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Sample records for ap-3 transport vesicles

  1. Directed vesicle transport by diffusio-osmosis

    NARCIS (Netherlands)

    Michler, D.; Shahidzadeh, N.; Sprik, R.; Bonn, D.

    2015-01-01

    We present a study on surfactant vesicles that spontaneously move towards an oil droplet that is deposited on a glass substrate. Tracer particles in the surfactant solution show that the motion is not self-propelled: the vesicles are entrained by a macroscopic hydrodynamic flow. Measurements of the

  2. Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

    International Nuclear Information System (INIS)

    Gracz, L.M.; Wang, W.; Parsons, S.M.

    1988-01-01

    Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP 1 synaptic vesicles banding at 1.055 g/mL actively transported [ 3 H]acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more [ 3 H]AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP 1 vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP 2 synaptic vesicle originally described in intact Torpedo marmorata electric organ. The properties of the receptor for vesamicol were studied by measuring binding of [ 3 H]vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP 1 vesicles the VP 2 vesicles had a ratio of [ 3 H]AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. The Hill coefficients α/sub H/ and equilibrium dissociation constants K for vesamicol binding to VP 1 and VP 2 vesicles were essentially the same. The positive Hill coefficient suggests that the vesamicol receptor exists as a homotropic oligomeric complex. The results demonstrate that VP 1 and VP 2 synaptic vesicles exhibit functional differences in the AcCh transport system, presumably as a result of regulatory phenomena

  3. Inositol depletion restores vesicle transport in yeast phospholipid flippase mutants.

    Science.gov (United States)

    Yamagami, Kanako; Yamamoto, Takaharu; Sakai, Shota; Mioka, Tetsuo; Sano, Takamitsu; Igarashi, Yasuyuki; Tanaka, Kazuma

    2015-01-01

    In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.

  4. Calcium and proton transport in membrane vesicles from barley roots

    International Nuclear Information System (INIS)

    DuPont, F.M.; Windle, J.J.; Bush, D.S.; Jones, R.L.

    1990-01-01

    Ca 2+ uptake by membrane fractions from barley (Hordeum vulgare L. cv CM72) roots was characterized. Uptake of 45 Ca 2+ was measured in membrane vesicles obtained from continuous and discontinuous sucrose gradients. A single, large peak of Ca 2+ uptake coincided with the peak of proton transport by the tonoplast H + -ATPase. Depending on the concentration of Ca 2+ in the assay, Ca 2+ uptake was inhibited 50 to 75% by those combinations of ionophores and solutes that eliminated the pH gradient and membrane potential. However, 25 to 50% of the Ca 2+ uptake in the tonoplast-enriched fraction was not sensitive to ionophores but was inhibited by vanadate. The results suggest that 45 Ca uptake was driven by the low affinity, high capacity tonoplast Ca 2+ /nH + antiporter and also by a high affinity, lower capacity Ca 2+ -ATPase. The Ca 2+ -ATPase may be associated with tonoplast, Golgi or contaminating vesicles of unknown origin. No Ca 2+ transport was specifically associated with the distinct peak of endoplasmic reticulum that was identified by NADH cytochrome c reductase, choline phosphotransferase, and dolichol-P-mannosyl synthase activities. A small shoulder of Ca 2+ uptake in the plasma membrane region of the gradient was inhibited by vanadate and erythrosin B and may represent the activity of a separate plasma membrane Ca 2+ -ATPase. Vesicle volumes were estimated using electron spin resonance techniques, and intravesicular Ca 2+ concentrations were estimated to be as high as 5 millimolar. ATP-driven uptake of Ca 2+ created 800- to 2,000-fold concentration gradients within minutes. Problems in interpreting the effects of Ca 2+ on ATP-generated pH gradients are discussed and the suggestion is made that Ca 2+ dissipates pH gradients by a different mechanism than is responsible for Ca 2+ uptake into tonoplast vesicles

  5. Rab proteins: The key regulators of intracellular vesicle transport

    International Nuclear Information System (INIS)

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-01-01

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future

  6. Rab proteins: The key regulators of intracellular vesicle transport

    Energy Technology Data Exchange (ETDEWEB)

    Bhuin, Tanmay [Cell and Developmental Biology Unit, Department of Zoology, The University of Burdwan, Golapbag 713104 (India); Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in [Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  7. Mineral-bearing vesicle transport in sea urchin embryos.

    Science.gov (United States)

    Vidavsky, Netta; Masic, Admir; Schertel, Andreas; Weiner, Steve; Addadi, Lia

    2015-12-01

    Sea urchin embryos sequester calcium from the sea water. This calcium is deposited in a concentrated form in granule bearing vesicles both in the epithelium and in mesenchymal cells. Here we use in vivo calcein labeling and confocal Raman spectroscopy, as well as cryo-FIB-SEM 3D structural reconstructions, to investigate the processes occurring in the internal cavity of the embryo, the blastocoel. We demonstrate that calcein stained granules are also present in the filopodial network within the blastocoel. Simultaneous fluorescence imaging and Raman spectroscopy show that these granules do contain a calcium mineral. By tracking the movements of these granules, we show that the granules in the epithelium and primary mesenchymal cells barely move, but those in the filopodial network move long distances. We could however not detect any unidirectional movement of the filopodial granules. We also show the presence of mineral containing multivesicular vesicles that also move in the filopodial network. We conclude that the filopodial network is an integral part of the mineral transport process, and possibly also for sequestering calcium and other ions. Although much of the sequestered calcium is deposited in the mineralized skeleton, a significant amount is used for other purposes, and this may be temporarily stored in these membrane-delineated intracellular deposits. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Transport Vesicle Tethering at the Trans Golgi Network: Coiled Coil Proteins in Action.

    Science.gov (United States)

    Cheung, Pak-Yan P; Pfeffer, Suzanne R

    2016-01-01

    The Golgi complex is decorated with so-called Golgin proteins that share a common feature: a large proportion of their amino acid sequences are predicted to form coiled-coil structures. The possible presence of extensive coiled coils implies that these proteins are highly elongated molecules that can extend a significant distance from the Golgi surface. This property would help them to capture or trap inbound transport vesicles and to tether Golgi mini-stacks together. This review will summarize our current understanding of coiled coil tethers that are needed for the receipt of transport vesicles at the trans Golgi network (TGN). How do long tethering proteins actually catch vesicles? Golgi-associated, coiled coil tethers contain numerous binding sites for small GTPases, SNARE proteins, and vesicle coat proteins. How are these interactions coordinated and are any or all of them important for the tethering process? Progress toward understanding these questions and remaining, unresolved mysteries will be discussed.

  9. Calcium transport in vesicles energized by cytochrome oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Rosier, Randy N. [Univ. of Rochester, NY (United States)

    1979-01-01

    Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K+ selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K+ flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interaction with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.

  10. 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.

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

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    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.

  12. Uptake of auxins into membrane vesicles isolated from pea stems: an in vitro auxin transport system

    International Nuclear Information System (INIS)

    Slone, J.H.

    1985-01-01

    The objective of this research was to test the applicability of the chemiosmotic theory of auxin transport to a subcellular system. Membrane vesicles were isolated from the basal portion of the third internode of etiolated pea plants (Pisum sativum L. var. Alaska) by differential centrifugation. Uptake of auxin was determined by adding 14 C-labeled indoleacetic acid (IAA) to vesicles. Nigericin, a monovalent cation ionophore, and the electrogenic protonophore, carbonyl-cyanide m-chlorophenylhydrazone (CCCP), at micromolar concentrations abolished saturable uptake. Bursting vesicles by sonication, osmotic shock and freeze/thawing also eliminated saturable uptake. As the temperature increased from 0 to 30 0 C, saturable uptake decreased markedly. Nonsaturable auxin uptake was less affected by these treatments. The pH gradient-dependent uptake of auxin appeared to be a transmembrane uptake of auxin into the vesicles rather than surface binding. Unlabeled IAA, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-naphthaleneacetic acid (NAA) at low concentrations reduced the saturable accumulation of [ 14 C]IAA in vesicles, while phenylacetic acid, benzoic acid, and 1-NAA were effective only at high concentrations. Kinetic analysis revealed two types of sites: a high affinity site with an uptake capacity of 25 to 40 pmoles/g tissue, and a low affinity site with an uptake capacity of 260 to 600 pmole/g tissue, fresh wt. In conclusion, several principal elements of an auxin transport system, as specific by the chemiosmotic theory of polar auxin transport, were present in membrane vesicles isolated from relatively mature pea stem tissue. However, one important aspect of the theory was not demonstrated in this in vitro system - a TIBA/NPA-sensitive auxin efflux. The kinetics and specificity of auxin uptake strongly suggested that this system was physiologically significant

  13. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes.

    Science.gov (United States)

    Dhonukshe, Pankaj; Grigoriev, Ilya; Fischer, Rainer; Tominaga, Motoki; Robinson, David G; Hasek, Jirí; Paciorek, Tomasz; Petrásek, Jan; Seifertová, Daniela; Tejos, Ricardo; Meisel, Lee A; Zazímalová, Eva; Gadella, Theodorus W J; Stierhof, York-Dieter; Ueda, Takashi; Oiwa, Kazuhiro; Akhmanova, Anna; Brock, Roland; Spang, Anne; Friml, Jirí

    2008-03-18

    Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this concept. However, despite the use of ATIs in plant research for many decades, the mechanism of ATI action has remained largely elusive. Using real-time live-cell microscopy, we show here that prominent ATIs such as 2,3,5-triiodobenzoic acid (TIBA) and 2-(1-pyrenoyl) benzoic acid (PBA) inhibit vesicle trafficking in plant, yeast, and mammalian cells. Effects on micropinocytosis, rab5-labeled endosomal motility at the periphery of HeLa cells and on fibroblast mobility indicate that ATIs influence actin cytoskeleton. Visualization of actin cytoskeleton dynamics in plants, yeast, and mammalian cells show that ATIs stabilize actin. Conversely, stabilizing actin by chemical or genetic means interferes with endocytosis, vesicle motility, auxin transport, and plant development, including auxin transport-dependent processes. Our results show that a class of ATIs act as actin stabilizers and advocate that actin-dependent trafficking of auxin transport components participates in the mechanism of auxin transport. These studies also provide an example of how the common eukaryotic process of actin-based vesicle motility can fulfill a plant-specific physiological role.

  14. Competition of energy between active transport and vesicle fusion at the origin of intracellular gradient fields.

    Science.gov (United States)

    Pelcé, Pierre

    2018-02-07

    It has been reported that the ionic patterns of hyphal growth can be explained by a weakening of the active transport at the tip at the expense of other biosynthesis processes, from which results energy transport from the proximal cells to the apical ones (Potapova et al. 1988). We present here a theory to support this hypothesis, whose extent is much more general than the initial frame where it has been formulated. It can be summarized in two basics mechanisms, one coupling active transport of the plasma membrane, electric potential and vesicle fusion, the other coupling the Ca 2+ -ATPase of the endoplasmic reticulum and vesicle fusion. For some values of parameters introduced in the theory, the uniform state of the cell becomes unstable, at the origin of intracellular gradient fields. Theoretical ionic patterns are spontaneously produced, which can be satisfactorily compared to several observed in and around tip-growing cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. H+ V-ATPase-Energized Transporters in Brush Border Membrane Vesicles from Whole Larvae of Aedes Aegypti

    Science.gov (United States)

    Brush Border Membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs ) contain an H+ V-ATPase (V), a Na+/H+ antiporter, NHA1 (A) and a Na+-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles...

  16. A model of Stokesian peristalsis and vesicle transport in a three-dimensional closed cavity.

    Science.gov (United States)

    Aranda, Vivian; Cortez, Ricardo; Fauci, Lisa

    2015-06-25

    The complexity of the mechanics involved in the mammalian reproductive process is evident. Neither an ovum nor an embryo is self-propelled, but move through the oviduct or uterus due to the peristaltic action of the tube walls, imposed pressure gradients, and perhaps ciliary motion. Here we use the method of regularized Stokeslets to model the transport of an ovum or an embryo within a peristaltic tube. We represent the ovum or the embryo as a spherical vesicle of finite volume - not a massless point particle. The outer membrane of the neutrally buoyant vesicle is discretized by nodes that are joined by a network of springs. The elastic moduli of these springs are chosen large enough so that a spherical shape is maintained. For simplicity, here we choose an axisymmetric tube where the geometry of the two-dimensional cross-section along the tube axis reflects that of the sagittal cross-section of the uterine cavity. Although the tube motion is axisymmetric, the presence of the vesicle within the tube requires a fully three-dimensional model. As was found in Yaniv et al. (2009, 2012) for a 2D closed channel, we find that the flow dynamics in a 3D peristaltic tube are strongly influenced by the closed end and the manner in which the peristaltic wave damps out towards the closure. In addition, we demonstrate that the trajectory of a vesicle of finite volume can greatly differ from the trajectory of a massless fluid particle initially placed at the vesicle׳s centroid. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes

    Science.gov (United States)

    Sitaram, Anand; Dennis, Megan K.; Chaudhuri, Rittik; De Jesus-Rojas, Wilfredo; Tenza, Danièle; Setty, Subba Rao Gangi; Wood, Christopher S.; Sviderskaya, Elena V.; Bennett, Dorothy C.; Raposo, Graça; Bonifacino, Juan S.; Marks, Michael S.

    2012-01-01

    Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine–based sorting signal in the pigment cell–specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1– and AP-3–favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs. PMID:22718909

  18. Effect of diet on insulin binding and glucose transport in rat sarcolemmal vesicles

    International Nuclear Information System (INIS)

    Grimditch, G.K.; Barnard, R.J.; Sternlicht, E.; Whitson, R.H.; Kaplan, S.A.

    1987-01-01

    The purpose of this study was to compare the effects of a high-fat, high-sucrose diet (HFS) and a low-fat, high-complex carbohydrate diet (LFC) on glucose tolerance, insulin binding, and glucose transport in rat skeletal muscle. During the intravenous glucose tolerance test, peak glucose values at 5 min were significantly higher in the HFS group; 0-, 20-, and 60-min values were similar. Insulin values were significantly higher in the HFS group at all time points (except 60 min), indicating whole-body insulin resistance. Skeletal muscle was responsible, in part, for this insulin resistance, because specific D-glucose transport in isolated sarcolemmal (SL) vesicles under basal conditions was similar between LFC and HFS rats, despite the higher plasma insulin levels. Scatchard analyses of insulin binding curves to sarcolemmal vesicles revealed that the K/sub a/ of the high-affinity binding sites was significantly reduced by the HFS diet; no other binding changes were noted. Specific D-glucose transport in SL vesicles after maximum insulin stimulation (1 U/kg) was significantly depressed in the HFS group, indicating that HFS feeding also caused a postbinding defect. These results indicate that the insulin resistance in skeletal muscle associated with a HFS diet is due to both a decrease in the K/sub a/ of the high-affinity insulin receptors and a postbinding defect

  19. 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.

  20. Energetics and molecular biology of active transport in bacterial membrane vesicles.

    Science.gov (United States)

    Kaback, H R; Ramos, S; Robertson, D E; Stroobant, P; Tokuda, H

    1977-01-01

    Bacterial membrane vesicles retain the same sidedness as the membrane in the intact cell and catalyze active transport of many solutes by a respiration-dependent mechanism that does not involve the generation of utilization of ATP or other high-energy phosphate compounds. In E. coli vesicles, most of these transport systems are coupled to an electrochemical gradient of protons (deltamuH+, interior negative and alkaline) generated primarily by the oxidation of D-lactate or reduced phenazine methosulfate via a membrane-bound respiratory chain. Oxygen or, under appropriate conditions, fumarate or nitrate can function as terminal electron acceptors, and the site at which deltamuH+ is generated is located before cytochrome b1 in the respiratory chain. Certain (N-dansyl)aminoalkyl-beta-D-galactopyranosides (Dns-gal) and N(2-nitro-4-azidophenyl)aminoalkyl 1-thio-beta-D-galactopyranosides (APG) are competitive inhibitors of lactose transport but are not transported themselves. Various fluorescence techniques, direct binding assays, and photoinactivation studies demonstrate that the great bulk of the lac carrier protein (ca. 95%) does not bind ligand in the absence of energy-coupling. Upon generation of a deltamuH+ (interior negative and alkaline), binding of Dns-gal and APG-dependent photoinactivation are observed. The data indicate that energy is coupled to the initial step in the transport process, and suggest that the lac carrier protein may be negatively charged.

  1. Biophysics of active vesicle transport, an intermediate step that couples excitation and exocytosis of serotonin in the neuronal soma.

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    Francisco F De-Miguel

    Full Text Available Transmitter exocytosis from the neuronal soma is evoked by brief trains of high frequency electrical activity and continues for several minutes. Here we studied how active vesicle transport towards the plasma membrane contributes to this slow phenomenon in serotonergic leech Retzius neurons, by combining electron microscopy, the kinetics of exocytosis obtained from FM1-43 dye fluorescence as vesicles fuse with the plasma membrane, and a diffusion equation incorporating the forces of local confinement and molecular motors. Electron micrographs of neurons at rest or after stimulation with 1 Hz trains showed cytoplasmic clusters of dense core vesicles at 1.5±0.2 and 3.7±0.3 µm distances from the plasma membrane, to which they were bound through microtubule bundles. By contrast, after 20 Hz stimulation vesicle clusters were apposed to the plasma membrane, suggesting that transport was induced by electrical stimulation. Consistently, 20 Hz stimulation of cultured neurons induced spotted FM1-43 fluorescence increases with one or two slow sigmoidal kinetics, suggesting exocytosis from an equal number of vesicle clusters. These fluorescence increases were prevented by colchicine, which suggested microtubule-dependent vesicle transport. Model fitting to the fluorescence kinetics predicted that 52-951 vesicles/cluster were transported along 0.60-6.18 µm distances at average 11-95 nms(-1 velocities. The ATP cost per vesicle fused (0.4-72.0, calculated from the ratio of the ΔG(process/ΔG(ATP, depended on the ratio of the traveling velocity and the number of vesicles in the cluster. Interestingly, the distance-dependence of the ATP cost per vesicle was bistable, with low energy values at 1.4 and 3.3 µm, similar to the average resting distances of the vesicle clusters, and a high energy barrier at 1.6-2.0 µm. Our study confirms that active vesicle transport is an intermediate step for somatic serotonin exocytosis by Retzius neurons and provides a

  2. Identification of retrograde transport vesicles containing nerve growth factor in vivo

    International Nuclear Information System (INIS)

    Weible, M.W.; Sandow, S.L.; Ozsarac, N.; Hendry, I.A.; Grimes, M.L.

    2002-01-01

    Full text: Survival, differentiation, and development of responsive neurons are regulated by neurotrophins secreted from the target cells that they innervate. These responsive neurons must meet the challenge of transporting the neurotrophins chemical message from the target tissue to the soma; the distance of which may be a few millimetres to many meters. One hypothesis involves the formation of a signalling organelle at the neurite tip and subsequent retrograde axonal transport to the soma. This signalling vesicle is derived from the endocytosis of the neurotrophin-receptor complex. By modifying a method developed to isolate signalling endosomes from PC12 cells, we are able to isolate signalling vesicles from rat and mouse sciatic tissue. Approximately, 4 mole of I 125 -labelled neurotrophin was injected into the rodent foot pad and the sciatic nerve ligated under 88 μ/g ketamine and 16 μ/g rompun (i.p.) anaesthetic. All experiments had the approval of the ANU animal ethics committee. We achieved a recovery of 23% and 34% in the mouse and rat respectively of total transported iodinated neurotrophin accumulating on the distal side of the ligation. The homogenized tissue was characterized via differential centrifugation, blotted, and probed using antibodies to the neurotrophin receptors. Electron microscopy confirmed that the membrane pellet containing the transported neurotrophin from this in vivo preparation contained vesicular structures. Copyright (2002) Australian Neuroscience Society

  3. Three-dimensional nanometry of vesicle transport in living cells using dual-focus imaging optics

    International Nuclear Information System (INIS)

    Watanabe, Tomonobu M.; Sato, Takashi; Gonda, Kohsuke; Higuchi, Hideo

    2007-01-01

    Dual-focus imaging optics for three-dimensional tracking of individual quantum dots has been developed to study the molecular mechanisms of motor proteins in cells. The new system has a high spatial and temporal precision, 2 nm in the x-y sample plane and 5 nm along the z-axis at a frame time of 2 ms. Three-dimensional positions of the vesicles labeled with quantum dots were detected in living cells. Vesicles were transported on the microtubules using 8-nm steps towards the nucleus. The steps had fluctuation of ∼20 nm which were perpendicular to the axis of the microtubule but with the constant distance from the microtubule. The most of perpendicular movement was not synchronized with the 8-nm steps, indicating that dynein moved on microtubules without changing the protofilaments. When the vesicles changed their direction of movement toward the cell membrane, they moved perpendicular with the constant distance from the microtubule. The present method is powerful tool to investigate three dimensional movement of molecules in cells with nanometer and millisecond accuracy

  4. The human synaptic vesicle protein, SV2A, functions as a galactose transporter in Saccharomyces cerevisiae.

    Science.gov (United States)

    Madeo, Marianna; Kovács, Attila D; Pearce, David A

    2014-11-28

    SV2A is a synaptic vesicle membrane protein expressed in neurons and endocrine cells and involved in the regulation of neurotransmitter release. Although the exact function of SV2A still remains elusive, it was identified as the specific binding site for levetiracetam, a second generation antiepileptic drug. Our sequence analysis demonstrates that SV2A has significant homology with several yeast transport proteins belonging to the major facilitator superfamily (MFS). Many of these transporters are involved in sugar transport into yeast cells. Here we present evidence showing, for the first time, that SV2A is a galactose transporter. We expressed human SV2A in hexose transport-deficient EBY.VW4000 yeast cells and demonstrated that these cells are able to grow on galactose-containing medium but not on other fermentable carbon sources. Furthermore, the addition of the SV2A-binding antiepileptic drug levetiracetam to the medium inhibited the galactose-dependent growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A. Most importantly, direct measurement of galactose uptake in the same strain verified that SV2A is able to transport extracellular galactose inside the cells. The newly identified galactose transport capability of SV2A may have an important role in regulating/modulating synaptic function. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. 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}}.

  6. Effect of vesicle traps on traffic jam formation in fast axonal transport.

    Science.gov (United States)

    Kuznetsov, A V

    2010-08-01

    The purpose of this paper is to develop a model for simulation of the formation of organelle traps in fast axonal transport. Such traps may form in the regions of microtubule polar mismatching. Depending on the orientation of microtubules pointing toward the trap region, these traps can accumulate either plus-end or minus-end oriented vesicles. The model predicts that the maximum concentrations of organelles occur at the boundaries of the trap regions; the overall concentration of organelles in the axon with traps is greatly increased compared to that in a healthy axon, which is expected to contribute to mechanical damages of the axon. The organelle traps induce hindrance to organelle transport down the axon; the total organelle flux down the axon with traps is found to be significantly reduced compared to that in a healthy axon. Copyright 2010 Elsevier Inc. All rights reserved.

  7. [Vesicular intracellular transport in the digestive organs. Membrane vesicle--the universal mechanism of the functional transport].

    Science.gov (United States)

    Morozov, I A

    2014-01-01

    On the basis of long-term research of the morpho-functional characteristics of the cells of the stomach, small intestine and gallbladder the mechanism and function of membrane vesicles in the implementation of the main functions of these organs sets out in this article: the secretion of hydrochloric acid by parietal cells, the absorption of nutrients in the small intestine and the fluid at a concentration of bile epitheliocytes of gallbladder. Proofs of the intracellular formation of hydrochloric acid in tubulovesicles of the parietal cells and turnover of its secretory membranes in the process of secretory cycle, that has ensured the re-use and explained the extraordinary life of these unique cells are presented. The credible mechanism of HCl output oppression by H(+)-K(+)-ATPase activity blockers has set out on this basis. The article provides detailed endocytosis mechanism of the ions and nutrients absorption by enterocytes. The mechanism of participation of the apical contractile complex of brush border of epithelial cells in the initiation of endocytosis and cytoplasmic microtubules in transport of membrane vesicles in the cytoplasm was analyzed. Based on our research and numerous of the world scientific proceedings the conclusion was done about the existence of two energy dependent types of transport in the absorptive epithelium of the digestive--transmembrane (ionic and nutritive) homeostatic type which is realized by the ATP-system of the basal plasmalemma, and vesicular (endocytosis) type which is impltmented by apical contractile complex of brush border and cytoplasmic microtubules. Both types of transport are interrelated and are under constant cellular control. This observation is relevant to the majority of cells, including those involved in the secretion of various substances: hydrochloric acid by parietal cells, enzymes by main cells of the gastric glands and exocrinocytes of the pancreas, hormone by endocrine cells of the APUD system and, finally

  8. Identification of myosin XI receptors in Arabidopsis defines a distinct class of transport vesicles.

    Science.gov (United States)

    Peremyslov, Valera V; Morgun, Eva A; Kurth, Elizabeth G; Makarova, Kira S; Koonin, Eugene V; Dolja, Valerian V

    2013-08-01

    To characterize the mechanism through which myosin XI-K attaches to its principal endomembrane cargo, a yeast two-hybrid library of Arabidopsis thaliana cDNAs was screened using the myosin cargo binding domain as bait. This screen identified two previously uncharacterized transmembrane proteins (hereinafter myosin binding proteins or MyoB1/2) that share a myosin binding, conserved domain of unknown function 593 (DUF593). Additional screens revealed that MyoB1/2 also bind myosin XI-1, whereas myosin XI-I interacts with the distantly related MyoB7. The in vivo interactions of MyoB1/2 with myosin XI-K were confirmed by immunoprecipitation and colocalization analyses. In epidermal cells, the yellow fluorescent protein-tagged MyoB1/2 localize to vesicles that traffic in a myosin XI-dependent manner. Similar to myosin XI-K, MyoB1/2 accumulate in the tip-growing domain of elongating root hairs. Gene knockout analysis demonstrated that functional cooperation between myosin XI-K and MyoB proteins is required for proper plant development. Unexpectedly, the MyoB1-containing vesicles did not correspond to brefeldin A-sensitive Golgi and post-Golgi or prevacuolar compartments and did not colocalize with known exocytic or endosomal compartments. Phylogenomic analysis suggests that DUF593 emerged in primitive land plants and founded a multigene family that is conserved in all flowering plants. Collectively, these findings indicate that MyoB are membrane-anchored myosin receptors that define a distinct, plant-specific transport vesicle compartment.

  9. 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.

  10. Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles.

    Science.gov (United States)

    Mandela, Prashant; Chandley, Michelle; Xu, Yao-Yu; Zhu, Meng-Yang; Ordway, Gregory A

    2010-01-01

    Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [(3)H]NE uptake capacity, an effect characterized by a robust decrease in the V(max) of the transport of [(3)H]NE. As expected, reserpine did not displace the binding of [(3)H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [(3)H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [(3)H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca(2+)/Ca(2+)-calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [(3)H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, alpha-methyl-p-tyrosine, increased [(3)H]NE uptake and eliminated the inhibitory effects of reserpine on [(3)H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca(2+)-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors. Copyright 2010 Elsevier Ltd. All rights reserved.

  11. Use of membrane vesicles as a simplified system for studying auxin transport of auxin: Progress report

    International Nuclear Information System (INIS)

    Goldsmith, M.H.M.

    1986-01-01

    Indoleacetic acid (IAA), the auxin regulating growth, is transported polarly in plants. IAA stimulates a rapid increase in the rate of electrogenic proton secretion by the plasma membrane. This not only increases the magnitude of the pH and electrical gradients providing the driving force for polar auxin transport and uptake of sugars, amino acids and inorganic ions, but, by acidifying the cell wall, also leads to growth. We find that auxin uptake by membrane vesicles isolated from actively growing plant tissues exhibits some of the same properties as by cells: the accumulation depends on the pH gradient, is saturable and specific for auxin, and enhanced by herbicides that inhibit polar auxin transport. We are using accumulation of a radioactive weak acid to quantify the pH gradient and distribution of fluorescent cyanine dyes to monitor the membrane potential. The magnitude of IAA accumulation exceeds that predicted from the pH gradient, and in the absence of a pH gradient, a membrane potential fails to support any auxin accumulation, leading to the conclusion that the transmembrane potential is not a significant driving force for auxin accumulation in this system. Since increasing the external ionic strength decreases saturable auxin accumulation, we are investigating how modifying the surface potential of the vesicles affects the interaction of the amphipathic IAA molecules with the membranes and whether protein modifying reagents affect the saturability and stimulation by NPA. These studies should provide information on the location and function of the auxin binding site and may enable us to identify the solubilized protein. 5 refs

  12. Transport of acidic amino acids by human jejunal brush-border membrane vesicles

    International Nuclear Information System (INIS)

    Rajendran, V.M.; Harig, J.M.; Adams, M.B.; Ramaswamy, K.

    1987-01-01

    This study characterizes the transport of radiolabeled acidic amino acids into brush-border membrane vesicles prepared from human jejunum. The uptakes of L-glutamic, L-aspartic, and D-aspartic acids were stimulated by a Na + gradient. Concentrative uptake (resulting in an overshoot phenomenon) of these dicarboxylic amino acids occurred when there was an outward K + gradient. In addition, increasing K + gradients resulted in enhanced uptake of L-glutamic acid. This K + requirement is somewhat specific as Rb + and Cs + could enhance uptake to a limited extent, whereas Li + and choline + showed no enhancement. The presence of a K + gradient did not affect the affinity of the carrier system for L-glutamic acid but it did increase the V/sub max/. The presence of extravesicular anions having differing membrane permeabilities did not altar L-glutamic acid uptake indicating an absence of an effect of membrane potential on the transport process. Finally, the human transport system for L-glutamic acid appears to be specific for acidic amino acids as demonstrated by inhibition studies. The studies demonstrate a transport system in human jejunum specific for acidic amino acids that is energized by an inward Na + gradient and an outward K + gradient

  13. Live-cell imaging of post-golgi transport vesicles in cultured hippocampal neurons

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Misonou, Hiroaki

    2015-01-01

    compartments of neurons. In the past two decades, the establishment and advancement of fluorescent protein technology have provided us with opportunities to study how proteins are trafficked in living cells. However, live imaging of trafficking processes in neurons necessitate imaging tools to distinguish......The subcellular localization of neuronal membrane signaling molecules such as receptors and ion channels depends on intracellular trafficking mechanisms. Essentially, vesicular trafficking mechanisms ensure that a large number of membrane proteins are correctly targeted to different subcellular...... the several different routes that neurons use for protein trafficking. Here we provide a novel protocol to selectively visualize post-Golgi transport vesicles carrying fluorescent-labeled ion channel proteins in living neurons. Further, we provide a number of analytical tools we developed to quantify...

  14. 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.

  15. ABC protein transport of MRI contrast agents in canalicular rat liver plasma vesicles and yeast vacuoles

    International Nuclear Information System (INIS)

    Pascolo, Lorella; Petrovic, Sinisa; Cupelli, Felicia; Bruschi, Carlo V.; Anelli, Pier Lucio; Lorusso, Vito; Visigalli, Massimo; Uggeri, Fulvio; Tiribelli, Claudio

    2001-01-01

    The mechanism of excretion into bile of hepatospecific magnetic resonance imaging (MRI) contrast media employed labeled Gd-reagents EOB.DTPA, BOPTA, B 20790 (iopanoate-linked), and B 21690 (glycocholate-linked) for measurement in rat liver canalicular plasma membrane vesicles and yeast vacuoles. The presence of ATP gave threefold greater transport of B 20790 and B 21690 than of EOB.DTPA and BOPTA. In yeast vacuoles the ATP stimulatory effect was eightfold with B 20790 and fivefold greater for B 21690, whereas in YCF1- or YLLO115w-deleted yeast cells the transport was significantly reduced and absent from double mutants, YCF1 and YLLO15w. The transport was similar in wild-type and deletant cells for B 21690; taurocholate gave 85% inhibition. These data suggest that bilary secretion of structurally related MRI agents depend on molecular structure. The findings are suggestive as of possible value for clinical diagnosis of inherited hyperbilirubinemias and other liver disorders

  16. Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles

    Science.gov (United States)

    Myint, Khine; Li, Yan; Paxton, James; McKeage, Mark

    2015-01-01

    The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In

  17. Regulation of vesicle transport and cell motility by Golgi-localized Dbs

    Science.gov (United States)

    Fitzpatrick, Ethan R; Hu, Tinghui; Ciccarelli, Bryan T; Whitehead, Ian P

    2014-01-01

    DBS/MCF2L has been recently identified as a risk locus for osteoarthritis. It encodes a guanine nucleotide exchange factor (Dbs) that has been shown to regulate both normal and tumor cell motility. In the current study, we have determined that endogenous Dbs is predominantly expressed as 2 isoforms, a 130 kDa form (Dbs-130) that is localized to the Golgi complex, and an 80 kDa form (Dbs-80) that is localized to the endoplasmic reticulum (ER). We have previously described an inhibitor that binds to the RhoGEF domain of Dbs and blocks its transforming activity. Here we show that the inhibitor localizes to the Golgi, where it specifically interacts with Dbs-130. Inhibition of endogenous Dbs-130 activity is associated with reduced levels of activated Cdc42, enlarged Golgi, and resistance to Brefeldin A-mediated Golgi dispersal, suggesting a role for Dbs in vesicle transport. Cells treated with the inhibitor exhibit normal protein transport from the ER to the Golgi, but are defective in transport from the Golgi to the plasma membrane. Inhibition of Dbs-130 in MDA-MB-231 human breast tumor cells limits motility in both transwell and wound healing assays, but appears to have no effect on the organization of the microtubule cytoskeleton. The reduced motility is associated with a failure to reorient the Golgi toward the leading edge. This is consistent with the Golgi localization, and suggests that the Dbs-130 regulates aspects of the secretory pathway that are required to support cell polarization during directed migration. PMID:25483302

  18. The retinitis pigmentosa protein RP2 links pericentriolar vesicle transport between the Golgi and the primary cilium.

    Science.gov (United States)

    Evans, R Jane; Schwarz, Nele; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe; Hardcastle, Alison J; Cheetham, Michael E

    2010-04-01

    Photoreceptors are complex ciliated sensory neurons. The basal body and periciliary ridge of photoreceptors function in association with the Golgi complex to regulate the export of proteins from the inner segment to the outer segment sensory axoneme. Here, we show that the retinitis pigmentosa protein RP2, which is a GTPase activating protein (GAP) for Arl3, localizes to the ciliary apparatus, namely the basal body and the associated centriole at the base of the photoreceptor cilium. Targeting to the ciliary base was dependent on N-terminal myristoylation. RP2 also localized to the Golgi and periciliary ridge of photoreceptors, which suggested a role for RP2 in regulating vesicle traffic and docking. To explore this hypothesis, we investigated the effect of RP2 depletion and the expression of a constitutively active form of Arl3 (Q71L) on pericentriolar vesicle transport. Kif3a, a component of intraflagellar transport (IFT), is important in cilia maintenance and transport of proteins through the connecting cilium in photoreceptors. Similar to Kif3a and Arl3 depletion, loss of RP2 led to fragmentation of the Golgi network. Depletion of RP2 and dysregulation of Arl3 resulted in dispersal of vesicles cycling cargo from the Golgi complex to the cilium, including the IFT protein IFT20. We propose that RP2 regulation of Arl3 is important for maintaining Golgi cohesion, facilitating the transport and docking of vesicles and thereby carrying proteins to the base of the photoreceptor connecting cilium for transport to the outer segment.

  19. Photosensitized electron transport across lipid vesicle walls: Enhancement of quantum yield by ionophores and transmembrane potentials

    Science.gov (United States)

    Laane, Colja; Ford, William E.; Otvos, John W.; Calvin, Melvin

    1981-01-01

    The photosensitized reduction of heptylviologen in the bulk aqueous phase of phosphatidylcholine vesicles containing EDTA inside and a membrane-bound tris(2,2′-bipyridine)ruthenium(2+) derivative is enhanced by a factor of 6.5 by the addition of valinomycin in the presence of K+. A 3-fold stimulation by gramicidin and carbonyl cyanide m-chlorophenylhydrazone is observed. The results suggest that, under these conditions, the rate of photoinduced electron transfer across vesicle walls in the absence of ion carriers is limited by cotransport of cations. The rate of electron transfer across vesicle walls could be influenced further by generating transmembrane potentials with K+ gradients in the presence of valinomycin. When vesicles are made with transmembrane potentials, interior more negative, the quantum yield of heptylviologen reduction is doubled, and, conversely, when vesicles are made with transmembrane potentials, interior more positive, the quantum yield is decreased and approaches the value found in the absence of valinomycin. PMID:16593002

  20. Dimethyltryptamine and other hallucinogenic tryptamines exhibit substrate behavior at the serotonin uptake transporter and the vesicle monoamine transporter.

    Science.gov (United States)

    Cozzi, Nicholas V; Gopalakrishnan, Anupama; Anderson, Lyndsey L; Feih, Joel T; Shulgin, Alexander T; Daley, Paul F; Ruoho, Arnold E

    2009-12-01

    N,N-dimethyltryptamine (DMT) is a potent plant hallucinogen that has also been found in human tissues. When ingested, DMT and related N,N-dialkyltryptamines produce an intense hallucinogenic state. Behavioral effects are mediated through various neurochemical mechanisms including activity at sigma-1 and serotonin receptors, modification of monoamine uptake and release, and competition for metabolic enzymes. To further clarify the pharmacology of hallucinogenic tryptamines, we synthesized DMT, N-methyl-N-isopropyltryptamine (MIPT), N,N-dipropyltryptamine (DPT), and N,N-diisopropyltryptamine. We then tested the abilities of these N,N-dialkyltryptamines to inhibit [(3)H]5-HT uptake via the plasma membrane serotonin transporter (SERT) in human platelets and via the vesicle monoamine transporter (VMAT2) in Sf9 cells expressing the rat VMAT2. The tryptamines were also tested as inhibitors of [(3)H]paroxetine binding to the SERT and [(3)H]dihydrotetrabenazine binding to VMAT2. Our results show that DMT, MIPT, DPT, and DIPT inhibit [(3)H]5-HT transport at the SERT with K ( I ) values of 4.00 +/- 0.70, 8.88 +/- 4.7, 0.594 +/- 0.12, and 2.32 +/- 0.46 microM, respectively. At VMAT2, the tryptamines inhibited [(3)H]5-HT transport with K ( I ) values of 93 +/- 6.8, 20 +/- 4.3, 19 +/- 2.3, and 19 +/- 3.1 muM, respectively. On the other hand, the tryptamines were very poor inhibitors of [(3)H]paroxetine binding to SERT and of [(3)H]dihydrotetrabenazine binding to VMAT2, resulting in high binding-to-uptake ratios. High binding-to-uptake ratios support the hypothesis that the tryptamines are transporter substrates, not uptake blockers, at both SERT and VMAT2, and also indicate that there are separate substrate and inhibitor binding sites within these transporters. The transporters may allow the accumulation of tryptamines within neurons to reach relatively high levels for sigma-1 receptor activation and to function as releasable transmitters.

