMPs).

    Science.gov (United States)

    Kuster, Aurelia; Nola, Sebastien; Dingli, Florent; Vacca, Barbara; Gauchy, Christian; Beaujouan, Jean-Claude; Nunez, Marcela; Moncion, Thomas; Loew, Damarys; Formstecher, Etienne; Galli, Thierry; Proux-Gillardeaux, Veronique

    2015-11-20

    SNAREs constitute the core machinery of intracellular membrane fusion, but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here, we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum (ER), and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with the trans-Golgi network VAMP4 and post-Golgi VAMP7 and -8. SNAP-47 also interacted with ER and Golgi syntaxin 5 and with syntaxin 1 in the absence of Munc18a, when syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Depolarization of ultracold neutrons during their storage in material bottles

    Science.gov (United States)

    Serebrov, A. P.; Lasakov, M. S.; Vassiljev, A. V.; Krasnoschekova, I. A.; Rudnev, Yu. P.; Fomin, A. K.; Varlamov, V. E.; Geltenbort, P.; Butterworth, J.; Young, A. R.; Pesavento, U.

    2003-07-01

    The depolarization of ultracold neutrons (UCN) during their storage in traps has been investigated. The neutron spin-flip probability for the materials studied amounts to ∼(1-2)×10 -5 per collision and does not depend on the temperature. The possible connection between the phenomenon of UCN depolarization and that of anomalous losses is discussed.

  18. Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins.

    Science.gov (United States)

    Jang, Yeongseon; Choi, Won Tae; Heller, William T; Ke, Zunlong; Wright, Elizabeth R; Champion, Julie A

    2017-09-01

    Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermal driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. These results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Amiodarone reduces depolarization-evoked glutamate release from hippocampual synaptosomes

    Directory of Open Access Journals (Sweden)

    Chia Yu Chang

    2017-03-01

    Full Text Available Decreased brain glutamate level has emerged as a new therapeutic approach for epilepsy. This study investigated the effect and mechanism of amiodarone, an anti-arrhythmic drug with antiepileptic activity, on glutamate release in the rat hippocampus. In a synaptosomal preparation, amiodarone reduced 4-aminopyridine-evoked Ca2+-dependent glutamate release and cytosolic Ca2+ concentration elevation. Amiodarone did not affect the 4-aminopyridine-evoked depolarization of the synaptosomal membrane potential or the Na+ channel activator veratridine-evoked glutamate release, indicating that the amiodarone-mediated inhibition of glutamate release is not caused by a decrease in synaptosomal excitability. The inhibitory effect of amiodarone on 4-aminopyridine-evoked glutamate release was markedly decreased in synaptosomes pretreated with the Cav2.2 (N-type and Cav2.1 (P/Q-type channel blocker ω-conotoxin MVIIC, the calmodulin antagonists W7 and calmidazolium, or the protein kinase A inhibitors H89 and KT5720. However, the intracellular Ca2+-release inhibitors dantrolene and CGP37157 had no effect on the amiodarone-mediated inhibition of glutamate release. Furthermore, amiodarone reduced the frequency of miniature excitatory postsynaptic currents without affecting their amplitude in hippocampal slices. Our data suggest that amiodarone reduces Ca2+ influx through N- and P/Q-type Ca2+ channels, subsequently reducing the Ca2+-calmodulin/protein kinase A cascade to inhibit the evoked glutamate release from rat hippocampal nerve terminals.

  20. Recognition and tethering of transport vesicles at the Golgi apparatus.

    Science.gov (United States)

    Witkos, Tomasz M; Lowe, Martin

    2017-08-01

    The Golgi apparatus occupies a central position within the secretory pathway where it is a hub for vesicle trafficking. Distinct classes of transport vesicles traffic diverse cargoes into and out of this organelle, as well as between the different Golgi subcompartments. A key feature of Golgi trafficking is the specific recognition of transport vesicles at the different regions of the Golgi apparatus, required for the correct cargo delivery. Specificity is ensured by coiled-coil golgins and multi-subunit tethering complexes (MTCs), which act together to capture vesicles and promote their subsequent fusion with the Golgi membrane. In this review we discuss our current understanding of how golgins and MTCs function together to mediate the specific recognition of vesicles at the Golgi apparatus. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

    Science.gov (United States)

    Michaelis, Mary L; Jiang, Lei; Michaelis, Elias K

    2017-01-01

    Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved

  2. Reconstitution of lipid vesicles associated with HVJ (Sendai virus) sikes. Purification and some properties of vesicles containing nontoxic fragment A of diphtheria toxin

    Science.gov (United States)

    1979-01-01

    A mixture of HVJ (Sendai virus) spike proteins, the nontoxic fragment A of diphtheria toxin, lecithin, and cholesterol was solubilized in sucrose solution containing a nonionic neutral detergent. The liposomal vesicles which formed on removal of the detergent by dialysis were purified by gel filtration and centrifugation on a sucrose gradient. The resulting purified vesicles had hemagglutinating activity, hemolytic activity and, after solubilization, the enzymic activity of fragment A. The vesicles had no cell fusion activity. Electron microscopy showed that both the outside and inside of membranes of the vesicles were associated with the spikes. When the vesicles were freeze- fractured, no large aggregates of particles were seen on either face. Such fragment A-containing lipid vesicles (liposomes) with HVJ spikes bound to mamalian cell membrane and released their fragment A into the cytoplasm causing cell death. Neither fragment A-containing liposomes without spikes nor empty liposomes with spikes were toxic. PMID:217880

  3. Low-resolution simulations of vesicle suspensions in 2D

    Science.gov (United States)

    Kabacaoğlu, Gökberk; Quaife, Bryan; Biros, George

    2018-03-01

    Vesicle suspensions appear in many biological and industrial applications. These suspensions are characterized by rich and complex dynamics of vesicles due to their interaction with the bulk fluid, and their large deformations and nonlinear elastic properties. Many existing state-of-the-art numerical schemes can resolve such complex vesicle flows. However, even when using provably optimal algorithms, these simulations can be computationally expensive, especially for suspensions with a large number of vesicles. These high computational costs can limit the use of simulations for parameter exploration, optimization, or uncertainty quantification. One way to reduce the cost is to use low-resolution discretizations in space and time. However, it is well-known that simply reducing the resolution results in vesicle collisions, numerical instabilities, and often in erroneous results. In this paper, we investigate the effect of a number of algorithmic empirical fixes (which are commonly used by many groups) in an attempt to make low-resolution simulations more stable and more predictive. Based on our empirical studies for a number of flow configurations, we propose a scheme that attempts to integrate these fixes in a systematic way. This low-resolution scheme is an extension of our previous work [51,53]. Our low-resolution correction algorithms (LRCA) include anti-aliasing and membrane reparametrization for avoiding spurious oscillations in vesicles' membranes, adaptive time stepping and a repulsion force for handling vesicle collisions and, correction of vesicles' area and arc-length for maintaining physical vesicle shapes. We perform a systematic error analysis by comparing the low-resolution simulations of dilute and dense suspensions with their high-fidelity, fully resolved, counterparts. We observe that the LRCA enables both efficient and statistically accurate low-resolution simulations of vesicle suspensions, while it can be 10× to 100× faster.