  1. Porewater methane transport within the gas vesicles of diurnally migrating Chaoborus spp.: An energetic advantage

    Science.gov (United States)

    McGinnis, Daniel F.; Flury, Sabine; Tang, Kam W.; Grossart, Hans-Peter

    2017-03-01

    Diurnally-migrating Chaoborus spp. reach populations of up to 130,000 individuals m-2 in lakes up to 70 meters deep on all continents except Antarctica. Linked to eutrophication, migrating Chaoborus spp. dwell in the anoxic sediment during daytime and feed in the oxic surface layer at night. Our experiments show that by burrowing into the sediment, Chaoborus spp. utilize the high dissolved gas partial pressure of sediment methane to inflate their tracheal sacs. This mechanism provides a significant energetic advantage that allows the larvae to migrate via passive buoyancy rather than more energy-costly swimming. The Chaoborus spp. larvae, in addition to potentially releasing sediment methane bubbles twice a day by entering and leaving the sediment, also transport porewater methane within their gas vesicles into the water column, resulting in a flux of 0.01-2 mol m-2 yr-1 depending on population density and water depth. Chaoborus spp. emerging annually as flies also result in 0.1-6 mol m-2 yr-1 of carbon export from the system. Finding the tipping point in lake eutrophication enabling this methane-powered migration mechanism is crucial for ultimately reconstructing the geographical expansion of Chaoborus spp., and the corresponding shifts in the lake’s biogeochemistry, carbon cycling and food web structure.

  2. Vesicle amine transport protein-1 (VAT-1) is upregulated in glioblastomas and promotes migration.

    Science.gov (United States)

    Mertsch, S; Becker, M; Lichota, A; Paulus, W; Senner, V

    2009-08-01

    Diffuse invasion of single-glioma cells is the main obstacle to successful therapy of these tumours. After identifying vesicle amine transport protein-1 (VAT-1) as being upregulated in invasive human gliomas, we study its possible function in glioblastoma cell migration. Based on data obtained from previous oligonucleotide arrays, we investigated expression of VAT-1 in glioblastoma tissue and cell lines on mRNA levels using reverse transcriptase PCR. Furthermore, we examined the amount and localization of VAT-1 protein using immunoblotting and immunohistochemistry. Using small interfering RNA technology we repressed VAT-1 expression in human glioma cell lines and analysed their migration using wound healing and transwell migration assays. Increased VAT-1 mRNA and protein levels were found in glioblastoma tissues and cell lines compared with normal human brain. Small interfering RNA-mediated VAT-1 knockdown led to significantly reduced migration of human glioma cells. VAT-1 is overexpressed in glioblastomas and functionally involved in glioma cell migration, representing a new component involved in glioma invasion

  3. Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

    International Nuclear Information System (INIS)

    Rajendran, V.M.; Kashgarian, M.; Binder, H.J.

    1989-01-01

    Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats

  4. Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules.

    Science.gov (United States)

    Jorge-Finnigan, Ana; Kleppe, Rune; Jung-Kc, Kunwar; Ying, Ming; Marie, Michael; Rios-Mondragon, Ivan; Salvatore, Michael F; Saraste, Jaakko; Martinez, Aurora

    2017-08-25

    Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Septin6 and Septin7 GTP binding proteins regulate AP-3- and ESCRT-dependent multivesicular body biogenesis.

    Directory of Open Access Journals (Sweden)

    Sofia Traikov

    Full Text Available Septins (SEPTs form a family of GTP-binding proteins implicated in cytoskeleton and membrane organization, cell division and host/pathogen interactions. The precise function of many family members remains elusive. We show that SEPT6 and SEPT7 complexes bound to F-actin regulate protein sorting during multivesicular body (MVB biogenesis. These complexes bind AP-3, an adapter complex sorting cargos destined to remain in outer membranes of maturing endosomes, modulate AP-3 membrane interactions and the motility of AP-3-positive endosomes. These SEPT-AP interactions also influence the membrane interaction of ESCRT (endosomal-sorting complex required for transport-I, which selects ubiquitinated cargos for degradation inside MVBs. Whereas our findings demonstrate that SEPT6 and SEPT7 function in the spatial, temporal organization of AP-3- and ESCRT-coated membrane domains, they uncover an unsuspected coordination of these sorting machineries during MVB biogenesis. This requires the E3 ubiquitin ligase LRSAM1, an AP-3 interactor regulating ESCRT-I sorting activity and whose mutations are linked with Charcot-Marie-Tooth neuropathies.

  6. APS 3D: a new benchmark in aspherical polishing

    Science.gov (United States)

    Gauch, Daniel; Mikulic, Dalibor; Veit, Christian

    2017-10-01

    The APS 3D system performs polishing and form correction in one step in order to reduce overall process time, reduce the number of polishing steps required and eliminate the need for highly skilled operators while providing a repeatable polishing process. This new 3D Polishing system yields better surface quality, and a better slope error, automatically determining the optimum speeds, feed rates and polish pressures to achieve a deterministic process based on the required quality parameters input by the operator. The process flow is always the same to ensure consistent quality and target quality values are defined before polishing begins.

  7. Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain.

    Directory of Open Access Journals (Sweden)

    Hiroaki Mochizuki

    2011-05-01

    Full Text Available Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II, an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM and No distributive disjunction (Nod, remains unaltered. Genetic analyses of kinesin light chain (Klc and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of

  8. Functional analysis of the two Brassica AP3 genes involved in apetalous and stamen carpelloid phenotypes.

    Directory of Open Access Journals (Sweden)

    Yanfeng Zhang

    Full Text Available The Arabidopsis homeotic genes APETALA3 (AP3 and PISTILLATA (PI are B genes which encode MADS-box transcription factors and specify petal and stamen identities. In the current study, the stamen carpelloid (SC mutants, HGMS and AMS, of B. rapa and B. napus were investigated and two types of AP3 genes, B.AP3.a and B.AP3.b, were functional characterized. B.AP3.a and B.AP3.b share high similarity in amino acid sequences except for 8 residues difference located at the C-terminus. Loss of this 8 residues in B.AP3.b led to the change of PI-derived motifs. Meanwhile, B.AP3.a specified petal and stamen development, whereas B.AP3.b only specified stamen development. In B. rapa, the mutations of both genes generated the SC mutant HGMS. In B. napus that contained two B.AP3.a and two B.AP3.b, loss of the two B.AP3.a functions was the key reason for the apetalous mutation, however, the loss-of-function in all four AP3 was related to the SC mutant AMS. We inferred that the 8 residues or the PI-derived motif in AP3 gene probably relates to petal formation.

  9. Huntingtin is required for ER-to-Golgi transport and for secretory vesicle fusion at the plasma membrane

    Directory of Open Access Journals (Sweden)

    Hemma Brandstaetter

    2014-12-01

    Full Text Available Huntingtin is a large membrane-associated scaffolding protein that associates with endocytic and exocytic vesicles and modulates their trafficking along cytoskeletal tracks. Although the progression of Huntington’s disease is linked to toxic accumulation of mutant huntingtin protein, loss of wild-type huntingtin function might also contribute to neuronal cell death, but its precise function is not well understood. Therefore, we investigated the molecular role of huntingtin in exocytosis and observed that huntingtin knockdown in HeLa cells causes a delay in endoplasmic reticulum (ER-to-Golgi transport and a reduction in the number of cargo vesicles leaving the trans-Golgi network. In addition, we found that huntingtin is required for secretory vesicle fusion at the plasma membrane. Similar defects in the early exocytic pathway were observed in primary fibroblasts from homozygous Htt140Q/140Q knock-in mice, which have the expansion inserted into the mouse huntingtin gene so lack wild-type huntingtin expression. Interestingly, heterozygous fibroblasts from a Huntington’s disease patient with a 180Q expansion displayed no obvious defects in the early secretory pathway. Thus, our results highlight the requirement for wild-type huntingtin at distinct steps along the secretory pathway.

  10. Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated

    NARCIS (Netherlands)

    Kok, JW; Babia, T; Klappe, K; Egea, G; Hoekstra, D

    1998-01-01

    Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degrees C, or in streptolysin O-permeabilized cells by

  11. 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

  12. The Human Synaptic Vesicle Protein, SV2A, Functions as a Galactose Transporter in Saccharomyces cerevisiae * ♦

    Science.gov (United States)

    Madeo, Marianna; Kovács, Attila D.; Pearce, David A.

    2014-01-01

    SV2A is a synaptic vesicle membrane protein expressed in neurons and endocrine cells and involved in the regulation of neurotransmitter release. Although the exact function of SV2A still remains elusive, it was identified as the specific binding site for levetiracetam, a second generation antiepileptic drug. Our sequence analysis demonstrates that SV2A has significant homology with several yeast transport proteins belonging to the major facilitator superfamily (MFS). Many of these transporters are involved in sugar transport into yeast cells. Here we present evidence showing, for the first time, that SV2A is a galactose transporter. We expressed human SV2A in hexose transport-deficient EBY.VW4000 yeast cells and demonstrated that these cells are able to grow on galactose-containing medium but not on other fermentable carbon sources. Furthermore, the addition of the SV2A-binding antiepileptic drug levetiracetam to the medium inhibited the galactose-dependent growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A. Most importantly, direct measurement of galactose uptake in the same strain verified that SV2A is able to transport extracellular galactose inside the cells. The newly identified galactose transport capability of SV2A may have an important role in regulating/modulating synaptic function. PMID:25326386

  13. 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...

  14. Restoration of active transport of solutes and oxidative phosphorylation by naphthoquinones in irradiated membrane vesicles from Mycobacterium phlei

    Science.gov (United States)

    Lee, Soon-Ho; Sutherland, Thomas O.; Deves, Rosa; Brodie, Arnold F.

    1980-01-01

    Irradiation of the inverted membrane vesicles of Mycobacterium phlei with light at 360 nm inactivated the natural menaquinone [MK9(II-H)] and resulted in a loss of substrate oxidation, pH gradient, membrane potential, active transport of proline or calcium ions, and oxidative phosphorylation. Restoration of the protonmotive force and active transport occurred on addition of naphthoquinones such as vitamin K1, menadione, or lapachol to the irradiated membrane vesicles. However, coupled phosphorylation was restored only by vitamin K1. Menadione and lapachol did not act as uncoupling agents. The magnitude of the pH gradient and membrane potential in the quinone-restored system was a reflection of the rate of oxidation and was correlated with the rate of uptake of proline or Ca2+. These results are consistent with the chemosmotic hypothesis proposed for the energy transducing mechanism for active transport and further demonstrate that the complete respiratory chain is not required to drive active transport. In contrast, the data suggest that in addition to the driving force (protonmotive force) necessary to establish oxidative phosphorylation, a specific spatial orientation of the respiratory components, such as the naphthaquinones, is essential for the utilization of the proton gradient or membrane potential or both. Bypass of electrons from the respiratory chain with menadione may explain the inability of this quinone to restore oxidative phosphorylation; however, lapachol restores oxidation by the same electron transport pathway as the natural menaquinone but fails to restore phosphorylation. Because all three quinones restore the protonmotive force, other factors that are discussed must be considered in understanding the mechanism of oxidative phosphorylation. PMID:6928606

  15. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

    Czech Academy of Sciences Publication Activity Database

    Dhonukshe, P.; Grigoriev, I.; Fischer, R.; Tominaga, M.; Robinson, D.G.; Hašek, Jiří; Paciorek, T.; Petrášek, Jan; Seifertová, Daniela; Tejos, R.; Meisel, L.A.; Zažímalová, Eva; Gadella, T.W.J.; Stierhof, Y. D.; Ueda, T.; Oiwa, K.; Akhmanova, A.; Brock, R.; Spang, A.; Friml, J.

    2008-01-01

    Roč. 105, č. 11 (2008), s. 4489-4494 ISSN 0027-8424 R&D Projects: GA ČR GA204/05/0838; GA AV ČR(CZ) IAA601630703; GA MŠk(CZ) LC06034; GA AV ČR KJB600380604 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50380511 Keywords : pin proteins * plant development * vesicle traffic Subject RIV: EE - Microbiology, Virology Impact factor: 9.380, year: 2008

  16. Diacylglycerol is required for the formation of COPI vesicles in the Golgi-to-ER transport pathway.

    Science.gov (United States)

    Fernández-Ulibarri, Inés; Vilella, Montserrat; Lázaro-Diéguez, Francisco; Sarri, Elisabet; Martínez, Susana E; Jiménez, Nuria; Claro, Enrique; Mérida, Isabel; Burger, Koert N J; Egea, Gustavo

    2007-09-01

    Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation.

  17. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

    NARCIS (Netherlands)

    Dhonukshe, P.; Grigoriev, I.; Fischer, R.; Tominaga, M.; Robinson, D.G.; Hašek, J.; Paciorek, T.; Petrášek, J.; Seifertová, D.; Tejos, R.; Meisel, L.A.; Zažímalová, E.; Gadella (jr.), T.W.J.; Stierhof, Y.-D.; Ueda, T.; Oiwa, K.; Akhmanova, A.; Brock, R.; Spang, A.; Friml, J.

    2008-01-01

    Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this

  18. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

    NARCIS (Netherlands)

    P. Dhonukshe (Pankaj); I. Grigoriev (Ilya); R. Fischer (Rainer); M. Tominaga (Motoki); D.G. Robinson (David); J. Hašek (Jiří); T. Paciorek (Tomasz); J. Petrášek (Jan); D. Seifertová (Daniela); R. Tejos (Ricardo); L.A. Meisel (Lee); E. Zažímalová (Eva); T.W.J. Gadella (Theodorus); Y.D. Stierhof; T. Ueda (Takashi); K. Oiwa (Kazuhiro); A.S. Akhmanova (Anna); R. Brock (Roland); A. Spang (Anne); J. Friml (Jiří)

    2008-01-01

    textabstractMany aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating

  19. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes.

    NARCIS (Netherlands)

    Dhonukshe, P.; Grigoriev, I.; Fischer, R.; Tominaga, M.; Robinson, D.G.; Hasek, J.; Paciorek, T.; Petrasek, J.; Seifertova, D.; Tejos, R.; Meisel, L.A.; Zazimalova, E.; Gadella, T.W.; Stierhof, Y.D.; Ueda, T.; Oiwa, K.; Akhmanova, A.; Brock, R.E.; Spang, A.; Friml, J.

    2008-01-01

    Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this

  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. Vesicles and vesicle fusion: coarse-grained simulations

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2010-01-01

    of vesicles that is crucial for this transport is their ability to fuse to target membranes and release their contents to the distal side. In industry, some personal care products contain vesicles to help transport reagents across the skin, and research on drug formulation shows that packaging active...

  2. Autoimmune polyglandular syndrome type 3 (APS-3) among patients with premature ovarian insufficiency (POI).

    Science.gov (United States)

    Szlendak-Sauer, Katarzyna; Jakubik, Daniel; Kunicki, Michał; Skórska, Jolanta; Smolarczyk, Roman

    2016-08-01

    Autoimmune polyglandular syndrome type 3 - (APS-3), is defined as the coexistence of autoimmune thyroiditis with other non-ovarian autoimmune diseases without primary adrenal insufficiency. Additionally the definition of APS-3 also includes primary ovarian insufficiency (POI) coexistence with autoimmune thyroiditis. The main goal of that study is to assess the prevalence of APS-3 defined as coexistence of autoimmune thyroiditis with POI in population of 46 XX karyotype women with primary ovarian insufficiency (POI). The second goal is to investigate hormonal profile and insulin sensitivity in women with POI and subgroups of women with APS-3 - POI/APS-3(+) and without APS 3 - POI/APS-3(-). Anthropometric measurements, coexistence of autoimmune diseases, androgens, fasting glucose and insulin, glucose and insulin at 60' and 120' of oral glucose tolerance test (OGTT) and homeostasis model for insulin resistance (HOMA-IR), were determine in 98 patients aged between 18 and 39 with spontaneous 46 XX primary ovarian insufficiency (POI), in 33 POI/APS-3(+), 65 POI/APS-3(-), and 75 healthy controls. Continuous data were summarized by the mean±standard deviation (SD), and categorical data by number (percentages). Data were checked for normality using Shapiro-Wilk test, the comparison between groups were performed using non-parametric Mann-Whitney or Kruskall-Wallis test. Pearson's correlation coefficient was used to assess the relationships between parameters. Statistical significance was defined as p values APS-3(+) and POI/APS-3(-) showed significantly lower serum androgens in comparison to controls. Additionally women with POI/APS-3(+) showed hyperinsulinemia after 1h of OGTT; No significant differences in serum fasting glucose, insulin and during 2h OGTT between groups were observed. The prevalence of APS-3 is 33.7% in patients with spontaneous 46 XX primary ovarian insufficiency. Women with POI, POI/APS-3(+) and POI/APS-3(-) feature lower testosterone, androstendione

  3. Effect of alpha interferon on glucose and alanine transport by rat renal brush border membrane vesicles

    International Nuclear Information System (INIS)

    Batuman, V.; Chadha, I.

    1990-01-01

    To investigate the pathogenetic mechanisms of interferon nephrotoxicity, we studied the effect of recombinant interferon alfa-2b on the uptake of 14 C-D-glucose and 14 C-L-alanine by rat renal brush-border-membrane vesicles. Interferon significantly inhibited 20 sec. sodium-dependent and 5 and 10 min. equilibrium uptake of both glucose and alanine. The inhibitory effect was dose dependent with maximum effect achieved at interferon concentration of 5 x 10 -8 M in the uptake media. The half-maximal inhibitory concentrations, IC 50 , of interferon on glucose uptake was 1.8 x 10 -8 M, and 5.4 x 10 -9 M on alanine uptake. Dixon plot analysis of uptake data was consistent with pure non-competitive inhibition. The inhibition constants, K i , 1.5 x 10 -8 M for glucose uptake, and 7.3 x 10 -9 M for alanine uptake, derived from Dixon plots were in close agreement with the IC 50 s calculated from the semilog dose response curves. These observations reveal that direct interactions at the proximal tubule cell membrane are involved in the pathogenesis of interferon nephrotoxicity, and that its mechanism of nephrotoxicity is similar to that of other low molecular weight proteins

  4. Reserpine-induced Reduction in Norepinephrine Transporter Function Requires Catecholamine Storage Vesicles

    OpenAIRE

    Mandela, Prashant; Chandley, Michelle; Xu, Yao-Yu; Zhu, Meng-Yang; Ordway, Gregory A.

    2010-01-01

    Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5 min decreased [3H]NE uptake capacity, an effect characterized by a robust decrease in the Vmax of the transport of [3H]NE. As expected, res...

  5. Properties of the mitochondrial carrier of adenine-nucleotide after purification. Study of the transport protein under isolated form and reincorporated form in phospho-lipidic vesicles

    International Nuclear Information System (INIS)

    Brandolin, Gerard

    1983-01-01

    The first part of this research thesis addresses the reconstitution of the ADP/ATP transport by incorporation of the specific carrier, isolated in presence of detergent, in phospholipids vesicles. Fundamental properties of the reconstituted transport are identical to that of transport in mitochondria, notably as far as the exchange stoichiometry, the turn over and the transport Km are concerned, as well as the asymmetric orientation of the carrier in the membrane. The second part of this research addresses the study of interactions of specific ligands with the ADP/ATP transport protein in presence of detergent. The study of the variations of the intrinsic fluorescence of the isolated ADP/ATP carrier highlights conformational changes exclusively induced by the presence of transportable nucleotides which are modulated in a different manner by carboxy-atractyloside or bongkrekic acid. Moreover, by using the isolated protein, a detailed analysis of binding parameters of fluorescent analogues of ATP is reported [fr

  6. CsAP3: a cucumber homolog to Arabidopsis APETALA3 with novel characteristics

    Directory of Open Access Journals (Sweden)

    Jinjing Sun

    2016-08-01

    Full Text Available In our previous efforts to understand the regulatory mechanisms of cucumber unisexual flower development, we observed a stamen-specific down-regulation of the ethylene receptor CsETR1 in stage 6 female flowers of cucumber (Cucumis sativus L. This down-regulation is correlated with the primordial anther-specific DNA damage that characterizes inappropriate stamen development in cucumber female flowers. To understand how CsETR1 is down regulated in the stamen, we characterized a cucumber MADS box gene homologous to Arabidopsis AP3, CsAP3. We demonstrated that CsAP3 is functionally equivalent to the Arabidopsis B-class MADS gene AP3. However, three novel characteristics of CsAP3 were found. These include firstly, binding and activating CsETR1 promoter in vitro and in vivo; secondly, containing a GV repeat in its C-terminus, which is conserved in cucurbits and required for the transcription activation; and thirdly, decreased expression as the node number increases, which is similar to that found for CsETR1. These findings revealed not only the conserved function of CsAP3 as a B-class floral identity gene, but also its unique functions in regulation of female flower development in cucumber.

  7. Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

    Science.gov (United States)

    Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

    2014-11-01

    Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people

  8. Methionine and glucose transport by isolated intestinal brush border membrane vesicles from pigs and lambs fed an Aspergillus product

    OpenAIRE

    Jang, Insurk

    1993-01-01

    This study was designed to determine whether feeding an Aspergillus product would influence growth or feed utilization and intestinal mucosal cell function as indicated by uptake of methionine and glucose by isolated intestinal brush border membrane vesicles (BBMV). In Experiment 1, 24 weanling pigs were paired by sex, BW, and litter and were allotted, within pairs, to either an 18% CP corn-soy diet (control) or the same diet supplemented (.15%) with an Aspergillus product. There were no diff...

  9. AP-3 and Rabip4’ Coordinately Regulate Spatial Distribution of Lysosomes

    Science.gov (United States)

    Ivan, Viorica; Martinez-Sanchez, Emma; Sima, Livia E.; Oorschot, Viola; Klumperman, Judith; Petrescu, Stefana M.; van der Sluijs, Peter

    2012-01-01

    The RUN and FYVE domain proteins rabip4 and rabip4’ are encoded by RUFY1 and differ in a 108 amino acid N-terminal extension in rabip4’. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing RUFY1 gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4’ yielded the adaptor protein complex AP-3, of which the hinge region in the β3 subunit bound directly to the FYVE domain of rabip4’. Rabip4’ colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4’ in regulating lysosome positioning through an interorganellar pathway. PMID:23144738

  10. UNC-16/JIP3 regulates early events in synaptic vesicle protein trafficking via LRK-1/LRRK2 and AP complexes.

    Directory of Open Access Journals (Sweden)

    Bikash Choudhary

    2017-11-01

    Full Text Available JIP3/UNC-16/dSYD is a MAPK-scaffolding protein with roles in protein trafficking. We show that it is present on the Golgi and is necessary for the polarized distribution of synaptic vesicle proteins (SVPs and dendritic proteins in neurons. UNC-16 excludes Golgi enzymes from SVP transport carriers and facilitates inclusion of specific SVPs into the same transport carrier. The SVP trafficking roles of UNC-16 are mediated through LRK-1, whose localization to the Golgi is reduced in unc-16 animals. UNC-16, through LRK-1, also enables Golgi-localization of the μ-subunit of the AP-1 complex. AP1 regulates the size but not the composition of SVP transport carriers. Additionally, UNC-16 and LRK-1 through the AP-3 complex regulates the composition but not the size of the SVP transport carrier. These early biogenesis steps are essential for dependence on the synaptic vesicle motor, UNC-104 for axonal transport. Our results show that UNC-16 and its downstream effectors, LRK-1 and the AP complexes function at the Golgi and/or post-Golgi compartments to control early steps of SV biogenesis. The UNC-16 dependent steps of exclusion, inclusion and motor recruitment are critical for polarized distribution of neuronal cargo.

  11. The AP-3 β adaptin mediates the biogenesis and function of lytic vacuoles in Arabidopsis.

    Science.gov (United States)

    Feraru, Elena; Paciorek, Tomasz; Feraru, Mugurel I; Zwiewka, Marta; De Groodt, Ruth; De Rycke, Riet; Kleine-Vehn, Jürgen; Friml, Jirí

    2010-08-01

    Plant vacuoles are essential multifunctional organelles largely distinct from similar organelles in other eukaryotes. Embryo protein storage vacuoles and the lytic vacuoles that perform a general degradation function are the best characterized, but little is known about the biogenesis and transition between these vacuolar types. Here, we designed a fluorescent marker-based forward genetic screen in Arabidopsis thaliana and identified a protein affected trafficking2 (pat2) mutant, whose lytic vacuoles display altered morphology and accumulation of proteins. Unlike other mutants affecting the vacuole, pat2 is specifically defective in the biogenesis, identity, and function of lytic vacuoles but shows normal sorting of proteins to storage vacuoles. PAT2 encodes a putative β-subunit of adaptor protein complex 3 (AP-3) that can partially complement the corresponding yeast mutant. Manipulations of the putative AP-3 β adaptin functions suggest a plant-specific role for the evolutionarily conserved AP-3 β in mediating lytic vacuole performance and transition of storage into the lytic vacuoles independently of the main prevacuolar compartment-based trafficking route.

  12. Phosphoenolpyruvate-dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides : The coupling between transport and phosphorylation in inside-out vesicles

    NARCIS (Netherlands)

    Lolkema, Juke S.; Robillard, George T.

    The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has however been questioned in the

  13. Phylogenetic utility of the AP3/DEF K-domain and its molecular evolution in Impatiens (Balsaminaceae)

    NARCIS (Netherlands)

    Janssens, S.; Geuten, K.; Viaene, T.; Yong-Ming, Y.; Yi, S.; Smets, E.

    2007-01-01

    APETALA3 (AP3)/DEFICIENS (DEF) is a MADS-box transcription factor that is involved in establishing the identity of petal and stamen floral organs. The AP3/DEF gene lineage has been extensively examined throughout the angiosperms in order to better understand its role in floral diversity and

  14. Controlled enlargement of the glycoprotein vesicle surrounding a volvox embryo requires the InvB nucleotide-sugar transporter and is required for normal morphogenesis.

    Science.gov (United States)

    Ueki, Noriko; Nishii, Ichiro

    2009-04-01

    Here, we report our analysis of a mutant of Volvox carteri, InvB, whose embryos fail to execute inversion, the process in which each Volvox embryo normally turns itself inside-out at the end of embryogenesis, thereby achieving the adult configuration. The invB gene encodes a nucleotide-sugar transporter that exhibits GDP-mannose transport activity when expressed in yeast. In wild-type embryos, the invB transcript is maximally abundant before and during inversion. A mannoside probe (fluorescent concanavalin A) stains the glycoprotein-rich gonidial vesicle (GV) surrounding wild-type embryos much more strongly than it stains the GV surrounding InvB embryos. Direct measurements revealed that throughout embryogenesis the GV surrounding a wild-type embryo increases in size much more than the GV surrounding an InvB embryo does, and the fully cleaved InvB embryo is much more tightly packed within its GV than a wild-type embryo is. To test the hypothesis that the restraint imposed by a smaller than normal GV directly causes the inversion defect in the mutant, we released InvB embryos from their GVs microsurgically. The resulting embryos inverted normally, demonstrating that controlled enlargement of the GV, by a process in which requires the InvB nucleotide-sugar transporter, is essential to provide the embryo sufficient space to complete inversion.

  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 va...

  16. Trafficking of astrocytic vesicles in hippocampal slices

    International Nuclear Information System (INIS)

    Potokar, Maja; Kreft, Marko; Lee, So-Young; Takano, Hajime; Haydon, Philip G.; Zorec, Robert

    2009-01-01

    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.

  17. The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage.

    Science.gov (United States)

    Roszniowski, Bartosz; Latka, Agnieszka; Maciejewska, Barbara; Vandenheuvel, Dieter; Olszak, Tomasz; Briers, Yves; Holt, Giles S; Valvano, Miguel A; Lavigne, Rob; Smith, Darren L; Drulis-Kawa, Zuzanna

    2017-02-01

    Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7-49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29-98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

  18. Characterization of sodium transport in Acholeplasma laidlawii B cells and in lipid vesicles containing purified A. laidlawii (Na+-Mg2+)-ATPase by using nuclear magnetic resonance spectroscopy and 22Na tracer techniques

    International Nuclear Information System (INIS)

    Mahajan, S.; Lewis, R.N.; George, R.; Sykes, B.D.; McElhaney, R.N.

    1988-01-01

    The active transport of sodium ions in live Acholeplasma laidlawii B cells and in lipid vesicles containing the (Na+-Mg2+)-ATPase from the plasma membrane of this microorganism was studied by 23Na nuclear magnetic resonance spectroscopic and 22 Na tracer techniques, respectively. In live A. laidlawii B cells, the transport of sodium was an active process in which metabolic energy was harnessed for the extrusion of sodium ions against a concentration gradient. The process was inhibited by low temperatures and by the formation of gel state lipid in the plasma membrane of this organism. In reconstituted proteoliposomes containing the purified (Na+-Mg2+)-ATPase, the hydrolysis of ATP was accompanied by the transport of sodium ions into the lipid vesicles, and the transport process was impaired by reagents known to inhibit ATPase activity. At the normal growth temperature (37 degrees C), this transport process required a maximum of 1 mol of ATP per mol of sodium ion transported. Together, these results provide direct experimental evidence that the (Na+-Mg2+)-ATPase of the Acholeplasma laidlawii B membrane is the cation pump which maintains the low levels of intracellular sodium characteristic of this microorganism

  19. Role of calcium in phosphoinositide metabolism and inhibition of norepinephrine transport into synaptic vesicles by amphetamine analogs

    International Nuclear Information System (INIS)

    Knepper, S.M.

    1985-01-01

    Norepinephrine-(NE) and calcium ionophore A23187-stimulated phosphoinositide (PIn) metabolism in rat brain slices was studied under varying calcium conditions. Tissue was labelled with 3 H-myo-inositol and 3 H-inositol phosphates (IPn), products of PIn metabolism were measured. In the absence of media calcium the response to NE was decreased while that to A23187 was little affected A23187 can release calcium from intracellular stores. Basal and stimulated accumulation of 3 H-IPn was reversibly antagonized with EGTA by addition of calcium. Using calcium buffers, approximately 10 -7 M free calcium was required to support hydrolysis. Free intracellular calcium is maintained at approximately this level. Thus calcium is required for PIn hydrolysis but appears to play a permissive role, basal levels being sufficient to support metabolism. Conformationally-defined (rigid) and -restricted (semi-rigid) analogs of the most stable conformations of amphetamine, antiperiplanar (exo) and gauche (endo), were utilized to probe the conformational requirements of vesicular NE transport. Analogs tested were 2-aminotetralin (2AT), 3-methyltetrahydroisoquinoline, anti- and syn-9-aminobenzobicyclo[2.2.1]heptene, and endo and exo conformers of 2-aminobenzobicyclo[2.2.1]heptene and 2-aminobenzobicyclo[2.2.2]octene

  20. Erv41p and Erv46p: New components of COPII vesicles involved in transport between the ER and Golgi complex

    DEFF Research Database (Denmark)

    Otte, S; Belden, W J; Heidtman, M

    2001-01-01

    , Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent......Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p...... uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation. Yeast strains lacking Erv41p and/or Erv46p are viable...

  1. Opposite effects of cell differentiation and apoptosis on Ap3A/Ap4A ratio in human cell cultures.

    Science.gov (United States)

    Vartanian, A; Prudovsky, I; Suzuki, H; Dal Pra, I; Kisselev, L

    1997-09-29

    The biological role of diadenosine oligophosphates (DAOP) remains obscure in spite of numerous attempts to solve this enigma. It is known that Ap3A contrary to Ap4A accumulates in human cultured cells treated with interferons (IFNs) alpha or gamma. Since IFNs are considered as antiproliferative regulators, we assumed that different cell status may be associated with varying intracellular levels of DAOP. Promyelocytic human cell line HL60 induced by phorbol ester (TPA) to differentiate to macrophage-like cells in culture exhibits a profound loss of proliferative potential. Here we have shown a 4-5-fold increase in Ap3A concentration in HL60 cells induced by TPA, similar to the effect of IFN, while the Ap4A concentration remained unchanged. On the contrary, in cells undergoing apoptosis induced by VP16, a topoisomerase II inhibitor, the Ap3A concentration considerably decreased, while the Ap4A concentration increased. These findings combined with earlier results suggest an involvement of the Ap3A/Ap4A ratio in signal transduction pathways controlling the cell status.

  2. A rare combination of type 3 autoimmune polyendocrine syndrome (APS-3) or multiple autoimmune syndrome (MAS-3).

    Science.gov (United States)

    Betterle, Corrado; Garelli, Silvia; Coco, Graziella; Burra, Patrizia

    2014-06-01

    Type 3 autoimmune polyendocrine syndrome (APS-3) is defined by the presence of an autoimmune thyroid disease and another autoimmune illness, excluding Addison's disease; this is a frequent combination. We report the case of a 55 years old female patient with APS-3, with seven clinical or latent autoimmune manifestations. At 49 years of age she was admitted at the General Hospital for leukopenia, weight loss, tremors, anxiety and diarrhea. The personal history revealed ulcerative colitis and, during the last year, episodes of fever with migrant arthralgia and cutaneous lesions. The patient was evaluated for thyroid function and imaging, mielobiopsy, glycaemic control, gastrointestinal and rheumatologic disorders with specific biochemical tests, imaging and endoscopic procedures. We concluded that the patient was affected by APS-3, characterized by the association of Graves' disease, autoimmune leukopenia, latent autoimmune diabetes of the adult (LADA), autoimmune gastritis, ulcerative colitis, Sjögren's and anti-phospholipid syndromes. The patient started low doses of corticosteroid drugs for leukopenia, underwent (131)I therapy for hyperthyroidism and later started substitutive thyroid therapy with l-thyroxine, insulin therapy for LADA, mesalazine for ulcerative colitis and artificial tears for Sjögren's syndrome. In this article we report a complex case of APS-3, characterized by the association of seven different autoimmune diseases, which required a complex therapeutic strategy.

  3. 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.

  4. Phospholipid Vesicles in Materials Science

    Energy Technology Data Exchange (ETDEWEB)

    Granick, Steve [Univ. of Illinois, Champaign, IL (United States)

    2016-05-11

    The objective of this research was to develop the science basis needed to deploy phospholipid vesicles as functional materials in energy contexts. Specifically, we sought to: (1) Develop an integrated molecular-level understanding of what determines their dynamical shape, spatial organization, and responsiveness to complex, time-varying environments; and (2) Develop understanding of their active transportation in crowded environments, which our preliminary measurements in cells suggest may hold design principles for targeting improved energy efficiency in new materials systems. The methods to do this largely involved fluorescence imaging and other spectroscopy involving single particles, vesicles, particles, DNA, and endosomes. An unexpected importance outcome was a new method to image light-emitting diodes during actual operation using super-resolution spectroscopy.

  5. Why do cells release vesicles?

    NARCIS (Netherlands)

    Nieuwland, Rienk; Sturk, Augueste

    2010-01-01

    Prokaryotic and eukaryotic cells release vesicles into their environment. To answer the question why eukaryotic cells release vesicles, we may learn from prokaryotes. Bacteria release outer membrane vesicles, resembling microparticles, which act as "multi-purpose carriers". They contain signalling

  6. 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...... around 20 C, but at temperatures above 26 C we observe an increase in the scattered intensity due to fusion. The system is unusually well suited for the study of basic mechanisms of vesicle fusion. The vesicles are flexible with a bending rigidity of only a few k(H)T. The monolayer spontaneous curvature......, Ho, depends strongly on temperature in a known way and is thus tunable. For temperatures where H-0 > 0 vesicles tyre long-term stable, while in the range H-0 fusion rate increases the more negative the Spontaneous curvature Through a quantitative;analysis of the fusion rate we arrive tit...

  7. 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.

  8. Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp.

    Science.gov (United States)

    Snowden, Christopher J; Thomas, Benjamin; Baxter, Charles J; Smith, J Andrew C; Sweetlove, Lee J

    2015-12-20

    This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al ., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development.

  9. Atomic force microscopy analysis of extracellular vesicles.

    Science.gov (United States)

    Parisse, P; Rago, I; Ulloa Severino, L; Perissinotto, F; Ambrosetti, E; Paoletti, P; Ricci, M; Beltrami, A P; Cesselli, D; Casalis, L

    2017-12-01

    Extracellular vesicles (EVs) are small vesicles ensuring transport of molecules between cells and throughout the body. EVs contain cell type-specific signatures and have been proposed as biomarkers in a variety of diseases. Their small size (vesicles exert their functions is still unknown and represents a great biomedical challenge. Moreover, because of their small dimensions, the quantification, size distribution and biophysical characterization of these particles are challenging and still subject to controversy. Here, we address the advantage of atomic force microscopy (AFM), for the characterization of isolated EVs. We review AFM imaging of EVs immobilized on different substrates (mica, glass) to identify the influence of isolation and deposition methods on the size distribution, morphology and mechanical properties of EVs.

  10. Human Immunodeficiency Virus Type 2 (HIV-2) Gag Is Trafficked in an AP-3 and AP-5 Dependent Manner.

    Science.gov (United States)

    Alford, Justine E; Marongiu, Michela; Watkins, Gemma L; Anderson, Emma C

    2016-01-01

    Although human immunodeficiency virus (HIV) types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HIV-2 may help to shed light on this issue. In contrast to HIV-1, little is known about the assembly of HIV-2 particles, and the trafficking of HIV-2 Gag, the structural component of the virus, within cells. We have established that HIV-2 Gag accumulates in intracellular CD63 positive compartments, from which it may be delivered or recycled to the cell surface, or degraded. HIV-2 particle release was dependent on the adaptor protein complex AP-3 and the newly identified AP-5 complex, but much less so on AP-1. In contrast, HIV-1 particle release required AP-1 and AP-3, but not AP-5. AP-2, an essential component of clathrin-mediated endocytosis, which was previously shown to be inhibitory to HIV-1 particle release, had no effect on HIV-2. The differential requirement for adaptor protein complexes confirmed that HIV-1 and HIV-2 Gag have distinct cellular trafficking pathways, and that HIV-2 particles may be more susceptible to degradation prior to release.

  11. Astrocytic Vesicle Mobility in Health and Disease

    Directory of Open Access Journals (Sweden)

    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. Adaptor Protein Complexes AP-1 and AP-3 Are Required by the HHV-7 Immunoevasin U21 for Rerouting of Class I MHC Molecules to the Lysosomal Compartment

    Science.gov (United States)

    Kimpler, Lisa A.; Glosson, Nicole L.; Downs, Deanna; Gonyo, Patrick; May, Nathan A.; Hudson, Amy W.

    2014-01-01

    The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes. PMID:24901711

  13. Direct imaging of RAB27B-enriched secretory vesicle biogenesis in lacrimal acinar cells reveals origins on a nascent vesicle budding site.

    Directory of Open Access Journals (Sweden)

    Lilian Chiang

    Full Text Available This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the "nascent vesicle site," from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150(Glued, a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules.

  14. Seminal vesicle cycts

    International Nuclear Information System (INIS)

    Alpern, M.B.; Dorfman, R.E.; Gross, B.H.; Gottlieb, C.A.; Sandler, M.A.

    1990-01-01

    PURPOSE: Adult polycystic kidney disease (APKCD), an autosomal dominant disorder, causes cyst formation in the kidney, liver, pancreas, esophagus, ovaries, uterus, and brain. This paper describes four APKCD patients with CT evidence of seminal vesicle cysts (SVCs). Four patients (aged 45-65 years) underwent abdominal/pelvic CT with oral and intravenous contrast material. Three were evaluated for possible renal transplantation and one for sepsis material. All seminal vesicles contained cystic masses with fluid that measured between 0 and 30 HU. Seminal vesicle thickness was 3-4 cm (normal, 1.5 cm). High-density walls separated the 3-12-mm diameter cysts. All patients demonstrated typical renal stigmata of APKCD. One patient had hepatic cysts, and none had cysts elsewhere. Postmortem examination in one patient confirmed the SVCs

  15. Interaction between silicon dioxide and dipalmitoylphosphatidylcholine (DPPC) vesicles

    Science.gov (United States)

    Mohd, Hur Munawar Kabir; Ahmad, Ainee Fatimah; Radiman, Shahidan; Mohamed, Faizal; Rosli, Nur Ratasha Alia Md; Ayob, Muhammad Taqiyuddin Mawardi; Rahman, Irman Abdul

    2014-09-01

    Many of the cellular process depend on the ability of the membrane to separate areas while allowing exchange and tightly regulated transport of material within and across the membrane to occur, which is the driving principle behind cell communication. The complexity of biological membranes has motivated the development of a wide variety of simpler model systems whose size, geometry and composition can be tailored with precision. This study was conducted to investigate the interactions between silica nanoparticles and Dipalmitoylphosphatidylcholine (DPPC) vesicles. The size range of DPPC vesicles formed was from 50 to 150 nm. Concentration of silica added to the vesicles was varied from 0.25 to 1.5 mg/ml. The change in vesicle size distribution, localization and positioning of silica nanoparticles in vesicles was studied via transmission electron microscopy (TEM) and differential scanning calorimetry (DSC).

  16. 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.

  17. Extracellular vesicles for drug delivery

    NARCIS (Netherlands)

    Vader, Pieter; Mol, Emma A; Pasterkamp, Gerard; Schiffelers, Raymond M

    2016-01-01

    Extracellular vesicles (EVs) are cell-derived membrane vesicles, and represent an endogenous mechanism for intercellular communication. Since the discovery that EVs are capable of functionally transferring biological information, the potential use of EVs as drug delivery vehicles has gained

  18. Energetics of arginine and lysine transport by whole cells and membrane vesicles of strain SR, a monensin-sensitive ruminal bacterium.

    OpenAIRE

    Van Kessel, J S; Russell, J B

    1992-01-01

    Strain SR, a monensin-sensitive, ammonia-producing ruminal bacterium, grew rapidly on arginine and lysine, but only if sodium was present. Arginine transport could be driven by either an electrical potential or a chemical gradient of sodium. Arginine was converted to ornithine, and it appeared that ornithine efflux created a sodium gradient which in turn drove arginine transport. There was a linear decline in arginine transport as pH was decreased from 7.5 to 5.5, and the cells did not grow a...

  19. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

    Science.gov (United States)

    Rodriguez-Fernandez, Imilce A; Dell'Angelica, Esteban C

    2015-01-01

    The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in

  20. Sorting of Dendritic and Axonal Vesicles at the Pre-axonal Exclusion Zone

    Directory of Open Access Journals (Sweden)

    Ginny G. Farías

    2015-11-01

    Full Text Available Polarized sorting of newly synthesized proteins to the somatodendritic and axonal domains of neurons occurs by selective incorporation into distinct populations of vesicular transport carriers. An unresolved issue is how the vesicles themselves are sorted to their corresponding neuronal domains. Previous studies concluded that the axon initial segment (AIS is an actin-based filter that selectively prevents passage of somatodendritic vesicles into the axon. We find, however, that most somatodendritic vesicles fail to enter the axon at a more proximal region in the axon hillock, herein referred to as the pre-axonal exclusion zone (PAEZ. Forced coupling of a somatodendritic cargo protein to an axonally directed kinesin is sufficient to drive transport of whole somatodendritic vesicles through the PAEZ toward the distal axon. Based on these findings, we propose that polarized sorting of transport vesicles occurs at the PAEZ and depends on the ability of the vesicles to acquire an appropriately directed microtubule motor.

  1. High energy irradiation of bacterial membrane vesicles

    International Nuclear Information System (INIS)

    De La Rosa, M.A.M.

    1977-01-01

    The interactions of membrane components and two well-defined transport systems in the E. coli ML 308-225 membrane vesicles with 60 Co gamma radiation were investigated. The results presented show that gamma radiation can monitor membrane components and functions of varying radiosensitivities. The possible application of high-energy radiation as a physical probe of membrane structure and functions is indeed promising

  2. Studies of matrix vesicle-induced mineralization in a gelatin gel

    Science.gov (United States)

    Boskey, A. L.; Boyan, B. D.; Doty, S. B.; Feliciano, A.; Greer, K.; Weiland, D.; Swain, L. D.; Schwartz, Z.

    1992-01-01

    Matrix vesicles isolated from fourth-passage cultures of chondrocytes were tested for their ability to induce hydroxyapatite formation in a gelatin gel in order to gain insight into the function of matrix vesicles in in situ mineralization. These matrix vesicles did not appear to be hydroxyapatite nucleators per se since the extent of mineral accumulation in the gel diffusion system was not altered by the presence of matrix vesicles alone, and in the vesicle containing gels, mineral crystals were formed whether associated with vesicles or not. In gels with these matrix vesicles and beta-glycerophosphate, despite the presence of alkaline phosphatase activity, there was no increase in mineral deposition. This suggested that in the gel system these culture-derived vesicles did not increase local phosphate concentrations. However, when known inhibitors of mineral crystal formation and growth (proteoglycan aggregates [4 mg/ml], or ATP [1 mM], or both proteoglycan and ATP) were included in the gel, more mineral was deposited in gels with the vesicles than in comparable gels without vesicles, indicating that enzymes within these vesicles were functioning to remove the inhibition. These data support the suggestion that one function of the extracellular matrix vesicles is to transport enzymes for matrix modification.

  3. 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.

  4. Vesicle Fusion Mediated by Solanesol-Anchored DNA.

    Science.gov (United States)

    Flavier, Kristina M; Boxer, Steven G

    2017-09-19

    Fusion between two lipid bilayers is one of the central processes in cell biology, playing a key role in endocytosis, exocytosis, and vesicle transport. We have previously developed a model system that uses the hybridization of complementary DNA strands to model the formation of the SNARE four-helix bundle that mediates synaptic vesicle fusion and used it to study vesicle fusion to a tethered lipid bilayer. Using single vesicle assays, 70% of observed fusion events in the DNA-lipid system are arrested at the hemifusion stage, whereas only 5% eventually go to full fusion. This may be because the diglycerol ether that anchors the DNA in the membrane spans only half the bilayer: upon hemifusion and mixing of the outer leaflets, the DNA-lipid is free to diffuse into the target membrane and away from the vesicle. Here, we test the hypothesis that the length of the membrane anchor may impact the outcome by comparing single leaflet-spanning DNA-lipid mediated vesicle fusion with fusion mediated by DNA anchored by solanesol, a C45 isoprenoid of sufficient length to span the bilayer. When the solanesol anchor was present on the incoming vesicles, target membrane, or both, ∼2-3 times as much full fusion was observed as in the DNA-lipid mediated system, as measured by lipid mixing or content transfer. These results indicate that a transmembrane anchor increases the efficiency of full fusion. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. Purification Protocols for Extracellular Vesicles.

    Science.gov (United States)

    Lane, Rebecca E; Korbie, Darren; Trau, Matt; Hill, Michelle M

    2017-01-01

    This chapter provides a description of some of the standard methods used for the isolation of extracellular vesicles (EVs) from a variety of biological fluids, including cell culture media, urine, plasma and serum. The methods presented include ultracentrifugation, ultrafiltration, proprietary polymer-based reagents, size exclusion chromatography, density gradient separation, and immunoaffinity capture. Ultracentrifugation methods use high speed centrifugation to pellet vesicles, whilst polymer-based reagents are added to the sample to facilitate vesicle precipitation using lower speeds. Ultrafiltration involves the concentration of vesicles from a large volume of biological fluid using a centrifugal filter unit. Size exclusion chromatography and density gradient separation are both designed to allow the separation of vesicles from other nonvesicular debris. Immunoaffinity capture methods use antibody-coated beads to selectively isolate vesicles displaying a surface marker of interest. Ultimately, the choice of purification method for an individual experiment is influenced by time, cost, and equipment considerations, as well as the sample requirements for any downstream analyses.

  6. Ca2+-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

    International Nuclear Information System (INIS)

    Stenovec, Matjaz; Kreft, Marko; Grilc, Sonja; Potokar, Maja; Kreft, Mateja Erdani; Pangrsic, Tina; Zorec, Robert

    2007-01-01

    Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca 2+ -dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca 2+ level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca 2+ -triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles

  7. 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.

  8. Ion content of synaptic vesicles

    International Nuclear Information System (INIS)

    Demeter, I.; Keszthelyi, L.; Szokefalvi-Nagy, Z.; Varga, L.; Hollos-Nagy, K.; Nagy, A.

    1977-09-01

    Proton induced X-ray emission analysis measurements were performed to determine the P, S, K, Ca, Fe, Ni, Cu, and Zn ion content of presynaptic vesicles prepared from guinea-pig cortex brain. The number of different ions per single vesicle is calculated using results of the additional protein content determinations. The ion content of cholinergic and adrenergic vesicles are compared. Some rough conclusions can be made regarding the biochemistry of vesicles on the basis of this measurements, but the elucidation of the meaning of the data needs further work which will demonstrate the value of PIXE-type investigations in similar studies. (D.P.)

  9. Plane partition vesicles

    International Nuclear Information System (INIS)

    Rensburg, E J Janse van; Ma, J

    2006-01-01

    We examine partitions and their natural three-dimensional generalizations, plane partitions, as models of vesicles undergoing an inflation-deflation transition. The phase diagrams of these models include a critical point corresponding to an inflation-deflation transition, and exhibits multicritical scaling in the vicinity of a multicritical point located elsewhere on the critical curve. We determine the locations of the multicritical points by analysing the generating functions using analytic and numerical means. In addition, we determine the numerical values of the multicritical scaling exponents associated with the multicritical scaling regimes in these models

  10. Modular endolysin of Burkholderia AP3 phage has the largest lysozyme-like catalytic subunit discovered to date and no catalytic aspartate residue.

    Science.gov (United States)

    Maciejewska, Barbara; Źrubek, Karol; Espaillat, Akbar; Wiśniewska, Magdalena; Rembacz, Krzysztof P; Cava, Felipe; Dubin, Grzegorz; Drulis-Kawa, Zuzanna

    2017-11-06

    Endolysins are peptidoglycan-degrading enzymes utilized by bacteriophages to release the progeny from bacterial cells. The lytic properties of phage endolysins make them potential antibacterial agents for medical and industrial applications. Here, we present a comprehensive characterization of phage AP3 modular endolysin (AP3gp15) containing cell wall binding domain and an enzymatic domain (DUF3380 by BLASTP), both widespread and conservative. Our structural analysis demonstrates the low similarity of an enzymatic domain to known lysozymes and an unusual catalytic centre characterized by only a single glutamic acid residue and no aspartic acid. Thus, our findings suggest distinguishing a novel class of muralytic enzymes having the activity and catalytic centre organization of DUF3380. The lack of amino acid sequence homology between AP3gp15 and other known muralytic enzymes may reflect the evolutionary convergence of analogous glycosidases. Moreover, the broad antibacterial spectrum, lack of cytotoxic effect on human cells and the stability characteristics of AP3 endolysin advocate for its future application development.

  11. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3 Mutants in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Imilce A Rodriguez-Fernandez

    Full Text Available The Adaptor Protein (AP-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with

  12. Effective Mechanism for Synthesis of Neurotransmitter Glutamate and its Loading into Synaptic Vesicles.

    Science.gov (United States)

    Takeda, Kouji; Ueda, Tetsufumi

    2017-01-01

    Glutamate accumulation into synaptic vesicles is a pivotal step in glutamate transmission. This process is achieved by a vesicular glutamate transporter (VGLUT) coupled to v-type proton ATPase. Normal synaptic transmission, in particular during intensive neuronal firing, would demand rapid transmitter re-filling of emptied synaptic vesicles. We have previously shown that isolated synaptic vesicles are capable of synthesizing glutamate from α-ketoglutarate (not from glutamine) by vesicle-bound aspartate aminotransferase for immediate uptake, in addition to ATP required for uptake by vesicle-bound glycolytic enzymes. This suggests that local synthesis of these substances, essential for glutamate transmission, could occur at the synaptic vesicle. Here we provide evidence that synaptosomes (pinched-off nerve terminals) also accumulate α-ketoglutarate-derived glutamate into synaptic vesicles within, at the expense of ATP generated through glycolysis. Glutamine-derived glutamate is also accumulated into synaptic vesicles in synaptosomes. The underlying mechanism is discussed. It is suggested that local synthesis of both glutamate and ATP at the presynaptic synaptic vesicle would represent an efficient mechanism for swift glutamate loading into synaptic vesicles, supporting maintenance of normal synaptic transmission.

  13. Agonists that increase [Ca2+]i halt the movement of cytoplasmatic vesicles in MDCK cells

    DEFF Research Database (Denmark)

    Bjælde, Randi Groslier; Árnadóttir, Sigrid Salling; Leipziger, Jens Georg

    2011-01-01

    of vesicles by 40%. Because all these perturbations increase [Ca²⁺]i, we speculated that this increase in [Ca²⁺]i was responsible for the vesicle arrest. Therefore, we tested the effect of the Ca²⁺ ionophore, ionomycin (1 μM), which in the presence of extracellular Ca²⁺ resulted in a considerable......Translocation of vesicles within the cytoplasm is essential to normal cell function. The vesicles are typically transported along the microtubules to their destination. The aim of this study was to characterize the vesicular movement in resting and stimulated renal epithelial cells. MDCK cells...... loaded with either quinacrine or acridine orange, dyes taken up by acidic vesicles, were observed at 37°C in semiopen perfusion chambers. Time-lapse series were analyzed by Imaris software. Our data revealed vigorous movement of stained vesicles in resting MDCK cells. These movements seem to require...

  14. Identification of a QTL in Mus musculus for alcohol preference, withdrawal, and Ap3m2 expression using integrative functional genomics and precision genetics.

    Science.gov (United States)

    Bubier, Jason A; Jay, Jeremy J; Baker, Christopher L; Bergeson, Susan E; Ohno, Hiroshi; Metten, Pamela; Crabbe, John C; Chesler, Elissa J

    2014-08-01

    Extensive genetic and genomic studies of the relationship between alcohol drinking preference and withdrawal severity have been performed using animal models. Data from multiple such publications and public data resources have been incorporated in the GeneWeaver database with >60,000 gene sets including 285 alcohol withdrawal and preference-related gene sets. Among these are evidence for positional candidates regulating these behaviors in overlapping quantitative trait loci (QTL) mapped in distinct mouse populations. Combinatorial integration of functional genomics experimental results revealed a single QTL positional candidate gene in one of the loci common to both preference and withdrawal. Functional validation studies in Ap3m2 knockout mice confirmed these relationships. Genetic validation involves confirming the existence of segregating polymorphisms that could account for the phenotypic effect. By exploiting recent advances in mouse genotyping, sequence, epigenetics, and phylogeny resources, we confirmed that Ap3m2 resides in an appropriately segregating genomic region. We have demonstrated genetic and alcohol-induced regulation of Ap3m2 expression. Although sequence analysis revealed no polymorphisms in the Ap3m2-coding region that could account for all phenotypic differences, there are several upstream SNPs that could. We have identified one of these to be an H3K4me3 site that exhibits strain differences in methylation. Thus, by making cross-species functional genomics readily computable we identified a common QTL candidate for two related bio-behavioral processes via functional evidence and demonstrate sufficiency of the genetic locus as a source of variation underlying two traits. Copyright © 2014 by the Genetics Society of America.

  15. Cellular uptake of extracellular vesicles is mediated by clathrin-independent endocytosis and macropinocytosis

    NARCIS (Netherlands)

    Costa Verdera, Helena; Francois, Jerney J. J. M.; Schiffelers, Raymond M.; Vader, Pieter

    2017-01-01

    Recent evidence has established that extracellular vesicles (EVs), including exosomes and microvesicles, form an endogenous transport system through which biomolecules, including proteins and RNA, are exchanged between cells. This endows EVs with immense potential for drug delivery and regenerative

  16. 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...

  17. Extracellular vesicles in renal disease.

    Science.gov (United States)

    Karpman, Diana; Ståhl, Anne-Lie; Arvidsson, Ida

    2017-09-01

    Extracellular vesicles, such as exosomes and microvesicles, are host cell-derived packages of information that allow cell-cell communication and enable cells to rid themselves of unwanted substances. The release and uptake of extracellular vesicles has important physiological functions and may also contribute to the development and propagation of inflammatory, vascular, malignant, infectious and neurodegenerative diseases. This Review describes the different types of extracellular vesicles, how they are detected and the mechanisms by which they communicate with cells and transfer information. We also describe their physiological functions in cellular interactions, such as in thrombosis, immune modulation, cell proliferation, tissue regeneration and matrix modulation, with an emphasis on renal processes. We discuss how the detection of extracellular vesicles could be utilized as biomarkers of renal disease and how they might contribute to disease processes in the kidney, such as in acute kidney injury, chronic kidney disease, renal transplantation, thrombotic microangiopathies, vasculitides, IgA nephropathy, nephrotic syndrome, urinary tract infection, cystic kidney disease and tubulopathies. Finally, we consider how the release or uptake of extracellular vesicles can be blocked, as well as the associated benefits and risks, and how extracellular vesicles might be used to treat renal diseases by delivering therapeutics to specific cells.

  18. Extracellular Vesicles in Renal Pathophysiology.

    Science.gov (United States)

    Pomatto, Margherita A C; Gai, Chiara; Bussolati, Benedetta; Camussi, Giovanni

    2017-01-01

    Extracellular vesicles are a heterogeneous population of microparticles released by virtually all living cells which have been recently widely investigated in different biological fields. They are typically composed of two primary types (exosomes and microvesicles) and are recently commanding increasing attention as mediators of cellular signaling. Indeed, these vesicles can affect recipient cells by carrying and delivering complex cargos of biomolecules (including proteins, lipids and nucleic acids), protected from enzymatic degradation in the environment. Their importance has been demonstrated in the pathophysiology of several organs, in particular in kidney, where different cell types secrete extracellular vesicles that mediate their communication with downstream urinary tract cells. Over the past few years, evidence has been shown that vesicles participate in kidney development and normal physiology. Moreover, EVs are widely demonstrated to be implicated in cellular signaling during renal regenerative and pathological processes. Although many EV mechanisms are still poorly understood, in particular in kidney, the discovery of their role could help to shed light on renal biological processes which are so far elusive. Lastly, extracellular vesicles secreted by renal cells gather in urine, thus becoming a great resource for disease or recovery markers and a promising non-invasive diagnostic instrument for renal disease. In the present review, we discuss the most recent findings on the role of extracellular vesicles in renal physiopathology and their potential implication in diagnosis and therapy.

  19. Active endocannabinoids are secreted on extracellular membrane vesicles.

    Science.gov (United States)

    Gabrielli, Martina; Battista, Natalia; Riganti, Loredana; Prada, Ilaria; Antonucci, Flavia; Cantone, Laura; Matteoli, Michela; Maccarrone, Mauro; Verderio, Claudia

    2015-02-01

    Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids. © 2015 The Authors.

  20. Transferring intercellular signals and traits between cancer cells: extracellular vesicles as "homing pigeons".

    Science.gov (United States)

    Cesi, Giulia; Walbrecq, Geoffroy; Margue, Christiane; Kreis, Stephanie

    2016-06-10

    Extracellular vesicles are cell-derived vesicles, which can transport various cargos out of cells. From their cell of origin, the content molecules (proteins, non-coding RNAs including miRNAs, DNA and others) can be delivered to neighboring or distant cells and as such extracellular vesicles can be regarded as vehicles of intercellular communication or "homing pigeons". Extracellular vesicle shuttling is able to actively modulate the tumor microenvironment and can partake in tumor dissemination. In various diseases, including cancer, levels of extracellular vesicle secretion are altered resulting in different amounts and/or profiles of detectable vesicular cargo molecules and these distinct content profiles are currently being evaluated as biomarkers. Apart from their potential as blood-derived containers of specific biomarkers, the transfer of extracellular vesicles to surrounding cells also appears to be involved in the propagation of phenotypic traits. These interesting properties have put extracellular vesicles into the focus of many recent studies.Here we review findings on the involvement of extracellular vesicles in transferring traits of cancer cells to their surroundings and briefly discuss new data on oncosomes, a larger type of vesicle. A pressing issue in cancer treatment is rapidly evolving resistance to many initially efficient drug therapies. Studies investigating the role of extracellular vesicles in this phenomenon together with a summary of the technical challenges that this field is still facing, are also presented. Finally, emerging areas of research such as the analysis of the lipid composition on extracellular vesicles and cutting-edge techniques to visualise the trafficking of extracellular vesicles are discussed.

  1. Extracellular Vesicles in Brain Tumors and Neurodegenerative Diseases

    Directory of Open Access Journals (Sweden)

    Federica Ciregia

    2017-08-01

    Full Text Available Extracellular vesicles (EVs can be classified into apoptotic bodies, microvesicles (MVs, and exosomes, based on their origin or size. Exosomes are the smallest and best characterized vesicles which derived from the endosomal system. These vesicles are released from many different cell types including neuronal cells and their functions in the nervous system are investigated. They have been proposed as novel means for intercellular communication, which takes part not only to the normal neuronal physiology but also to the transmission of pathogenic proteins. Indeed, exosomes are fundamental to assemble and transport proteins during development, but they can also transfer neurotoxic misfolded proteins in pathogenesis. The present review will focus on their roles in neurological diseases, specifically brain tumors, such as glioblastoma (GBM, neuroblastoma (NB, medulloblastoma (MB, and metastatic brain tumors and chronic neurodegenerative diseases, such as Alzheimer, Parkinson, multiple sclerosis (MS, amyotrophic lateral sclerosis (ALS, Huntington, and Prion diseseases highlighting their involvement in spreading neurotoxicity, in therapeutics, and in pathogenesis.

  2. Souffle/Spastizin Controls Secretory Vesicle Maturation during Zebrafish Oogenesis

    Science.gov (United States)

    Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

    2014-01-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research. PMID:24967841

  3. Souffle/Spastizin controls secretory vesicle maturation during zebrafish oogenesis.

    Directory of Open Access Journals (Sweden)

    Palsamy Kanagaraj

    2014-06-01

    Full Text Available During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP gene SPASTIZIN (SPG15. We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.

  4. 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.

  5. Cystadenoma of the seminal vesicle

    Directory of Open Access Journals (Sweden)

    Gil Antônio O.

    2003-01-01

    Full Text Available Primary tumors of the seminal vesicle are extremely rare. Among them, there is a spectrum of tumors derived from both epithelium and stroma and so classified as epithelial-stromal tumors. Herein, we report a case of a cystadenoma in a 49-year-old asymptomatic man, detected in a routine ultrasonography for liver disease follow-up. The digital rectal examination detected a large mass anterior to rectum and posterior to bladder. Computed tomography scan and magnetic resonance imaging showed a normal prostate and a 9.0 cm cystic tumor, replacing the left seminal vesicle. The gross appearance and microscopic aspect was compatible with cystadenoma of seminal vesicle. Patient's postoperative recovery was uneventful. He is currently alive, 3 years after the diagnosis, with no signs of recurrence.

  6. When to biopsy seminal vesicles.

    Science.gov (United States)

    Panach-Navarrete, J; García-Morata, F; Hernández-Medina, J A; Martínez-Jabaloyas, J M

    2015-05-01

    The involvement of seminal vesicles in prostate cancer can affect the prognosis and determine the treatment. The objective of this study was to determine whether we could predict its infiltration at the time of the prostate biopsy to know when to indicate the biopsy of the seminal vesicles. observational retrospective study of 466 patients who underwent seminal vesicle biopsy. The indication for this biopsy was a prostate-specific antigen (PSA) level greater than 10 ng/ml or an asymmetric or obliterated prostatoseminal angle. The following variables were included in the analysis: PSA level, PSA density, prostate volume, number of cores biopsied, suspicious rectal examination, and preservation of the prostatoseminal angle, studying its relationship with the involvement of the seminal vesicles. Forty-one patients (8.8%) had infiltrated seminal vesicles and 425 (91.2%) had no involvement. In the univariate analysis, the cases with infiltration had a higher mean PSA level (P 19.60 ng/dL (P < .01) and 2.95 times higher if there is a suspicious rectal examination (P = .014). Furthermore, this probability increases by 1.04 times for each unit of prostate volume lower (P < .01). The ROC curves showed maximum sensitivity and specificity at 19.6 ng/mL for PSA and 0.39 for PSA density. In this series, greater involvement of seminal vesicles was associated with a PSA level ≥20 ng/ml, a suspicious rectal examination and a lack of prostatoseminal angle preservation. Copyright © 2014 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

  7. 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

  8. GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with TGN

    DEFF Research Database (Denmark)

    Ralston, E; Ploug, Thorkil

    1996-01-01

    There is little consensus on the nature of the storage compartment of the glucose transporter GLUT4, in non-stimulated cells of muscle and fat. More specifically, it is not known whether GLUT4 is localized to unique, specialized intracellular storage vesicles, or to vesicles that are part of the ...

  9. Extracellular vesicles in physiological and pathological conditions

    NARCIS (Netherlands)

    Yuana, Yuana; Sturk, Auguste; Nieuwland, Rienk

    2013-01-01

    Body fluids contain surprising numbers of cell-derived vesicles which are now thought to contribute to both physiology and pathology. Tools to improve the detection of vesicles are being developed and clinical applications using vesicles for diagnosis, prognosis, and therapy are under investigation.

  10. 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 ...

  11. The Arabidopsis adaptor protein AP-3µ interacts with the G-protein β subunit AGB1 and is involved in abscisic acid regulation of germination and post-germination development

    Science.gov (United States)

    Tsugama, Daisuke; Takano, Tetsuo

    2013-01-01

    Heterotrimeric G-proteins (G-proteins) have been implicated in ubiquitous signalling mechanisms in eukaryotes. In plants, G-proteins modulate hormonal and stress responses and regulate diverse developmental processes. However, the molecular mechanisms of their functions are largely unknown. A yeast two-hybrid screen was performed to identify interacting partners of the Arabidopsis G-protein β subunit AGB1. One of the identified AGB1-interacting proteins is the Arabidopsis adaptor protein AP-3µ. The interaction between AGB1 and AP-3µ was confirmed by an in vitro pull-down assay and bimolecular fluorescence complementation assay. Two ap-3µ T-DNA insertional mutants were found to be hyposensitive to abscisic acid (ABA) during germination and post-germination growth, whereas agb1 mutants were hypersensitive to ABA. During seed germination, agb1/ap-3µ double mutants were more sensitive to ABA than the wild type but less sensitive than agb1 mutants. However, in post-germination growth, the double mutants were as sensitive to ABA as agb1 mutants. These data suggest that AP-3µ positively regulates the ABA responses independently of AGB1 in seed germination, while AP-3µ does require AGB1 to regulate ABA responses during post-germination growth. PMID:24098050

  12. Formation of Oligovesicular Vesicles by Micromanipulation

    Directory of Open Access Journals (Sweden)

    Yukihisa Okumura

    2011-09-01

    Full Text Available Cell-sized lipid bilayer membrane vesicles (giant vesicles, GVs or semi-vesicles were formed from egg yolk phosphatidylcholine on a platinum electrode under applied electric voltage by electroformation. Micromanipulation of the semi-vesicle by first pressing its membrane with a glass microneedle and then withdrawing the needle left a GV in the interior of the vesicle. During the process, an aqueous solution of Ficoll that filled the needle was introduced into the newly formed inner vesicle and remained encapsulated. Approximately 50% of attempted micromanipulation resulted in the formation of an inner daughter vesicle, “microvesiculation”. By repeating the microvesiculation process, multiple inner GVs could be formed in a single parent semi-vesicle. A semi-vesicle with inner GVs could be detached from the electrode by scraping with a microneedle, yielding an oligovesicular vesicle (OVV with desired inner aqueous contents. Microvesiculation of a GV held on the tip of a glass micropipette was also possible, and this also produced an OVV. Breaking the membrane of the parent semi-vesicle by micromanipulation with a glass needle after microvesiculation, released the inner GVs. This protocol may be used for controlled formation of GVs with desired contents.

  13. Mechanics and stability of vesicles and droplets in confined spaces

    Science.gov (United States)

    Benet, Eduard; Vernerey, Franck J.

    2016-12-01

    The permeation and trapping of soft colloidal particles in the confined space of porous media are of critical importance in cell migration studies, design of drug delivery vehicles, and colloid separation devices. Our current understanding of these processes is however limited by the lack of quantitative models that can relate how the elasticity, size, and adhesion properties of the vesicle-pore complex affect colloid transport. We address this shortcoming by introducing a semianalytical model that predicts the equilibrium shapes of a soft vesicle driven by pressure in a narrow pore. Using this approach, the problem is recast in terms of pressure and energy diagrams that characterize the vesicle stability and permeation pressures in different conditions. We particularly show that the critical permeation pressure for a vesicle arises from a compromise between the critical entry pressure and exit pressure, both of which are sensitive to geometrical features, mechanics, and adhesion. We further find that these results can be leveraged to rationally design microfluidic devices and diodes that can help characterize, select, and separate colloids based on physical properties.

  14. [Myasthenia gravis, Graves-Basedow disease and other autoimmune diseases in patient with diabetes type 1 - APS-3 case report, therapeutic complications].

    Science.gov (United States)

    Klenczar, Karolina; Deja, Grażyna; Kalina-Faska, Barbara; Jarosz-Chobot, Przemysława

    2017-01-01

    Diabetes type 1(T1D) is the most frequent form of diabetes in children and young people, which essence is autoimmune destruction of pancreatic B cells islet. Co-occurrence of other autoimmune diseases is observed in children with T1D, the most often are: Hashimoto disease or coeliac disease. We report the case of the patient, who presents coincidence of T1D with other rare autoimmune diseases such as: Graves - Basedow disease, myasthenia gravis, vitiligo and IgA deficiency. All mentioned diseases significantly complicated both endocrine and diabetic treatment of our patient and they negatively contributed her quality of life. The clinical picture of the case allows to recognize one of the autoimmune polyendocrine syndromes: APS-3 and is associated with still high risk of developing another autoimmune disease. © Polish Society for Pediatric Endocrinology and Diabetology.

  15. 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.

  16. Effect of Gamma Radiation on Amino Acid Based Vesicle Carrying Radiosensitizer

    International Nuclear Information System (INIS)

    Nur Ratasha Alia Mohd Rosli; Faizal Mohamed; Muhammad Amir Syafiq Mohd Sah; Irman Abdul Rahman

    2014-01-01

    Vesicles has been developed and studied to be used as a medium to transport radiosensitizer in treating cancer cells by increasing its sensitivity effectively towards the radiation given during radiotherapy. This study was conducted to investigate the effect of gamma radiation on amino acid-based vesicle carrying radiosensitizer. Amino acid based vesicles carrying radiosensitizer were synthesized using sonication method with sodium N-lauroylsarcosinate hydrate and decanol being the primary surfactant, while hydrogen peroxide and sodium hyaluronate as the encapsulated radiosensitizer. The synthesized vesicle was then irradiated at radiation doses equivalent to those given during radiotherapy. Irradiated vesicle carrying radiosensitizer were then characterized using Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR) and Polarized Light Microscope. Results obtained shows that there were no significant changes in morphology and molecular conformation of the synthesized vesicle after irradiation. Even at higher radiation dose of 100 Gray and 200 Gray, the results remained unchanged. This indicates that the synthesized vesicle carrying radiosensitizer is morphologically and spectroscopically stable even at high radiation doses. (author)

  17. Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

    Directory of Open Access Journals (Sweden)

    Daungruthai Jarukanont

    Full Text Available Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We

  18. Ultrasound-responsive ultrathin multiblock copolyamide vesicles

    Science.gov (United States)

    Huang, Lei; Yu, Chunyang; Huang, Tong; Xu, Shuting; Bai, Yongping; Zhou, Yongfeng

    2016-02-01

    This study reports the self-assembly of novel polymer vesicles from an amphiphilic multiblock copolyamide, and the vesicles show a special structure with an ultrathin wall thickness of about 4.5 nm and a combined bilayer and monolayer packing model. Most interestingly, the vesicles are ultrasound-responsive and can release the encapsulated model drugs in response to ultrasonic irradiation.This study reports the self-assembly of novel polymer vesicles from an amphiphilic multiblock copolyamide, and the vesicles show a special structure with an ultrathin wall thickness of about 4.5 nm and a combined bilayer and monolayer packing model. Most interestingly, the vesicles are ultrasound-responsive and can release the encapsulated model drugs in response to ultrasonic irradiation. Electronic supplementary information (ESI) available: Details of experiments and characterization, and FT-IR, TEM, DPD, FL and micro-DSC results. See DOI: 10.1039/c5nr08596a

  19. 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 ...

  20. 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...

  1. Exosomes and Other Extracellular Vesicles: The New Communicators in Parasite Infections

    Science.gov (United States)

    Coakley, Gillian; Maizels, Rick M.; Buck, Amy H.

    2015-01-01

    Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism for transferring information between cells and organisms across all three kingdoms of life. In addition to their roles in normal physiology, vesicles also transport molecules from pathogens to hosts and can spread antigens as well as infectious agents. Although initially described in the host–pathogen context for their functions in immune surveillance, vesicles enable multiple modes of communication by, and between, parasites. Here we review the literature demonstrating that EVs are secreted by intracellular and extracellular eukaryotic parasites, as well as their hosts, and detail the functional properties of these vesicles in maturation, pathogenicity and survival. We further describe the prospects for targeting or exploiting these complexes in therapeutic and vaccine strategies. PMID:26433251

  2. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

    OpenAIRE

    L?sser, Cecilia; Th?ry, Clotilde; Buz?s, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; L?tvall, Jan

    2016-01-01

    The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field co...

  3. Extracellular vesicles in cartilage homeostasis and osteoarthritis.

    Science.gov (United States)

    Miyaki, Shigeru; Lotz, Martin K

    2018-01-01

    Extracellular vesicles carry bioactive molecules that can be transferred between cells and tissues. The purpose of this review is to describe how extracellular vesicles regulate functions of cells in cartilage and other joint tissues. The potential application of extracellular vesicles in the treatment of osteoarthritis and as biomarkers will also be discussed. Extracellular vesicles are found in synovial fluid, in articular cartilage and in the supernatants of synoviocytes and chondrocytes. Extracellular vesicles in cartilage have been proposed to be involved in cross talk between cells in joint tissues and to affect extracellular matrix turnover and inflammation. Extracellular vesicles from arthritic joints can promote abnormal gene expression and changes in cartilage extracellular matrix, including abnormal mineralization. Promising results were obtained in the therapeutic application of mesenchymal stem cell-derived extracellular vesicles for cartilage repair and experimental osteoarthritis. Extracellular vesicles have emerged as vehicles for the exchange of bioactive signaling molecules within cartilage and between joint tissues to promote joint homeostasis and arthritis pathogenesis. As the molecular content of extracellular vesicles can be customized, they offer utility in therapeutic applications.

  4. Extracellular vesicles in cardiovascular homeostasis and disease.

    Science.gov (United States)

    Hutcheson, Joshua D; Aikawa, Elena

    2018-05-01

    Extracellular vesicles have emerged as one of the most important means through which cells interact with each other and the extracellular environment, but extracellular vesicle research remains challenging due to their small size, limited amount of material required for traditional molecular biology assays and inconsistency in the methods of their isolation. The advent of new technologies and standards in the field, however, have led to increased mechanistic insight into extracellular vesicle function. Herein, the latest studies on the role of extracellular vesicles in cardiovascular physiology and disease are discussed. Extracellular vesicles help control cardiovascular homeostasis and remodelling by mediating communication between cells and directing alterations in the extracellular matrix to respond to changes in the environment. The message carried from the parent cell to extracellular space can be intended for both local (within the same tissue) and distal (downstream of blood flow) targets. Pathological cargo loaded within extracellular vesicles could further result in various diseases. On the contrary, new studies indicate that injection of extracellular vesicles obtained from cultured cells into diseased tissues can promote restoration of normal tissue function. Extracellular vesicles are an integral part of cell and tissue function, and harnessing the properties inherent to extracellular vesicles may provide a therapeutic strategy to promote tissue regeneration.

  5. Reversibly formed bilayer vesicles: Energetics and polydispersity

    DEFF Research Database (Denmark)

    Bergstöm, M.

    1997-01-01

    orders of magnitude larger than where the local free energy minima of the equilibrium vesicle actually occur. Moreover, according to our analysis, the relative width of a vesicle size distribution, sigma(R)/R-max, is generally at full equilibrium equal to 0.283, independently of the energetic vesicle....... and a statistical-mechanical factor that accounts for the fluctuations in composition, chain packing density and shape. We demonstrate that the free energy required to form a spherical vesicle is made up of two main contributions: the (size-independent) work of bending the constituent monolayers and the work...

  6. Extracellular Vesicles in Hematological Disorders

    Directory of Open Access Journals (Sweden)

    Anat Aharon

    2014-10-01

    Full Text Available Extracellular vesicles (EVs, comprised of exosomes, microparticles, apoptotic bodies, and other microvesicles, are shed from a variety of cells upon cell activation or apoptosis. EVs promote clot formation, mediate pro-inflammatory processes, transfer proteins and miRNA to cells, and induce cell signaling that regulates cell differentiation, proliferation, migration, invasion, and apoptosis. This paper will review the contribution of EVs in hematological disorders, including hemoglobinopathies (sickle cell disease, thalassemia, paroxysmal nocturnal hemoglobinuria, and hematological malignancies (lymphomas, myelomas, and acute and chronic leukemias.

  7. Further characterization of cadmium uptake by rat liver sinusoidal plasma membrane vesicles as a carrier mediated process

    International Nuclear Information System (INIS)

    Eastman, H.B.; Frazier, J.M.