  4. Proteomic Analysis of Blood Extracellular Vesicles in Cardiovascular Disease by LC-MS/MS Analysis.

    Science.gov (United States)

    Baldan-Martin, Montserrat; de la Cuesta, Fernando; Alvarez-Llamas, Gloria; Ruiz-Hurtado, Gema; Ruilope, Luis M; Barderas, Maria G

    2017-01-01

    Extracellular vesicles are membrane vesicles related to cell communication. These vesicles consist of proteins, RNA, and microRNA and are an interesting and important tool to understand the processes taking place in the secreting cell, especially in diseases in which its release is often enhanced. The used of blood extracellular vesicles in cardiovascular disease as a low invasive, easily accessible source of circulating markers could give us important information related to pathological process even more with the use of proteomic analysis. In this chapter, we describe a protocol to isolate and proteomic analyze extracellular vesicles from blood associated with cardiovascular disease.

  5. Extracellular Vesicles in Cardiovascular Theranostics.

    Science.gov (United States)

    Bei, Yihua; Das, Saumya; Rodosthenous, Rodosthenis S; Holvoet, Paul; Vanhaverbeke, Maarten; Monteiro, Marta Chagas; Monteiro, Valter Vinicius Silva; Radosinska, Jana; Bartekova, Monika; Jansen, Felix; Li, Qian; Rajasingh, Johnson; Xiao, Junjie

    2017-01-01

    Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells play essential roles in pathophysiological processes such as cardiac hypertrophy, cardiomyocyte survival and apoptosis, cardiac fibrosis, and angiogenesis in relation to CVDs. In this review, we will first outline the current knowledge about the physical characteristics, biological contents, and isolation methods of EVs. We will then focus on the functional roles of cardiovascular EVs and their pathophysiological effects in CVDs, as well as summarize the potential of EVs as therapeutic agents and biomarkers for CVDs. Finally, we will discuss the specific application of EVs as a novel drug delivery system and the utility of EVs in the field of regenerative medicine.

  6. Membranes

    OpenAIRE

    Junbo Hou; Min Yang

    2012-01-01

    Lithium ion batteries have proven themselves the main choice of power sources for portable electronics. Besides consumer electronics, lithium ion batteries are also growing in popularity for military, electric vehicle, and aerospace applications. The present review attempts to summarize the knowledge about some selected membranes in lithium ion batteries. Based on the type of electrolyte used, literature concerning ceramic-glass and polymer solid ion conductors, microporous filter type separa...

  7. Vesicle shape, molecular tilt, and the suppression of necks

    Science.gov (United States)

    Jiang, Hongyuan; Huber, Greg; Pelcovits, Robert A.; Powers, Thomas R.

    2007-09-01

    Can the presence of molecular-tilt order significantly affect the shapes of lipid bilayer membranes, particularly membrane shapes with narrow necks? Motivated by the propensity for tilt order and the common occurrence of narrow necks in the intermediate stages of biological processes such as endocytosis and vesicle trafficking, we examine how tilt order inhibits the formation of necks in the equilibrium shapes of vesicles. For vesicles with a spherical topology, point defects in the molecular order with a total strength of +2 are required. We study axisymmetric shapes and suppose that there is a unit-strength defect at each pole of the vesicle. The model is further simplified by the assumption of tilt isotropy: invariance of the energy with respect to rotations of the molecules about the local membrane normal. This isotropy condition leads to a minimal coupling of tilt order and curvature, giving a high energetic cost to regions with Gaussian curvature and tilt order. Minimizing the elastic free energy with constraints of fixed area and fixed enclosed volume determines the allowed shapes. Using numerical calculations, we find several branches of solutions and identify them with the branches previously known for fluid membranes. We find that tilt order changes the relative energy of the branches, suppressing thin necks by making them costly, leading to elongated prolate vesicles as a generic family of tilt-ordered membrane shapes.

  8. A readily retrievable pool of synaptic vesicles

    OpenAIRE

    Hua, Y; Sinha, R.; Thiel, C.; Schmidt, R.; Hueve, J.; Martens, H.; Hell, S.; Egner, A.; Klingauf, J.

    2011-01-01

    Abstract Although clathrin-mediated endocytosis (CME) is thought to be the predominant mechanism of synaptic vesicle (SV) recycling, it seems to be too slow for fast recycling. Therefore, it was suggested that a pre-sorted and pre-assembled pool of SV proteins on the presynaptic membrane might support a first wave of fast CME. In this study we monitored the temporal dynamics of such a 'readily retrievable pool' of SV proteins in rat hippocampal neurons using a novel probe. Applying...

  9. Tomosyn inhibits synaptic vesicle priming in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Elena O Gracheva

    2006-07-01

    Full Text Available Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.

  10. TNF-? promotes extracellular vesicle release in mouse astrocytes through glutaminase

    OpenAIRE

    Wang, Kaizhe; Ye, Ling; Lu, Hongfang; Chen, Huili; Zhang, Yanyan; Huang, Yunlong; Zheng, Jialin C.

    2017-01-01

    Background Extracellular vesicles (EVs) are membrane-contained vesicles shed from cells. EVs contain proteins, lipids, and nucleotides, all of which play important roles in intercellular communication. The release of EVs is known to increase during neuroinflammation. Glutaminase, a mitochondrial enzyme that converts glutamine to glutamate, has been implicated in the biogenesis of EVs. We have previously demonstrated that TNF-? promotes glutaminase expression in neurons. However, the expressio...

  11. Production of outer membrane vesicles (OMV in batch cultivation of Neisseria meningitidis serogroup B Produção de vesículas da membrana externa (OMV em cultivo batelada de Neisseria meningitidis sorogrupo B

    Directory of Open Access Journals (Sweden)

    Silvia Santos

    2006-12-01

    Full Text Available Meningococcal disease is an important cause of death and morbidity throughout the world. Nearly 330,000 cases and 35,000 deaths occur yearly. Neisseria meningitidis, serogroup B strain N.44/89, is prevalent in Brazil. Its outer membrane vesicles (OMV with iron regulated proteins (IRP are released to the culture medium and are used as antigen for vaccine production. In order to have knowledge about the kinetic parameters, especially the final OMV concentration values, 20-h batch cultivations were carried out in Catlin medium with iron restriction. Process conditions comprised: 7 L bioreactor, 36ºC, 0.5 atm, overlay air flowrate of 1 L/min, agitation varying from 250 rpm to 850 rpm and dissolved oxygen control set at 10% of saturation condition. Biomass was determined by optical density at 540 nm and dry weight. Glycerol, lactate, pH and dissolved oxygen were measured from samples taken during cultivation. Outer membrane vesicle (OMV concentration was determined by Lowry's method after ultracentrifugation. IRP presence was verified by SDS-PAGE. Highest biomass value, corresponding to the highest initial lactate concentration (7.84 g/L was achieved at the 9th hour process time corresponding to 1.0 g/L dry biomass and 2.3 optical density at 540 nm. Lactate consumption was directly related to cell growth (yield factor: 0.24 g dry biomass / g lactate. Glycerol concentration in the medium did not change significantly during the process. OMV concentration reached the highest value of 80 mg/L at end cultivation time. The obtained results suggest that lactate is a main limiting growth factor and the maximum amount of antigen is obtained during stationary growth and cell death phases.A doença meningocócica é uma causa importante de morte a nível mundial. Aproximadamente 330.000 casos e 35.000 mortes ocorrem anualmente. A cepa N.44/89 do sorogrupo B de Neisseria meningitidis é prevalente no Brasil. Suas vesículas de membrana externa (OMV - "outer