    1990-01-01

    Previously we have reported that cadmium (Cd) transport by rat hepatic sinusoidal plasma membrane vesicles (SPMV's) occurs by both carrier mediated process and simple diffusion. This study was undertaken in order to further characterize the carrier mediated component of Cd transport as a carrier mediated process. Efflux of Cd from SPMV's was measured by first loading the vesicles with 1 μM Cd, containing 109 Cd (Amersham, 0.25 mCi/ml, carrier free) as a tracer, and then diluting the vesicles 1 to 5 into efflux buffer containing 0.25 M sucrose, 150 mM NaCl and 50 mM Tris/HCl (pH 7.4). Under standard conditions, no efflux of Cd from the vesicles was observed. However, the presence of 4mM CdCl 2 or 4.0% BSA in the efflux buffer was able to release 109 Cd from the vesicles. When the vesicles were lysed with 0.1% Triton X-100, approximately 75% of the internalized Cd could be released from the vesicles. Efflux of Cd from the vesicles was also determined to be a temperature dependent process. At 0 C the efflux of Cd from the vesicles, in the presence of a 4 mM CdCl 2 or 4.0% BSA chase, was blocked. The specificity of the carrier mediated component of Cd transport for Cd was investigated by determining whether other metals could compete for Cd uptake. Zinc was a competitive inhibitor of the carrier mediated component of Cd uptake while calcium had no effect on Cd uptake. Using this system, we have demonstrated that one component of Cd transport exhibits the basic characteristics of a carrier mediated process: saturation, reversibility, specificity and temperature dependence

  8. Fully collapsed (kippah) vesicles: preparation and characterization.

    Science.gov (United States)

    Azzam, Tony; Eisenberg, Adi

    2010-07-06

    A study is presented of the formation of a kippah or hemispherical dome structure, a new morphology generated when a vesicle completely collapses to a hollow hemisphere. Justification for the new name is given in the Introduction. Relatively large vesicles of ca. approximately 500 nm in diameter were prepared from poly(acrylic acid)-block-polystyrene (PAA-b-PS) amphiphilic copolymer in the dioxane/water system. The vesicle specimens for transmission electron microscopy (TEM) were prepared using four different methods: drying under ambient conditions, freeze-drying, freeze-drying and subsequent resuspension in water, and drying under vacuum. The formation of the kippah was found to be strongly influenced by the method of preparation. When the vesicles were allowed to dry on the grid, either by drying under ambient conditions or by direct freeze-drying, "normal" vesicles (i.e., not kippah) with the classical indentation pattern were the only structures to be observed. Kippah vesicles, on the other hand, were obtained only by freeze-drying and subsequent rehydration in water or by direct drying under vacuum where no freezing is involved. The cause of the kippah vesicle formation is not yet completely understood for all methods of preparation; however, it was postulated to be strongly influenced by one or more of the following parameters: the relative flexibility of the vesicle wall, pressure gradient, and surface tension. Unlike "normal" vesicles, which exhibit, in TEM, a classical indentation pattern, kippah vesicles appear nearly round but with average wall thickness twice as large as in the "normal" vesicles. The study illustrates also the usefulness of specimen tilting in the analysis of the kippah. In addition, specimen tilting was found to allow the unambiguous determination of the orientation of the kippah on the surface (i.e., open-side-up or open-side-down).

  9. Transportation

    International Nuclear Information System (INIS)

    Anon.

    1998-01-01

    Here is the decree of the thirtieth of July 1998 relative to road transportation, to trade and brokerage of wastes. It requires to firms which carry out a road transportation as well as to traders and to brokers of wastes to declare their operations to the prefect. The declaration has to be renewed every five years. (O.M.)

  10. Illuminating the physiology of extracellular vesicles

    OpenAIRE

    Choi, Hongyoon; Lee, Dong Soo

    2016-01-01

    Extracellular vesicles play a crucial role in intercellular communication by transmitting biological materials from donor cells to recipient cells. They have pathophysiologic roles in cancer metastasis, neurodegenerative diseases, and inflammation. Extracellular vesicles also show promise as emerging therapeutics, with understanding of their physiology including targeting, distribution, and clearance therefore becoming an important issue. Here, we review recent advances in methods for trackin...

  11. 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.

  12. Feruloyl Dioleoyglycerol Antioxidant Capacity in Phospholipid Vesicles

    Science.gov (United States)

    Ferulic acid and its esters are known to be effective antioxidants. Feruloyl dioleoylglycerol was assessed for its ability to serve as an antioxidant in model membrane phospholipid vesicles. The molecule was incorporated into single-lamellar vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine at ...

  13. Amyloglucosidase enzymatic reactivity inside lipid vesicles

    Directory of Open Access Journals (Sweden)

    Kim Jin-Woo

    2007-10-01

    Full Text Available Abstract Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG (EC 3.2.1.3 from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC multilamellar vesicles (MLVs and large unilamellar vesicles (LUVs was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, Vmax and Km, were determined by initial velocity measurements, and Ki was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations.

  14. Heparin affinity purification of extracellular vesicles

    NARCIS (Netherlands)

    Balaj, Leonora; Atai, Nadia A.; Chen, Weilin; Mu, Dakai; Tannous, Bakhos A.; Breakefield, Xandra O.; Skog, Johan; Maguire, Casey A.

    2015-01-01

    Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely

  15. Detection of extracellular vesicles: size does matter

    NARCIS (Netherlands)

    van der Pol, E.

    2015-01-01

    Cells release small sacks filled with fluid, which are called "extracellular vesicles". The diameter of extracellular vesicles (EV) typically ranges from 30 nm to 1 µm. Because cells release EV into their environment, our body fluids contain numerous EV. Cells release EV to remove waste and to

  16. Extracellular vesicles: new players in cardiovascular diseases.

    Science.gov (United States)

    Gaceb, Abderahim; Martinez, Maria Carmen; Andriantsitohaina, Ramaroson

    2014-05-01

    Extracellular vesicles, particles released by all cell types, represent a new way to convey information between cells such as proteins, second messengers, and genetic information to modify the phenotype and function of the target cells. Recent data suggest that extracellular vesicles play a crucial role in both physiology and pathology, including coagulation, angiogenesis, cell survival, modulation of the immune response, and inflammation. Thus extracellular vesicles participate in the processes of cardiovascular diseases from atherosclerosis, myocardial infarction to heart failure. Consequently, extracellular vesicles can potentially be exploited for therapy, prognosis, and biomarkers for health and disease. This review focuses on the role of extracellular vesicles in the development of cardiovascular diseases, as well as the deleterious and beneficial effects that they may provide in vascular cells and myocardium. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Distribution, formation and regulation of gas vesicles.

    Science.gov (United States)

    Pfeifer, Felicitas

    2012-10-01

    A range of bacteria and archaea produce intracellular gas-filled proteinaceous structures that function as flotation devices in order to maintain a suitable depth in the aqueous environment. The wall of these gas vesicles is freely permeable to gas molecules and is composed of a small hydrophobic protein, GvpA, which forms a single-layer wall. In addition, several minor structural, accessory or regulatory proteins are required for gas vesicle formation. In different organisms, 8-14 genes encoding gas vesicle proteins have been identified, and their expression has been shown to be regulated by environmental factors. In this Review, I describe the basic properties of gas vesicles, the genes that encode them and how their production is regulated. I also discuss the function of these vesicles and the initial attempts to exploit them for biotechnological purposes.

  18. Spontaneous unilamellar polymer vesicles in aqueous solution.

    Science.gov (United States)

    Kim, Tae-Hwan; Song, Chaeyeon; Han, Young-Soo; Jang, Jong-Dae; Choi, Myung Chul

    2014-01-21

    A unilamellar polymeric vesicle is a self-assembled structure of a block copolymer that forms a spherical single bilayer structure with a hydrophobic interlayer and a hydrophilic surface. Due to their enhanced colloidal stability and mechanical property, controllable surface functionality, or tunable membrane thickness, polymeric vesicles are useful in nano and bio-science, providing potential applications as nanosized carriers for catalysts, drugs, and enzymes. For fabrication of a unilamellar vesicle, however, preparative procedures with a few steps are inherently required. Herein, without complicated preparative procedures, we report spontaneous unilamellar polymeric vesicles with nanometer sizes (small angle neutron scattering and cryo-TEM, resulting in a phase diagram drawn as a function of temperature and the 5mS concentration. Interestingly the critical temperature for the micelle-to-vesicle phase transition was easily controlled by varying the 5mS concentration, i.e. it was decreased with increasing the 5mS concentration.

  19. 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.

  20. 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.

  1. Responsive Polydiacetylene Vesicles for Biosensing Microorganisms

    Directory of Open Access Journals (Sweden)

    Estelle Lebègue

    2018-02-01

    Full Text Available Polydiacetylene (PDA inserted in films or in vesicles has received increasing attention due to its property to undergo a blue-to-red colorimetric transition along with a change from non-fluorescent to fluorescent upon application of various stimuli. In this review paper, the principle for the detection of various microorganisms (bacteria, directly detected or detected through the emitted toxins or through their DNA, and viruses and of antibacterial and antiviral peptides based on these responsive PDA vesicles are detailed. The analytical performances obtained, when vesicles are in suspension or immobilized, are given and compared to those of the responsive vesicles mainly based on the vesicle encapsulation method. Many future challenges are then discussed.

  2. Polymer Vesicles: Modular Platforms for Cancer Theranostics.

    Science.gov (United States)

    Wang, Fangyingkai; Xiao, Jiangang; Chen, Shuai; Sun, Hui; Yang, Bo; Jiang, Jinhui; Zhou, Xue; Du, Jianzhong

    2018-02-16

    As an emerging field that is receiving an increasing amount of interest, theranostics is becoming increasingly important in the field of nanomedicine. Among the various smart platforms that have been proposed for use in theranostics, polymer vesicles (or polymersomes) are among the most promising candidates for integration of designated functionalities and modalities. Here, a brief summary of typical theranostic platforms is presented with a focus on modular polymer vesicles. To highlight modularity, the different methodologies for designing therapeutic and diagnostic modules are classified and current examples of theranostic vesicles that excel in both performance and design principle are provided. Finally, future prospects for theranostic polymer vesicles that can be readily prepared with functional modules are proposed. Overall, theranostic polymer vesicles with modular modalities and functions are more promising in nanomedicine than simply being "over-engineered". © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Transportation

    National Research Council Canada - National Science Library

    Allshouse, Michael; Armstrong, Frederick Henry; Burns, Stephen; Courts, Michael; Denn, Douglas; Fortunato, Paul; Gettings, Daniel; Hansen, David; Hoffman, Douglas; Jones, Robert

    2007-01-01

    .... The ability of the global transportation industry to rapidly move passengers and products from one corner of the globe to another continues to amaze even those wise to the dynamics of such operations...

  4. Extracellular vesicles and a novel form of communication in the brain

    Directory of Open Access Journals (Sweden)

    Manuela eBasso

    2016-03-01

    Full Text Available In numerous neurodegenerative diseases, the interplay between neurons and glia modulates the outcome and progression of pathology. One particularly intriguing mode of interaction between neurons, astrocytes, microglia, and oligodendrocytes is characterized by the release of extracellular vesicles that transport proteins, lipids, and nucleotides from one cell to another. Notably, several proteins that cause disease, including the prion protein and mutant SOD1, have been detected in glia-derived extracellular vesicles and observed to fuse with neurons and trigger pathology in vitro. Here we review the structural and functional characterization of such extracellular vesicles in neuron-glia interactions. Furthermore, we discuss possible mechanisms of extracellular vesicle biogenesis and release from activated glia and microglia, and their effects on neurons. Given that exosomes, the smallest type of extracellular vesicles, have been reported to recognize specific cellular populations and act as carriers of very specialized cargo, a thorough analysis of these vesicles may aid in their engineering in vitro and targeted delivery in vivo, opening opportunities for therapeutics.

  5. Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System.

    Science.gov (United States)

    Haraszti, Reka A; Coles, Andrew; Aronin, Neil; Khvorova, Anastasia; Didiot, Marie-Cécile

    2017-06-20

    Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. This natural trafficking ability gives extracellular vesicles the potential to be used as delivery vehicles for oligonucleotides, i.e., siRNAs. However, robust and scalable methods for loading of extracellular vesicles with oligonucleotide cargo are lacking. We describe a detailed protocol for the loading of hydrophobically modified siRNAs into extracellular vesicles upon simple co-incubation. We detail methods of the workflow from purification of extracellular vesicles to data analysis. This method may advance extracellular vesicles-based therapies for the treatment of a broad range of neurological disorders.

  6. Long-term, repeated dose in vitro neurotoxicity of the glutamate receptor antagonist L-AP3, demonstrated in rat hippocampal slice cultures by using continuous propidium iodide incubation

    DEFF Research Database (Denmark)

    Kristensen, Bjarne W; Blaabjerg, Morten; Noraberg, Jens

    2007-01-01

    Most in vitro models are only used to assess short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3...

  7. Interaction of Cryptococcus neoformans extracellular vesicles with the cell wall.

    Science.gov (United States)

    Wolf, Julie M; Espadas-Moreno, Javier; Luque-Garcia, Jose L; Casadevall, Arturo

    2014-12-01

    Cryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to "trap" vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. Extracellular vesicles in the hematopoietic microenvironment

    Science.gov (United States)

    Butler, John T.; Abdelhamed, Sherif; Kurre, Peter

    2018-01-01

    Self-renewal and differentiation are defining characteristics of hematopoietic stem and progenitor cells, and their balanced regulation is central to lifelong function of both blood and immune systems. In addition to cell-intrinsic programs, hematopoietic stem and progenitor cell fate decisions are subject to extrinsic cues from within the bone marrow microenvironment and systemically. Yet, many of the paracrine and endocrine mediators that shape hematopoietic function remain to be discovered. Extracellular vesicles serve as evolutionarily conserved, constitutive regulators of cell and tissue homeostasis, with several recent reports supporting a role for extracellular vesicles in the regulation of hematopoiesis. We review the physiological and pathophysiological effects that extracellular vesicles have on bone marrow compartmental function while highlighting progress in understanding vesicle biogenesis, cargo incorporation, differential uptake, and downstream effects of vesicle internalization. This review also touches on the role of extracellular vesicles in hematopoietic stem and progenitor cell fate regulation and recent advances in therapeutic and diagnostic applications of extracellular vesicles in hematologic disorders. PMID:29439185

  9. 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.

  10. On the Computing Potential of Intracellular Vesicles.

    Science.gov (United States)

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing.

  11. MIXING SOLUTIONS IN INKJET FORMED VESICLES

    Science.gov (United States)

    Li, Thomas H.; Stachowiak, Jeanne C.

    2010-01-01

    Controlling the contents of liposomes and vesicles is essential for their use in medicine, biotechnology, and basic research. Cargos such as proteins, DNA, and RNA are of growing interest for therapeutic applications as well as for fundamental studies of cellular organization and function, but controlled encapsulation and mixing of biomolecules within vesicles has been a challenge. Recently, micro fluidic encapsulation has been shown to efficiently load arbitrary solutions of biomolecules into unilamellar vesicles. This method utilizes a piezo-electrically driven liquid jet to deform a planar bilayer and form a vesicle, with the fluid vortex formed by the jet mixing the solution in the jet with the surrounding solution. Here, we describe the equipment and protocol used for loading mixtures within unilamellar vesicles by microfluidic encapsulation, and we measure the encapsulated fraction to be 79 ± 5% using a falling vesicle technique. Additionally, we find that the presence of a continuous flow from the nozzle and changes in actuation voltage polarity do not significantly affect the encapsulated fraction. These results help to guide current applications and future development of this microfluidic encapsulation technique for forming and loading unilamellar vesicles. PMID:19913162

  12. MR imaging of the seminal vesicles

    International Nuclear Information System (INIS)

    Edson, S.B.; Hricak, H.; Chun-Fang Chang, Y.

    1987-01-01

    The seminal vesicles of 56 healthy males and 23 males with pathologic conditions were studied with a .35-T magnet and spin-echo (SE) techniques (repetition time/echo time [msec] = 500/30 and 2,000/60). The authors analyzed (1) the size and relative signal intensity of seminal vesicles compared to surrounding fat, muscle, or urine; (2) the effect of aging on the size and signal intensity of the vesicles, and (3) the appearance of the seminal vesicles in different pathologic conditions. In the transverse plane, the normal seminal vesicle measures 31 +- 7 mm in length and 17 +- 4 mm in width. Its size or signal intensity did not change significantly with age. On SE = 500/30 images the seminal vesicles were isointense with muscle; on SE = 2,000/60 images they were isointense or slightly hypointense relative to fat. MR imaging was highly sensitive for displaying seminal vesicle pathology, based on asymmetry in size and changes in signal intensities. MR imaging provides unique information but its role in pathologic conditions needs to be further explored

  13. Structure of Amphiphilic Terpolymer Raspberry Vesicles

    Directory of Open Access Journals (Sweden)

    Yingying Guo

    2017-07-01

    Full Text Available Terpolymer raspberry vesicles contain domains of different chemical affinities. They are potential candidates as multi-compartment cargo carriers. Their efficacy depends on their stability and load capacity. Using a model star terpolymer system in an aqueous solution, a dissipative particle dynamic (DPD simulation is employed to investigate how equilibrium aggregate structures are affected by polymer concentration and pairwise interaction energy in a solution. It is shown that a critical mass of polymer is necessary for vesicle formation. The free energy of the equilibrium aggregates are calculated and the results show that the transition from micelles to vesicles is governed by the interactions between the longest solvophobic block and the solvent. In addition, the ability of vesicles to encapsulate solvent is assessed. It is found that reducing the interaction energy favours solvent encapsulation, although solvent molecules can permeate through the vesicle’s shell when repulsive interactions among monomers are low. Thus, one can optimize the loading capacity and the release rate of the vesicles by turning pairwise interaction energies of the polymer and the solvent. The ability to predict and control these aspects of the vesicles is an essential step towards designing vesicles for specific purposes.

  14. Illuminating the physiology of extracellular vesicles.

    Science.gov (United States)

    Choi, Hongyoon; Lee, Dong Soo

    2016-04-16

    Extracellular vesicles play a crucial role in intercellular communication by transmitting biological materials from donor cells to recipient cells. They have pathophysiologic roles in cancer metastasis, neurodegenerative diseases, and inflammation. Extracellular vesicles also show promise as emerging therapeutics, with understanding of their physiology including targeting, distribution, and clearance therefore becoming an important issue. Here, we review recent advances in methods for tracking and imaging extracellular vesicles in vivo and critically discuss their systemic distribution, targeting, and kinetics based on up-to-date evidence in the literature.

  15. Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation

    NARCIS (Netherlands)

    Yuana, Yuana; Levels, Johannes; Grootemaat, Anita; Sturk, Auguste; Nieuwland, Rienk

    2014-01-01

    Extracellular vesicles (EVs) facilitate intercellular communication by carrying bioactive molecules such as proteins, messenger RNA, and micro (mi)RNAs. Recently, high-density lipoproteins (HDL) isolated from human plasma were also reported to transport miRNA to other cells. HDL, when isolated from

  16. Analysing the distribution of synaptic vesicles using a spatial point process model

    DEFF Research Database (Denmark)

    Khanmohammadi, Mahdieh; Waagepetersen, Rasmus; Nava, Nicoletta

    2014-01-01

    Stress can affect the brain functionality in many ways. As the synaptic vesicles have a major role in nervous signal transportation in synapses, their distribution in relationship to the active zone is very important in studying the neuron responses. We study the effect of stress on brain functio...

  17. The class V myosin motor, myosin 5c, localizes to mature secretory vesicles and facilitates exocytosis in lacrimal acini.

    Science.gov (United States)

    Marchelletta, Ronald R; Jacobs, Damon T; Schechter, Joel E; Cheney, Richard E; Hamm-Alvarez, Sarah F

    2008-07-01

    We investigated the role of the actin-based myosin motor, myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Lacrimal gland acinar cells (LGAC) are the major source for the regulated secretion of proteins from the lacrimal gland into the tear film. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles in primary rabbit LGAC. Upon stimulation of secretion with the muscarinic agonist, carbachol, Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of green fluorescent protein (GFP) fused to the tail domain of Myo5c (Ad-GFP-Myo5c-tail) showed that this protein was localized to secretory vesicles. Furthermore, its expression induced a significant (P < or = 0.05) decrease in carbachol-stimulated release of two secretory vesicle content markers, secretory component and syncollin-GFP. Adenovirus-mediated expression of GFP appended to the full-length Myo5c (Ad-GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGAC to compare various parameters of secretory vesicles labeled with either GFP-labeled protein in resting and stimulated LGAC. These studies revealed that the carbachol-stimulated increase in secretory vesicle diameter associated with compound fusion of secretory vesicles that was also exhibited by vesicles labeled with GFP-Myo5c-full was impaired in vesicles labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFP-Myo5c-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myo5c participates in apical exocytosis of secretory vesicles.

  18. Classification, Functions, and Clinical Relevance of Extracellular Vesicles

    NARCIS (Netherlands)

    van der Pol, Edwin; Böing, Anita N.; Harrison, Paul; Sturk, Augueste; Nieuwland, Rienk

    2012-01-01

    Both eukaryotic and prokaryotic cells release small, phospholipid-enclosed vesicles into their environment. Why do cells release vesicles? Initial studies showed that eukaryotic vesicles are used to remove obsolete cellular molecules. Although this release of vesicles is beneficial to the cell, the

  19. Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer

    OpenAIRE

    Torrano, Veronica; Royo, Felix; Peinado, Héctor; Loizaga-Iriarte, Ana; Unda, Miguel; Falcón-Perez, Juan M.; Carracedo, Arkaitz

    2016-01-01

    Normal and tumor cells shed vesicles to the environment. Within the large family of extracellular vesicles, exosomes and microvesicles have attracted much attention in the recent years. Their interest ranges from mediators of cancer progression, inflammation, immune regulation and metastatic niche regulation, to non-invasive biomarkers of disease. In this respect, the procedures to purify and analyze extracellular vesicles have quickly evolved and represent a source of variability for data in...

  20. 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.

  1. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

    NARCIS (Netherlands)

    Kieselbach, Thomas; Zijnge, Vincent; Granstrom, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and

  2. Liver extracellular vesicles in health and disease

    OpenAIRE

    Royo, Felix; Falcon-Perez, Juan M.

    2012-01-01

    Extracellular vesicles (EVs) play an important role in cell-to-cell communication. Although there are different kinds of vesicles, each with their own secretion and capture biology, all of them carry a cargo of proteins, lipids, metabolites and nucleic acids. They act as vehicles for exchange of biological materials and signals and are involved in the regulation of various physiological processes. Liver is an essential organ containing different cell populations fulfilling various functions, ...

  3. Bmp4 from the Optic Vesicle Specifies Murine Retina Formation

    OpenAIRE

    Huang, Jie; Liu, Ying; Oltean, Alina; Beebe, David C.

    2015-01-01

    Previous studies of mouse embryos concluded that after the optic vesicle evaginates from the ventral forebrain and contacts the surface ectoderm, signals from the ectoderm specify the distal region of the optic vesicle to become retina and signals from the optic vesicle induce the lens. Germline deletion of Bmp4 resulted in failure of lens formation. We performed conditional deletion of Bmp4 from the optic vesicle to test the function of Bmp4 in murine eye development. The optic vesicle evagi...

  4. Elastic energy of polyhedral bilayer vesicles.

    Science.gov (United States)

    Haselwandter, Christoph A; Phillips, Rob

    2011-06-01

    In recent experiments [M. Dubois, B. Demé, T. Gulik-Krzywicki, J.-C. Dedieu, C. Vautrin, S. Désert, E. Perez, and T. Zemb, Nature (London) 411, 672 (2001)] the spontaneous formation of hollow bilayer vesicles with polyhedral symmetry has been observed. On the basis of the experimental phenomenology it was suggested [M. Dubois, V. Lizunov, A. Meister, T. Gulik-Krzywicki, J. M. Verbavatz, E. Perez, J. Zimmerberg, and T. Zemb, Proc. Natl. Acad. Sci. USA 101, 15082 (2004)] that the mechanism for the formation of bilayer polyhedra is minimization of elastic bending energy. Motivated by these experiments, we study the elastic bending energy of polyhedral bilayer vesicles. In agreement with experiments, and provided that excess amphiphiles exhibiting spontaneous curvature are present in sufficient quantity, we find that polyhedral bilayer vesicles can indeed be energetically favorable compared to spherical bilayer vesicles. Consistent with experimental observations we also find that the bending energy associated with the vertices of bilayer polyhedra can be locally reduced through the formation of pores. However, the stabilization of polyhedral bilayer vesicles over spherical bilayer vesicles relies crucially on molecular segregation of excess amphiphiles along the ridges rather than the vertices of bilayer polyhedra. Furthermore, our analysis implies that, contrary to what has been suggested on the basis of experiments, the icosahedron does not minimize elastic bending energy among arbitrary polyhedral shapes and sizes. Instead, we find that, for large polyhedron sizes, the snub dodecahedron and the snub cube both have lower total bending energies than the icosahedron.

  5. 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.

  6. Endothelial Extracellular Vesicles-Promises and Challenges.

    Science.gov (United States)

    Hromada, Carina; Mühleder, Severin; Grillari, Johannes; Redl, Heinz; Holnthoner, Wolfgang

    2017-01-01

    Extracellular vesicles, including exosomes, microparticles, and apoptotic bodies, are phospholipid bilayer-enclosed vesicles that have once been considered as cell debris lacking biological functions. However, they have recently gained immense interest in the scientific community due to their role in intercellular communication, immunity, tissue regeneration as well as in the onset, and progression of various pathologic conditions. Extracellular vesicles of endothelial origin have been found to play a versatile role in the human body, since they are on the one hand known to contribute to cardiovascular diseases, but on the other hand have also been reported to promote endothelial cell survival. Hence, endothelial extracellular vesicles hold promising therapeutic potential to be used as a new tool to detect as well as treat a great number of diseases. This calls for clinically approved, standardized, and efficient isolation and characterization protocols to harvest and purify endothelial extracellular vesicles. However, such methods and techniques to fulfill stringent requirements for clinical trials have yet to be developed or are not harmonized internationally. In this review, recent advances and challenges in the field of endothelial extracellular vesicle research are discussed and current problems and limitations regarding isolation and characterization are pointed out.

  7. Extracellular vesicles in parasitic diseases

    Directory of Open Access Journals (Sweden)

    Antonio Marcilla

    2014-12-01

    Full Text Available Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs in parasitic diseases, acting both in parasite–parasite inter-communication as well as in parasite–host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens.

  8. Reversible inactivation of vectorial phosphorylation by hydroxybutynoate in Escherichia coli membrane vesicles.

    Science.gov (United States)

    Kaczorowski, G; Kaback, H R; Walsh, C

    1975-08-26

    The acetylenic hydroxy acid 2-hydroxy-3-butynoate causes irreversible inactivation of the Escherichia coli membrane-bound flavoenzyme D-lactic dehyrogenase, and thus blocks D-lactate dependent active transport in isolated membrane vesicles [Walsh, C. T., Abeles, R. H., and Kaback H. R. (1972), J. Biol. Chem. 247, 7858]. The inactivator is a suicide substrate for the dehydrogenase, undergoing a small number of turnovers before partitioning between oxidation and inactiviation. It is now demonstrated that reactive product molecules of 2-keto-3-butynoate can diffuse in the membranes to a component of the phosphotransferase system and cause time-dependent and covalent inactivation of phosphoenolpyruvate-dependent hexose uptake. Membrane vesicles from double mutants with low levels of both D- and L-lactic dehydrogenase lose only 30 percent of their hexose uptake capacity on exposure to hydroxybutynoate under conditions sufficient to fully inactivate hexose transport in wild type vesicles. Transport of 1-[14C]hydroxybutynoate into vesicles is followed by rapid covalent labeling of membrane proteins by the reactive, enzymatically generating keto acid oxidation product. Incubation of hydroxybutynoate-inactivated vesicles (5% residual activity) for 20 min in buffer with 10 mM dithiothreitol results in reactivation of 63% of the hexose transport activity, a 12-fold increase in activity. No reactivation occurs if the vesicular phosphotransferase system is inactivated by keto acid derived from membrane oxidation of the olefinic congener 2-hydroxy-3-butenoate. In contrast to thiol reactivation of acetylenic-blocked glucose transport, blockage of D-lactate-stimulated proline uptake is not alleviated, stressing different modes of inactivation of the phosphotransferase system compared to the membranous lactate dehydrogenases.

  9. Size distribution and radial density profile of synaptic vesicles by SAXS and light scattering

    Energy Technology Data Exchange (ETDEWEB)

    Castorph, Simon; Salditt, Tim [Institute for X-ray Physics, Goettingen (Germany); Holt, Matthew; Jahn, Reinhard [Max Plank Institute for Biophysical Chemistry, Goettingen (Germany); Sztucki, Michael [European Synchrotron Radiation Facility, Grenoble (France)

    2008-07-01

    Synaptic vesicles are small membraneous organelles within the nerve terminal, encapsulating neurotransmitters by a lipid bilayer. The transport of the neurotransmitter, the fusion at the plasma membrane, and the release of the stored neurotransmitters into the synaptic cleft are since long know as essential step in nerve conduction of the chemical synapse. A detailed structural view of these molecular mechanisms is still lacking, not withstanding the enormous progress in the field during recent years. From measurements and quantitative fitting of small angle X-ray scattering curves and dynamic light scattering the averaged structural properties of synaptic vesicles can be determined. We present SAXS measurements and fits revealing the width of the size distribution function and details of the radial scattering length profile of synaptic vesicles from rat brain. Representative values for the inner and outer radius and the size polydispersity as well as the density and width of the outer protein layer are obtained.

  10. Large GLUT4 vesicles are stationary while locally and reversibly depleted during transient insulin stimulation of skeletal muscle of living mice: imaging analysis of GLUT4-enhanced green fluorescent protein vesicle dynamics

    DEFF Research Database (Denmark)

    Lauritzen, Hans P M M; Galbo, Henrik; Brandauer, Josef

    2007-01-01

    OBJECTIVE: Insulin stimulates glucose transport in skeletal muscle by GLUT4 translocation from intracellular compartments to sarcolemma and t-tubules. We studied in living animals the recruitment of GLUT4 vesicles in more detail than previously done and, for the first time, analyzed the steady......-state recycling and subsequent re-internalization of GLUT4 on an insulin bolus. RESEARCH DESIGN AND METHODS: A confocal imaging technique was used in GLUT4-enhanced green fluorescent protein-transfected superficial muscle fibers in living mice. RESULTS: During the first 30 min of insulin stimulation, very few...... superficially or deeply located GLUT4 storage vesicles (>1 microm) moved in toto. Rather, big vesicles were stationary in their original position at sarcolemma or t-tubules and were locally depleted of GLUT4 by budding off of smaller vesicles. Photobleaching experiments revealed that during initial...

  11. Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Verdin, Anthony [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France); Lounes-Hadj Sahraoui, Anissa [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France)]. E-mail: lounes@univ-littoral.fr; Newsam, Ray [Department of Biosciences, University of Kent, Canterbury CT2 7NJ (United Kingdom); Robinson, Gary [Department of Biosciences, University of Kent, Canterbury CT2 7NJ (United Kingdom); Durand, Roger [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France)

    2005-01-01

    Accumulation and elimination of polycyclic aromatic hydrocarbons (PAHs) were studied in the fungus Fusarium solani. When the fungus was grown on a synthetic medium containing benzo[a]pyrene, hyphae of F. solani contained numerous lipid vesicles which could be stained by the lipid-specific dyes: Sudan III and Rhodamine B. The fluorescence produced by Rhodamine B and PAH benzo[a]pyrene were at the same locations in the fungal hyphae, indicating that F. solani stored PAH in pre-existing lipid vesicles. A passive temperature-independent process is involved in the benzo[a]pyrene uptake and storage. Sodium azide, a cytochrome c oxidation inhibitor, and the two cytoskeleton inhibitors colchicine and cytochalasin did not prevent the transport and accumulation of PAH in lipid vesicles of F. solani hyphae. F. solani degraded a large range of PAHs at different rates. PAH intracellular storage in lipid vesicles was not necessarily accompanied by degradation and was common to numerous other fungi. - Fungi can store PAHs intracellularly in lipid vesicles independently of their PAH degradation abilities.

  12. Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles

    International Nuclear Information System (INIS)

    Verdin, Anthony; Lounes-Hadj Sahraoui, Anissa; Newsam, Ray; Robinson, Gary; Durand, Roger

    2005-01-01

    Accumulation and elimination of polycyclic aromatic hydrocarbons (PAHs) were studied in the fungus Fusarium solani. When the fungus was grown on a synthetic medium containing benzo[a]pyrene, hyphae of F. solani contained numerous lipid vesicles which could be stained by the lipid-specific dyes: Sudan III and Rhodamine B. The fluorescence produced by Rhodamine B and PAH benzo[a]pyrene were at the same locations in the fungal hyphae, indicating that F. solani stored PAH in pre-existing lipid vesicles. A passive temperature-independent process is involved in the benzo[a]pyrene uptake and storage. Sodium azide, a cytochrome c oxidation inhibitor, and the two cytoskeleton inhibitors colchicine and cytochalasin did not prevent the transport and accumulation of PAH in lipid vesicles of F. solani hyphae. F. solani degraded a large range of PAHs at different rates. PAH intracellular storage in lipid vesicles was not necessarily accompanied by degradation and was common to numerous other fungi. - Fungi can store PAHs intracellularly in lipid vesicles independently of their PAH degradation abilities

  13. 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.

  14. Ascent of atomic force microscopy as a nanoanalytical tool for exosomes and other extracellular vesicles

    Science.gov (United States)

    Sharma, S.; LeClaire, M.; Gimzewski, J. K.

    2018-04-01

    Over the last 30 years, atomic force microscopy (AFM) has made several significant contributions to the field of biology and medicine. In this review, we draw our attention to the recent applications and promise of AFM as a high-resolution imaging and force sensing technology for probing subcellular vesicles: exosomes and other extracellular vesicles. Exosomes are naturally occurring nanoparticles found in several body fluids such as blood, saliva, cerebrospinal fluid, amniotic fluid and urine. Exosomes mediate cell-cell communication, transport proteins and genetic content between distant cells, and are now known to play important roles in progression of diseases such as cancers, neurodegenerative disorders and infectious diseases. Because exosomes are smaller than 100 nm (about 30-120 nm), the structural and molecular characterization of these vesicles at the individual level has been challenging. AFM has revealed a new degree of complexity in these nanosized vesicles and generated growing interest as a nanoscale tool for characterizing the abundance, morphology, biomechanics, and biomolecular make-up of exosomes. With the recent interest in exosomes for diagnostic and therapeutic applications, AFM-based characterization promises to contribute towards improved understanding of these particles at the single vesicle and sub-vesicular levels. When coupled with complementary methods like optical super resolution STED and Raman, AFM could further unlock the potential of exosomes as disease biomarkers and as therapeutic agents.

  15. Evidence of Extracellular Vesicles Biogenesis and Release in Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Cruz, Lilian; Arevalo Romero, Jenny Andrea; Brandão Prado, Mariana; Santos, Tiago G; Hohmuth Lopes, Marilene

    2017-10-14

    Extracellular vesicles (EVs) released by mouse embryonic stem cells (mESCs) are considered a source of bioactive molecules that modulate their microenvironment by acting on intercellular communication. Either intracellular endosomal machinery or their derived EVs have been considered a relevant system of signal circuits processing. Herein, we show that these features are found in mESCs. Ultrastructural analysis revealed structures and organelles of the endosomal system such as coated pits and endocytosis-related vesicles, prominent rough endoplasmic reticulum and Golgi apparatus, and multivesicular bodies (MVBs) containing either few or many intraluminal vesicles (ILVs) that could be released as exosomes to extracellular milieu. Besides, budding vesicles shed from the plasma membrane to the extracellular space is suggestive of microvesicle biogenesis in mESCs. mESCs and mouse blastocyst express specific markers of the Endosomal Sorting Complex Required for Transport (ESCRT) system. Ultrastructural analysis and Nanoparticle Tracking Analysis (NTA) of isolated EVs revealed a heterogeneous population of exosomes and microvesicles released by mESCs. These vesicles contain Wnt10b and the Notch ligand Delta-like 4 (DLL4) and also the co-chaperone stress inducible protein 1 (STI1) and its partner Hsp90. Wnt10b and Dll4 colocalize with EVs biogenesis markers in mESCs. Overall, the present study supports the function of the mESCs endocytic network and their EVs as players in stem cell biology.

  16. EXTRACELLULAR VESICLES: CLASSIFICATION, FUNCTIONS AND CLINICAL RELEVANCE

    Directory of Open Access Journals (Sweden)

    A. V. Oberemko

    2014-12-01

    Full Text Available This review presents a generalized definition of vesicles as bilayer extracellular organelles of all celular forms of life: not only eu-, but also prokaryotic. The structure and composition of extracellular vesicles, history of research, nomenclature, their impact on life processes in health and disease are discussed. Moreover, vesicles may be useful as clinical instruments for biomarkers, and they are promising as biotechnological drug. However, many questions in this area are still unresolved and need to be addressed in the future. The most interesting from the point of view of practical health care represents a direction to study the effect of exosomes and microvesicles in the development and progression of a particular disease, the possibility of adjusting the pathological process by means of extracellular vesicles of a particular type, acting as an active ingredient. Relevant is the further elucidation of the role and importance of exosomes to the surrounding cells, tissues and organs at the molecular level, the prospects for the use of non-cellular vesicles as biomarkers of disease.

  17. Feruloyl dioleoylglycerol antioxidant capacity in phospholipid vesicles.

    Science.gov (United States)

    Laszlo, Joseph A; Evans, Kervin O; Vermillion, Karl E; Appell, Michael

    2010-05-12

    Ferulic acid and its esters are known to be effective antioxidants. Feruloyl dioleoylglycerol was assessed for its ability to serve as an antioxidant in model membrane phospholipid vesicles. The molecule was incorporated into single-lamellar vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine at 1 and 5 mol fractions. Employing a lipid peroxidation inhibition assay, feruloyl dioleoylglycerol was demonstrated to express an oxidation protection ratio relative to Trolox of 0.94 and 0.74 at the 1% and 5% incorporation levels, respectively. The impact of feruloyl dioleoylglycerol incorporation on vesicle integrity was examined by determining calcein-cobalt complex leakage rates. Vesicle leakage was not influenced at 22 or 37 degrees C with 5% feruloyl dioleoylglycerol incorporation in comparison to that of vesicles lacking feruloyl dioleoylglycerol. Resonance energy transfer analysis showed that the closest approach distance between feruloyl dioleoylglycerol and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) was approximately 31 A, which indicated that feruloyl dioleoylglycerol was thoroughly distributed throughout the bilayer plane. Conformational analysis determined that feruloyl dioleoylglycerol has a splayed conformation in which its feruloyl moiety is not closely contacted by its oleoyl groups. Feruloyl dioleoylglycerol integrates into the bilayer with its feruloyl moiety oriented close to the hydrophilic/lipophilic interface and its oleoyl groups extended deeply in the membrane. These findings indicate that feruloyl dioleoylglycerol expresses antioxidant activity by intercepting aqueous-phase free radicals as they penetrate the bilayer.

  18. 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.

  19. Pathogenic or Therapeutic Extracellular Vesicles in Rheumatic Diseases: Role of Mesenchymal Stem Cell-Derived Vesicles.

    Science.gov (United States)

    Cosenza, Stella; Ruiz, Maxime; Maumus, Marie; Jorgensen, Christian; Noël, Danièle

    2017-04-22

    Extracellular vesicles (EVs) are important mediators of cell-to-cell communication pathways via the transport of proteins, mRNA, miRNA and lipids. There are three main types of EVs, exosomes, microparticles and apoptotic bodies, which are classified according to their size and biogenesis. EVs are secreted by all cell types and their function reproduces that of the parental cell. They are involved in many biological processes that regulate tissue homeostasis and physiopathology of diseases. In rheumatic diseases, namely osteoarthritis (OA) and rheumatoid arthritis (RA), EVs have been isolated from synovial fluid and shown to play pathogenic roles contributing to progression of both diseases. By contrast, EVs may have therapeutic effect via the delivery of molecules that may stop disease evolution. In particular, EVs derived from mesenchymal stem cells (MSCs) reproduce the main functions of the parental cells and therefore represent the ideal type of EVs for modulating the course of either disease. The aim of this review is to discuss the role of EVs in OA and RA focusing on their potential pathogenic effect and possible therapeutic options. Special attention is given to MSCs and MSC-derived EVs for modulating OA and RA progression with the perspective of developing innovative therapeutic strategies.

  20. Rapid quantification of cardiolipin and DOPC lipid and vesicle concentration.

    Science.gov (United States)

    Elmer-Dixon, Margaret M; Bowler, Bruce E

    2017-03-01

    A novel approach to quantification of cardiolipin and DOPC lipid and vesicle concentration that is rapid and inexpensive is described. Traditional approaches to quantifying vesicle concentration destroy sample and are often time consuming. Using common laboratory equipment and software, lipid vesicles were reliably quantified allowing for immediate use without significant sample loss. Once calibrated, only absorbance measurements with a UV-Vis spectrophotometer are necessary as input into a Matlab program, which calculates the corresponding vesicle and lipid concentration. Fast and accurate concentration determination for preparations of vesicles is essential for analytical titration experiments necessary for protein/vesicle binding curves. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Membrane Trafficking and Vesicle Fusion

    Indian Academy of Sciences (India)

    IAS Admin

    investigators were awarded the Nobel Prize in Physiology or. Medicine in 2013. Introduction. Membrane Transport: In the eukaryotic cell, a majority of proteins are made in the cytosol. But the transmembrane and secretory proteins are synthesized in an organelle called the rough endoplasmic reticulum (ER). These proteins ...

  2. 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.

  3. Congenital cystic disease of the seminal vesicle

    International Nuclear Information System (INIS)

    King, B.F.; Hattery, R.; Lieber, M.; Williamson, B.; Hartman, G.; Berquist, T.

    1988-01-01

    A review of case material (1970-1987) from the Mayo Clinic revealed a variety of seminal vesicle abnormalities (and associated genitourinary anomalies). Sixteen cases of congenital seminal vesicle cysts were studied. Ipsilateral renal agenesis or dysgenesis was associated with 11 of the 16 cases. Other associated genitourinary anomalies included ipsilateral ureteral ectopy, agenesis of the vas deferens, and cryptorchidism. Diagnostic imaging studies were reviewed and the results tabulated. The spectrum of urographic and surgical findings in patients is presented and compared with those in cases previously reported in the literature

  4. Primary adenocarcinoma of the seminal vesicle

    Directory of Open Access Journals (Sweden)

    Safae Terrisse

    2017-10-01

    Full Text Available Primary adenocarcinoma of the seminal vesicle is a rare condition with only about 60 cases described in the literature. The unusual characteristics of this disease makes diagnosis difficult and treatment strategies differ as there are no specific guidelines available. This report presents a case of adenocarcinoma of the seminal vesicle with lung metastases in which surgical and chemotherapeutic treatments have been carried out. The MVAC dose dense regimen following local resection seems effective in this scenario and may be used in the treatment of this disease.

  5. Extracellular vesicles in the pathogenesis of rheumatoid arthritis and osteoarthritis.

    Science.gov (United States)

    Withrow, Joseph; Murphy, Cameron; Liu, Yutao; Hunter, Monte; Fulzele, Sadanand; Hamrick, Mark W

    2016-12-01

    Osteoarthritis (OA) and rheumatoid arthritis (RA) are both debilitating diseases that cause significant morbidity in the US population. Extracellular vesicles (EVs), including exosomes and microvesicles, are now recognized to play important roles in cell-to-cell communication by transporting various proteins, microRNAs (miRNAs), and mRNAs. EV-derived proteins and miRNAs impact cell viability and cell differentiation, and are likely to play a prominent role in the pathophysiology of both OA and RA. Some of the processes by which these membrane-bound vesicles can alter joint tissue include extracellular matrix degradation, cell-to-cell communication, modulation of inflammation, angiogenesis, and antigen presentation. For example, EVs from IL-1β-stimulated fibroblast-like synoviocytes have been shown to induce osteoarthritic changes in chondrocytes. RA models have shown that EVs stimulated with inflammatory cytokines are capable of inducing apoptosis resistance in T cells, presenting antigen to T cells, and causing extracellular damage with matrix-degrading enzymes. EVs derived from rheumatoid models have also been shown to induce secretion of COX-2 and stimulate angiogenesis. Additionally, there is evidence that synovium-derived EVs may be promising biomarkers of disease in both OA and RA. The characterization of EVs in the joint space has also opened up the possibility for delivery of small molecules. This article reviews current knowledge on the role of EVs in both RA and OA, and their potential role as therapeutic targets for modulation of these debilitating diseases.

  6. 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.

  7. 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.

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

    International Nuclear Information System (INIS)

    Meier, P.J.; Valantinas, J.; Hugentobler, G.; Rahm, I.

    1987-01-01

    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 [ 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'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (IC 50 , ∼40 μM). 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

  9. Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer.

    Science.gov (United States)

    Torrano, Veronica; Royo, Felix; Peinado, Héctor; Loizaga-Iriarte, Ana; Unda, Miguel; Falcón-Perez, Juan M; Carracedo, Arkaitz

    2016-08-01

    Normal and tumor cells shed vesicles to the environment. Within the large family of extracellular vesicles, exosomes and microvesicles have attracted much attention in the recent years. Their interest ranges from mediators of cancer progression, inflammation, immune regulation and metastatic niche regulation, to non-invasive biomarkers of disease. In this respect, the procedures to purify and analyze extracellular vesicles have quickly evolved and represent a source of variability for data integration in the field. In this review, we provide an updated view of the potential of exosomes and microvesicles as biomarkers and the available technologies for their isolation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. The role of extracellular vesicles in malaria biology and pathogenesis.

    Science.gov (United States)

    Sampaio, Natalia Guimaraes; Cheng, Lesley; Eriksson, Emily M

    2017-06-09

    In the past decade, research on the functions of extracellular vesicles in malaria has expanded dramatically. Investigations into the various vesicle types, from both host and parasite origin, has revealed important roles for extracellular vesicles in disease pathogenesis and susceptibility, as well as cell-cell communication and immune responses. Here, work relating to extracellular vesicles in malaria is reviewed, and the areas that remain unknown and require further investigations are highlighted.

  11. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers?

    OpenAIRE

    Erdbrügger, Uta; Le, Thu H.

    2015-01-01

    Extracellular vesicles from the urine and circulation have gained significant interest as potential diagnostic biomarkers in renal diseases. Urinary extracellular vesicles contain proteins from all sections of the nephron, whereas most studied circulating extracellular vesicles are derived from platelets, immune cells, and the endothelium. In addition to their diagnostic role as markers of kidney and vascular damage, extracellular vesicles may have functional significance in renal health and ...

  12. Sodium dodecyl sulfate/β-cyclodextrin vesicles embedded in chitosan gel for insulin delivery with pH-selective release

    Directory of Open Access Journals (Sweden)

    Zhuo Li

    2016-07-01

    Full Text Available In an answer to the challenge of enzymatic instability and low oral bioavailability of proteins/peptides, a new type of drug-delivery vesicle has been developed. The preparation, based on sodium dodecyl sulfate (SDS and β-cyclodextrin (β-CD embedded in chitosan gel, was used to successfully deliver the model drug-insulin. The self-assembled SDS/β-CD vesicles were prepared and characterized by particle size, zeta potential, appearance, microscopic morphology and entrapment efficiency. In addition, both the interaction of insulin with vesicles and the stability of insulin loaded in vesicles in the presence of pepsin were investigated. The vesicles were crosslinked into thermo-sensitive chitosan/β-glycerol phosphate solution for an in-situ gel to enhance the dilution stability. The in vitro release characteristics of insulin from gels in media at different pH values were investigated. The insulin loaded vesicles–chitosan hydrogel (IVG improved the dilution stability of the vesicles and provided pH-selective sustained release compared with insulin solution–chitosan hydrogel (ISG. In vitro, IVG exhibited slow release in acidic solution and relatively quick release in neutral solutions to provide drug efficacy. In simulated digestive fluid, IVG showed better sustained release and insulin protection properties compared with ISG. Thus IVG might improve the stability of insulin during its transport in vivo and contribute to the bioavailability and therapeutic effect of insulin.

  13. Methodological Guidelines to Study Extracellular Vesicles

    NARCIS (Netherlands)

    Coumans, Frank A. W.; Brisson, Alain R.; Buzas, Edit I.; Dignat-George, Françoise; Drees, Esther E. E.; El-Andaloussi, Samir; Emanueli, Costanza; Gasecka, Aleksandra; Hendrix, An; Hill, Andrew F.; Lacroix, Romaric; Lee, Yi; van Leeuwen, Ton G.; Mackman, Nigel; Mäger, Imre; Nolan, John P.; van der Pol, Edwin; Pegtel, D. Michiel; Sahoo, Susmita; Siljander, Pia R. M.; Sturk, Guus; de Wever, Olivier; Nieuwland, Rienk

    2017-01-01

    Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the

  14. 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 August 2015 pp 711-725. Fulltext. Click here to view fulltext PDF. Permanent link:

  15. 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.

  16. 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.

  17. Functional transferred DNA within extracellular vesicles

    International Nuclear Information System (INIS)

    Cai, Jin; Wu, Gengze; Jose, Pedro A.; Zeng, Chunyu

    2016-01-01

    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.

  18. Bulk immunoassays for analysis of extracellular vesicles

    NARCIS (Netherlands)

    Coumans, Frank A. W.; Gool, Elmar L.; Nieuwland, Rienk

    2017-01-01

    There is increasing clinical interest in extracellular vesicles (EV) for diagnostic and treatment purposes. This review provides an overview of bulk immunoassays to analyse EV. Western blot and enzyme-linked immunosorbent assay are still the two predominant bulk immunoassays. Recently, new assays

  19. Towards traceable size determination of extracellular vesicles

    NARCIS (Netherlands)

    Varga, Zoltán; Yuana, Yuana; Grootemaat, Anita E.; van der Pol, Edwin; Gollwitzer, Christian; Krumrey, Michael; Nieuwland, Rienk

    2014-01-01

    Extracellular vesicles (EVs) have clinical importance due to their roles in a wide range of biological processes. The detection and characterization of EVs are challenging because of their small size, low refractive index, and heterogeneity. In this manuscript, the size distribution of an

  20. Role of Outer Membrane Vesicles of Bacteria

    Indian Academy of Sciences (India)

    IAS Admin

    However, not all the surface-associated bacterial toxins mediate binding and internal- ization of the vesicles. Role in Pathogenesis. OMVs are important for pathogenicity and virulence of bacteria. Studies involving various pathogenic bacteria clearly reveal that they produce OMVs within the infected host tissues. Body fluids.

  1. Nanoplasmonic ruler to measure lipid vesicle deformation

    Czech Academy of Sciences Publication Activity Database

    Jackman, J.A.; Špačková, Barbora; Linardy, E.; Kim, M.C.; Yoon, B.K.; Homola, Jiří; Cho, N.J.

    2016-01-01

    Roč. 52, č. 1 (2016), s. 76-79 ISSN 1359-7345 R&D Projects: GA ČR(CZ) GBP205/12/G118 Institutional support: RVO:67985882 Keywords : nanomaterial * silicon * lipid vesicle Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 6.319, year: 2016

  2. Endosomal accumulation of APP in wobbler motor neurons reflects impaired vesicle trafficking: implications for human motor neuron disease.

    Science.gov (United States)

    Palmisano, Ralf; Golfi, Panagiota; Heimann, Peter; Shaw, Christopher; Troakes, Claire; Schmitt-John, Thomas; Bartsch, Jörg W

    2011-03-07

    The cause of sporadic amyotrophic lateral sclerosis (ALS) is largely unknown but hypotheses about disease mechanisms include oxidative stress, defective axonal transport, mitochondrial dysfunction and disrupted RNA processing. Whereas familial ALS is well represented by transgenic mutant SOD1 mouse models, the mouse mutant wobbler (WR) develops progressive motor neuron degeneration due to a point mutation in the Vps54 gene, and provides an animal model for sporadic ALS. VPS54 protein as a component of a protein complex is involved in vesicular Golgi trafficking; impaired vesicle trafficking might also be mechanistic in the pathogenesis of human ALS. In motor neurons of homozygous symptomatic WR mice, a massive number of endosomal vesicles significantly enlarged (up to 3 μm in diameter) were subjected to ultrastructural analysis and immunohistochemistry for the endosome-specific small GTPase protein Rab7 and for amyloid precursor protein (APP). Enlarged vesicles were neither detected in heterozygous WR nor in transgenic SOD1(G93A) mice; in WR motor neurons, numerous APP/Rab7-positive vesicles were observed which were mostly LC3-negative, suggesting they are not autophagosomes. We conclude that endosomal APP/Rab7 staining reflects impaired vesicle trafficking in WR mouse motor neurons. Based on these findings human ALS tissues were analysed for APP in enlarged vesicles and were detected in spinal cord motor neurons in six out of fourteen sporadic ALS cases. These enlarged vesicles were not detected in any of the familial ALS cases. Thus our study provides the first evidence for wobbler-like aetiologies in human ALS and suggests that the genes encoding proteins involved in vesicle trafficking should be screened for pathogenic mutations.

  3. Long-term, repeated dose in vitro neurotoxicity of the glutamate receptor antagonist L-AP3, demonstrated in rat hippocampal slice cultures by using continuous propidium iodide incubation

    DEFF Research Database (Denmark)

    Kristensen, Bjarne W; Blaabjerg, Morten; Noraberg, Jens

    2007-01-01

    Most in vitro models are only used to assess short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3......), which is known to be toxic in vivo after subchronic, but not acute, administration. Degenerative effects were monitored by measuring the cellular uptake of propidium iodide (PI; continuously present in the medium) and lactate dehydrogenase (LDH) leakage, and by using a panel of histological stains...

  4. Bacterial membrane vesicles as novel nanosystems for drug delivery

    Directory of Open Access Journals (Sweden)

    Jain S

    2017-08-01

    Full Text Available Sapna Jain, Jonathan Pillai Implants, Devices and Drug Delivery Systems Laboratory, Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute, Faridabad, Haryana, India Abstract: Bacterial membrane vesicles (BMVs are closed spherical nanostructures that are shed naturally and ubiquitously by most bacterial species both in vivo and in vitro. Researchers have elucidated their roles in long-distance transport of a wide array of cargoes, such as proteins, toxins, antigens, virulence factors, microbicidal agents and antibiotics. Given that these natural carriers are important players in intercellular communication, it has been hypothesized that they are equally well attuned for transport and delivery of exogenous therapeutic cargoes. Additionally, BMVs appear to possess specific properties that enable their utilization as drug delivery vehicles. These include their ability to evade the host immune system, protection of the therapeutic payload and natural stability. Using bioengineering approaches, BMVs have been applied as carriers of therapeutic moieties in vaccines and for targeted delivery in cancer. In this article, we explore BMVs from the perspective of understanding their applicability to drug delivery. BMV biology, including biogenesis, physiology and pathology, is briefly reviewed. Practical issues related to bioprocessing, loading of therapeutic moieties and characterization for enabling scalability and commercial viability are evaluated. Finally, challenges to clinical translation and rational design approaches for novel BMV formulations are presented. Although the realization of the full potential of BMVs in drug delivery hinges on the development of scalable approaches for their production as well as the refinement of targeting and loading methods, they are promising candidates for development of a novel generation of drug delivery vehicles in future. Keywords: bacteria, membrane vesicles, immune system

  5. Surface degassing and modifications to vesicle size distributions in active basalt flows

    Science.gov (United States)

    Cashman, K.V.; Mangan, M.T.; Newman, S.

    1994-01-01

    The character of the vesicle population in lava flows includes several measurable parameters that may provide important constraints on lava flow dynamics and rheology. Interpretation of vesicle size distributions (VSDs), however, requires an understanding of vesiculation processes in feeder conduits, and of post-eruption modifications to VSDs during transport and emplacement. To this end we collected samples from active basalt flows at Kilauea Volcano: (1) near the effusive Kupaianaha vent; (2) through skylights in the approximately isothermal Wahaula and Kamoamoa tube systems transporting lava to the coast; (3) from surface breakouts at different locations along the lava tubes; and (4) from different locations in a single breakout from a lava tube 1 km from the 51 vent at Pu'u 'O'o. Near-vent samples are characterized by VSDs that show exponentially decreasing numbers of vesicles with increasing vesicle size. These size distributions suggest that nucleation and growth of bubbles were continuous during ascent in the conduit, with minor associated bubble coalescence resulting from differential bubble rise. The entire vesicle population can be attributed to shallow exsolution of H2O-dominated gases at rates consistent with those predicted by simple diffusion models. Measurements of H2O, CO2 and S in the matrix glass show that the melt equilibrated rapidly at atmospheric pressure. Down-tube samples maintain similar VSD forms but show a progressive decrease in both overall vesicularity and mean vesicle size. We attribute this change to open system, "passive" rise and escape of larger bubbles to the surface. Such gas loss from the tube system results in the output of 1.2 ?? 106 g/day SO2, an output representing an addition of approximately 1% to overall volatile budget calculations. A steady increase in bubble number density with downstream distance is best explained by continued bubble nucleation at rates of 7-8/cm3s. Rates are ???25% of those estimated from the vent

  6. Determination of the Vesicular Systems Parameters: Parallel Implementation and Analysis of the PTNS Vesicle Structure

    Science.gov (United States)

    Bashashin, Maxim; Zemlyanaya, Elena; Zhabitskaya, Evgeniya; Kiselev, Mikhail; Sapozhnikova, Tatyana

    2018-02-01

    Parameters of the phospholipid vesicular systems are determined by means of minimization of the discrepancy between experimental data on the small angle scattering intensity and the numerical results within the frame of the separated form factors method. Effectiveness of parallel implementation is tested at the cluster HybriLIT. Results of numerical analysis of small angle neutron scattering data on polydispersed population of unilamellar vesicles of phospholipid transport nanosystem are presented.

  7. Determination of the Vesicular Systems Parameters: Parallel Implementation and Analysis of the PTNS Vesicle Structure

    Directory of Open Access Journals (Sweden)

    Bashashin Maxim

    2018-01-01

    Full Text Available Parameters of the phospholipid vesicular systems are determined by means of minimization of the discrepancy between experimental data on the small angle scattering intensity and the numerical results within the frame of the separated form factors method. Effectiveness of parallel implementation is tested at the cluster HybriLIT. Results of numerical analysis of small angle neutron scattering data on polydispersed population of unilamellar vesicles of phospholipid transport nanosystem are presented.

  8. Model-free thermodynamics of fluid vesicles.

    Science.gov (United States)

    Diamant, Haim

    2011-12-01

    Motivated by a long-standing debate concerning the nature and interrelations of surface-tension variables in fluid membranes, we reformulate the thermodynamics of a membrane vesicle as a generic two-dimensional finite system enclosing a three-dimensional volume. The formulation is shown to require two tension variables, conjugate to the intensive constraints of area per molecule and volume-to-area ratio. We obtain the relation between these two variables in various scenarios, as well as their correspondence to other definitions of tension variables for membranes. Several controversies related to membrane tension are thereby resolved on a model-free thermodynamic level. The thermodynamic formulation may be useful also for treating large-scale properties of vesicles that are insensitive to the membrane's detailed statistical mechanics and interactions.

  9. Signaling by Extracellular Vesicles Advances Cancer Hallmarks.

    Science.gov (United States)

    Kanada, Masamitsu; Bachmann, Michael H; Contag, Christopher H

    2016-02-01

    Mammalian cells secrete various extracellular vesicles (EVs; exosomes, microvesicles, and apoptotic bodies) that differ in biogenesis, composition, and function. Each vesicle type can originate from normal or cancerous cells, transfer molecular cargo to both neighboring and distant cells, and modulate cellular behaviors involved in eubiology and pathology, such as tumor development. Here, we review evidence for the role of EVs in the establishment and maintenance of cancer hallmarks, including sustaining proliferative signaling, evading growth suppression, resisting cell death, reprogramming energy metabolism, acquiring genomic instability, and remodeling the tumor microenvironment. We also discuss how EVs are implicated in the induction of angiogenesis, control of cellular invasion, initiation of premetastatic niches, maintenance of inflammation, and evasion of immune surveillance. The deeper understanding of the biology of EVs and their contribution to the development and progression of tumors is leading to new opportunities in the diagnosis and treatment of cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Towards traceable size determination of extracellular vesicles

    OpenAIRE

    Varga, Zoltán; Yuana, Yuana; Grootemaat, Anita E.; van der Pol, Edwin; Gollwitzer, Christian; Krumrey, Michael; Nieuwland, Rienk

    2014-01-01

    Background: Extracellular vesicles (EVs) have clinical importance due to their roles in a wide range of biological processes. The detection and characterization of EVs are challenging because of their small size, low refractive index, and heterogeneity.Methods: In this manuscript, the size distribution of an erythrocyte-derived EV sample is determined using state-of-the-art techniques such as nanoparticle tracking analysis, resistive pulse sensing, and electron microscopy, and novel technique...

  11. ATP: The crucial component of secretory vesicles.

    Science.gov (United States)

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  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. Simulation of lipid vesicle breakup in extensional flows*

    Science.gov (United States)

    Banton, Rohan; Eggleton, Charles

    2001-11-01

    Lipid molecules in solution can spontaneously form vesicles encapsulating the solvent. Vesicles can be used to manufacture artificial cells and as a novel means of drug delivery. Unlike biological cells, vesicles do not possess a cytoskeleton (scaffolding for structural rigidity) and cannot resist in-plane shearing forces. The Evans-Skalak strain energy function is used to model the interfacial stresses in the vesicles due to deformation using measured values of the area dilatational and bending moduli. An understanding of vesicle breakup in fluid flow is required in order to effectively design vesicles for particular applications. The Boundary Integral Method is employed to simulate the deformation of the model membrane in both uniaxial and bi-axial extensional flows. The flow field is characterized by a capillary number based on the area dilatational modulus of the vesicle. The response of an initially spherical vesicle to the external flow field was simulated for internal to external viscosity ratios of 0.1,1,10 and capillary numbers in the approximate range of 0.005-0.025. The perturbation analysis developed by Barthes-Biesel (1980) was adapted to predict the deformation of lipid vesicles and compared to the simulations at small capillary numbers. At large capillary numbers, the break-up of the vesicle is simulated and the critical capillary number is determined.

  14. Extracellular vesicles in cardiovascular disease: are they Jedi or Sith?

    Science.gov (United States)

    Osteikoetxea, Xabier; Németh, Andrea; Sódar, Barbara W; Vukman, Krisztina V; Buzás, Edit Irén

    2016-06-01

    In the recent past, extracellular vesicles have become recognized as important players in cell biology and biomedicine. Extracellular vesicles, including exosomes, microvesicles and apoptotic bodies, are phospholipid bilayer-enclosed structures found to be secreted by most if not all cells. Extracellular vesicle secretion represents a universal and highly conserved active cellular function. Importantly, increasing evidence supports that extracellular vesicles may serve as biomarkers and therapeutic targets or tools in human diseases. Cardiovascular disease undoubtedly represents one of the most intensely studied and rapidly growing areas of the extracellular vesicle field. However, in different studies related to cardiovascular disease, extracellular vesicles have been shown to exert diverse and sometimes discordant biological effects. Therefore, it might seem a puzzle whether these vesicles are in fact beneficial or detrimental to cardiovascular health. In this review we provide a general introduction to extracellular vesicles and an overview of their biological roles in cardiovascular diseases. Furthermore, we aim to untangle the various reasons for the observed discrepancy in biological effects of extracellular vesicles in cardiovascular diseases. To this end, we provide several examples that demonstrate that the observed functional diversity is in fact due to inherent differences among various types of extracellular vesicles. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  15. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers?

    Science.gov (United States)

    Erdbrügger, Uta; Le, Thu H

    2016-01-01

    Extracellular vesicles from the urine and circulation have gained significant interest as potential diagnostic biomarkers in renal diseases. Urinary extracellular vesicles contain proteins from all sections of the nephron, whereas most studied circulating extracellular vesicles are derived from platelets, immune cells, and the endothelium. In addition to their diagnostic role as markers of kidney and vascular damage, extracellular vesicles may have functional significance in renal health and disease by facilitating communication between cells and protecting against kidney injury and bacterial infection in the urinary tract. However, the current understanding of extracellular vesicles has derived mostly from studies with very small numbers of patients or in vitro data. Moreover, accurate assessment of these vesicles remains a challenge, in part because of a lack of consensus in the methodologies to measure extracellular vesicles and the inability of most techniques to capture the entire size range of these vesicles. However, newer techniques and standardized protocols to improve the detection of extracellular vesicles are in development. A clearer understanding of the composition and biology of extracellular vesicles will provide insights into their pathophysiologic, diagnostic, and therapeutic roles. Copyright © 2016 by the American Society of Nephrology.

  16. Bioinspired vesicle restraint and mobilization using a biopolymer scaffold.

    Science.gov (United States)

    Zhu, Chao; Lee, Jae-Ho; Raghavan, Srinivasa R; Payne, Gregory F

    2006-03-28

    Biology employs vesicles to package molecules (e.g., neurotransmitters) for their targeted delivery in response to specific spatiotemporal stimuli. Biology is also capable of employing localized stimuli to exert an additional control on vesicle trafficking; intact vesicles can be restrained (or mobilized) by association with (or release from) a cytoskeletal scaffold. We mimic these capabilities by tethering vesicles to a biopolymer scaffold that can undergo (i) stimuli-responsive network formation (for vesicle restraint) and (ii) enzyme-catalyzed network cleavage (for vesicle mobilization). Specifically, we use the aminopolysaccharide chitosan as our scaffold and graft a small number of hydrophobic moieties onto its backbone. These grafted hydrophobes can insert into the bilayer to tether vesicles to the scaffold. Under acidic conditions, the vesicles are not restrained by the hydrophobically modified chitosan (hm-chitosan) because this scaffold is soluble. Increasing the pH to neutral or basic conditions allows chitosan to form interpolymer associations that yield a strong, insoluble restraining network. Enzymatic hydrolysis of this scaffold by chitosanase cleaves the network and mobilizes intact vesicles. Potentially, this approach will provide a controllable means to store and liberate vesicle-based reagents/therapeutics for microfluidic/medical applications.

  17. Inhibition of the coated vesicle proton pump and labeling of a 17,000-dalton polypeptide by N,N'-dicyclohexylcarbodiimide

    International Nuclear Information System (INIS)

    Arai, H.; Berne, M.; Forgac, M.

    1987-01-01

    N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits 100% of proton transport and 80-85% of (Mg2+)-ATPase activity in clathrin-coated vesicles. Half-maximum inhibition of proton transport is observed at 10 microM DCCD after 30 min. Although treatment of the coated vesicle (H+)-ATPase with DCCD has no effect on ATP hydrolysis in the detergent-solubilized state, sensitivity of proton transport and ATPase activity to DCCD is restored following reconstitution into phospholipid vesicles. In addition, treatment of the detergent-solubilized enzyme with DCCD followed by reconstitution gives a preparation that is blocked in both proton transport and ATP hydrolysis. These results suggest that although the coated vesicle (H+)-ATPase can react with DCCD in either a membrane-bound or detergent-solubilized state, inhibition of ATPase activity is only manifested when the pump is present in sealed membrane vesicles. To identify the subunit responsible for inhibition of the coated vesicle (H+)-ATPase by DCCD, we have labeled the partially purified enzyme with [ 14 C]DCCD. A single polypeptide of molecular weight 17,000 is labeled. The extremely hydrophobic nature of this polypeptide is indicated by its extraction with chloroform:methanol. The 17,000-dalton protein can be labeled to a maximum stoichiometry of 0.99 mol of DCCD/mol of protein with 100% inhibition of proton transport occurring at a stoichiometry of 0.15-0.20 mol of DCCD/mol of protein. Amino acid analysis of the chloroform:methanol extracted 17,000-dalton polypeptide reveals a high percentage of nonpolar amino acids. The similarity in properties of this protein and the DCCD-binding subunit of the coupling factor (H+)-ATPases suggests that the 17,000-dalton polypeptide may function as part of a proton channel in the coated vesicle proton pump

  18. Identification of a transport mechanism for NH4+ in the symbiosome membrane of pea root nodules

    DEFF Research Database (Denmark)

    Mouritzen, P.; Rosendahl, L.

    1997-01-01

    Symbiosome membrane vesicles, facing bacteroid-side-out, were purified from pea (Pisum sativum L.) root nodules and used to study NH4+ transport across the membrane by recording vesicle uptake of the NH4+ analog [C-14]methylamine (MA). Membrane potentials (Delta psi) were imposed on the vesicles ...

  19. Counting and dynamic studies of the small unilamellar phospholipid vesicle translocation with single conical glass nanopores.

    Science.gov (United States)

    Chen, Lizhen; He, Haili; Jin, Yongdong

    2015-01-06

    Phospholipid vesicles are ubiquitous cellular organelles that perform vital functions including materials transport and information transmission and have found promising biomedical applications. Although the transmembrane translocation (via nanopores) of phospholipid vesicles, especially small unilamellar phospholipid vesicles (SUVs), is recognized to be very important for these processes and applications, the details and dynamics remain not very clear. Herein, we use single conical glass nanopores as a model platform to systematically investigate the translocation dynamics of SUVs (∼50-60 nm in diameter) through small nanopores with orifice diameters ranging from ∼14 to 72 nm. Dynamic translocation of individual SUVs one by one through the nanopores was clearly observed and was analyzed by the occurrence of periodic oscillation in ionic current blockage signal under a negatively applied voltage. Translocation behaviors of the SUVs, in terms of magnitude and duration of ionic current blockage signal, varied and can be modulated by changing nanopore size, solution pH, vesicle concentration, applied voltage, and inner surface charge properties of the nanopores. The translocation rate of the SUVs through an ∼72 nm nanopore is typically on a time scale of a few seconds (per SUV translocation event) and found nonlinearly proportional to the concentration of the SUVs. Moreover, the electrophoretic force has been verified as a main force to drive the SUVs through the nanopore since there is a nearly linear relationship between the current blockage frequency of SUVs translocation and the applied bias potentials ranging from -0.6 to -1 V. The findings provide fundamental insights into the translocation and interactions of SUVs with nanopores, and the reported nanopore platform may find potential useful bioapplications in single-cell and single-vesicle studies.

  20. Characterization of extracellular vesicles isolated by size exclusion chromatography

    DEFF Research Database (Denmark)

    Kristensen, Anne Flou; Pedersen, Shona; Jørgensen, Malene

    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...... is observed all over the gradient. The variety of the membrane vesicles is currently being investigated further by several means. Summary/conclusion: A new procedure for easy and gentle isolation of bovine milk membrane vesicles encompassing ultracentrifugation and size-exclusion chromatography has been...... established. The resulting vesicle isolate exhibits the general membrane vesicle characteristics and provides an appropriate start material from which the variety of milk vesicles can be investigated...

  1. Human mammospheres secrete hormone-regulated active extracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Esperanza Gonzalez

    Full Text Available Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important prognostic factors for survival is the early detection of the disease. Recent studies indicate that extracellular vesicles may provide diagnostic information for cancer management. We demonstrate the secretion of extracellular vesicles by primary breast epithelial cells enriched for stem/progenitor cells cultured as mammospheres, in non-adherent conditions. Using a proteomic approach we identified proteins contained in these vesicles whose expression is affected by hormonal changes in the cellular environment. In addition, we showed that these vesicles are capable of promoting changes in expression levels of genes involved in epithelial-mesenchymal transition and stem cell markers. Our findings suggest that secreted extracellular vesicles could represent potential diagnostic and/or prognostic markers for breast cancer and support a role for extracellular vesicles in cancer progression.

  2. 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.

  3. 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.

  4. Membrane steroid binding protein 1 (MSBP1) stimulates tropism by regulating vesicle trafficking and auxin redistribution.

    Science.gov (United States)

    Yang, Xi; Song, Li; Xue, Hong-Wei

    2008-11-01

    Overexpression of membrane steroid binding protein 1 (MSBP1) stimulates the root gravitropism and anti-gravitropism of hypocotyl, which is mainly due to the enhanced auxin redistribution in the bending regions of hypocotyls and root tips. The inhibitory effects by 1-N-naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, are suppressed under the MSBP1 overexpression, suggesting the positive effects of MSBP1 on polar auxin transport. Interestingly, sub-cellular localization studies showed that MSBP1 is also localized in endosomes and observations of the membrane-selective dye FM4-64 revealed the enhanced vesicle trafficking under MSBP1 overexpression. MSBP1-overexpressing seedlings are less sensitive to brefeldin A (BFA) treatment, whereas the vesicle trafficking was evidently reduced by suppressed MSBP1 expression. Enhanced MSBP1 does not affect the polar localization of PIN2, but stimulates the PIN2 cycling and enhances the asymmetric PIN2 redistribution under gravi-stimulation. These results suggest that MSBP1 could enhance the cycling of PIN2-containing vesicles to stimulate the auxin redistribution under gravi-stimulation, providing informative hints on interactions between auxin and steroid binding protein.

  5. Spin State As a Probe of Vesicle Self-Assembly.

    Science.gov (United States)

    Kim, Sanghoon; Bellouard, Christine; Eastoe, Julian; Canilho, Nadia; Rogers, Sarah E; Ihiawakrim, Dris; Ersen, Ovidiu; Pasc, Andreea

    2016-03-02

    A novel system of paramagnetic vesicles was designed using ion pairs of iron-containing surfactants. Unilamellar vesicles (diameter ≈ 200 nm) formed spontaneously and were characterized by cryogenic transmission electron microscopy, nanoparticle tracking analysis, and light and small-angle neutron scattering. Moreover, for the first time, it is shown that magnetization measurements can be used to investigate self-assembly of such functionalized systems, giving information on the vesicle compositions and distribution of surfactants between the bilayers and the aqueous bulk.

  6. Spin State As a Probe of Vesicle Self-Assembly

    OpenAIRE

    Kim, Sanghoon; Bellouard, Christine; Eastoe, Julian; Canilho, Nadia; Rogers, Sarah E; Ihiawakrim, Dris; Ersen, Ovidiu; Pasc, Andreea

    2016-01-01

    A novel system of paramagnetic vesicles was designed using ion pairs of iron-containing surfactants. Unilamellar vesicles (diameter ≈ 200 nm) formed spontaneously and were characterized by cryogenic transmission electron microscopy, nanoparticle tracking analysis, and light and small-angle neutron scattering. Moreover, for the first time, it is shown that magnetization measurements can be used to investigate self-assembly of such functionalized systems, giving information on the vesicle compo...

  7. Lipid-targeting peptide probes for extracellular vesicles

    OpenAIRE

    Flynn, Aaron D.; Yin, Hang

    2016-01-01

    Extracellular vesicles released from cells are under intense investigation for their roles in cell-cell communication and cancer progression. However, individual vesicles have been difficult to probe as their small size renders them invisible by conventional light microscopy. However, as a consequence of their small size these vesicles possess highly curved lipid membranes that offer an unconventional target for curvature-sensing probes. In this article, we present a strategy for using peptid...

  8. 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.

  9. The motion of a train of vesicles in channel flow

    Science.gov (United States)

    Barakat, Joseph; Shaqfeh, Eric

    2017-11-01

    The inertialess motion of a train of lipid-bilayer vesicles flowing through a channel is simulated using a 3D boundary integral equation method. Steady-state results are reported for vesicles positioned concentrically inside cylindrical channels of circular, square, and rectangular cross sections. The vesicle translational velocity U and excess channel pressure drop Δp+ depend strongly on the ratio of the vesicle radius to the hydraulic radius λ and the vesicle reduced volume υ. ``Deflated vesicles'' of lower reduced volume υ are more streamlined and translate with greater velocity U relative to the mean flow velocity V. Increasing the vesicle size (λ) increases the wall friction force and extra pressure drop Δp+, which in turn reduces the vesicle velocity U. Hydrodynamic interactions between vesicles in a periodic train are largely screened by the channel walls, in accordance with previous results for spheres and drops. The hydraulic resistance is compared across different cross sections, and a simple correction factor is proposed to unify the results. Nonlinear effects are observed when β - the ratio of membrane bending elasticity to viscous traction - is changed. The simulation results show excellent agreement with available experimental measurements as well as a previously reported ``small-gap theory'' valid for large values of λ. NSF CBET 1066263/1066334.

  10. Dynamic Properties of the Alkaline Vesicle Population at Hippocampal Synapses

    Science.gov (United States)

    Röther, Mareike; Brauner, Jan M.; Ebert, Katrin; Welzel, Oliver; Jung, Jasmin; Bauereiss, Anna; Kornhuber, Johannes; Groemer, Teja W.

    2014-01-01

    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. PMID:25079223

  11. Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate

    DEFF Research Database (Denmark)

    Schotten, Sebastiaan; Meijer, Marieke; Walter, Alexander Matthias

    2015-01-01

    supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced......-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca2+-dependent release....

  12. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Cecilia Lässer

    2016-12-01

    Full Text Available The International Society for Extracellular Vesicles (ISEV has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs. This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge.

  13. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles.

    Science.gov (United States)

    Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

    2016-01-01

    The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, "Basics of Extracellular Vesicles," uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform "Coursera" and is free of charge.

  14. The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

    Science.gov (United States)

    Lässer, Cecilia; Théry, Clotilde; Buzás, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; Lötvall, Jan

    2016-01-01

    The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field covering the nomenclature and history of EVs. Module 2 focuses on the biogenesis and uptake mechanisms of EVs, as well as their RNA, protein and lipid cargo. Module 3 covers the collection and processing of cell culture media and body fluids such as blood, breast milk, cerebrospinal fluid and urine prior to isolation of EVs. Modules 4 and 5 present different isolation methods and characterisation techniques utilised in the EV field. Here, differential ultracentrifugation, size-exclusion chromatography, density gradient centrifugation, kit-based precipitation, electron microscopy, cryo-electron microscopy, flow cytometry, atomic-force microscopy and nanoparticle-tracking analysis are covered. This first massive open online course (MOOC) on EVs was launched on 15 August 2016 at the platform “Coursera” and is free of charge. PMID:27989272

  15. 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.

  16. Morphometric image analysis of giant vesicles

    DEFF Research Database (Denmark)

    Husen, Peter Rasmussen; Arriaga, Laura; Monroy, Francisco

    2012-01-01

    We have developed a strategy to determine lengths and orientations of tie lines in the coexistence region of liquid-ordered and liquid-disordered phases of cholesterol containing ternary lipid mixtures. The method combines confocal-fluorescence-microscopy image stacks of giant unilamellar vesicles...... (GUVs), a dedicated 3D-image analysis, and a quantitative analysis based in equilibrium thermodynamic considerations. This approach was tested in GUVs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-palmitoyl-sn-glycero-3-phosphocholine/cholesterol. In general, our results show a reasonable...