  12. Routes and mechanisms of extracellular vesicle uptake

    Directory of Open Access Journals (Sweden)

    Laura Ann Mulcahy

    2014-08-01

    Full Text Available Extracellular vesicles (EVs are small vesicles released by donor cells that can be taken up by recipient cells. Despite their discovery decades ago, it has only recently become apparent that EVs play an important role in cell-to-cell communication. EVs can carry a range of nucleic acids and proteins which can have a significant impact on the phenotype of the recipient. For this phenotypic effect to occur, EVs need to fuse with target cell membranes, either directly with the plasma membrane or with the endosomal membrane after endocytic uptake. EVs are of therapeutic interest because they are deregulated in diseases such as cancer and they could be harnessed to deliver drugs to target cells. It is therefore important to understand the molecular mechanisms by which EVs are taken up into cells. This comprehensive review summarizes current knowledge of EV uptake mechanisms. Cells appear to take up EVs by a variety of endocytic pathways, including clathrin-dependent endocytosis, and clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft–mediated internalization. Indeed, it seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route. The uptake mechanism used by a given EV may depend on proteins and glycoproteins found on the surface of both the vesicle and the target cell. Further research is needed to understand the precise rules that underpin EV entry into cells.

  13. Formation and structural properties of multi-block copolymer vesicles

    Science.gov (United States)

    Wang, Rong; Ma, Shiying

    2014-03-01

    Due to the unique structure, vesicles have attracted considerable attention for their potential applications, such as gene and drug delivery, microcapsules, nanoreactors, cell membrane mimetic, synthetic organelles, etc. By using dissipative particle dynamics, we studied the self-assembly of amphiphilic multi-block copolymer. The phase diagram was constructed by varying the interaction parameters and the composition of the block copolymers. The results show that the vesicles are stable in a large region which is different from the diblock copolymer or triblock copolymer. The structural properties of vesicles can be controlled by varying the interaction parameters and the length of the hydrophobic block. The relationship between the hydrophilic and hydrophobic block length vs the aqueous cavity size and vesicle size are revealed. The copolymers with shorter hydrophobic blocks length or the higher hydrophilicity are more likely to form vesicles with larger aqueous cavity size and vesicle size as well as thinner wall thickness. However, the increase in hydrophobic-block length results to form vesicles with smaller aqueous cavity size and larger vesicle size. Acknowledgments. This work has been supported by NNSFC (No. 21074053) and NBRPC (No. 2010CB923303).

  14. Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation.

    Science.gov (United States)

    Takaoka, Rina; Kurosaki, Haruko; Nakao, Hiroyuki; Ikeda, Keisuke; Nakano, Minoru

    2018-02-01

    Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7M serine and 0.3M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. The Vesicle Priming Factor CAPS Functions as a Homodimer via C2 Domain Interactions to Promote Regulated Vesicle Exocytosis.

    Science.gov (United States)

    Petrie, Matt; Esquibel, Joseph; Kabachinski, Greg; Maciuba, Stephanie; Takahashi, Hirohide; Edwardson, J Michael; Martin, Thomas F J

    2016-09-30

    Neurotransmitters and peptide hormones are secreted by regulated vesicle exocytosis. CAPS (also known as CADPS) is a 145-kDa cytosolic and peripheral membrane protein required for vesicle docking and priming steps that precede Ca 2+ -triggered vesicle exocytosis. CAPS binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming. We characterized purified soluble CAPS as mainly monomer in equilibrium with small amounts of dimer. However, the active form of CAPS bound to PC12 cell membranes or to liposomes containing PI(4,5)P 2 and Q-SNARE proteins was mainly dimer. CAPS dimer formation required its C2 domain based on mutation or deletion studies. Moreover, C2 domain mutations or deletions resulted in a loss of CAPS function in regulated vesicle exocytosis, indicating that dimerization is essential for CAPS function. Comparison of the CAPS C2 domain to a structurally defined Munc13-1 C2A domain dimer revealed conserved residues involved in CAPS dimerization. We conclude that CAPS functions as a C2 domain-mediated dimer in regulated vesicle exocytosis. The unique tandem C2-PH domain of CAPS may serve as a PI(4,5)P 2 -triggered switch for dimerization. CAPS dimerization may be coupled to oligomeric SNARE complex assembly for vesicle docking and priming. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Role of extracellular vesicles in autoimmune diseases.

    Science.gov (United States)

    Turpin, Delphine; Truchetet, Marie-Elise; Faustin, Benjamin; Augusto, Jean-François; Contin-Bordes, Cécile; Brisson, Alain; Blanco, Patrick; Duffau, Pierre

    2016-02-01

    Extracellular vesicles (EVs) consist of exosomes released upon fusion of multivesicular bodies with the cell plasma membrane and microparticles shed directly from the cell membrane of many cell types. EVs can mediate cell-cell communication and are involved in many processes including inflammation, immune signaling, angiogenesis, stress response, senescence, proliferation, and cell differentiation. Accumulating evidence reveals that EVs act in the establishment, maintenance and modulation of autoimmune processes among several others involved in cancer and cardiovascular complications. EVs could also present biomedical applications, as disease biomarkers and therapeutic targets or agents for drug delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Axonal GABAA receptors depolarize presynaptic terminals and facilitate transmitter release in cerebellar Purkinje cells.

    Science.gov (United States)

    Zorrilla de San Martin, Javier; Trigo, Federico F; Kawaguchi, Shin-Ya

    2017-10-26

    In neurons of the adult brain, somatodendritic GABAA Rs mediate fast synaptic inhibition and play a crucial role in synaptic integration. GABAA Rs are not only present in the somatodendritic compartment, but also in the axonal compartment where they modulate action potential (AP) propagation and transmitter release. Although presynaptic GABAA Rs have been reported in various brain regions, their mechanisms of action and physiological roles remain obscure, particularly at GABAergic boutons. Here, using a combination of direct whole-bouton or perforated patch-clamp recordings and local GABA photolysis in single axonal varicosities of cerebellar Purkinje cells, we investigate the subcellular localization and functional role of axonal GABAA Rs both in primary cultures and acute slices. Our results indicate that presynaptic terminals of PCs carry GABAA Rs that behave as auto-receptors; their activation leads to a depolarization of the terminal membrane after an AP due to the relatively high cytoplasmic Cl(-) concentration in the axon, but do not modulate the AP itself. Paired recordings from different terminals of the same axon show that the GABAA R-mediated local depolarizations propagate substantially to neighboring varicosities. Finally, the depolarization mediated by presynaptic GABAA R activation augmented Ca(2+) influx and transmitter release, resulting in a marked effect on short-term plasticity. Altogether, our results reveal a mechanism by which presynaptic GABAA Rs influence neuronal computation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  18. Characterization of extracellular vesicles in whole blood: Influence of pre-analytical parameters and visualization of vesicle-cell interactions using imaging flow cytometry.