  17. Comparative proteomic analysis of extracellular vesicles isolated by acoustic trapping or differential centrifugation

    NARCIS (Netherlands)

    Rezeli, Melinda; Gidlöf, Olof; Evander, Mikael; Bryl-Górecka, Paulina; Sathanoori, Ramasri; Gilje, Patrik; Pawlowski, Krzysztof; Horvatovich, Péter; Erlinge, David; Marko-Varga, György; Laurell, Thomas

    2016-01-01

    Extracellular vesicles (ECVs), including microparticles (MPs) and exosomes, are submicron membrane vesicles released by diverse cell types upon activation or stress. Circulating ECVs are potential reservoirs of disease biomarkers, and the complexity of these vesicles is significantly lower compared

  18. Role of Extracellular Vesicles in Hematological Malignancies

    Directory of Open Access Journals (Sweden)

    Stefania Raimondo

    2015-01-01

    Full Text Available In recent years the role of tumor microenvironment in the progression of hematological malignancies has been widely recognized. Recent studies have focused on how cancer cells communicate within the microenvironment. Among several factors (cytokines, growth factors, and ECM molecules, a key role has been attributed to extracellular vesicles (EV, released from different cell types. EV (microvesicles and exosomes may affect stroma remodeling, host cell functions, and tumor angiogenesis by inducing gene expression modulation in target cells, thus promoting cancer progression and metastasis. Microvesicles and exosomes can be recovered from the blood and other body fluids of cancer patients and contain and deliver genetic and proteomic contents that reflect the cell of origin, thus constituting a source of new predictive biomarkers involved in cancer development and serving as possible targets for therapies. Moreover, due to their specific cell-tropism and bioavailability, EV can be considered natural vehicles suitable for drug delivery. Here we will discuss the recent advances in the field of EV as actors in hematological cancer progression, pointing out the role of these vesicles in the tumor-host interplay and in their use as biomarkers for hematological malignancies.

  19. BACTERIAL OUTER MEMBRANE VESICLES AND VACCINE APPLICATIONS

    Directory of Open Access Journals (Sweden)

    Reinaldo eAcevedo

    2014-03-01

    Full Text Available Vaccines based on outer membrane vesicles (OMV were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of self meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA, serogroup W (dOMVW and serogroup X (dOMVX were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC, Bordetella pertussis (dOMVBP, Mycobacterium smegmatis (dOMVSM and BCG (dOMVBCG. The immunogenicity of the OMV have been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice have shown their protective potential. dOMVB has been evaluated with non-self neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates.

  20. Bioinformatics Tools for Extracellular Vesicles Research.

    Science.gov (United States)

    Keerthikumar, Shivakumar; Gangoda, Lahiru; Gho, Yong Song; Mathivanan, Suresh

    2017-01-01

    Extracellular vesicles (EVs) are a class of membranous vesicles that are released by multiple cell types into the extracellular environment. This unique class of extracellular organelles which play pivotal role in intercellular communication are conserved across prokaryotes and eukaryotes. Depending upon the cell origin and the functional state, the molecular cargo including proteins, lipids, and RNA within the EVs are modulated. Owing to this, EVs are considered as a subrepertoire of the host cell and are rich reservoirs of disease biomarkers. In addition, the availability of EVs in multiple bodily fluids including blood has created significant interest in biomarker and signaling research. With the advancement in high-throughput techniques, multiple EV studies have embarked on profiling the molecular cargo. To benefit the scientific community, existing free Web-based resources including ExoCarta, EVpedia, and Vesiclepedia catalog multiple datasets. These resources aid in elucidating molecular mechanism and pathophysiology underlying different disease conditions from which EVs are isolated. Here, the existing bioinformatics tools to perform integrated analysis to identify key functional components in the EV datasets are discussed.

  1. Tetraspanins in Extracellular Vesicle formation and function

    Directory of Open Access Journals (Sweden)

    Zoraida Andreu Martínez

    2014-09-01

    Full Text Available Extracellular vesicles (EVs represent a novel mechanism of intercellular communication as vehicles for intercellular transfer of functional membrane and cytosolic proteins, lipids, and RNAs. Microvesicles, ectosomes, shedding vesicles, microparticles and exosomes are the most common terms to refer to the different kinds of EVs based on their origin, composition, size and density. Exosomes have an endosomal origin and are released by many different cell types, participating in different physiological and/or pathological processes. Depending on their origin, they can alter the fate of recipient cells according to the information transferred. In the last two decades, EVs have become the focus of many studies because of their putative use as non-invasive biomarkers and their potential in bioengineering and clinical applications. In order to exploit this ability of EVs many aspects of their biology should be deciphered. Here, we review the mechanisms involved in EV biogenesis, assembly, recruitment of selected proteins and genetic material as well as the uptake mechanisms by target cells in an effort to understand EV functions and their utility in clinical applications. In these contexts, the role of proteins from the tetraspanin superfamily, which are among the most abundant membrane proteins of EVs, will be highlighted.

  2. The endocytosis gene END3 is essential for the glucose-induced rapid decline of small vesicles in the extracellular fraction in Saccharomyces cerevisiae.

    Science.gov (United States)

    Giardina, Bennett J; Stein, Kathryn; Chiang, Hui-Ling

    2014-01-01

    Protein secretion is a fundamental process in all living cells. Gluconeogenic enzymes are secreted when Saccharomyces cerevisiae are grown in media containing low glucose. However, when cells are transferred to media containing high glucose, they are internalized. We investigated whether or not gluconeogenic enzymes were associated with extracellular vesicles in glucose-starved cells. We also examined the role that the endocytosis gene END3 plays in the internalization of extracellular proteins/vesicles in response to glucose addition. Transmission electron microscopy was performed to determine the presence of extracellular vesicles in glucose-starved wild-type cells and the dynamics of vesicle transport in cells lacking the END3 gene. Proteomics was used to identify extracellular proteins that associated with these vesicles. Total extracts prepared from glucose-starved cells consisted of about 95% small vesicles (30-50 nm) and 5% large structures (100-300 nm). The addition of glucose caused a rapid decline in small extracellular vesicles in wild-type cells. However, most of the extracellular vesicles were still observed in cells lacking the END3 gene following glucose replenishment. Proteomics was used to identify 72 extracellular proteins that may be associated with these vesicles. Gluconeogenic enzymes fructose-1,6-bisphosphatase, malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase, as well as non-gluconeogenic enzymes glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A, were distributed in the vesicle-enriched fraction in total extracts prepared from cells grown in low glucose. Distribution of these proteins in the vesicle-enriched fraction required the integrity of the membranes. When glucose was added to glucose-starved wild-type cells, levels of extracellular fructose-1,6-bisphosphatase, malate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxykinase, glyceraldehyde-3-phosphate dehydrogenase, and cyclophilin A were

  3. Advances in transport phenomena 2011

    CERN Document Server

    2014-01-01

    This new volume of the annual review “Advances in Transport Phenomena” series contains three in-depth review articles on the microfluidic fabrication of vesicles, the dielectrophoresis field-flow fractionation for continuous-flow separation of particles and cells in microfluidic devices, and the thermodynamic analysis and optimization of heat exchangers, respectively.

  4. Generic sorting of raft lipids into secretory vesicles in yeast

    DEFF Research Database (Denmark)

    Surma, Michal A; Klose, Christian; Klemm, Robin W

    2011-01-01

    a complete lipid overview of the yeast late secretory pathway. We could show that vesicles captured with different baits carry the same cargo and have almost identical lipid compositions; being highly enriched in ergosterol and sphingolipids. This finding indicates that lipid raft sorting is a generic...... feature of vesicles carrying PM cargo and suggests a common lipid-based mechanism for their formation....

  5. 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

  6. Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation

    NARCIS (Netherlands)

    H. Kalra (Hina); R.J. Simpson (Richard); H. Ji (Hong); M. Aikawa (Masanori); P. Altevogt (Peter); P. Askenase (Philip); V.C. Bond (Vincent); F.E. Borràs (Francesc); X. Breakefield (Xandra); V. Budnik (Vivian); E. Buzas (Edit); G. Camussi (Giovanni); A. Clayton (Aled); E. Cocucci (Emanuele); J.M. Falcon-Perez (Juan); S. Gabrielsson (Susanne); Y.S. Gho (Yong Song); D. Gupta (Dwijendra); H.C. Harsha (H.); A. Hendrix (An); A.F. Hill (Andrew); J.M. Inal (Jameel); G.W. Jenster (Guido); E.-M. Krämer-Albers (Eva-Maria); S.K. Lim (Sai Kiang); A. Llorente (Alicia); J. Lötvall; A. Marcilla (Antonio); L. Mincheva-Nilsson (Lucia); I. Nazarenko (Irina); C.C.M. van Nieuwland (Carolien); E.N.M. Nolte-'t Hoen (Esther); A. Pandey (Akhilesh); T. Patel (Tushar); M.D. Piper; S. Pluchino (Stefano); T.S.K. Prasad (T. S. Keshava); L. Rajendran (Lawrence); L. Raposo (Luís); M. Record (Michel); G.E. Reid (Gavin); F. Sánchez-Madrid (Francisco); R.M. Schiffelers (Raymond); P. Siljander (Pia); A. Stensballe (Allan); W. Stoorvogel (Willem); D. Taylor (Deborah); C. Thery; H. Valadi (Hadi); B.W.M. van Balkom (Bas); M. Vidal (Michel); M.H.M. Wauben (Marca); M. Yáñez-Mó (María); M. Zoeller (Margot); S. Mathivanan (Suresh)

    2012-01-01

    textabstractExtracellular 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.

  7. 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).

  8. Interaction of Phenol-Soluble Modulins with Phosphatidylcholine Vesicles

    Directory of Open Access Journals (Sweden)

    Anthony C. Duong

    2012-07-01

    Full Text Available Several members of the staphylococcal phenol-soluble modulin (PSM peptide family exhibit pronounced capacities to lyse eukaryotic cells, such as neutrophils, monocytes, and erythrocytes. This is commonly assumed to be due to the amphipathic, α-helical structure of PSMs, giving PSMs detergent-like characteristics and allowing for a relatively non-specific destruction of biological membranes. However, the capacities of PSMs to lyse synthetic phospholipid vesicles have not been investigated. Here, we analyzed lysis of synthetic phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC vesicles by all Staphylococcus aureus and S. epidermidis PSMs. In addition, we investigated the lytic capacities of culture filtrates obtained from different S. aureus PSM deletion mutants toward POPC vesicles. Our results show that all staphylococcal PSMs have phospholipid vesicle-lysing activity and the capacity of S. aureus culture filtrate to lyse POPC vesicles is exclusively dependent on PSMs. Notably, we observed largely differing capacities among PSM peptides to lyse POPC vesicles. Interestingly, POPC vesicle-lytic capacities did not correlate with those previously seen for the lysis of eukaryotic cells. For example, the β-type PSMs were strongly lytic for POPC vesicles, but are known to exhibit only very low lytic capacities toward neutrophils and erythrocytes. Thus our results also suggest that the interaction between PSMs and eukaryotic membranes is more specific than previously assumed, potentially depending on additional structural features of those membranes, such as phospholipid composition or yet unidentified docking molecules.

  9. Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles

    NARCIS (Netherlands)

    Jackson, R.L.; Verkleij, A.J.; Zoelen, E.J.J. van; Lane, L.K.; Schwartz, A.; Deenen, L.L.M. van

    Purified lamb kidney Na+, K+-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the Mτ- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles

  10. Encapsulation of antitumor drug methotrexate in liposome vesicles

    International Nuclear Information System (INIS)

    Xu Bo; Sun Qixun; Zhang Nianbao; Xie Binghua; Zhang Jiong

    1990-01-01

    Liposome vesicles containing antitumor drug methotrexate (MTX) were prepared. MTX was labelled by the tritium ion beam method. After purification by TLC, the specific radioactivity of 3 H-MTX was 1.19 GBq/mmol with radiochemical purity orver 95%. Under various forming conditions of liposome vesicles, the efficiency of encapsulation was 21-53%

  11. ASSOCIATION OF LYSOZYME TO PHOSPHOLIPID SURFACES AND VESICLE FUSION

    NARCIS (Netherlands)

    ARNOLD, K; HOEKSTRA, D; OHKI, S

    1992-01-01

    Lysozyme-induced fusion of phosphatidylserine (PS) vesicles was studied as a function of pH. Fusion, monitored by lipid-mixing, was measured by following the dilution of pyrene-labelled phosphatidylcholine, incorporated in PS vesicles, into unlabelled bilayers. It is demonstrated that

  12. VESICLE-ENCAPSULATED CORTICOSTEROIDS FOR THE TREATMENT OF CANCER

    NARCIS (Netherlands)

    Schiffelers, Raymond M.; Metselaar, J.M.; Molema, Grietje; Storm, Gerrit

    2004-01-01

    The invention relates to the use of a composition comprising a corticosteroid encapsulated in a vesicle for the manufacture of a medicament for treating cancer, such as the use of a composition comprising a corticosteroid and liposomes, the liposomes comprising a non-charged vesicle-forming lipid,

  13. Vesicle-encapsulated corticosteroids for the treatment of cancer

    NARCIS (Netherlands)

    Schiffelers, Raymond M.; Metselaar, J.M.; Molema, Grietje; Storm, Gerrit

    2005-01-01

    The invention relates to the use of a composition comprising a corticosteroid encapsulated in a vesicle for the manufacture of a medicament for treating cancer, such as the use of a composition comprising a corticosteroid and liposomes, the liposomes comprising a non-charged vesicle-forming lipid

  14. Molecular dynamics simulations of lipid vesicle fusion in atomic detail

    NARCIS (Netherlands)

    Knecht, Volker; Marrink, Siewert-Jan

    The fusion of a membrane-bounded vesicle with a target membrane is a key step in intracellular trafficking, exocytosis, and drug delivery. Molecular dynamics simulations have been used to study the fusion of small unilamellar vesicles composed of a dipalmitoyl-phosphatidylcholine (DPPC)/palmitic

  15. The freezing process of small lipid vesicles at molecular resolution

    NARCIS (Netherlands)

    Risselada, H. Jelger; Marrink, Siewert J.

    2009-01-01

    At present very little is known about the kinetic barriers which a small vesicle will face during the transformation from the liquid-crystalline to the gel phase, and what the structure of frozen vesicles looks like at the molecular level. The formation of gel domains in the strongly curved bilayer

  16. Gas vesicles in actinomycetes : old buoys in novel habitats?

    NARCIS (Netherlands)

    Keulen, Geertje van; Hopwood, David A.; Dijkhuizen, Lubbert; Sawers, R. Gary

    2005-01-01

    Gas vesicles are gas-filled prokaryotic organelles that function as flotation devices. This enables planktonic cyanobacteria and halophilic archaea to position themselves within the water column to make optimal use of light and nutrients. Few terrestrial microbes are known to contain gas vesicles.

  17. Adsorption of DOPC vesicles on hydrophobic substrates in the ...

    Indian Academy of Sciences (India)

    Administrator

    Na2SO4, KCl of ANALAR grade and HPLC grade ethanol were obtained from Merck, India. 2.2 Vesicle preparation. The lipid ... (Brookhaven Instruments Co.), suggesting the for- mation of multilamellar vesicles (MLVs). ... were measured with a custom built instrument before each measurement. The average contact angle.

  18. ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Y.C.; Liu, C.

    2010-12-28

    Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.

  19. Shedding light on the cell biology of extracellular vesicles.

    Science.gov (United States)

    van Niel, Guillaume; D'Angelo, Gisela; Raposo, Graça

    2018-04-01

    Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively. They are present in biological fluids and are involved in multiple physiological and pathological processes. Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material. Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis. However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles.

  20. Extracellular vesicles as new pharmacological targets to treat atherosclerosis.

    Science.gov (United States)

    Yin, Min; Loyer, Xavier; Boulanger, Chantal M

    2015-09-15

    Extracellular vesicles released by most cell types, include apoptotic bodies (ABs), microvesicles (MVs) and exosomes. They play a crucial role in physiology and pathology, contributing to "cell-to-cell" communication by modifying the phenotype and the function of target cells. Thus, extracellular vesicles participate in the key processes of atherosclerosis from endothelial dysfunction, vascular wall inflammation to vascular remodeling. The purpose of this review is to summarize recent findings on extracellular vesicle formation, structure, release and clearance. We focus on the deleterious and beneficial effects of extracellular vesicles in the development of atherosclerosis. The potential role of extracellular vesicles as biomarkers and pharmacological targets, their innate therapeutic capacity, or their use for novel drug delivery devices in atherosclerotic cardiovascular diseases will also be discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Placental Extracellular Vesicles and Feto-Maternal Communication

    Science.gov (United States)

    Tong, M.; Chamley, L.W.

    2015-01-01

    The human placenta is an anatomically unique structure that extrudes a variety of extracellular vesicles into the maternal blood (including syncytial nuclear aggregates, microvesicles, and nanovesicles). Large quantities of extracellular vesicles are produced by the placenta in both healthy and diseased pregnancies. Since their first description more than 120 years ago, placental extracellular vesicles are only now being recognized as important carriers for proteins, lipids, and nucleic acids, which may play a crucial role in feto-maternal communication. Here, we summarize the current literature on the cargos of placental extracellular vesicles and the known effects of such vesicles on maternal cells/systems, especially those of the maternal immune and vascular systems. PMID:25635060

  2. ISEV position paper: extracellular vesicle RNA analysis and bioinformatics

    Directory of Open Access Journals (Sweden)

    Andrew F. Hill

    2013-12-01

    Full Text Available Extracellular vesicles (EVs are the collective term for the various vesicles that are released by cells into the extracellular space. Such vesicles include exosomes and microvesicles, which vary by their size and/or protein and genetic cargo. With the discovery that EVs contain genetic material in the form of RNA (evRNA has come the increased interest in these vesicles for their potential use as sources of disease biomarkers and potential therapeutic agents. Rapid developments in the availability of deep sequencing technologies have enabled the study of EV-related RNA in detail. In October 2012, the International Society for Extracellular Vesicles (ISEV held a workshop on “evRNA analysis and bioinformatics.” Here, we report the conclusions of one of the roundtable discussions where we discussed evRNA analysis technologies and provide some guidelines to researchers in the field to consider when performing such analysis.

  3. Dual and Direction-Selective Mechanisms of Phosphate Transport by the Vesicular Glutamate Transporter

    Directory of Open Access Journals (Sweden)

    Julia Preobraschenski

    2018-04-01

    Full Text Available Summary: Vesicular glutamate transporters (VGLUTs fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (Pi. It is still unclear how VGLUTs accommodate glutamate transport coupled to an electrochemical proton gradient ΔμH+ with inversely directed Pi transport coupled to the Na+ gradient and the membrane potential. Using both functional reconstitution and heterologous expression, we show that VGLUT transports glutamate and Pi using a single substrate binding site but different coupling to cation gradients. When facing the cytoplasm, both ions are transported into synaptic vesicles in a ΔμH+-dependent fashion, with glutamate preferred over Pi. When facing the extracellular space, Pi is transported in a Na+-coupled manner, with glutamate competing for binding but at lower affinity. We conclude that VGLUTs have dual functions in both vesicle transmitter loading and Pi homeostasis within glutamatergic neurons. : Preobraschenski et al. show that the vesicular glutamate transporter functions as a bi-directional phosphate transporter that is coupled with different cations in each direction and hence may play a key role in neuronal phosphate homeostasis. Keywords: VGLUT, SLC17 family, type I Na+-dependent inorganic phosphate transporter, ATPase, proteoliposomes, hybrid vesicles, anti-VGLUT1 nanobody

  4. 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.

  5. Mesenchymal stem cell-derived extracellular vesicles attenuate kidney inflammation.

    Science.gov (United States)

    Eirin, Alfonso; Zhu, Xiang-Yang; Puranik, Amrutesh S; Tang, Hui; McGurren, Kelly A; van Wijnen, Andre J; Lerman, Amir; Lerman, Lilach O

    2017-07-01

    Mesenchymal stem/stromal cells (MSCs) have distinct capability for renal repair, but may have safety concerns. MSC-derived extracellular vesicles emerged as a novel noncellular alternative. Using a porcine model of metabolic syndrome and renal artery stenosis we tested whether extracellular vesicles attenuate renal inflammation, and if this capacity is mediated by their cargo of the anti-inflammatory cytokine interleukin (IL) 10. Pigs with metabolic syndrome were studied after 16 weeks of renal artery stenosis untreated or treated four weeks earlier with a single intrarenal delivery of extracellular vesicles harvested from adipose tissue-derived autologous MSCs. Lean and sham metabolic syndrome animals served as controls (seven each). Five additional pigs with metabolic syndrome and renal artery stenosis received extracellular vesicles with pre-silenced IL10 (IL10 knock-down). Single-kidney renal blood flow, glomerular filtration rate, and oxygenation were studied in vivo and renal injury pathways ex vivo. Retention of extracellular vesicles in the stenotic kidney peaked two days after delivery and decreased thereafter. Four weeks after injection, extracellular vesicle fragments colocalized with stenotic-kidney tubular cells and macrophages, indicating internalization or fusion. Extracellular vesicle delivery attenuated renal inflammation, and improved medullary oxygenation and fibrosis. Renal blood flow and glomerular filtration rate fell in metabolic syndrome and renal artery stenosis compared to metabolic syndrome, but was restored in pigs treated with extracellular vesicles. These renoprotective effects were blunted in pigs treated with IL10-depleted extracellular vesicles. Thus, extracellular vesicle-based regenerative strategies might be useful for patients with metabolic syndrome and renal artery stenosis. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  6. Microfluidic filtration system to isolate extracellular vesicles from blood.

    Science.gov (United States)

    Davies, Ryan T; Kim, Junho; Jang, Su Chul; Choi, Eun-Jeong; Gho, Yong Song; Park, Jaesung

    2012-12-21

    Extracellular vesicles are released by various cell types, particularly tumor cells, and may be potential targets for blood-based cancer diagnosis. However, studies performed on blood-borne vesicles to date have been limited by lack of effective, standardized purification strategies. Using in situ prepared nanoporous membranes, we present a simple strategy employing a microfluidic filtration system to isolate vesicles from whole blood samples. This method can be applied to purify nano-sized particles from blood allowing isolation of intact extracellular vesicles, avoiding the need for laborious and potentially damaging centrifugation steps or overly specific antibody-based affinity purification. Porous polymer monoliths were integrated as membranes into poly(methyl methacrylate) microfluidic chips by benchtop UV photopolymerization through a mask, allowing precise positioning of membrane elements while preserving simplicity of device preparation. Pore size could be manipulated by changing the ratio of porogenic solvent to prepolymer solution, and was tuned to a size proper for extraction of vesicles. Using the membrane as a size exclusion filter, we separated vesicles from cells and large debris by injecting whole blood under pressure through the microfluidic device. To enhance isolation purity, DC electrophoresis was employed as an alternative driving force to propel particles across the filter and increase the separation efficiency of vesicles from proteins. From the whole blood of melanoma-grown mice, we isolated extracellular vesicles and performed RT-PCR to verify their contents of RNA. Melan A mRNA derived from melanoma tumor cells were found enriched in filtered samples, confirming the recovery of vesicles via their cargo. This filtration system can be incorporated into other on-chip processes enabling integrated sample preparation for the downstream analysis of blood-based extracellular vesicles.

  7. The launch of Journal of Extracellular Vesicles (JEV), the official journal of the International Society for Extracellular Vesicles - about microvesicles, exosomes, ectosomes and other extracellular vesicles

    OpenAIRE

    Lötvall, Jan; Rajendran, Lawrence; Gho, Yong Song; Théry, Clotilde; Wauben, Marca; Raposo, Graca; Sjöstrand, Margareta; Taylor, Douglas; Telemo, Esbjörn; Breakefield, Xandra O.

    2012-01-01

    In 2011, researchers around the world interested in extracellular vesicles (EV) joined forces and founded the International Society for Extracellular Vesicles (ISEV). Membership has grown to approximately 750 in eight months, and the Society’s first meeting will take place in Gothenburg, Sweden, on 18-21 April 2012. Already approximately 500 participants have been attracted to this event. These are signs of rapid expansion in global research in the field of EV.(Published: 16 April 2012)Citati...

  8. Remotely controlled fusion of selected vesicles and living cells: a key issue review

    Science.gov (United States)

    Bahadori, Azra; Moreno-Pescador, Guillermo; Oddershede, Lene B.; Bendix, Poul M.

    2018-03-01

    Remote control over fusion of single cells and vesicles has a great potential in biological and chemical research allowing both transfer of genetic material between cells and transfer of molecular content between vesicles. Membrane fusion is a critical process in biology that facilitates molecular transport and mixing of cellular cytoplasms with potential formation of hybrid cells. Cells precisely regulate internal membrane fusions with the aid of specialized fusion complexes that physically provide the energy necessary for mediating fusion. Physical factors like membrane curvature, tension and temperature, affect biological membrane fusion by lowering the associated energy barrier. This has inspired the development of physical approaches to harness the fusion process at a single cell level by using remotely controlled electromagnetic fields to trigger membrane fusion. Here, we critically review various approaches, based on lasers or electric pulses, to control fusion between individual cells or between individual lipid vesicles and discuss their potential and limitations for present and future applications within biochemistry, biology and soft matter.

  9. Microfluidic approaches for isolation, detection, and characterization of extracellular vesicles: Current status and future directions.

    Science.gov (United States)

    Gholizadeh, Shima; Shehata Draz, Mohamed; Zarghooni, Maryam; Sanati-Nezhad, Amir; Ghavami, Saeid; Shafiee, Hadi; Akbari, Mohsen

    2017-05-15

    Extracellular vesicles (EVs) are cell-derived vesicles present in body fluids that play an essential role in various cellular processes, such as intercellular communication, inflammation, cellular homeostasis, survival, transport, and regeneration. Their isolation and analysis from body fluids have a great clinical potential to provide information on a variety of disease states such as cancer, cardiovascular complications and inflammatory disorders. Despite increasing scientific and clinical interest in this field, there are still no standardized procedures available for the purification, detection, and characterization of EVs. Advances in microfluidics allow for chemical sampling with increasingly high spatial resolution and under precise manipulation down to single molecule level. In this review, our objective is to give a brief overview on the working principle and examples of the isolation and detection methods with the potential to be used for extracellular vesicles. This review will also highlight the integrated on-chip systems for isolation and characterization of EVs. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Uptake and binding of 125I-calmodulin by isolated rat renal brush border membrane vesicles

    International Nuclear Information System (INIS)

    Meezan, E.; Elgavish, A.; Wallace, R.W.

    1986-01-01

    The authors have investigated the interaction of 125 I-calmodulin with isolated rat renal brush border membrane vesicles (BBV) using an experimental protocol which allows us to distinguish between ligand binding to the outside of the vesicles vs. uptake and possible binding to the vesicle interior. By examining the association of 125 I-calmodulin with BBV as a function of medium osmolarity (300-1100 mosm) to alter intravesicular space, virtually all ligand interaction with BBV was found to represent uptake of intact 125 I-calmodulin into the intravesicular space. Uptake appeared specific by the following criteria: (1) it was largely calcium dependent (2) it was inhibited in a dose dependent fashion by calmodulin and the homologous protein troponin C, but not by unrelated proteins (lysozyme, cytochrome C, insulin) (3) it was inhibited by known calmodulin antagonists (calmidazolium, mellitin, trifluoperazine). Calmodulin uptake may be followed by binding of 125 I-calmodulin to intravesicular BBV proteins; calmodulin-binding proteins in BBV with molecular weights of 143K, 118K, 50K, 47.5K, 46.5K and 35K were identified by Western blotting techniques. The specific association of 125 I-calmodulin with isolated BBV is of interest in regard to the possible role of this calcium regulatory protein in the protein reabsorptive and ion transport functions of this renal tubular membrane fraction

  11. Construction of vesicle CdSe nano-semiconductors photocatalysts with improved photocatalytic activity: Enhanced photo induced carriers separation efficiency and mechanism insight.

    Science.gov (United States)

    Wen, Jiangsu; Ma, Changchang; Huo, Pengwei; Liu, Xinlin; Wei, Maobin; Liu, Yang; Yao, Xin; Ma, Zhongfei; Yan, Yongsheng

    2017-10-01

    Visible-light-driven photocatalysis as a green technology has attracted a lot of attention due to its potential applications in environmental remediation. Vesicle CdSe nano-semiconductor photocatalyst are successfully prepared by a gas template method and characterized by a variety of methods. The vesicle CdSe nano-semiconductors display enhanced photocatalytic performance for the degradation of tetracycline hydrochloride, the photodegradation rate of 78.824% was achieved by vesicle CdSe, which exhibited an increase of 31.779% compared to granular CdSe. Such an exceptional photocatalytic capability can be attributed to the unique structure of the vesicle CdSe nano-semiconductor with enhanced light absorption ability and excellent carrier transport capability. Meanwhile, the large surface area of the vesicle CdSe nano-semiconductor can increase the contact probability between catalyst and target and provide more surface-active centers. The photocatalytic mechanisms are analyzed by active species quenching. It indicates that h + and O 2 - are the main active species which play a major role in catalyzing environmental toxic pollutants. Simultaneously, the vesicle CdSe nano-semiconductor had high efficiency and stability. Copyright © 2017. Published by Elsevier B.V.

  12. Seminal vesicle intrafraction motion analysed with cinematic magnetic resonance imaging

    International Nuclear Information System (INIS)

    Gill, Suki; Dang, Kim; Fox, Chris; Bressel, Mathias; Kron, Tomas; Bergen, Noelene; Ferris, Nick; Owen, Rebecca; Chander, Sarat; Tai, Keen Hun; Foroudi, Farshad

    2014-01-01

    This study analyses seminal vesicle displacement relative to the prostate and in relation to treatment time. A group of eleven patients undergoing prostate cancer radiotherapy were imaged with a continuous 3 T cine-MRI in the standard treatment setup position. Four images were recorded every 4 seconds for 15 minutes in the sagittal plane and every 6.5 seconds for 12 minutes in the coronal plane. The prostate gland and seminal vesicles were contoured on each MRI image. The coordinates of the centroid of the prostate and seminal vesicles on each image was analysed for displacement against time. Displacements between the 2.5 percentile and 97.5 percentile (i.e. the 2.5% trimmed range) for prostate and seminal vesicle centroid displacements were measured for 3, 5, 10 and 15 minutes time intervals in the anterior-posterior (AP), left-right (LR) and superior-inferior (SI) directions. Real time prostate and seminal vesicle displacement was compared for individual patients. The 2.5% trimmed range for 3, 5, 10 and 15 minutes for the seminal vesicle centroids in the SI direction measured 4.7 mm; 5.8 mm; 6.5 mm and 7.2 mm respectively. In the AP direction, it was 4.0 mm, 4.5 mm, 6.5 mm, and 7.0 mm. In the LR direction for 3, 5 and 10 minutes; for the left seminal vesicle, it was 2.7 mm, 2.8 mm, 3.4 mm and for the right seminal vesicle, it was 3.4 mm, 3.3 mm, and 3.4 mm. The correlation between the real-time prostate and seminal vesicle displacement varied substantially between patients indicating that the relationship between prostate displacement and seminal vesicles displacement is patient specific with the majority of the patients not having a strong relationship. Our study shows that seminal vesicle motion increases with treatment time, and that the prostate and seminal vesicle centroids do not move in unison in real time, and that an additional margin is required for independent seminal vesicle motion if treatment localisation is to the prostate

  13. Role of extracellular vesicles in rheumatoid arthritis.

    Science.gov (United States)

    Fu, Haitao; Hu, Die; Zhang, Licheng; Tang, Peifu

    2018-01-01

    Cell-derived extracellular vesicles (EVs) are involved in the pathogenesis of rheumatoid arthritis (RA), playing important roles in antigen presentation, inflammation, angiogenesis, cell-cell signal communication, thrombosis, and articular cartilage extracellular matrix degradation. Understanding the pathogenic mechanism of RA is important for developing therapies. The pathogenic indicators of RA, such as submicron-sized EVs, represent promising biomarkers for evaluating RA activity. This review summarizes the recent advances in understanding the pathogenesis of RA, and sheds light on the pathogenic as well as anti-inflammatory or immunosuppressive roles of EVs. We suggest that EVs could be harnessed as tools for drug delivery or targets for RA therapies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Molecular Signatures Underlying Synaptic Vesicle Cargo Retrieval

    Science.gov (United States)

    Mori, Yasunori; Takamori, Shigeo

    2018-01-01

    Efficient retrieval of the synaptic vesicle (SV) membrane from the presynaptic plasma membrane, a process called endocytosis, is crucial for the fidelity of neurotransmission, particularly during sustained neural activity. Although multiple modes of endocytosis have been identified, it is clear that the efficient retrieval of the major SV cargos into newly formed SVs during any of these modes is fundamental for synaptic transmission. It is currently believed that SVs are eventually reformed via a clathrin-dependent pathway. Various adaptor proteins recognize SV cargos and link them to clathrin, ensuring the efficient retrieval of the cargos into newly formed SVs. Here, we summarize our current knowledge of the molecular signatures within individual SV cargos that underlie efficient retrieval into SV membranes, as well as discuss possible contributions of the mechanisms under physiological conditions. PMID:29379416

  15. Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation.

    Science.gov (United States)

    Couch, Yvonne; Akbar, Naveed; Davis, Simon; Fischer, Roman; Dickens, Alex M; Neuhaus, Ain A; Burgess, Annette I; Rothwell, Peter M; Buchan, Alastair M

    2017-08-01

    Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs. EVs were quantified and analyzed in the sera of patients after an acute stroke (size, is significantly increased in stroke patients when compared to age-matched controls. Proteomic analysis reveals an overall increase in acute phase proteins, including C-reactive protein. EV fractions applied to monocyte-differentiated macrophage cultures induced inflammatory gene expression. Together these data show that EVs from stroke patients are proinflammatory in nature and are capable of inducing inflammation in immune cells. © 2017 American Heart Association, Inc.

  16. Bulk immunoassays for analysis of extracellular vesicles.

    Science.gov (United States)

    Coumans, Frank A W; Gool, Elmar L; Nieuwland, Rienk

    2017-05-01

    There is increasing clinical interest in extracellular vesicles (EV) for diagnostic and treatment purposes. This review provides an overview of bulk immunoassays to analyse EV. Western blot and enzyme-linked immunosorbent assay are still the two predominant bulk immunoassays. Recently, new assays have become available that can detect exposure to EV concentrations that are up to 10,000-fold lower. This is advantageous for applications that detect rare EV. Other important parameters are the detectable concentration range, the required sample volume, whether simultaneous presence of different antigens on a single EV can be detected, size selectivity of each assay and practical considerations. In this review, we will explain the working principles of the traditional and novel assays together with their performance parameters. The most sensitive assays are micro-nuclear magnetic resonance, surface plasmon resonance, and time-resolved fluorescent immunoassay.

  17. Methods to isolate extracellular vesicles for diagnosis

    Science.gov (United States)

    Kang, Hyejin; Kim, Jiyoon; Park, Jaesung

    2017-12-01

    Extracellular vesicles (EVs) are small membrane-bound bodies that are released into extracellular space by diverse cells, and are found in body fluids like blood, urine and saliva. EVs contain RNA, DNA and proteins, which can be biomarkers for diagnosis. EVs can be obtained by minimally-invasive biopsy, so they are useful in disease diagnosis. High yield and purity contribute to precise diagnosis of disease, but damaged EVs and impurities can cause confu sed results. However, EV isolation methods have different yields and purities. Furthermore, the isolation method that is most suitable to maximize EV recovery efficiency depends on the experimental conditions. This review focuses on merits and demerits of several types of EV isolation methods, and provides examples of how to diagnose disease by exploiting information obtained by analysis of EVs.

  18. Extracellular vesicles in obesity and diabetes mellitus.

    Science.gov (United States)

    Pardo, Fabián; Villalobos-Labra, Roberto; Sobrevia, Bastián; Toledo, Fernando; Sobrevia, Luis

    2018-04-01

    Cell-to-cell communication happens via diverse mechanisms including the synthesis, release and transfer to target cells of extracellular vesicles (EVs). EVs include nanovesicles (i.e., exosomes) and microvesicles, including apoptotic bodies. The amount and cargo of released EVs, which consist of microRNAs (miRNAs), mRNA, proteins, DNA, among other molecules, are altered in obesity and diabetes mellitus. EVs from these diseases show with altered cargo including several miRNAs and the enrichment with molecules involved in inflammation, immune efficiency, and cell activation. The role of EVs in obesity regards with adipocytes-released vesicles that may end in a systemic insulin resistance. In diabetes mellitus, the exosomes cargo may signal to transform a normal phenotype into a diabetic phenotype in endothelial cells. The evidence of EVs as modulators of cell function is increasing; however, it is still unclear whether exosomes or microvesicles are a trustable and useful marker for the diagnose or early detection of obesity or diabetes mellitus. In this review, we summarise the reported information regarding EVs involvement in obesity, T1 and T2 diabetes mellitus, and gestational diabetes mellitus. We emphasise the fact that studies addressing a potential effect of obesity or diabetes mellitus on cell function and the severity of the diseases are done in patients suffering simultaneously with both of these diseases, i.e., diabesity. Unfortunately, the lack of information regarding the biological effects and the potential involved mechanisms makes difficult to understand the role of the EVs as a marker of these and perhaps other diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. In vitro toxicology studies of extracellular vesicles.

    Science.gov (United States)

    Maji, Sayantan; Yan, Irene K; Parasramka, Mansi; Mohankumar, Swathi; Matsuda, Akiko; Patel, Tushar

    2017-03-01

    Extracellular vesicles (EVs) are membrane-bound vesicles released from cells into the extracellular environment. There is emerging interest in the use of EVs as potential therapeutic interventions. We sought to evaluate the safety of EVs that may be therapeutically used by performing in vitro toxicological assessments. EVs were obtained from mesenchymal stem cells (MSC-EV) or from bovine milk (BM-EV) by differential ultracentrifugation, and quantitated using nanoparticle tracking analysis. Genotoxic effects, hematological effects, immunological effects and endotoxin production were evaluated at two dose levels. Neither MSC-EVs nor BM-EVs elicited detectable genotoxic effects using either the alkaline comet assay or micronucleus assay. Hemolysis was observed with BM-EVs but not with MSC-EVs. MSC-EVs did not have any significant effect on either spontaneous or collagen-induced platelet aggregation. In contrast, BM-EVs were noted to increase collagen-induced platelet aggregation, even though no spontaneous increase in platelet aggregation was noted. Both types of EVs induced leukocyte proliferation, which was greater with BM-EV. Neither MSC-EVs nor BM-EVs induced HL-60 phagocytosis, although BM-EVs decreased zymosan-induced phagocytosis. Furthermore, neither MSC-EVs nor BM-EVs induced nitric oxide production. Unlike MSC-EVs, BM-EVs tested positive for endotoxin and induced complement activation. There are significant differences in toxicological profiles between MSC-EVs and BM-EVs that may reflect variations in techniques for EV isolation, EV content or cross-species differences. The safety of MSC-EV supports their use for disease therapeutics, whereas detailed safety and toxicological assessment will be necessary before the use of BM-EVs. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  20. [EXTRACELLULAR VESICLES: INTERCELLULAR INFORMATION FLOW AND MEDICAL APPLICATIONS].