    Science.gov (United States)

    Fendl, Birgit; Weiss, René; Fischer, Michael B; Spittler, Andreas; Weber, Viktoria

    2016-09-09

    Extracellular vesicles are central players in intercellular communication and are released from the plasma membrane under tightly regulated conditions, depending on the physiological and pathophysiological state of the producing cell. Their heterogeneity requires a spectrum of methods for isolation and characterization, where pre-analytical parameters have profound impact on vesicle analysis, particularly in blood, since sampling, addition of anticoagulants, as well as post-sampling vesicle generation may influence the outcome. Here, we characterized microvesicles directly in whole blood using a combination of flow cytometry and imaging flow cytometry. We assessed the influence of sample agitation, anticoagulation, and temperature on post-sampling vesicle generation, and show that vesicle counts remained stable over time in samples stored without agitation. Storage with gentle rolling mimicking agitation, in contrast, resulted in strong release of platelet-derived vesicles in blood anticoagulated with citrate or heparin, whereas vesicle counts remained stable upon anticoagulation with EDTA. Using imaging flow cytometry, we could visualize microvesicles adhering to blood cells and revealed an anticoagulant-dependent increase in vesicle-cell aggregates over time. We demonstrate that vesicles adhere preferentially to monocytes and granulocytes in whole blood, while no microvesicles could be visualized on lymphocytes. Our data underscore the relevance of pre-analytical parameters in vesicle analysis and demonstrate that imaging flow cytometry is a suitable tool to study the interaction of extracellular vesicles with their target cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Gender Differences in Histamine-Induced Depolarization and Inward Currents in Vagal Ganglion Neurons in Rats

    Science.gov (United States)

    Li, Jun-Nan; Qian, Zhao; Xu, Wen-Xiao; Xu, Bing; Lu, Xiao-Long; Yan, Zhen-Yu; Han, Li-Min; Liu, Yang; Yuan, Mei; Schild, John; Qiao, Guo-Fen; Li, Bai-Yan

    2013-01-01

    Evidence has shown gender differences regarding the critical roles of histamine in the prevalence of asthma, anaphylaxis, and angina pectoris. Histamine depolarizes unmyelinated C-type neurons without any effects on myelinated A-type vagal ganglion neurons (VGNs) in male rats. However, little is known if VGNs from females react to histamine in a similar manner. Membrane depolarization and inward currents were tested in VGNs isolated from adult rats using a whole-cell patch technique. Results from males were consistent with the literature. Surprisingly, histamine-induced depolarization and inward currents were observed in both unmyelinated C-type and myelinated A- and Ah-type VGNs from female rats. In Ah-type neurons, responses to 1.0 μM histamine were stronger in intact females than in males and significantly reduced in ovariectomized (OVX) females. In C-type neurons, histamine-induced events were significantly smaller (pA/pF) in intact females compared with males and this histamine-induced activity was dramatically increased by OVX. Female A-types responded to histamine, which was further increased following ovariectomy. Histamine at 300 nM depolarized Ah-types in females, but not Ah-types in OVX females. In contrast, the sensitivity of A- and C-types to histamine was upregulated by OVX. These data demonstrate gender differences in VGN chemosensitivity to histamine for the first time. Myelinated Ah-types showed the highest sensitivity to histamine across female populations, which was changed by OVX. These novel findings improve the understanding of gender differences in the prevalence of asthma, anaphylaxis, and pain. Changes in sensitivity to histamine by OVX may explain alterations in the prevalence of certain pathophysiological conditions when women reach a postmenopausal age. PMID:24339729

  20. Commercial cow milk contains physically stable extracellular vesicles expressing immunoregulatory TGF-β.

    Science.gov (United States)

    Pieters, Bartijn C H; Arntz, Onno J; Bennink, Miranda B; Broeren, Mathijs G A; van Caam, Arjan P M; Koenders, Marije I; van Lent, Peter L E M; van den Berg, Wim B; de Vries, Marieke; van der Kraan, Peter M; van de Loo, Fons A J

    2015-01-01

    Extracellular vesicles, including exosomes, have been identified in all biological fluids and rediscovered as an important part of the intercellular communication. Breast milk also contains extracellular vesicles and the proposed biological function is to enhance the antimicrobial defense in newborns. It is, however, unknown whether extracellular vesicles are still present in commercial milk and, more importantly, whether they retained their bioactivity. Here, we characterize the extracellular vesicles present in semi-skimmed cow milk available for consumers and study their effect on T cells. Extracellular vesicles from commercial milk were isolated and characterized. Milk-derived extracellular vesicles contained several immunomodulating miRNAs and membrane protein CD63, characteristics of exosomes. In contrast to RAW 267.4 derived extracellular vesicles the milk-derived extracellular vesicles were extremely stable under degrading conditions, including low pH, boiling and freezing. Milk-derived extracellular vesicles were easily taken up by murine macrophages in vitro. Furthermore, we found that they can facilitate T cell differentiation towards the pathogenic Th17 lineage. Using a (CAGA)12-luc reporter assay we showed that these extracellular vesicles carried bioactive TGF-β, and that anti-TGF-β antibodies blocked Th17 differentiation. Our findings show that commercial milk contains stable extracellular vesicles, including exosomes, and carry immunoregulatory cargo. These data suggest that the extracellular vesicles present in commercial cow milk remains intact in the gastrointestinal tract and exert an immunoregulatory effect.

  1. Spreading convulsions, spreading depolarization and epileptogenesis in human cerebral cortex

    DEFF Research Database (Denmark)

    Dreier, Jens P; Major, Sebastian; Pannek, Heinz-Wolfgang

    2012-01-01

    channels initiates spreading depression of brain activity. In contrast, epileptic seizures show modest ion translocation and sustained depolarization below the inactivation threshold for action potential generating channels. Such modest sustained depolarization allows synchronous, highly frequent neuronal...... firing; ictal epileptic field potentials being its electrocorticographic and epileptic seizure its clinical correlate. Nevertheless, Leão in 1944 and Van Harreveld and Stamm in 1953 described in animals that silencing of brain activity induced by spreading depolarization changed during minimal electrical......, monopolar recordings here provided unequivocal evidence of spreading convulsions in patients. Hence, practically all major pathological cortical network events in animals have now been observed in people. Early spreading depolarizations may indicate a risk for late post-haemorrhagic seizures....

  2. Depolarizing collisions with hydrogen: Neutral and singly ionized alkaline earths

    Energy Technology Data Exchange (ETDEWEB)

    Manso Sainz, Rafael; Ramos, Andrés Asensio; Bueno, Javier Trujillo [Instituto de Astrofísica de Canarias, Vía Láctea s/n, E-38205 La Laguna, Tenerife (Spain); Roncero, Octavio; Sanz-Sanz, Cristina [Instituto de Física Fundamental (IFF-CSIC), CSIC, Serrano 123, E-28006 Madrid (Spain); Aguado, Alfredo, E-mail: octavio.roncero@csic.es [Departamento de Química Física, Unidad Asociada UAM-CSIC, Facultad de Ciencias M-14, Universidad Autónoma de Madrid, E-28049 Madrid (Spain)

    2014-06-20

    Depolarizing collisions are elastic or quasielastic collisions that equalize the populations and destroy the coherence between the magnetic sublevels of atomic levels. In astrophysical plasmas, the main depolarizing collider is neutral hydrogen. We consider depolarizing rates on the lowest levels of neutral and singly ionized alkali earths Mg I, Sr I, Ba I, Mg II, Ca II, and Ba II, due to collisions with H°. We compute ab initio potential curves of the atom-H° system and solve the quantum mechanical dynamics. From the scattering amplitudes, we calculate the depolarizing rates for Maxwellian distributions of colliders at temperatures T ≤ 10,000 K. A comparative analysis of our results and previous calculations in the literature is completed. We discuss the effect of these rates on the formation of scattering polarization patterns of resonant lines of alkali earths in the solar atmosphere, and their effect on Hanle effect diagnostics of solar magnetic fields.

  3. Extracellular vesicles: fundamentals and clinical relevance

    Directory of Open Access Journals (Sweden)

    Wael Nassar

    2015-01-01

    Full Text Available All types of cells of eukaryotic organisms produce and release small nanovesicles into their extracellular environment. Early studies have described these vesicles as ′garbage bags′ only to remove obsolete cellular molecules. Valadi and colleagues, in 2007, were the first to discover the capability of circulating extracellular vesicles (EVs to horizontally transfer functioning gene information between cells. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, chemoresistance, genetic exchange, and signaling pathway activation through growth factor/receptor transfer. EVs represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, signaling proteins, and RNAs. They contribute to physiology and pathology, and they have a myriad of potential clinical applications in health and disease. Moreover, vesicles can pass the blood-brain barrier and may perhaps even be considered as naturally occurring liposomes. These cell-derived EVs not only represent a central mediator of the disease microenvironment, but their presence in the peripheral circulation may serve as a surrogate for disease biopsies, enabling real-time diagnosis and disease monitoring. In this review, we′ll be addressing the characteristics of different types of extracellular EVs, as well as their clinical relevance and potential as diagnostic markers, and also define therapeutic options.

  4. Functional transferred DNA within extracellular vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Jin [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China); Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Jiangsu Province (China); Wu, Gengze [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China); Jose, Pedro A. [Division of Nephrology, Department of Medicine and Physiology, University of Maryland, School of Medicine, Baltimore, MD 21201 (United States); Zeng, Chunyu, E-mail: Chunyuzeng01@163.com [Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing 400042 (China)

    2016-11-15

    Extracellular vesicles (EVs) are small membrane vesicles including exosomes and shedding vesicles that mediated a cell-to-cell communication. EVs are released from almost all cell types under both physiological and pathological conditions and incorporate nuclear and cytoplasmic molecules for intercellular delivery. Besides protein, mRNA, and microRNA of these molecules, as recent studies show, specific DNA are prominently packaged into EVs. It appears likely that some of exosomes or shedding vesicles, bearing nuclear molecules are released upon bubble-like blebs. Specific interaction of EVs with susceptible recipients performs the uptake of EVs into the target cells, discharging their cargo including nuclear and cytoplasmic macromolecules into the cytosol. These findings expand the nucleic acid content of EVs to include increased levels of specific DNA. Thus, EVs contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer and atherosclerosis. In this review, the focus is on the characteristics, biological functions, and roles in diseases of DNA within EVs. - Highlights: • This review is focused on the DNA within EVs including its characteristics, biological functions, and roles in diseases. • It is clear that DNA within EVs might have important physiological and pathological roles in various diseases. • Knowledge in this area may provides us alternative methods for disease diagnosis or therapy in the future.

  5. Wolbachia bacteria reside in host Golgi-related vesicles whose position is regulated by polarity proteins.

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Cho

    Full Text Available Wolbachia pipientis are intracellular symbiotic bacteria extremely common in various organisms including Drosophila melanogaster, and are known for their ability to induce changes in host reproduction. These bacteria are present in astral microtubule-associated vesicular structures in host cytoplasm, but little is known about the identity of these vesicles. We report here that Wolbachia are restricted only to a group of Golgi-related vesicles concentrated near the site of membrane biogenesis and minus-ends of microtubules. The Wolbachia vesicles were significantly mislocalized in mutant embryos defective in cell/planar polarity genes suggesting that cell/tissue polarity genes are required for apical localization of these Golgi-related vesicles. Furthermore, two of the polarity proteins, Van Gogh/Strabismus and Scribble, appeared to be present in these Golgi-related vesicles. Thus, establishment of polarity may be closely linked to the precise insertion of Golgi vesicles into the new membrane addition site.

  6. Vesicle dynamics in a confined Poiseuille flow: From steady state to chaos

    Science.gov (United States)

    Aouane, Othmane; Thiébaud, Marine; Benyoussef, Abdelilah; Wagner, Christian; Misbah, Chaouqi

    2014-09-01

    Red blood cells (RBCs) are the major component of blood, and the flow of blood is dictated by that of RBCs. We employ vesicles, which consist of closed bilayer membranes enclosing a fluid, as a model system to study the behavior of RBCs under a confined Poiseuille flow. We extensively explore two main parameters: (i) the degree of confinement of vesicles within the channel and (ii) the flow strength. Rich and complex dynamics for vesicles are revealed, ranging from steady-state shapes (in the form of parachute and slipper shapes) to chaotic dynamics of shape. Chaos occurs through a cascade of multiple periodic oscillations of the vesicle shape. We summarize our results in a phase diagram in the parameter plane (degree of confinement and flow strength). This finding highlights the level of complexity of a flowing vesicle in the small Reynolds number where the flow is laminar in the absence of vesicles and can be rendered turbulent due to elasticity of vesicles.

  7. Amiodarone reduces depolarization-evoked glutamate release from hippocampual synaptosomes.

    Science.gov (United States)

    Chang, Chia Yu; Hung, Chi Feng; Huang, Shu Kuei; Kuo, Jinn Rung; Wang, Su Jane

    2017-03-01

    Decreased brain glutamate level has emerged as a new therapeutic approach for epilepsy. This study investigated the effect and mechanism of amiodarone, an anti-arrhythmic drug with antiepileptic activity, on glutamate release in the rat hippocampus. In a synaptosomal preparation, amiodarone reduced 4-aminopyridine-evoked Ca2+-dependent glutamate release and cytosolic Ca2+ concentration elevation. Amiodarone did not affect the 4-aminopyridine-evoked depolarization of the synaptosomal membrane potential or the Na+ channel activator veratridine-evoked glutamate release, indicating that the amiodarone-mediated inhibition of glutamate release is not caused by a decrease in synaptosomal excitability. The inhibitory effect of amiodarone on 4-aminopyridine-evoked glutamate release was markedly decreased in synaptosomes pretreated with the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, the calmodulin antagonists W7 and calmidazolium, or the protein kinase A inhibitors H89 and KT5720. However, the intracellular Ca2+-release inhibitors dantrolene and CGP37157 had no effect on the amiodarone-mediated inhibition of glutamate release. Furthermore, amiodarone reduced the frequency of miniature excitatory postsynaptic currents without affecting their amplitude in hippocampal slices. Our data suggest that amiodarone reduces Ca2+ influx through N- and P/Q-type Ca2+ channels, subsequently reducing the Ca2+-calmodulin/protein kinase A cascade to inhibit the evoked glutamate release from rat hippocampal nerve terminals. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  8. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles

    Science.gov (United States)

    Hill, Andrew F.; Hochberg, Fred; Buzás, Edit I.; Di Vizio, Dolores; Gardiner, Christopher; Gho, Yong Song; Kurochkin, Igor V.; Mathivanan, Suresh; Quesenberry, Peter; Sahoo, Susmita; Tahara, Hidetoshi; Wauben, Marca H.; Witwer, Kenneth W.; Théry, Clotilde

    2014-01-01

    Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs. PMID:25536934

  9. Role of Outer Membrane Vesicles of Bacteria

    Indian Academy of Sciences (India)

    IAS Admin

    nificant role in cell-to-cell communication, virulence, nutri- tion and protection of the bacterial cells from ... communication with other microorganisms and also with the host cells. It is also essential for detoxification of ..... are known to evolve with different types of strategies that make them immune to the growth-inhibitory or.