    Science.gov (United States)

    Pupyshev, A B

    2015-01-01

    The major features of extracellular vesicles secreted by mammalian cells are considered. Cell activation caused by formation of pathology stimulates the secretion acutely. The vesicles (exosomes, microvesicles) are enriched with annexin V, tetraspanin, miRNA. Exosomes are enriched especially by integrins, heat shock proteins. Microvesicles contain elevated amounts of tissue factors, phosphatidylserine, mRNA. The vesicles carry information about the pathological process, and microvesicles contain more proteins characteristic of inflammation and death than exosomes. They are important mediators of inflammation and infection in the body, have different effects on the immune system and the processes of carcinogenesis and neurodegeneration. However, antigenic profiles of extracellular vesicles differ not profoundly in various pathologies and so far they help diagnostics limitedly. The vesicles carry signals of genetic reprogramming of the cells and epigenetic stimulation, connected with both protein factors and mRNA and miRNA. Profiles of miRNA vesicles produced by the various pathological sources are studied actively and are useful as indicators of source and stage of cancer. Some ways of therapeutic use of the vesicles are also considered.

  1. 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.

  2. Formation and size distribution of self-assembled vesicles.

    Science.gov (United States)

    Huang, Changjin; Quinn, David; Sadovsky, Yoel; Suresh, Subra; Hsia, K Jimmy

    2017-03-14

    When detergents and phospholipid membranes are dispersed in aqueous solutions, they tend to self-assemble into vesicles of various shapes and sizes by virtue of their hydrophobic and hydrophilic segments. A clearer understanding of such vesiculation processes holds promise for better elucidation of human physiology and disease, and paves the way to improved diagnostics, drug development, and drug delivery. Here we present a detailed analysis of the energetics and thermodynamics of vesiculation by recourse to nonlinear elasticity, taking into account large deformation that may arise during the vesiculation process. The effects of membrane size, spontaneous curvature, and membrane stiffness on vesiculation and vesicle size distribution were investigated, and the critical size for vesicle formation was determined and found to compare favorably with available experimental evidence. Our analysis also showed that the critical membrane size for spontaneous vesiculation was correlated with membrane thickness, and further illustrated how the combined effects of membrane thickness and physical properties influenced the size, shape, and distribution of vesicles. These findings shed light on the formation of physiological extracellular vesicles, such as exosomes. The findings also suggest pathways for manipulating the size, shape, distribution, and physical properties of synthetic vesicles, with potential applications in vesicle physiology, the pathobiology of cancer and other diseases, diagnostics using in vivo liquid biopsy, and drug delivery methods.

  3. Extracellular Vesicles in Luminal Fluid of the Ovine Uterus

    Science.gov (United States)

    Burns, Gregory; Brooks, Kelsey; Wildung, Mark; Navakanitworakul, Raphatphorn; Christenson, Lane K.; Spencer, Thomas E.

    2014-01-01

    Microvesicles and exosomes are nanoparticles released from cells and can contain small RNAs, mRNA and proteins that affect cells at distant sites. In sheep, endogenous beta retroviruses (enJSRVs) are expressed in the endometrial epithelia of the uterus and can be transferred to the conceptus trophectoderm. One potential mechanism of enJSRVs transfer from the uterus to the conceptus is via exosomes/microvesicles. Therefore, studies were conducted to evaluate exosomes in the uterine luminal fluid (ULF) of sheep. Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Transmission electron microscopy and nanoparticle tracking analysis found the isolates contained vesicles that ranged from 50 to 200 nm in diameter. The isolated extracellular vesicles were positive for two common markers of exosomes (CD63 and HSP70) by Western blot analysis. Proteins in the extracellular vesicles were determined by mass spectrometry and Western blot analysis. Extracellular vesicle RNA was analyzed for small RNAs by sequencing and enJSRVs RNA by RT-PCR. The ULF extracellular vesicles contained a large number of small RNAs and miRNAs including 81 conserved mature miRNAs. Cyclic and pregnant ULF extracellular vesicles contained enJSRVs env and gag RNAs that could be delivered to heterologous cells in vitro. These studies support the hypothesis that ULF extracellular vesicles can deliver enJSRVs RNA to the conceptus, which is important as enJSRVs regulate conceptus trophectoderm development. Importantly, these studies support the idea that extracellular vesicles containing select miRNAs, RNAs and proteins are present in the ULF and likely have a biological role in conceptus-endometrial interactions important for the establishment and maintenance of pregnancy. PMID:24614226

  4. Genetically Controlled Fusion, Exocytosis and Fission of Artificial Vesicles

    DEFF Research Database (Denmark)

    Bönzli, Eva; Hadorn, Maik; De Lucrezia, Davide

    to induce in a next step fusion of adjacent vesicles, fission and exocytosis of nested vesicles. Second, we will replace the peptides by an enclosed cell-free expression system to internally synthesize fusion peptides. To control the gene expression, different mechanisms are available, e.g. addition...... of transcription factors. Changes in the pH are reported to control the activity of the fusion peptides. So far, we successfully enclosed a commercially available cell-free system and expressed eGFP in vesicles as a proof of principle. Furthermore, we optimized the already established protocol to produce nested...

  5. Extracellular vesicles are the Trojan horses of viral infection.

    Science.gov (United States)

    Altan-Bonnet, Nihal

    2016-08-01

    Extracellular vesicles have recently emerged as a novel mode of viral propagation exploited by both enveloped and non-enveloped viruses. In particular non-enveloped viruses utilize the hosts' production of extracellular vesicles to exit from cells non-lytically and to hide and manipulate the immune system. Moreover, challenging the long held idea that viruses behave as independent genetic units, extracellular vesicles enable multiple viral particles and genomes to collectively traffic in and out of cells, which can promote genetic cooperativity among viral quasispecies and enhance the fitness of the overall viral population. Published by Elsevier Ltd.

  6. Colocalization of synapsin and actin during synaptic vesicle recycling

    DEFF Research Database (Denmark)

    Bloom, Ona; Evergren, Emma; Tomilin, Nikolay

    2003-01-01

    activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin......-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known...

  7. Ex vivo skin delivery of diclofenac by transcutol containing liposomes and suggested mechanism of vesicle-skin interaction.

    Science.gov (United States)

    Manconi, Maria; Caddeo, Carla; Sinico, Chiara; Valenti, Donatella; Mostallino, Maria Cristina; Biggio, Giovanni; Fadda, Anna Maria

    2011-05-01

    Recently, we described a novel family of liposomes, the Penetration Enhancer-containing Vesicles (PEVs), as carriers for enhanced (trans)dermal drug delivery. In this study, to go deeply into the potential of these new vesicles and suggest the possible mechanism of vesicle-skin interaction, we investigated transcutol containing PEVs as carriers for diclofenac, in the form of either acid or sodium salt. PEVs, prepared with soy phosphatidylcholine and aqueous solutions containing different concentrations of transcutol, were characterized by size distribution, zeta potential, incorporation efficiency, thermotropic behavior, and stability. (Trans)dermal diclofenac delivery from PEVs was investigated ex vivo through new born pig skin using conventional liposomes and a commercial gel as controls. The mode of action of the vesicles was also studied by performing a pre-treatment test and confocal laser scanning microscopy (CLSM) analyses. Results of the all skin permeation experiments showed an improved diclofenac (both acid and sodium salt) delivery to and through the skin when PEVs were used (especially in comparison with the commercial gel) thus suggesting intact PEVs' penetration through the pig skin. Images of the qualitative CLSM analyses support this conclusion. Thus, this work shows the superior ability of the PEVs to enhance ex vivo drug transport of both hydrophilic and lipophilic diclofenac forms. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Modeling the self-assembly of lipids and nanotubes in solution: forming vesicles and bicelles with transmembrane nanotube channels.

    Science.gov (United States)

    Dutt, Meenakshi; Kuksenok, Olga; Nayhouse, Michael J; Little, Steven R; Balazs, Anna C

    2011-06-28

    Via dissipative particle dynamics (DPD), we simulate the self-assembly of end-functionalized, amphiphilic nanotubes and lipids in a hydrophilic solvent. Each nanotube encompasses a hydrophobic stalk and two hydrophilic ends, which are functionalized with end-tethered chains. With a relatively low number of the nanotubes in solution, the components self-assemble into stable lipid-nanotube vesicles. As the number of nanotubes is increased, the system exhibits a vesicle-to-bicelle transition, resulting in stable hybrid bicelle. Moreover, our results reveal that the nanotubes cluster into distinct tripod-like structures within the vesicles and aggregate into a ring-like assembly within the bicelles. For both the vesicles and bicelles, the nanotubes assume trans-membrane orientations, with the tethered hairs extending into the surrounding solution or the encapsulated fluid. Thus, the hairs provide a means of regulating the transport of species through the self-assembled structures. Our findings provide guidelines for creating nanotube clusters with distinctive morphologies that might be difficult to achieve through more conventional means. The results also yield design rules for creating synthetic cell-like objects or microreactors that can exhibit biomimetic functionality.

  9. Propeptide of aminopeptidase 1 protein mediates aggregation and vesicle formation in cytoplasm-to-vacuole targeting pathway.

    Science.gov (United States)

    Morales Quinones, Mariana; Winston, Jared T; Stromhaug, Per E

    2012-03-23

    Misfolded protein aggregation causes disease and aging; autophagy counteracts this by eliminating damaged components, enabling cells to survive starvation. The cytoplasm-to-vacuole targeting pathway in yeast encompasses the aggregation of the premature form of aminopeptidase 1 (prApe1) in cytosol and its sequestration by autophagic proteins into a vesicle for vacuolar transport. We show that the propeptide of Ape1 is important for aggregation and vesicle formation and that it is sufficient for binding to prApe1 and Atg19. Defective aggregation disrupts vacuolar transport, suggesting that aggregate shape is important in vesicle formation, whereas Atg19 binding is not sufficient for vacuolar transport. Aggregation involves hydrophobicity, whereas Atg19 binding requires additional electrostatic interactions. Ape1 dodecamerization may cluster propeptides into trimeric structures, with sufficient affinity to form propeptide hexamers by binding to other dodecamers, causing aggregation. We show that Ape1 aggregates bind Atg19 and Atg8 in vitro; this could be used as a scaffold for an in vitro assay of autophagosome formation to elucidate the mechanisms of autophagy.

  10. Glioblastoma extracellular vesicles: reservoirs of potential biomarkers

    Directory of Open Access Journals (Sweden)

    Redzic JS

    2014-02-01

    Full Text Available Jasmina S Redzic,1 Timothy H Ung,2 Michael W Graner2 1Skaggs School of Pharmacy and Pharmaceutical Sciences, 2Department of Neurosurgery, School of Medicine, University of Colorado Denver, Aurora, CO, USA Abstract: Glioblastoma multiforme (GBM is the most frequent and most devastating of the primary central nervous system tumors, with few patients living beyond 2 years postdiagnosis. The damage caused by the disease and our treatments for the patients often leave them physically and cognitively debilitated. Generally, GBMs appear after very short clinical histories and are discovered by imaging (using magnetic resonance imaging [MRI], and the diagnosis is validated by pathology, following surgical resection. The treatment response and diagnosis of tumor recurrence are also tracked by MRI, but there are numerous problems encountered with these monitoring modalities, such as ambiguous interpretation and forms of pseudoprogression. Diagnostic, prognostic, and predictive biomarkers would be an immense boon in following treatment schemes and in determining recurrence, which often requires an invasive intracranial biopsy to verify imaging data. Extracellular vesicles (EVs are stable, membrane-enclosed, virus-sized particles released from either the cell surface or from endosomal pathways that lead to the systemic release of EVs into accessible biofluids, such as serum/plasma, urine, cerebrospinal fluid, and saliva. EVs carry a wide variety of proteins, nucleic acids, lipids, and other metabolites, with many common features but with enough individuality to be able to identify the cell of origin of the vesicles. These components, if properly interrogated, could allow for the identification of tumor-derived EVs in biofluids, indicating tumor progression, relapse, or treatment failure. That knowledge would allow clinicians to continue with treatment regimens that were actually effective or to change course if the therapies were failing. Here, we review

  11. Nanoparticle-triggered release from lipid membrane vesicles.

    Science.gov (United States)

    Reimhult, Erik

    2015-12-25

    Superparamagnetic iron oxide nanoparticles are used in a rapidly expanding number of research and practical applications in biotechnology and biomedicine. We highlight how recent developments in iron oxide nanoparticle design and understanding of nanoparticle membrane interactions have led to applications in magnetically triggered, liposome delivery vehicles with controlled structure. Nanoscale vesicles actuated by incorporated nanoparticles allow for controlling location and timing of compound release, which enables e.g. use of more potent drugs in drug delivery as the interaction with the right target is ensured. This review emphasizes recent results on the connection between nanoparticle design, vesicle assembly and the stability and release properties of the vesicles. While focused on lipid vesicles magnetically actuated through iron oxide nanoparticles, these insights are of general interest for the design of capsule and cell delivery systems for biotechnology controlled by nanoparticles. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. 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

  13. Biological properties of extracellular vesicles and their physiological functions

    NARCIS (Netherlands)

    Yáñez-Mó, María; Siljander, Pia R-M; Andreu, Zoraida; Zavec, Apolonija Bedina; Borràs, Francesc E; Buzas, Edit I; Buzas, Krisztina; Casal, Enriqueta; Cappello, Francesco; Carvalho, Joana; Colás, Eva; Cordeiro-da Silva, Anabela; Fais, Stefano; Falcon-Perez, Juan M; Ghobrial, Irene M; Giebel, Bernd; Gimona, Mario; Graner, Michael; Gursel, Ihsan; Gursel, Mayda; Heegaard, Niels H H; Hendrix, An; Kierulf, Peter; Kokubun, Katsutoshi; Kosanovic, Maja; Kralj-Iglic, Veronika; Krämer-Albers, Eva-Maria; Laitinen, Saara; Lässer, Cecilia; Lener, Thomas; Ligeti, Erzsébet; Linē, Aija; Lipps, Georg; Llorente, Alicia; Lötvall, Jan; Manček-Keber, Mateja; Marcilla, Antonio; Mittelbrunn, Maria; Nazarenko, Irina; Nolte-'t Hoen, Esther N M; Nyman, Tuula A; O'Driscoll, Lorraine; Olivan, Mireia; Oliveira, Carla; Pállinger, Éva; Del Portillo, Hernando A; Reventós, Jaume; Rigau, Marina; Rohde, Eva; Sammar, Marei; Sánchez-Madrid, Francisco; Santarém, N; Schallmoser, Katharina; Ostenfeld, Marie Stampe; Stoorvogel, Willem|info:eu-repo/dai/nl/074352385; Stukelj, Roman; Van der Grein, Susanne G|info:eu-repo/dai/nl/412755211; Vasconcelos, M Helena; Wauben, Marca H M|info:eu-repo/dai/nl/112675735; De Wever, Olivier

    2015-01-01

    In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological

  14. EVpedia : A community web portal for extracellular vesicles research

    NARCIS (Netherlands)

    Kim, Dae Kyum; Lee, Jaewook; Kim, Sae Rom; Choi, Dong Sic; Yoon, Yae Jin; Kim, Ji Hyun; Go, Gyeongyun; Nhung, Dinh; Hong, Kahye; Jang, Su Chul; Kim, Si Hyun; Park, Kyong Su; Kim, Oh Youn; Park, Hyun Taek; Seo, Ji Hye; Aikawa, Elena; Baj-Krzyworzeka, Monika; Van Balkom, Bas W M; Belting, Mattias; Blanc, Lionel; Bond, Vincent; Bongiovanni, Antonella; Borràs, Francesc E.; Buée, Luc; Buzás, Edit I.; Cheng, Lesley; Clayton, Aled; Cocucci, Emanuele; Dela Cruz, Charles S.; Desiderio, Dominic M.; Di Vizio, Dolores; Ekström, Karin; Falcon-Perez, Juan M.; Gardiner, Chris; Giebel, Bernd; Greening, David W.; Christina Gross, Julia; Gupta, Dwijendra; Hendrix, An; Hill, Andrew F.; Hill, Michelle M.; Nolte-'T Hoen, Esther; Hwang, Do Won; Inal, Jameel; Jagannadham, Medicharla V.; Jayachandran, Muthuvel; Jee, Young Koo; Jørgensen, Malene; Kim, Kwang Pyo; Kim, Yoon Keun; Kislinger, Thomas; Lässer, Cecilia; Lee, Dong Soo; Lee, Hakmo; Van Leeuwen, Johannes; Lener, Thomas; Liu, Ming Lin; Lötvall, Jan; Marcilla, Antonio; Mathivanan, Suresh; Möller, Andreas; Morhayim, Jess; Mullier, Francois; Nazarenko, Irina; Nieuwland, Rienk; Nunes, Diana N.; Pang, Ken; Park, Jaesung; Patel, Tushar; Pocsfalvi, Gabriella; Del Portillo, Hernando; Putz, Ulrich; Ramirez, Marcel I.; Rodrigues, Marcio L.; Roh, Tae Young; Royo, Felix; Sahoo, Susmita; Schiffelers, Raymond; Sharma, Shivani; Siljander, Pia; Simpson, Richard J.; Soekmadji, Carolina; Stahl, Philip; Stensballe, Allan; Stepień, Ewa; Tahara, Hidetoshi; Trummer, Arne; Valadi, Hadi; Vella, Laura J.; Wai, Sun Nyunt; Witwer, Kenneth; Yánez-Mó, Maria; Youn, Hyewon; Zeidler, Reinhard; Gho, Yong Song

    2015-01-01

    Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. Results: We

  15. Radiological seminal vesicle stones may actually be in the ureter

    Directory of Open Access Journals (Sweden)

    Brusabhanu Nayak

    2013-01-01

    Full Text Available Calculi in blind-ending ureters are uncommon. We describe a rare case of calculi in the diverticulum of a blind-ending ureter associated with ipsilateral renal agenesis, which masqueraded as seminal vesicle calculi.

  16. Assembly of cells and vesicles for organ engineering

    Directory of Open Access Journals (Sweden)

    Tetsushi Taguchi

    2011-01-01

    Full Text Available The development of materials and technologies for the assembly of cells and/or vesicles is a key for the next generation of tissue engineering. Since the introduction of the tissue engineering concept in 1993, various types of scaffolds have been developed for the regeneration of connective tissues in vitro and in vivo. Cartilage, bone and skin have been successfully regenerated in vitro, and these regenerated tissues have been applied clinically. However, organs such as the liver and pancreas constitute numerous cell types, contain small amounts of extracellular matrix, and are highly vascularized. Therefore, organ engineering will require the assembly of cells and/or vesicles. In particular, adhesion between cells/vesicles will be required for regeneration of organs in vitro. This review introduces and discusses the key technologies and materials for the assembly of cells/vesicles for organ regeneration.

  17. 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.

  18. A scenario for a genetically controlled fission of artificial vesicles

    DEFF Research Database (Denmark)

    Bönzli, Eva; Hadorn, Maik; Jørgensen, Mikkel Girke

    2011-01-01

    construction of a cell in a bottom-up manner. Numerous efforts to build an artificial cell that bridge the living and non-living world will most presumably represent one of the main goals of science in the 21st century. It was shown that artificial genetic programs and the required cellular machinery can...... to vesicles (Hanczyc et al. 2003). In the present work, we developed a scenario how a genetically controlled fission of vesicles may be achieved by the synthesis of a special class of viral proteins within artificial vesicles. Because the authors already have a lot of experience in the water-in-oil emulsion...... transfer method to prepare vesicles, the base for the development of a protocol to induce fission in artificial cell may be available....

  19. Plasma membrane aquaporins mediates vesicle stability in broccoli.

    Science.gov (United States)

    Martínez-Ballesta, Maria Del Carmen; García-Gomez, Pablo; Yepes-Molina, Lucía; Guarnizo, Angel L; Teruel, José A; Carvajal, Micaela

    2018-01-01

    The use of in vitro membrane vesicles is attractive because of possible applications in therapies. Here we aimed to compare the stability and functionality of plasma membrane vesicles extracted from control and salt-treated broccoli. The impact of the amount of aquaporins was related to plasma membrane osmotic water permeability and the stability of protein secondary structure. Here, we describe for first time an increase in plant aquaporins acetylation under high salinity. Higher osmotic water permeability in NaCl vesicles has been related to higher acetylation, upregulation of aquaporins, and a more stable environment to thermal denaturation. Based on our findings, we propose that aquaporins play an important role in vesicle stability.

  20. Small-angle scattering studies on clathrin-coated vesicles

    International Nuclear Information System (INIS)

    Bauer, R.; Hansen, S.; Oegendal, L.; Behan, M.; Jones, G.; Mortensen, K.; Saermark, T.

    1991-01-01

    Structural information of clathrin-coated vesicles has been achieved by small-angle X-ray, neutron and dynamic light scattering studies. A characteristic peak in the X-ray and neutron scattering profile (in D 2 O) from intact coated vesicles is consistent with the polygonic structure of the clathrin coat. Neutron as well as dynamic light scattering gives a coated vesicle size close to 1000A. Dynamic light scattering detects a distribution of sizes for the coated vesicles demonstrating polydispersity of the samples. Quick freezing and slow thawing cause breakdown of the polygonic coat and production of large aggregates, as observed by dynamic light scattering and the reduction of the peak in the X-ray scattering profile as well as an increase in the scattering intensity at the lowest angles in the neutron scattering profile. (orig.)

  1. Shape Control of Vesicle by Reverse Process Method of Relaxation

    Science.gov (United States)

    Kohyama, Tamotsu

    2018-03-01

    We consider a reverse process of relaxation and obtain a necessary condition under which the reverse process proceeds. Applying this method to fluid vesicle dynamics, which is derived by Onsager's variational principle, we derive the necessary equations for spontaneous curvature and anisotropic bending rigidity distributions that are induced by the proteins attached to the membrane of the vesicle. Numerical schemes to obtain the distributions of the protein properties are proposed and applied to spheroid vesicles. It is shown that proteins with an anisotropic spontaneous curvature deviator are effective in forming a long prolate spheroid. We conclude that the proposed method may become a useful tool to study the protein distribution in the membrane of a vesicle with a measured shape.

  2. Cysteine String Protein α: A New Role in Vesicle Recycling

    OpenAIRE

    Sheng, Jiansong; Wu, Ling-Gang

    2012-01-01

    Zhang et al. (2012) and Rozas et al. (2012) find that cysteine string protein α, a protein involved in neurodegeneration, regulates vesicle endocytosis via interaction with dynamin 1, which may participate in regulating synaptic transmission and possibly in maintaining synapses.

  3. Improved Methods of Producing and Administering Extracellular Vesicles | Poster

    Science.gov (United States)

    An efficient method of producing purified extracellular vesicles (EVs), in conjunction with a method that blocks liver macrophages from clearing EVs from the body, has produced promising results for the use of EVs in cancer therapy.

  4. Extracellular vesicles in human follicular fluid do not promote coagulation.

    Science.gov (United States)

    Franz, Cordula; Böing, Anita N; Montag, Markus; Strowitzki, Thomas; Markert, Udo R; Mastenbroek, Sebastiaan; Nieuwland, Rienk; Toth, Bettina

    2016-11-01

    Body fluids contain extracellular vesicles expressing tissue factor on their surface and serve as an additional trigger for coagulation. During the menstrual cycle ovarian tissue restoration is mandatory and it is unknown whether follicular fluid might provide procoagulant substances. Within an observational study, follicular fluid from women undergoing IVF/intracytoplasmic sperm injection (ICSI) was analysed by fluorescence-activated cell sorting (FACS), electron microscopy, resistive pulse sensing (RPS), nanoparticle-tracking analysis (NTA) and fibrin generation tests (FGT). The presence of extracellular vesicles, especially CD9-positive extracellular vesicles in follicular fluid, was proven. However, clotting tests revealed no procoagulant properties of the detected extracellular vesicles. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  5. Sortilin mediates vascular calcification via its recruitment into extracellular vesicles

    DEFF Research Database (Denmark)

    Goettsch, Claudia; Hutscheson, JD; Aikawa, M

    2016-01-01

    Vascular calcification is a common feature of major cardiovascular diseases. Extracellular vesicles participate in the formation of microcalcifications that are implicated in atherosclerotic plaque rupture; however, the mechanisms that regulate formation of calcifying extracellular vesicles remain...... obscure. Here, we have demonstrated that sortilin is a key regulator of smooth muscle cell (SMC) calcification via its recruitment to extracellular vesicles. Sortilin localized to calcifying vessels in human and mouse atheromata and participated in formation of microcalcifications in SMC culture. Sortilin...... regulated the loading of the calcification protein tissue nonspecific alkaline phosphatase (TNAP) into extracellular vesicles, thereby conferring its calcification potential. Furthermore, SMC calcification required Rab11-dependent trafficking and FAM20C/casein kinase 2-dependent C-terminal phosphorylation...

  6. Assembly of cells and vesicles for organ engineering

    Science.gov (United States)

    Taguchi, Tetsushi

    2011-12-01

    The development of materials and technologies for the assembly of cells and/or vesicles is a key for the next generation of tissue engineering. Since the introduction of the tissue engineering concept in 1993, various types of scaffolds have been developed for the regeneration of connective tissues in vitro and in vivo. Cartilage, bone and skin have been successfully regenerated in vitro, and these regenerated tissues have been applied clinically. However, organs such as the liver and pancreas constitute numerous cell types, contain small amounts of extracellular matrix, and are highly vascularized. Therefore, organ engineering will require the assembly of cells and/or vesicles. In particular, adhesion between cells/vesicles will be required for regeneration of organs in vitro. This review introduces and discusses the key technologies and materials for the assembly of cells/vesicles for organ regeneration.

  7. Assembly of cells and vesicles for organ engineering

    Energy Technology Data Exchange (ETDEWEB)

    Taguchi, Tetsushi, E-mail: taguchi.tetsushi@nims.go.jp [Biofunctional Materials Unit, Nano-Bio Field, Materials Nanoarchitectonics (MANA), National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan)

    2011-12-15

    The development of materials and technologies for the assembly of cells and/or vesicles is a key for the next generation of tissue engineering. Since the introduction of the tissue engineering concept in 1993, various types of scaffolds have been developed for the regeneration of connective tissues in vitro and in vivo. Cartilage, bone and skin have been successfully regenerated in vitro, and these regenerated tissues have been applied clinically. However, organs such as the liver and pancreas constitute numerous cell types, contain small amounts of extracellular matrix, and are highly vascularized. Therefore, organ engineering will require the assembly of cells and/or vesicles. In particular, adhesion between cells/vesicles will be required for regeneration of organs in vitro. This review introduces and discusses the key technologies and materials for the assembly of cells/vesicles for organ regeneration. (topical review)

  8. Lipid-Targeting Peptide Probes for Extracellular Vesicles.

    Science.gov (United States)

    Flynn, Aaron D; Yin, Hang

    2016-11-01

    Extracellular vesicles released from cells are under intense investigation for their roles in cell-cell communication and cancer progression. However, individual vesicles have been difficult to probe as their small size renders them invisible by conventional light microscopy. However, as a consequence of their small size these vesicles possess highly curved lipid membranes that offer an unconventional target for curvature-sensing probes. In this article, we present a strategy for using peptide-based biosensors to detect highly curved membranes and the negatively charged membrane lipid phosphatidylserine, we delineate several assays used to validate curvature- and lipid-targeting mechanisms, and we explore potential applications in probing extracellular vesicles released from sources such as apoptotic cells, cancer cells, or activated platelets. J. Cell. Physiol. 231: 2327-2332, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates.

    Science.gov (United States)

    Sotillo, Javier; Pearson, Mark; Potriquet, Jeremy; Becker, Luke; Pickering, Darren; Mulvenna, Jason; Loukas, Alex

    2016-01-01

    Herein we show for the first time that Schistosoma mansoni adult worms secrete exosome-like extracellular vesicles ranging from 50 to 130nm in size. Extracellular vesicles were collected from the excretory/secretory products of cultured adult flukes and purified by Optiprep density gradient, resulting in highly pure extracellular vesicle preparations as confirmed by transmission electron microscopy and Nanosight tracking analysis. Extracellular vesicle proteomic analysis showed numerous known vaccine candidates, potential virulence factors and molecules implicated in feeding. These findings provide new avenues for the exploration of host-schistosome interactions and offer a potential mechanism by which some vaccine antigens exert their protective efficacy. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. EVpedia : a community web portal for extracellular vesicles research

    NARCIS (Netherlands)

    Kim, Dae-Kyum; Lee, Jaewook; Kim, Sae Rom; Choi, Dong-Sic; Yoon, Yae Jin; Kim, Ji Hyun; Go, Gyeongyun; Nhung, Dinh; Hong, Kahye; Jang, Su Chul; Kim, Si-Hyun; Park, Kyong-Su; Kim, Oh Youn; Park, Hyun Taek; Seo, Ji Hye; Aikawa, Elena; Baj-Krzyworzeka, Monika; van Balkom, Bas W M; Belting, Mattias; Blanc, Lionel; Bond, Vincent; Bongiovanni, Antonella; Borràs, Francesc E; Buée, Luc; Buzás, Edit I; Cheng, Lesley; Clayton, Aled; Cocucci, Emanuele; Dela Cruz, Charles S; Desiderio, Dominic M; Di Vizio, Dolores; Ekström, Karin; Falcon-Perez, Juan M; Gardiner, Chris; Giebel, Bernd; Greening, David W; Gross, Julia Christina; Gupta, Dwijendra; Hendrix, An; Hill, Andrew F; Hill, Michelle M; Nolte-'t Hoen, Esther; Hwang, Do Won; Inal, Jameel; Jagannadham, Medicharla V; Jayachandran, Muthuvel; Jee, Young-Koo; Jørgensen, Malene; Kim, Kwang Pyo; Kim, Yoon-Keun; Kislinger, Thomas; Lässer, Cecilia; Lee, Dong Soo; Lee, Hakmo; van Leeuwen, Johannes; Lener, Thomas; Liu, Ming-Lin; Lötvall, Jan; Marcilla, Antonio; Mathivanan, Suresh; Möller, Andreas; Morhayim, Jess; Mullier, François; Nazarenko, Irina; Nieuwland, Rienk; Nunes, Diana N; Pang, Ken; Park, Jaesung; Patel, Tushar; Pocsfalvi, Gabriella; Del Portillo, Hernando; Putz, Ulrich; Ramirez, Marcel I; Rodrigues, Marcio L; Roh, Tae-Young; Royo, Felix; Sahoo, Susmita; Schiffelers, Raymond|info:eu-repo/dai/nl/212909509; Sharma, Shivani; Siljander, Pia; Simpson, Richard J; Soekmadji, Carolina; Stahl, Philip; Stensballe, Allan; Stępień, Ewa; Tahara, Hidetoshi; Trummer, Arne; Valadi, Hadi; Vella, Laura J; Wai, Sun Nyunt; Witwer, Kenneth; Yáñez-Mó, María; Youn, Hyewon; Zeidler, Reinhard; Gho, Yong Song; Nolte - t Hoen, Esther|info:eu-repo/dai/nl/261632175

    2014-01-01

    MOTIVATION: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS: We

  11. EVpedia: a community web portal for extracellular vesicles research

    NARCIS (Netherlands)

    Kim, Dae-Kyum; Lee, Jaewook; Kim, Sae Rom; Choi, Dong-Sic; Yoon, Yae Jin; Kim, Ji Hyun; Go, Gyeongyun; Nhung, Dinh; Hong, Kahye; Jang, Su Chul; Kim, Si-Hyun; Park, Kyong-Su; Kim, Oh Youn; Park, Hyun Taek; Seo, Ji Hye; Aikawa, Elena; Baj-Krzyworzeka, Monika; van Balkom, Bas W. M.; Belting, Mattias; Blanc, Lionel; Bond, Vincent; Bongiovanni, Antonella; Borràs, Francesc E.; Buée, Luc; Buzás, Edit I.; Cheng, Lesley; Clayton, Aled; Cocucci, Emanuele; Dela Cruz, Charles S.; Desiderio, Dominic M.; Di Vizio, Dolores; Ekström, Karin; Falcon-Perez, Juan M.; Gardiner, Chris; Giebel, Bernd; Greening, David W.; Gross, Julia Christina; Gupta, Dwijendra; Hendrix, An; Hill, Andrew F.; Hill, Michelle M.; Nolte-'t Hoen, Esther; Hwang, Do Won; Inal, Jameel; Jagannadham, Medicharla V.; Jayachandran, Muthuvel; Jee, Young-Koo; Jørgensen, Malene; Kim, Kwang Pyo; Kim, Yoon-Keun; Kislinger, Thomas; Lässer, Cecilia; Lee, Dong Soo; Lee, Hakmo; van Leeuwen, Johannes; Lener, Thomas; Liu, Ming-Lin; Lötvall, Jan; Marcilla, Antonio; Mathivanan, Suresh; Möller, Andreas; Morhayim, Jess; Mullier, François; Nazarenko, Irina; Nieuwland, Rienk; Nunes, Diana N.; Pang, Ken; Park, Jaesung; Patel, Tushar; Pocsfalvi, Gabriella; del Portillo, Hernando; Putz, Ulrich; Ramirez, Marcel I.; Rodrigues, Marcio L.; Roh, Tae-Young; Royo, Felix; Sahoo, Susmita; Schiffelers, Raymond; Sharma, Shivani; Siljander, Pia; Simpson, Richard J.; Soekmadji, Carolina; Stahl, Philip; Stensballe, Allan; Stępień, Ewa; Tahara, Hidetoshi; Trummer, Arne; Valadi, Hadi; Vella, Laura J.; Wai, Sun Nyunt; Witwer, Kenneth; Yáñez-Mó, María; Youn, Hyewon; Zeidler, Reinhard; Gho, Yong Song

    2015-01-01

    Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. We present an improved

  12. Coiled coil driven membrane fusion between cyclodextrin vesicles and liposomes

    NARCIS (Netherlands)

    Versluis, Frank; Voskuhl, Jens; Vos, Jan; Friedrich, Heiner; Ravoo, Bart Jan; Bomans, Paul H H; Stuart, Marc C A; Sommerdijk, Nico A J M; Kros, Alexander

    2014-01-01

    Controlled fusion events between natural membranes composed of phospholipids with synthetic unnatural membranes will yield valuable fundamental information on the mechanism of membrane fusion. Here, fusion between vastly different phospholipid liposomes and cyclodextrin amphiphile based vesicles

  13. Determination of the Strength of Adhesion between Lipid Vesicles

    Directory of Open Access Journals (Sweden)

    Tomáš Mareš

    2012-01-01

    Full Text Available A commonly used method to determine the strength of adhesion between adhering lipid vesicles is measuring their effective contact angle from experimental images. The aim of this paper is to estimate the interobserver variations in vesicles effective contact angle measurements and to propose a new method for estimating the strength of membrane vesicle adhesion. Theoretical model shows for the old and for the new measure a monotonic dependence on the strength of adhesion. Results obtained by both measuring techniques show statistically significant correlation and high interobserver reliability for both methods. Therefore the conventional method of measuring the effective contact angle gives qualitatively relevant results as the measure of the lipid vesicle adhesion. However, the new measuring technique provides a lower variation of the measured values than the conventional measures using the effective contact angle. Moreover, obtaining the adhesion angle can be automatized more easily than obtaining the effective contact angle.

  14. Cystadenoma of the seminal vesicle. A case report

    DEFF Research Database (Denmark)

    Lundhus, E; Bundgaard, N; Sørensen, Flemming Brandt

    1984-01-01

    Cystadenomas of the seminal vesicle are extremely rare benign tumours, which only have been reported seven times earlier in the literature. The first Danish case is reported with discussion of symptomatology, pathology and treatment.......Cystadenomas of the seminal vesicle are extremely rare benign tumours, which only have been reported seven times earlier in the literature. The first Danish case is reported with discussion of symptomatology, pathology and treatment....

  15. Interaction and rheology of vesicle suspensions in confined shear flow

    Science.gov (United States)

    Shen, Zaiyi; Farutin, Alexander; Thiébaud, Marine; Misbah, Chaouqi

    2017-10-01

    Dynamics and rheology of a confined suspension of vesicles (a model for red blood cells) are studied numerically in two dimensions by using an immersed boundary lattice Boltzmann method. We pay particular attention to the link between the spatiotemporal organization and the rheology of the suspension. Besides confinement, we analyze the effect of concentration of the suspension, ϕ (defined as the area fraction occupied by the vesicles in the simulation domain), as well as the viscosity contrast λ (defined as the ratio between the viscosity of the fluid inside the vesicles, ηint, and that of the suspending fluid, ηext). The hydrodynamic interaction between two vesicles is shown to play a key role in determining the spatial organization. For λ =1 , the pair of vesicles settles into an equilibrium state with constant interdistance, which is regulated by the confinement. The equilibrium interdistance increases with the gap between walls, following a linear relationship. However, no stable equilibrium interdistance between two tumbling vesicles is observed for λ =10 . A quite ordered suspension is observed concomitant with the existence of an equilibrium interdistance between a vesicle pair. However, a disordered suspension prevails when no pair equilibrium interdistance exists, as occurs for tumbling vesicles. We then analyze the rheology, focusing on the effective viscosity, denoted as η , as well as on normalized viscosity, defined as [η ] =(η -ηext) /(ηextϕ ) . Ordering of the suspension is accompanied by a nonmonotonic behavior of [η ] with ϕ , while η exhibits plateaus. The nonmonotonic behavior of [η ] is suppressed when a disordered pattern prevails.

  16. Development, characterization, and skin delivery studies of related ultradeformable vesicles: transfersomes, ethosomes, and transethosomes

    Directory of Open Access Journals (Sweden)

    Ascenso A

    2015-09-01

    Full Text Available Andreia Ascenso,1 Sara Raposo,1 Cátia Batista,2 Pedro Cardoso,2 Tiago Mendes,2 Fabíola Garcia Praça,3 Maria Vitória Lopes Badra Bentley,3 Sandra Simões1 1Instituto de Investigação do Medicamento (iMed.ULisboa, 2Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal; 3Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Monte Alegre, Ribeirão Preto, São Paulo, Brazil Abstract: Ultradeformable vesicles (UDV have recently become a promising tool for the development of improved and innovative dermal and transdermal therapies. The aim of this work was to study three related UDV: transfersomes, ethosomes, and transethosomes for the incorporation of actives of distinct polarities, namely, vitamin E and caffeine, and to evaluate the effect of the carrier on skin permeation and penetration. These actives were incorporated in UDV formulations further characterized for vesicles imaging by transmission electron microscopy; mean vesicle size and polydispersity index by photon correlation spectroscopy; zeta potential by laser-Doppler anemometry; deformability by pressure-driven transport; and incorporation efficiency (IE after actives quantification by high-performance liquid chromatography. Topical delivery studies were performed in order to compare UDV formulations regarding the release, skin permeation, and penetration profiles. All UDV formulations showed size values within the expected range, except transethosomes prepared by “transfersomal method”, for which size was smaller than 100 nm in contrast to that obtained for vesicles prepared by “ethosomal method”. Zeta potential was negative and higher for formulations containing sodium cholate. The IE was much higher for vitamin E- than caffeine-loaded UDV as expected. For flux measurements, the following order was obtained: transethosomes (TE > ethosomes (E ≥ transfersomes (T. This result was consistent with the release and skin penetration

  17. Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

    Science.gov (United States)

    Silvas, Jesus A.; Popov, Vsevolod L.; Paulucci-Holthauzen, Adriana

    2015-01-01

    that extracellular vesicles produced by SFTS virus-infected cells harbor infectious virions. We sought to determine whether these “infectious” extracellular vesicles can mediate transmission of the virus and confirmed that the SFTS virions were efficiently transported by these secreted structures into uninfected cells and were able to sustain efficient replication of SFTS virus. These results have significant impact on our understanding of how the novel tick-borne phleboviruses hijack cellular machineries to establish infection and point toward a novel mechanism for virus replication among arthropod-borne viruses. PMID:26512089

  18. Extracellular Membrane Vesicles and Phytopathogenicity of Acholeplasma laidlawii PG8

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    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.