  10. Outer membrane vesicles and soluble factors released by probiotic Escherichia coli Nissle 1917 and commensal ECOR63 enhance barrier function by regulating expression of tight junction proteins in intestinal epithelial cells

    Directory of Open Access Journals (Sweden)

    Carina Shianya Alvarez

    2016-12-01

    Full Text Available The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Escherichia coli Nissle 1917 (EcN is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the

  11. Biogenesis and function of ESCRT-dependent extracellular vesicles.

    Science.gov (United States)

    Juan, Thomas; Fürthauer, Maximilian

    2018-02-01

    From bacteria to humans, cells secrete a large variety of membrane-bound extracellular vesicles. Only relatively recently has it however started to become clear that the exovesicular transport of proteins and RNAs is important for normal physiology and numerous pathological conditions. Extracellular vesicles can be formed through the release of the intralumenal vesicles of multivesicular endosomes as so-called exosomes, or through direct, ectosomal, budding from the cell surface. Through their ability to promote the bending of membranes away from the cytoplasm, the components of the Endosomal Sorting Complex Required for Transport (ESCRT) have been implicated in both exo- and ectosomal biogenesis. Studies of the ESCRT machinery may therefore provide important insights into the formation and function of extracellular vesicles. In the present review, we first describe the cell biological mechanisms through which ESCRT components contribute to the biogenesis of different types of extracellular vesicles. We then discuss how recent functional studies have started to uncover important roles of ESCRT-dependent extracellular vesicles in a wide variety of processes, including the transport of developmental signaling molecules and embryonic morphogenesis, the regulation of social behavior and host-pathogen interactions, as well as the etiology and progression of neurodegenerative pathologies and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Extracellular Vesicles Produced by the Gram-positive Bacterium Bacillus subtilis are Disrupted by the Lipopeptide Surfactin

    Science.gov (United States)

    Brown, Lisa; Kessler, Anne; Cabezas-Sanchez, Pablo; Luque-Garcia, Jose L.; Casadevall, Arturo

    2014-01-01

    Summary Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harboring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin. PMID:24826903

  13. Removal of Vesicle Structures From Transmission Electron Microscope Images

    Science.gov (United States)

    Jensen, Katrine Hommelhoff; Sigworth, Fred J.; Brandt, Sami Sebastian

    2016-01-01

    In this paper, we address the problem of imaging membrane proteins for single-particle cryo-electron microscopy reconstruction of the isolated protein structure. More precisely, we propose a method for learning and removing the interfering vesicle signals from the micrograph, prior to reconstruction. In our approach, we estimate the subspace of the vesicle structures and project the micrographs onto the orthogonal complement of this subspace. We construct a 2d statistical model of the vesicle structure, based on higher order singular value decomposition (HOSVD), by considering the structural symmetries of the vesicles in the polar coordinate plane. We then propose to lift the HOSVD model to a novel hierarchical model by summarizing the multidimensional HOSVD coefficients by their principal components. Along with the model, a solid vesicle normalization scheme and model selection criterion are proposed to make a compact and general model. The results show that the vesicle structures are accurately separated from the background by the HOSVD model that is also able to adapt to the asymmetries of the vesicles. This is a promising result and suggests even wider applicability of the proposed approach in learning and removal of statistical structures. PMID:26642456

  14. Spermatozoa as a transport system of large unilamellar lipid vesicles into the oocyte.

    Science.gov (United States)

    Geerts, N; McGrath, J; Stronk, J N; Vanderlick, T K; Huszar, G

    2014-04-01

    In addition to their role as man-made membranes, vesicles continue to be investigated as carriers for drug delivery. While most research focuses on their injectable properties, here a new delivery strategy is proposed. It is shown that spermatozoa can transport vesicles of variable composition. For human spermatozoa, the vesicles started to show binding after 20 mol% of the nonbinding vesicle backbone lipids were substituted with positive, negative, cerebroside or ganglioside lipids. Vesicle binding is a dynamic process with constant 'on' and 'off' binding. The physiological and motility attributes of the spermatozoa are not affected by the attached vesicles. Sperm swimming characteristics changed only marginally. Also, the activation status of the acrosomal membrane, tested with the fluorescent probe Pisum sativum agglutinin, was not affected by vesicle binding. Moreover, the hyaluronic acid-binding test showed that viable, fully developed spermatozoa will attach and remain bound to hyaluronic acid-coated slides regardless of vesicle binding. Therefore a new 'hybrid' delivery system was created with human spermatozoa, and tested with a mouse IVF system. Large unilamellar vesicles physisorbed to mouse spermatozoa can not only penetrate the mouse oocytes in these proof-of-principle experiments, but also deliver the cargo placed within the vesicles. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  15. Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery.

    Science.gov (United States)

    Merchant, Michael L; Rood, Ilse M; Deegens, Jeroen K J; Klein, Jon B

    2017-12-01

    Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies. This classification is based on the mechanisms by which membrane vesicles are formed: fusion of multivesicular bodies with the plasma membranes (exosomes), budding of vesicles directly from the plasma membrane (microvesicles) or those shed from dying cells (apoptotic bodies). During their formation, urinary extracellular vesicles incorporate various cell-specific components (proteins, lipids and nucleic acids) that can be transferred to target cells. The rigour needed for comparative studies has fueled the search for optimal approaches for their isolation, purification, and characterization. RNA, the newest extracellular vesicle component to be discovered, has received substantial attention as an extracellular vesicle therapeutic, and compelling evidence suggests that ex vivo manipulation of microRNA composition may have uses in the treatment of kidney disorders. The results of these studies are building the case that urinary extracellular vesicles act as mediators of renal pathophysiology. As the field of extracellular vesicle studies is burgeoning, this Review focuses on primary data obtained from studies of human urine rather than on data from studies of laboratory animals or cultured immortalized cells.

  16. How pure are your vesicles?

    Science.gov (United States)

    Webber, Jason; Clayton, Aled

    2013-01-01

    We propose a straightforward method to estimate the purity of vesicle preparations by comparing the ratio of nano-vesicle counts to protein concentration, using tools such as the increasingly available NanoSight platform and a colorimetric protein assay such as the BCA-assay. Such an approach is simple enough to apply to every vesicle preparation within a given laboratory, assisting researchers as a routine quality control step. Also, the approach may aid in comparing/standardising vesicle purity across diverse studies, and may be of particular importance in evaluating vesicular biomarkers. We herein propose some criteria to aid in the definition of pure vesicles. PMID:24009896

  17. Synthetic lipid (DOPG) vesicles accumulate in the cell plate region but do not fuse.

    NARCIS (Netherlands)

    Esseling-Ozdoba, A.; Vos, J.W.; Lammeren, AA van; Emons, A.M.C.

    2008-01-01

    The cell plate is the new cell wall, with bordering plasma membrane, that is formed between two daughter cells in plants, and it is formed by fusion of vesicles (approximately 60 nm). To start to determine physical properties of cell plate forming vesicles for their transport through the

  18. Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression.

    Science.gov (United States)

    Pi, Fengmei; Binzel, Daniel W; Lee, Tae Jin; Li, Zhefeng; Sun, Meiyan; Rychahou, Piotr; Li, Hui; Haque, Farzin; Wang, Shaoying; Croce, Carlo M; Guo, Bin; Evers, B Mark; Guo, Peixuan

    2018-01-01

    Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

  19. Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

    Science.gov (United States)

    Pi, Fengmei; Binzel, Daniel W.; Lee, Tae Jin; Li, Zhefeng; Sun, Meiyan; Rychahou, Piotr; Li, Hui; Haque, Farzin; Wang, Shaoying; Croce, Carlo M.; Guo, Bin; Evers, B. Mark; Guo, Peixuan

    2018-01-01

    Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

  20. Three-channel single-wavelength lidar depolarization calibration

    Directory of Open Access Journals (Sweden)

    E. M. McCullough

    2018-02-01

    Full Text Available Linear depolarization measurement capabilities were added to the CANDAC Rayleigh–Mie–Raman lidar (CRL at Eureka, Nunavut, in the Canadian High Arctic in 2010. This upgrade enables measurements of the phases (liquid versus ice of cold and mixed-phase clouds throughout the year, including during polar night. Depolarization measurements were calibrated according to existing methods using parallel- and perpendicular-polarized profiles as discussed in . We present a new technique that uses the polarization-independent Rayleigh elastic channel in combination with one of the new polarization-dependent channels, and we show that for a lidar with low signal in one of the polarization-dependent channels this method is superior to the traditional method. The optimal procedure for CRL is to determine the depolarization parameter using the traditional method at low resolution (from parallel and perpendicular signals and then to use this value to calibrate the high-resolution new measurements (from parallel and polarization-independent Rayleigh elastic signals. Due to its use of two high-signal-rate channels, the new method has lower statistical uncertainty and thus gives depolarization parameter values at higher spatial–temporal resolution by up to a factor of 20 for CRL. This method is easily adaptable to other lidar systems which are considering adding depolarization capability to existing hardware.

  1. Dynamics of multicomponent vesicles in a viscous fluid

    Science.gov (United States)

    Sohn, Jin Sun; Tseng, Yu-Hau; Li, Shuwang; Voigt, Axel; Lowengrub, John S.

    2010-01-01

    We develop and investigate numerically a thermodynamically consistent model of two-dimensional multicomponent vesicles in an incompressible viscous fluid. The model is derived using an energy variation approach that accounts for different lipid surface phases, the excess energy (line energy) associated with surface phase domain boundaries, bending energy, spontaneous curvature, local inextensibility and fluid flow via the Stokes equations. The equations are high-order (fourth order) nonlinear and nonlocal due to incompressibil-ity of the fluid and the local inextensibility of the vesicle membrane. To solve the equations numerically, we develop a nonstiff, pseudo-spectral boundary integral method that relies on an analysis of the equations at small scales. The algorithm is closely related to that developed very recently by Veerapaneni et al. [81] for homogeneous vesicles although we use a different and more efficient time stepping algorithm and a reformulation of the inextensibility equation. We present simulations of multicomponent vesicles in an initially quiescent fluid and investigate the effect of varying the average surface concentration of an initially unstable mixture of lipid phases. The phases then redistribute and alter the morphology of the vesicle and its dynamics. When an applied shear is introduced, an initially elliptical vesicle tank-treads and attains a steady shape and surface phase distribution. A sufficiently elongated vesicle tumbles and the presence of different surface phases with different bending stiffnesses and spontaneous curvatures yields a complex evolution of the vesicle morphology as the vesicle bends in regions where the bending stiffness and spontaneous curvature are small. PMID:20808718

  2. AFM/TIRF force clamp measurements of neurosecretory vesicle tethers reveal characteristic unfolding steps.

    Directory of Open Access Journals (Sweden)

    Mark C Harris

    Full Text Available Although several proteins have been implicated in secretory vesicle tethering, the identity and mechanical properties of the components forming the physical vesicle-plasma membrane link remain unknown. Here we present the first experimental measurements of nanomechanical properties of secretory vesicle-plasma membrane tethers using combined AFM force clamp and TIRF microscopy on membrane sheets from PC12 cells expressing the vesicle marker ANF-eGFP. Application of pulling forces generated tether extensions composed of multiple steps with variable length. The frequency of short (<10 nm tether extension events was markedly higher when a fluorescent vesicle was present at the cantilever tip and increased in the presence of GTPγS, indicating that these events reflect specifically the properties of vesicle-plasma membrane tethers. The magnitude of the short tether extension events is consistent with extension lengths expected from progressive unfolding of individual helices of the exocyst complex, supporting its direct role in forming the physical vesicle-plasma membrane link.

  3. AFM/TIRF force clamp measurements of neurosecretory vesicle tethers reveal characteristic unfolding steps.

    Science.gov (United States)

    Harris, Mark C; Cislo, Dillon; Lenz, Joan S; Umbach, Christopher; Lindau, Manfred

    2017-01-01

    Although several proteins have been implicated in secretory vesicle tethering, the identity and mechanical properties of the components forming the physical vesicle-plasma membrane link remain unknown. Here we present the first experimental measurements of nanomechanical properties of secretory vesicle-plasma membrane tethers using combined AFM force clamp and TIRF microscopy on membrane sheets from PC12 cells expressing the vesicle marker ANF-eGFP. Application of pulling forces generated tether extensions composed of multiple steps with variable length. The frequency of short (<10 nm) tether extension events was markedly higher when a fluorescent vesicle was present at the cantilever tip and increased in the presence of GTPγS, indicating that these events reflect specifically the properties of vesicle-plasma membrane tethers. The magnitude of the short tether extension events is consistent with extension lengths expected from progressive unfolding of individual helices of the exocyst complex, supporting its direct role in forming the physical vesicle-plasma membrane link.

  4. α-Synuclein can inhibit SNARE-mediated vesicle fusion through direct interactions with lipid bilayers.

    Science.gov (United States)

    DeWitt, David C; Rhoades, Elizabeth

    2013-04-09

    The native function of α-synuclein is thought to involve regulation of synaptic vesicle trafficking. Recent work has also implicated a role in neurotransmission, possibly through interactions with the proteins involved in synaptic vesicle fusion. Here, we demonstrate that α-synuclein inhibits SNARE-mediated vesicle fusion through binding the membrane, without a direct interaction between α-synuclein and any of the SNARE proteins. This work supports a model in which α-synuclein plays a role in the regulation of vesicle fusion by modulating properties of the lipid bilayer.

  5. The continuum of spreading depolarizations in acute cortical lesion development

    DEFF Research Database (Denmark)

    Hartings, Jed A; Shuttleworth, C William; Kirov, Sergei A

    2017-01-01

    A modern understanding of how cerebral cortical lesions develop after acute brain injury is based on Aristides Leão's historic discoveries of spreading depression and asphyxial/anoxic depolarization. Treated as separate entities for decades, we now appreciate that these events define a continuum....... The causal role of these waves in lesion development has been proven by real-time monitoring of electrophysiology, blood flow, and cytotoxic edema. The spreading depolarization continuum further applies to other models of acute cortical lesions, suggesting that it is a universal principle of cortical lesion...... development. These pathophysiologic concepts establish a working hypothesis for translation to human disease, where complex patterns of depolarizations are observed in acute brain injury and appear to mediate and signal ongoing secondary damage....

  6. Effect of sodium deoxycholate and sodium cholate on DPPC vesicles

    Indian Academy of Sciences (India)

    TECS

    monitor different stages of interaction of bile salts with DPPC vesicles. NaDC induced significant changes in the ... more hydrophilic NaC does not interact with the membrane efficiently. Complete solubilisation of phos- pholipids .... the temperature was controlled by circulating water through a jacketted cuvette holder from a ...

  7. Growth and instability of a phospholipid vesicle in a bath of fatty acids

    Science.gov (United States)

    Dervaux, J.; Noireaux, V.; Libchaber, A. J.

    2017-06-01

    Using a microfluidic trap, we study the behavior of individual phospholipid vesicles in contact with fatty acids. We show that spontaneous fatty acids insertion inside the bilayer is controlled by the vesicle size, osmotic pressure difference across the membrane and fatty acids concentration in the external bath. Depending on these parameters, vesicles can grow spherically or become unstable and fragment into several daughter vesicles. We establish the phase diagram for vesicle growth and we derive a simple thermodynamic model that reproduces the time evolution of the vesicle volume. Finally, we show that stable growth can be achieved on an artificial cell expressing a simple set of bacterial cytoskeletal proteins, paving the way toward artificial cell reproduction.

  8. Salt, shake, fuse--giant hybrid polymer/lipid vesicles through mechanically activated fusion.

    Science.gov (United States)

    Henderson, Ian M; Paxton, Walter F

    2014-03-24

    Large (200 nm) poly(ethylene oxide)-b-poly(butadiene) polymer vesicles fuse into giant (>1 μm) vesicles with mild agitation in dilute aqueous NaCl solutions. This unusual effect is attributed to the salt-induced contraction of the poly(ethylene oxide) corona, reducing steric resistance between vesicles and, with agitation, increasing the probability of contact between the hydrophobic cores of adjacent membranes. In addition, NaCl and agitation facilitated the creation of giant hybrid vesicles from much smaller homogeneous polymersomes and liposomes. Whereas lipid vesicles do not readily fuse with each other under the same circumstances, they did fuse with polymersomes to produce hybrid polymer/lipid vesicles. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. The biology and function of extracellular vesicles in nasopharyngeal carcinoma (Review).

    Science.gov (United States)

    You, Bo; Shan, Ying; Bao, Lili; Chen, Jing; Yang, Liu; Zhang, Qicheng; Zhang, Wei; Zhang, Zhenxin; Zhang, Jie; Shi, Si; You, Yiwen

    2018-01-01

    Extracellular vesicles are a heterogeneous group of membrane-enclosed vesicles, which play an important role in intercellular communication. Increasing number of studies have shown that tumor-derived extracellular vesicles might be involved in the transfer of oncogenic cargo (proteins, lipids, messenger RNA, microRNA, non-coding RNAs and DNA) through which cancer cells could shape the tumor microenvironment and influence tumor progression. Nasopharyngeal carcinoma-derived extracellular vesicles have also reported to facilitate tumor proliferation, metastasis and immune escape. Moreover, nasopharyngeal carcinoma-derived extracellular vesicles might serve as biomarkers for early diagnosis and therapeutic targets. The present review provides information on the biological and clinical significance of extracellular vesicles in tumors, especially in nasopharyngeal carcinoma.

  10. Deformation analysis of vesicles in an alternating-current electric field.

    Science.gov (United States)

    Tang, Yu-Gang; Liu, Ying; Feng, Xi-Qiao

    2014-08-01

    In this paper the shape equation for axisymmetric vesicles subjected to an ac electric field is derived on the basis of the liquid-crystal model. The equilibrium morphology of a lipid vesicle is determined by the minimization of its free energy in coupled mechanical and ac electric fields. Besides elastic bending, the effects of the osmotic pressure difference, surface tension, Maxwell pressure, and flexoelectric and dielectric properties of phospholipid membrane as well are taken into account. The influences of elastic bending, osmotic pressure difference, and surface tension on the frequency-dependent behavior of a vesicle membrane in an ac electric field are examined. The singularity of the ac electric field is also investigated. Our theoretical results of vesicle deformation agree well with previous experimental and numerical results. The present study provides insights into the physical mechanisms underpinning the frequency-dependent morphological evolution of vesicles in the electric and mechanical fields.

  11. Active elastohydrodynamics of vesicles in narrow blind constrictions

    Science.gov (United States)

    Fai, T. G.; Kusters, R.; Harting, J.; Rycroft, C. H.; Mahadevan, L.

    2017-11-01

    Fluid-resistance limited transport of vesicles through narrow constrictions is a recurring theme in many biological and engineering applications. Inspired by the motor-driven movement of soft membrane-bound vesicles into closed neuronal dendritic spines, here we study this problem using a combination of passive three-dimensional simulations and a simplified semianalytical theory for the active transport of vesicles forced through constrictions by molecular motors. We show that the motion of these objects is characterized by two dimensionless quantities related to the geometry and to the strength of forcing relative to the vesicle elasticity. We use numerical simulations to characterize the transit time for a vesicle forced by fluid pressure through a constriction in a channel and find that relative to an open channel, transport into a blind end leads to the formation of a smaller forward-flowing lubrication layer that strongly impedes motion. When the fluid pressure forcing is complemented by forces due to molecular motors that are responsible for vesicle trafficking into dendritic spines, we find that the competition between motor forcing and fluid drag results in multistable dynamics reminiscent of the real system. Our study highlights the role of nonlocal hydrodynamic effects in determining the kinetics of vesicular transport in constricted geometries.

  12. Biological reactivity of nanoparticles: mosaics from optical microscopy videos of giant lipid vesicles

    Science.gov (United States)

    Zupanc, Jernej; Dobnikar, Andrej; Drobne, Damjana; Valant, Janez; Erdogmus, Deniz; Bas, Erhan

    2011-02-01

    Emerging fields such as nanomedicine and nanotoxicology, demand new information on the effects of nanoparticles on biological membranes and lipid vesicles are suitable as an experimental model for bio-nano interaction studies. This paper describes image processing algorithms which stitch video sequences into mosaics and recording the shapes of thousands of lipid vesicles, which were used to assess the effect of CoFe2O4 nanoparticles on the population of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipid vesicles. The applicability of this methodology for assessing the potential of engineered nanoparticles to affect morphological properties of lipid membranes is discussed.

  13. Differential regulation of synaptic vesicle tethering and docking by UNC-18 and TOM-1

    Directory of Open Access Journals (Sweden)

    Elena O Gracheva

    2010-10-01

    Full Text Available The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18, unc-64(syntaxin and tom-1(tomosyn. We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin

  14. Emergent properties of extracellular vesicles: a holistic approach to decode the complexity of intercellular communication networks.

    Science.gov (United States)

    Gho, Yong Song; Lee, Changjin

    2017-06-27

    Shedding of nano-sized bilayered extracellular vesicles and extracellular vesicle-mediated intercellular communication are evolutionarily conserved biological processes. Communication between cells and the environment is an essential process in living organisms and dysregulation of intercellular communication leads to various diseases. Thus, systematic studies on extracellular vesicles, also known as exosomes, microvesicles, and outer membrane vesicles, are critical for a deeper understanding of intercellular communication networks that are crucial for decoding the exact causes of various difficult-to-cure diseases. Recent progress in this emerging field reveals that extracellular vesicles are endogenous carriers of specific subsets of proteins, mRNAs, miRNAs, and other bioactive materials, as well as play diverse pa