  19. 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.......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......) with a lipid‐containing solvent phase. We made GPVs by using n‐decane and squalene as solvents, and applied generalized polarization (GP) imaging to monitor the polarity around the protein transmembrane region of aquaporins labeled with the polarity‐sensitive probe Badan. Specifically, we created GPVs...

  20. Extracellular vesicles in Alzheimer's disease: friends or foes? Focus on aβ-vesicle interaction.

    Science.gov (United States)

    Joshi, Pooja; Benussi, Luisa; Furlan, Roberto; Ghidoni, Roberta; Verderio, Claudia

    2015-03-03

    The intercellular transfer of amyloid-β (Aβ) and tau proteins has received increasing attention in Alzheimer's disease (AD). Among other transfer modes, Aβ and tau dissemination has been suggested to occur through release of Extracellular Vesicles (EVs), which may facilitate delivery of pathogenic proteins over large distances. Recent evidence indicates that EVs carry on their surface, specific molecules which bind to extracellular Aβ, opening the possibility that EVs may also influence Aβ assembly and synaptotoxicity. In this review we focus on studies which investigated the impact of EVs in Aβ-mediated neurodegeneration and showed either detrimental or protective role for EVs in the pathology.

  1. Extracellular Vesicles and Autophagy in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Tianyang Gao

    2016-01-01

    Full Text Available Osteoarthritis (OA is a type of chronic joint disease that is characterized by the degeneration and loss of articular cartilage and hyperplasia of the synovium and subchondral bone. There is reasonable knowledge about articular cartilage physiology, biochemistry, and chondrocyte metabolism. However, the etiology and pathogenesis of OA remain unclear and need urgent clarification to guide the early diagnosis and treatment of OA. Extracellular vesicles (EVs are small membrane-linking particles that are released from cells. In recent decades, several special biological properties have been found in EV, especially in terms of cartilage. Autophagy plays a critical role in the regulation of cellular homeostasis. Likewise, more and more research has gradually focused on the effect of autophagy on chondrocyte proliferation and function in OA. The synthesis and release of EV are closely associated with autophagy. At the same time, both EV and autophagy play a role in OA development. Based on the mechanism of EV and autophagy in OA development, EV may be beneficial in the early diagnosis of OA; on the other hand, the combination of EV and autophagy-related regulatory drugs may provide insight into possible OA therapeutic strategies.

  2. Association of vancomycin with lipid vesicles

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    Xiaohui Hu

    2017-09-01

    Full Text Available Antibiotics play a pivotal role in modern medicine for the treatment of bacterial infection in patients. Membrane defines the boundary between single cell and its environment and is a main target for antibacterial agents. To better understand the mechanism of antibiotics action on microbes, we utilized liposome as membrane mimic model to study antibiotics interaction with bacterial membrane by variety of biophysical methods. Isothermal calorimetry and fluorescence photometry experiments were performed to examine interaction between antibiotics and liposome. We found that vancomycin, one of the most important antibiotics for the treatment of serious infections by gram-positive bacteria, binds to the liposome. The association between the drug and the liposome does not involve the tail part of the lipids. Moreover, the binding affinity increases along with the increment of liposome size. Of three major lipid components, phosphatidylglycerol is the preferential target for vancomycin binding. We also showed that vancomycin associates with vesicle derived from Staphylococcus aureus membrane in a similar manner as the binding to liposome. Our data suggested that vancomycin associates with bacterial membrane through direct interaction with lipid head groups with the extent of the association depending very much on specific type of lipids and curvature of local membrane structure.

  3. Characterization and biological role of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Aneta Wójtowicz

    2014-12-01

    Full Text Available Extracellular vesicles (EV form a heterogeneous population of mostly spherical membrane structures released by almost all cells, including tumour cells, both in vivo and in vitro. Their size varies from 30 nm to 1 μm, and size is one of the main criteria of the selection of two categories of EV: small (30-100 nm, more homogeneous exosomes and larger fragments (0.1-1 μm called membrane microvesicles or ectosomes. The presence of EV has already been detected in many human body fluids: blood, urine, saliva, semen and amniotic fluid. Formation of EV is tightly controlled, and their function and biochemical composition depend on the cell type they originate from. EV are the “vehicles” of bioactive molecules, such as proteins, mRNA and microRNA, and may play an important role in intercellular communication and modulation of e.g. immune system cell activity. In addition, on the surface of tumour-derived microvesicles (TMV, called oncosomes, several markers specific for cancer cells were identified, which indicates a role of TMV in tumour growth and cancer development. On the other hand, TMV may be an important source of tumour-associated antigens (TAA which can be potentially useful as biomarkers with prognostic value, as well as in development of new forms of targeted immunotherapy of cancer.

  4. Towards traceable size determination of extracellular vesicles.

    Science.gov (United States)

    Varga, Zoltán; Yuana, Yuana; Grootemaat, Anita E; van der Pol, Edwin; Gollwitzer, Christian; Krumrey, Michael; Nieuwland, Rienk

    2014-01-01

    Extracellular vesicles (EVs) have clinical importance due to their roles in a wide range of biological processes. The detection and characterization of EVs are challenging because of their small size, low refractive index, and heterogeneity. In this manuscript, the size distribution of an erythrocyte-derived EV sample is determined using state-of-the-art techniques such as nanoparticle tracking analysis, resistive pulse sensing, and electron microscopy, and novel techniques in the field, such as small-angle X-ray scattering (SAXS) and size exclusion chromatography coupled with dynamic light scattering detection. The mode values of the size distributions of the studied erythrocyte EVs reported by the different methods show only small deviations around 130 nm, but there are differences in the widths of the size distributions. SAXS is a promising technique with respect to traceability, as this technique was already applied for traceable size determination of solid nanoparticles in suspension. To reach the traceable measurement of EVs, monodisperse and highly concentrated samples are required.

  5. Towards traceable size determination of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Zoltán Varga

    2014-02-01

    Full Text Available Background: Extracellular vesicles (EVs have clinical importance due to their roles in a wide range of biological processes. The detection and characterization of EVs are challenging because of their small size, low refractive index, and heterogeneity. Methods: In this manuscript, the size distribution of an erythrocyte-derived EV sample is determined using state-of-the-art techniques such as nanoparticle tracking analysis, resistive pulse sensing, and electron microscopy, and novel techniques in the field, such as small-angle X-ray scattering (SAXS and size exclusion chromatography coupled with dynamic light scattering detection. Results: The mode values of the size distributions of the studied erythrocyte EVs reported by the different methods show only small deviations around 130 nm, but there are differences in the widths of the size distributions. Conclusion: SAXS is a promising technique with respect to traceability, as this technique was already applied for traceable size determination of solid nanoparticles in suspension. To reach the traceable measurement of EVs, monodisperse and highly concentrated samples are required.

  6. 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; 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

  7. 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.

  8. Sugar-based gemini surfactant with a vesicle-to-micelle transition at acidic pH and a reversible vesicle flocculation near neutral pH

    NARCIS (Netherlands)

    Johnsson, M; Wagenaar, A; Engberts, JBFN

    2003-01-01

    A sugar-based (reduced glucose) gemini surfactant forms vesicles in dilute aqueous solution near neutral pH. At lower pH, there is a vesicle-to-micelle transition within a narrow pH region (pH 6.0-5.6). The vesicles are transformed into large cylindrical micelles that in turn are transformed into

  9. Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition are mediated by the packing parameter of phospholipid-detergent systems

    NARCIS (Netherlands)

    Stuart, Marc C. A.; Boekema, Egbert J.

    2007-01-01

    The detergent solubilization and reformation of phospholipid vesicles was studied for various detergents. Two distinct mechanisms of vesicle-to-micelle and micelle-to-vesicle transition were observed by turbidimetry and cryo-electron microscopy. The first mechanism involves fast solubilization of

  10. The effect of cadmium chloride in vitro on sodium-glutamate cotransport in brush border membrane vesicles isolated from rabbit kidney.

    Science.gov (United States)

    Kinne, R K; Schütz, H; Kinne-Saffran, E

    1995-12-01

    To further elucidate the mechanism of cadmium inhibition of renal amino acid transport, brush border membrane vesicles were isolated from rabbit renal cortex and the effect of cadmium on the uptake of L-glutamate into the vesicles was investigated. Preincubation of the membranes with CdCl2 decreased sodium-dependent L-glutamate uptake at concentrations higher than 10(-6)M. In the presence of 20 mM potassium inside the vesicles a half-maximal inhibition was observed at 0.5 to 1 x 10(-4) M. Kinetic analysis revealed a strong reduction of Vmax by cadmium but only minor changes in Km for glutamate. The inhibition required preincubation of the vesicles with cadmium, was not elicited by cadmium metallothionein, and was not reversed by ethylenediaminetetraacetic acid. These findings suggest an action of cadmium at the cytoplasmic face of the brush border membrane. Furthermore, the sensitivity of the transport system for cadmium was lower in the absence of potassium. Inhibition increased in a saturable manner when intravesicular potassium was augmented, indicating that the transporter interacts with cadmium most avidly when potassium is bound to the carrier.

  11. 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.

  12. 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.

  13. Extracellular Vesicles from Hypoxic Adipocytes and Obese Subjects Reduce Insulin‐Stimulated Glucose Uptake

    Science.gov (United States)

    Mleczko, Justyna; Ortega, Francisco J.; Falcon‐Perez, Juan Manuel; Wabitsch, Martin; Fernandez‐Real, Jose Manuel

    2018-01-01

    Scope We investigate the effects of extracellular vesicles (EVs) obtained from in vitro adipocyte cell models and from obese subjects on glucose transport and insulin responsiveness. Methods and results EVs are isolated from the culture supernatant of adipocytes cultured under normoxia, hypoxia (1% oxygen), or exposed to macrophage conditioned media (15% v/v). EVs are isolated from the plasma of lean individuals and subjects with obesity. Cultured adipocytes are incubated with EVs and activation of insulin signalling cascades and insulin‐stimulated glucose transport are measured. EVs released from hypoxic adipocytes impair insulin‐stimulated 2‐deoxyglucose uptake and reduce insulin mediated phosphorylation of AKT. Insulin‐mediated phosphorylation of extracellular regulated kinases (ERK1/2) is not affected. EVs from individuals with obesity decrease insulin stimulated 2‐deoxyglucose uptake in adipocytes (p = 0.0159). Conclusion EVs released by stressed adipocytes impair insulin action in neighboring adipocytes. PMID:29292863

  14. Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. Studies using a fast-reaction apparatus

    International Nuclear Information System (INIS)

    Chase, H.S. Jr.; Al-Awqati, Q.

    1983-01-01

    Regulation of the sodium permeability of the luminal membrane is the major mechanism by which the net rate of sodium transport across tight epithelia is varied. Previous evidence has suggested that the permeability of the luminal membrane might be regulated by changes in intracellular sodium or calcium activities. To test this directly, we isolated a fraction of the plasma membrane from the toad urinary bladder, which contains a fast, amiloride-sensitive sodium flux with characteristics similar to those of the native luminal membrane. Using a flow-quench apparatus to measure the initial rate of sodium efflux from these vesicles in the millisecond time range, we have demonstrated that the isotope exchange permeability of these vesicles is very sensitive to calcium. Calcium reduces the sodium permeability, and the half-maximal inhibitory concentration is 0.5 microM, well within the range of calcium activity found in cells. Also, the permeability of the luminal membrane vesicles is little affected by the ambient sodium concentration. These results, when taken together with studies on whole tissue, suggest that cell calcium may be an important regulator of transepithelial sodium transport by its effect on luminal sodium permeability. The effect of cell sodium on permeability may be mediated by calcium rather than by sodium itself

  15. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

    Science.gov (United States)

    Widdows, Kate L.; Panitchob, Nuttanont; Crocker, Ian P.; Please, Colin P.; Hanson, Mark A.; Sibley, Colin P.; Johnstone, Edward D.; Sengers, Bram G.; Lewis, Rohan M.; Glazier, Jocelyn D.

    2015-01-01

    Uptake of system L amino acid substrates into isolated placental plasma membrane vesicles in the absence of opposing side amino acid (zero-trans uptake) is incompatible with the concept of obligatory exchange, where influx of amino acid is coupled to efflux. We therefore hypothesized that system L amino acid exchange transporters are not fully obligatory and/or that amino acids are initially present inside the vesicles. To address this, we combined computational modeling with vesicle transport assays and transporter localization studies to investigate the mechanisms mediating [14C]l-serine (a system L substrate) transport into human placental microvillous plasma membrane (MVM) vesicles. The carrier model provided a quantitative framework to test the 2 hypotheses that l-serine transport occurs by either obligate exchange or nonobligate exchange coupled with facilitated transport (mixed transport model). The computational model could only account for experimental [14C]l-serine uptake data when the transporter was not exclusively in exchange mode, best described by the mixed transport model. MVM vesicle isolates contained endogenous amino acids allowing for potential contribution to zero-trans uptake. Both L-type amino acid transporter (LAT)1 and LAT2 subtypes of system L were distributed to MVM, with l-serine transport attributed to LAT2. These findings suggest that exchange transporters do not function exclusively as obligate exchangers.—Widdows, K. L., Panitchob, N., Crocker, I. P., Please, C. P., Hanson, M. A., Sibley, C. P., Johnstone, E. D., Sengers, B. G., Lewis, R. M., Glazier, J. D. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms. PMID:25761365

  16. Surface glycosylation profiles of urine extracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Jared Q Gerlach

    Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

  17. Organic Nano vesicular Cargoes for Sustained Drug Delivery: Synthesis, Vesicle Formation, Controlling “Pearling” States, and Terfenadine Loading/Release Studies

    International Nuclear Information System (INIS)

    Botcha, A.K.; Chandrasekar, R.; Dulla, B.; Reddy, E.R.; Rajadurai, M.S.; Chennubhotla, K.S.; Kulkarni, P.; Kulkarni, P.

    2014-01-01

    “Sustained drug delivery systems” which are designed to accomplish long-lasting therapeutic effect are one of the challenging topics in the area of nano medicine. We developed an innovative strategy to prepare nontoxic and polymer stabilized organic nano vesicles (diameter: 200 nm) from a novel bolaamphiphile, where two hydrogen bonding acetyl cytosine molecules connected to 4,4′′-positions of the 2,6-bispyrazolylpyridine through two flexible octyne chains. The nano vesicles behave like biological membrane by spontaneously self-assembling into “pearl-like” chains and subsequently forming long nano tubes (diameter: 150 nm), which further develop into various types of network-junctions through self-organization. For drug loading and delivery applications, the nano vesicles were externally protected with biocompatible poly(ethyleneglycol)-2000 to prevent them from fusion and ensuing tube formation. Nontoxic nature of the nano vesicles was demonstrated by zebra fish teratogenicity assay. Biocompatible nano vesicles were loaded with “terfenadine” drug and successfully utilized to transport and release drug in sustained manner (up to 72 h) in zebra fish larvae, which is recognized as an emerging in vivo model system Synthetic nano

  18. Melanoma affects the composition of blood cell-derived extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Nina Koliha

    2016-07-01

    Full Text Available Extracellular vesicles are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of extracellular vesicles reflects the type and status of the originating cell and extracellular vesicles in melanoma patient’s plasma could be indicative for the tumor. Likewise, extracellular vesicles might influence tumor progression by regulating immune responses. We performed a broad protein characterization of extracellular vesicles from plasma of melanoma patients and healthy donors as well as from T cells, B cells, natural killer cells, monocytes, monocyte-derived dendritic cells and platelets using a multiplex bead-based platform. Using this method, we succeeded in analyzing 58 proteins that were differentially displayed on extracellular vesicles. Hierarchal clustering of protein intensity patterns grouped extracellular vesicles according to their originating cell type. The analysis of extracellular vesicles from stimulated B cells and monocyte-derived dendritic cells revealed the transfer of surface proteins to vesicles depending on the cell status. The protein profiles of plasma vesicles resembled the protein profiles of extracellular vesicles from platelets, antigen presenting cells and natural cells as shown by platelet markers, costimulatory proteins, and a natural killer cell subpopulation marker. In comparison to healthy plasma vesicles, melanoma plasma vesicles showed altered signals for platelet markers indicating a changed vesicle secretion or protein loading of extracellular vesicles by platelets and a lower CD8 signal that might be associated with a diminished activity of natural killer cells or T cells. As we hardly detected melanoma-derived vesicles in patient’s plasma, we concluded that blood cells induced the observed differences. In summary, our results question a direct effect of melanoma cells on the composition of extracellular vesicles in melanoma plasma, but rather argue

  19. Preparation of artificial plasma membrane mimicking vesicles with lipid asymmetry.

    Directory of Open Access Journals (Sweden)

    Qingqing Lin

    Full Text Available Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM and/or phosphatidylcholine (PC outside/phosphatidylethanolamine (PE and phosphatidylserine (PS inside, and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes "artificial plasma membrane mimicking" ("PMm" vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.

  20. Preparation of artificial plasma membrane mimicking vesicles with lipid asymmetry.

    Science.gov (United States)

    Lin, Qingqing; London, Erwin

    2014-01-01

    Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes "artificial plasma membrane mimicking" ("PMm") vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.

  1. Surfactant Effects on Lipid-Based Vesicles Properties.

    Science.gov (United States)

    Bnyan, Ruba; Khan, Iftikhar; Ehtezazi, Touraj; Saleem, Imran; Gordon, Sarah; O'Neill, Francis; Roberts, Matthew

    2018-01-11

    Understanding the effect of surfactant properties is critical when designing vesicular delivery systems. This review evaluates previous studies to explain the influence of surfactant properties on the behavior of lipid vesicular systems, specifically their size, charge, stability, entrapment efficiency, pharmacokinetics, and pharmacodynamics. Generally, the size of vesicles decreases by increasing the surfactant concentration, carbon chain length, the hydrophilicity of the surfactant head group, and the hydrophilic-lipophilic balance. Increasing surfactant concentration can also lead to an increase in charge, which in turn reduces vesicle aggregation and enhances the stability of the system. The vesicles' entrapment efficiency not only depends on the surfactant properties but also on the encapsulated drug. For example, the encapsulation of a lipophilic drug could be enhanced by using a surfactant with a low hydrophilic-lipophilic balance value. Moreover, the membrane permeability of vesicles depends on the surfactant's carbon chain length and transition temperature. In addition, surfactants have a clear influence on pharmacokinetics and pharmacodynamics such as sustaining drug release, enhancing the circulation time of vesicles, improving targeting and cellular uptake. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  2. Extracellular vesicles and their immunomodulatory functions in pregnancy.

    Science.gov (United States)

    Nair, Soumyalekshmi; Salomon, Carlos

    2018-04-03

    Extracellular vesicles (EVs) are membrane-bound vesicles released into the extracellular space by almost all types of cells. EVs can cross the physiological barriers, and a variety of biological fluids are enriched in them. EVs are a heterogeneous population of vesicles, including exosomes, microvesicles, and apoptotic bodies. The different subpopulations of vesicles can be differentiated by size and origin, in which exosomes (~100 nm and from endocytic origin) are the most studied so far. EVs have essential roles in cell-to-cell communication and are critical modulators of immune response under normal and pathological conditions. Pregnancy is a unique situation of immune-modulation in which the maternal immune system protects the fetus from allogenic rejection and maintains the immunosurveillance. The placenta is a vital organ that performs a multitude of functions to support the pregnancy. The EVs derived from the human placenta have crucial roles in regulating the maternal immune response for successful pregnancy outcome. Placenta-derived vesicles perform a myriad of functions like suppression of immune reaction to the developing fetus and establishment and maintenance of a systemic inflammatory response to combat infectious intruders. A fine-tuning of these mechanisms is quintessential for successful completion of pregnancy and healthy outcome for mother and fetus. Dysregulation in the mechanisms mentioned above can lead to several pregnancy disorders. In this review, we summarize the current literature regarding the critical roles played by the EVs in immunomodulation during pregnancy with particular attention to the placenta-derived exosomes.

  3. Overall energy conversion efficiency of a photosynthetic vesicle

    Energy Technology Data Exchange (ETDEWEB)

    Sener, Melih [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States; Strumpfer, Johan [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States; Singharoy, Abhishek [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Hunter, C. Neil [Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom; Schulten, Klaus [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States

    2016-08-26

    The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.

  4. The phospholipid vesicles coating on metal chelated inorganic surfaces

    International Nuclear Information System (INIS)

    Chang, Jeong Ho; Cho, Min Ae; Son, Hong Ha; Lee, Cheon Koo; Kim, Kyung Ja

    2007-01-01

    This work showed the formation of phospholipid vesicle coating on inorganic sericite surface with characterization by combining electron microscopy of FE-SEM, TEM, AFM, and qualitatively evaluated the coated phospholipid vesicle by XPS as a function of etching time. The possibility of phospholipid vesicle mobility on the surface was restrained by the chelation effect of magnesium cation. The stabilization properties of phospholipid vesicles on sericite surface were demonstrated by the various concentration of magnesium cation. The presence of magnesium was found to have a much more pronounced influence on the lipid deposition process. The Mg cation plays an important role for attaching the phospholipids with optimum concentration of 7 mM. Totally, the phospholipid vesicles coating on inorganic powder could be useful for bio-related fields such as cosmetics and drug delivery system as the key functional compounds. We hope this basic result lead to a general and simple approach to prepare a wide a range of controlled releasing materials including an encapsulation with cosmetics or drugs

  5. JIP3 localises to exocytic vesicles and focal adhesions in the growth cones of differentiated PC12 cells.

    Science.gov (United States)

    Caswell, Patrick T; Dickens, Martin

    2017-11-20

    The JNK-interacting protein 3 (JIP3) is a molecular scaffold, expressed predominantly in neurons, that serves to coordinate the activation of the c-Jun N-terminal kinase (JNK) by binding to JNK and the upstream kinases involved in its activation. The JNK pathway is involved in the regulation of many cellular processes including the control of cell survival, cell death and differentiation. JIP3 also associates with microtubule motor proteins such as kinesin and dynein and is likely an adapter protein involved in the tethering of vesicular cargoes to the motors involved in axonal transport in neurons. We have used immunofluorescence microscopy and biochemical fractionation to investigate the subcellular distribution of JIP3 in relation to JNK and to vesicular and organelle markers in rat pheochromocytoma cells (PC12) differentiating in response to nerve growth factor. In differentiated PC12 cells, JIP3 was seen to accumulate in growth cones at the tips of developing neurites where it co-localised with both JNK and the JNK substrate paxillin. Cellular fractionation of PC12 cells showed that JIP3 was associated with a subpopulation of vesicles in the microsomal fraction, distinct from synaptic vesicles, likely to be an anterograde-directed exocytic vesicle pool. In differentiated PC12 cells, JIP3 did not appear to associate with retrograde endosomal vesicles thought to be involved in signalling axonal injury. Together, these observations indicate that JIP3 may be involved in transporting vesicular cargoes to the growth cones of PC12 cells, possibly targeting JNK to its substrate paxillin, and thus facilitating neurite outgrowth.

  6. Mechanism of mercurial inhibition of sodium-coupled alanine uptake in liver plasma membrane vesicles from Raja erinacea

    International Nuclear Information System (INIS)

    Sellinger, M.; Ballatori, N.; Boyer, J.L.

    1991-01-01

    In mammalian hepatocytes the L-alanine carrier contains a sulfhydryl group that is essential for its activity and is inhibited by mercurials. In hepatocytes of the evolutionarily primitive little skate (Raja erinacea), HgCl2 inhibits Na(+)-dependent alanine uptake and Na+/K(+)-ATPase and increase K+ permeability. To distinguish between direct effects of HgCl2 on the Na(+)-alanine cotransporter and indirect effects on membrane permeability, [3H]alanine transport was studied in plasma membrane vesicles. [3H]Alanine uptake was stimulated by an out-to-in Na+ but not K+ gradient and was saturable confirming the presence of Na(+)-alanine cotransport in liver plasma membranes from this species. Preincubation of the vesicles with HgCl2 for 5 min reduced initial rates of Na(+)-dependent but not Na(+)-independent alanine uptake in a dose-dependent manner (10-200 microM). In the presence of equal concentrations of NaCl or KCl inside and outside of the vesicles, 75 microM HgCl2 directly inhibited sodium-dependent alanine-[3H]alanine exchange, demonstrating that HgCl2 directly affected the alanine cotransporter. Inhibition of Na(+)-dependent alanine uptake by 30 microM HgCl2 was reversed by dithiothreitol (1 mM). HgCl2 (10-30 microM) also increased initial rates of 22Na uptake (at 5 sec), whereas 22Na uptake rates were decreased at HgCl2 concentrations greater than 50 microM. Higher concentrations of HgCl2 (100-200 microM) produced nonspecific effects on vesicle integrity. These studies indicate that HgCl2 inhibits Na(+)-dependent alanine uptake in skate hepatocytes by three different concentration-dependent mechanisms: direct interaction with the transporters, dissipation of the driving force (Na+ gradient), and loss of membrane integrity

  7. 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.

  8. Serotonin-induced down-regulation of cell surface serotonin transporter

    DEFF Research Database (Denmark)

    Jørgensen, Trine Nygaard; Christensen, Peter Møller; Gether, Ulrik

    2014-01-01

    The serotonin transporter (SERT) terminates serotonergic signaling and enables refilling of synaptic vesicles by mediating reuptake of serotonin (5-HT) released into the synaptic cleft. The molecular and cellular mechanisms controlling SERT activity and surface expression are not fully understood...

  9. In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors.

    Science.gov (United States)

    Fort, Alfredo G; Murray, John W; Dandachi, Nadine; Davidson, Michael W; Dermietzel, Rolf; Wolkoff, Allan W; Spray, David C

    2011-07-01

    Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 μm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.

  10. Decreased intestinal calcium absorption in vivo and normal brush border membrane vesicle calcium uptake in cortisol-treated chickens: evidence for dissociation of calcium absorption from brush border vesicle uptake.

    Science.gov (United States)

    Shultz, T D; Bollman, S; Kumar, R

    1982-06-01

    The influence of cortisol on intestinal calcium transport was studied in isolated duodenal loops and brush border membrane (BBM) vesicles of vitamin D-deficient or replete chickens. Four- to five-week-old vitamin D-deficient cockerels were dosed intraperitoneally with 1 microgram of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] alone 15 hr before sacrifice or in combination with 1, 3, or 5 mg of cortisol 24 and 48 hr before sacrifice. After a 1-microgram dose of 1,25-)OH)2D3 the in situ intestinal ligated loop technique revealed a 60% increase in calcium absorption compared to control birds (P less than or equal to 0.001). However, the administration of cortisol in various doses (3 and 5 mg) to chickens given 1,25-(OH)2D3 resulted in significant decreases in intestinal calcium transport in vivo (P less than or equal to 0.05; P less than or equal to 0.05). When intestinal BBM vesicles were prepared from birds treated in a manner identical with that described above, there was no observable difference between calcium uptake in BBM vesicles of the 1,25-(OH)2D3-treated birds and that of the cortisol plus 1,25-(OH)2D3-treated birds. 1,25-(OH)2D3-treated and 1,25-(OH)2D3 plus cortisol-treated chicks had intestinal BBM vesicle uptakes that were significantly greater than those of vitamin D-deficient controls (P less than or equal to 0.02; P less than or equal to 0.025). These data show that in vivo intestinal calcium transport may be markedly reduced in the presence of normal intestinal BBM vesicle calcium uptake. This suggest that factors other than BBM calcium uptake (e.g., protein synthesis or contraluminal membrane events) play an important role in the movement of calcium from the intestinal lumen into the bloodstream and extracellular fluid of the organism.

  11. 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 ... nm diameter. OMVs are not formed by fragmentation of the cell or accidental detachment of some quantities of the cell membrane. They are the outcome of the normal turnover of the .... transport of small metabolites. (e.g sugars, amino ...

  12. Exosomes and other extracellular vesicles in host-pathogen interactions.

    Science.gov (United States)

    Schorey, Jeffrey S; Cheng, Yong; Singh, Prachi P; Smith, Victoria L

    2015-01-01

    An effective immune response requires the engagement of host receptors by pathogen-derived molecules and the stimulation of an appropriate cellular response. Therefore, a crucial factor in our ability to control an infection is the accessibility of our immune cells to the foreign material. Exosomes-which are extracellular vesicles that function in intercellular communication-may play a key role in the dissemination of pathogen- as well as host-derived molecules during infection. In this review, we highlight the composition and function of exosomes and other extracellular vesicles produced during viral, parasitic, fungal and bacterial infections and describe how these vesicles could function to either promote or inhibit host immunity. © 2014 The Authors.

  13. Dynamics of Shape Fluctuations of Quasi-spherical Vesicles Revisited

    DEFF Research Database (Denmark)

    Miao, L.; Lomholt, Michael Andersen; Kleis, J.

    2002-01-01

    In this paper, the dynamics of spontaneous shape fluctuations of a single, giant quasi-spherical vesicle formed from a single lipid species is revisited theoretically. A coherent physical theory for the dynamics is developed based on a number of fundamental principles and considerations, and a sy......In this paper, the dynamics of spontaneous shape fluctuations of a single, giant quasi-spherical vesicle formed from a single lipid species is revisited theoretically. A coherent physical theory for the dynamics is developed based on a number of fundamental principles and considerations......, and a systematic formulation of the theory is also established. From-the systematic theoretical formulation, an analytical description of the dynamics of shape fluctuations of quasi-spherical vesicles is derived. In particular, in developing the theory we have made a new interpretation of some...

  14. Immobilization of stable thylakoid vesicles in conductive nanofibers by electrospinning.

    Science.gov (United States)

    Bedford, Nicholas M; Winget, G Douglas; Punnamaraju, Srikoundinya; Steckl, Andrew J

    2011-03-14

    Electrospun fibers consisting of poly(3,4-ethylenedioxythiophene)/poly(styrene sulfonate) (PEDOT/PSS) and poly(ethylene oxide) (PEO) have been used to successfully encapsulate and stabilize thylakoid membrane vesicles isolated from spinach. Light-driven electronic properties were measured. Fibers with immobilized thylakoids show higher electrical conductivity compared with fibers without thylakoids under white light conditions. This is attributed to the electron-generating photosynthetic reactions from the thylakoids. Electron and optical microscopy show the presence of thylakoid vesicles within the fibers using lipid-specific stains. After electrospinning into fibers, the thylakoid vesicles still exhibit an ability to produce a light-driven electron gradient, indicating that activity is preserved during the electrospinning process. These electrospun fibers provide an excellent example of incorporating photosynthetic function into an artificial system.

  15. Exosomes and other extracellular vesicles in host–pathogen interactions

    Science.gov (United States)

    Schorey, Jeffrey S; Cheng, Yong; Singh, Prachi P; Smith, Victoria L

    2015-01-01

    An effective immune response requires the engagement of host receptors by pathogen-derived molecules and the stimulation of an appropriate cellular response. Therefore, a crucial factor in our ability to control an infection is the accessibility of our immune cells to the foreign material. Exosomes—which are extracellular vesicles that function in intercellular communication—may play a key role in the dissemination of pathogen- as well as host-derived molecules during infection. In this review, we highlight the composition and function of exosomes and other extracellular vesicles produced during viral, parasitic, fungal and bacterial infections and describe how these vesicles could function to either promote or inhibit host immunity. PMID:25488940

  16. Extracellular vesicles as mediators of immunopathology in infectious diseases.

    Science.gov (United States)

    Beheshti, Elham Hosseini; Grau, Georges Emile Raymond

    2018-03-25

    During last decades, extracellular vesicles have emerged as important elements in cell-cell communication and as key players in disease pathogenesis via transmission of their cargo between different cells. Various works have described different subpopulations of these membrane structures, based on their cell of origin, biogenesis, size, biophysical properties, cargo, etc. In addition to their pathophysiological role in the development and progression of different diseases including infectious diseases, neurodegenerative disorders and cancer, extracellular vesicles are now recognized for their potential as novel therapeutic target and intelligent drug delivery system. Here, we have reviewed the most recent data on different subtypes of extracellular vesicles, focusing on microvesicles and exosomes and their subpopulations, their involvement in immune-mediated pathogenesis of various infectious diseases and their role as potential therapeutic targets. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  17. Lipids, lipid bilayers and vesicles as seen by neutrons

    International Nuclear Information System (INIS)

    Seto, Hideki

    2011-01-01

    Lipid molecules self-assemble into bilayers in water with their hydrocarbon chains facing inward due to their amphiphilic nature. The structural and dynamical properties of lipids and lipid bilayers have been studied by neutron scattering intensively. In this article, 3 topics are shown as typical examples. 1) a time-resolved small-angle neutron scattering on uni-lamellar vesicles composed of deuterated and protonated lipids to determine lipid kinetics, 2) small-angle neutron scattering to investigate spontaneous formation of nanopores on uni-lamellar vesicles, and 3) neutron spin echo study to determine bending modulus of lipid bilayers. (author)

  18. Tension-induced vesicle fusion: pathways and pore dynamics

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2008-01-01

    The dynamics of tension-induced fusion of two vesicles is studied using dissipative particle dynamics (DPD) simulations. The vesicle membranes use an improved DPD parameter set that results in their sustaining only a 10–30% relative area stretch before rupturing on the microsecond timescale...... fusion time on membrane tension implies that the fusion process is completed by overcoming two energy barriers with scales of 13kBT and 11kBT. The fusion pore radius as a function of time has also been extracted from the simulations, and provides a quantitative measure of the fusion dynamics which...

  19. Proteomic analysis of cerebrospinal fluid extracellular vesicles: a comprehensive dataset.

    Science.gov (United States)

    Chiasserini, Davide; van Weering, Jan R T; Piersma, Sander R; Pham, Thang V; Malekzadeh, Arjan; Teunissen, Charlotte E; de Wit, Heidi; Jiménez, Connie R

    2014-06-25

    Extracellular vesicles (EVs) are present in human cerebrospinal fluid (CSF), yet little is known about their protein composition. The aim of this study is to provide a comprehensive analysis of the proteome of CSF EVs by electron microscopy and high resolution tandem mass spectrometry (MS/MS) in conjunction with bioinformatics. We report an extensive catalog of 1315 proteins identified in EVs isolated from two different CSF pools by ultracentrifugation, including 230 novel EV proteins. Out of 1315 proteins, 760 were identified in both CSF pools and about 30% of those were also quantitatively enriched in the EV fraction versus the soluble CSF fraction. The proteome of CSF EVs was enriched in exosomal markers such as alix and syntenin-1, heat shock proteins and tetraspanins and contained a high proportion of brain-derived proteins (n=373). Interestingly, several known biomarkers for neurodegenerative diseases such as the amyloid precursor protein, the prion protein and DJ-1 were identified in the EV fractions. Our dataset represents the first comprehensive inventory of the EV proteome in CSF, underscoring the biomarker potential of this organelle. Further comparative studies on CSF EVs isolated from patients diagnosed with neurological disorders are warranted. Data are available via ProteomeXchange with identifier PXD000608. Biological significance In this study we analyzed the protein composition of extracellular vesicles isolated from pooled samples of human cerebrospinal fluid (CSF). CSF is a colorless fluid surrounding the brain and the spinal cord, important for the physiology of the central nervous system, ensuing mechanical protection, regulation of brain blood flow and elimination of byproducts of the brain. Since brain (patho)physiology is reflected in CSF, this biological fluid represents an ideal source of soluble and vesicle-based biomarkers for neurological diseases. Here we confirm the presence of exosome-like extracellular vesicles in CSF, underscoring

  20. Pretransitional effects in dimyristoylphosphatidylcholine vesicle membranes: optical dynamometry study.

    OpenAIRE

    Dimova, R; Pouligny, B; Dietrich, C

    2000-01-01

    We used micron-sized latex spheres to probe the phase state and the viscoelastic properties of dimyristoylphosphatidylcholine (DMPC) bilayers as a function of temperature. One or two particles were manipulated and stuck to a DMPC giant vesicle by means of an optical trap. Above the fluid-gel main transition temperature, T(m) congruent with 23.4 degrees C, the particles could move on the surface of the vesicle, spontaneously (Brownian motion) or driven by an external force, either gravity or t...

  1. Extracellular Vesicles as Biomarkers of Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Javier Perez-Hernandez

    2015-01-01

    Full Text Available Systemic lupus erythematosus is an autoimmune disease that predominantly affects women and typically manifests in multiple organs. The damage caused by this disorder is characterized by a chronic inflammatory state. Extracellular vesicles (EVs, including microvesicles (also known as microparticles, apoptotic bodies, and exosomes, are recognized vehicles of intercellular communication, carrying autoantigens, cytokines, and surface receptors. Therefore, the evidence of EVs and their cargo as biomarkers of autoimmune disease is rapidly expanding. This review will focus on biogenesis of extracellular vesicles, their pathophysiological roles, and their potential as biomarkers and therapeutics in inflammatory disease, especially in systemic lupus erythematosus.

  2. Postcoital Hemorrhage of a Recurrent Seminal Vesicle Cyst Requiring Embolization

    Directory of Open Access Journals (Sweden)

    Eric Royston

    2014-09-01

    Full Text Available Herein is a case of a 23-year-old man with recurrence of a seminal vesicle cyst after percutaneous drainage and laparoscopic excision complicated by hemorrhage requiring embolization. He presented to the emergency department for pain after ejaculation. Computed tomographic scan of his pelvis revealed extravasation of contrast near his cyst and pelvic fluid collection suspicious for a hematoma. The patient had steadily decreasing hemoglobin and hematocrit levels. An interventional radiologist performed an embolization of the left seminal vesicle cystic arteries. Hemoglobin and hematocrit values improved and he was discharged. Hemorrhage resolved with embolization procedure and pain dissipated over the course of follow up care.

  3. 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.

  4. Lactobacillus-derived extracellular vesicles enhance host immune responses against vancomycin-resistant enterococci.

    Science.gov (United States)

    Li, Ming; Lee, Kiho; Hsu, Min; Nau, Gerard; Mylonakis, Eleftherios; Ramratnam, Bharat

    2017-03-14

    Probiotic bacteria are known to modulate host immune responses against various pathogens. Recently, extracellular vesicles (EVs) have emerged as potentially important mediators of host-pathogen interactions. In this study, we explored the role of L. plantarum derived EVs in modulating host responses to vancomycin-resistant Enterococcus faecium (VRE) using both Caenorhabditis elegans and human cells. Our previous work has shown that probiotic conditioning C. elegans with L. acidophilus NCFM prolongs the survival of nematodes exposed to VRE. Similarly, L. plantarum WCFS1 derived extracellular vesicles (LDEVs) also significantly protected the worms against VRE infection. To dissect the molecular mechanisms of this EV-induced protection, we found that treatment of C. elegans with LDEVs significantly increased the transcription of host defense genes, cpr-1 and clec-60. Both cpr-1 and clec-60 have been previously reported to have protective roles against bacterial infections. Incubating human colon-derived Caco-2 cells with fluorescent dye-labeled LDEVs confirmed that LDEVs could be transported into the mammalian cells. Furthermore, LDEV uptake was associated with significant upregulation of CTSB, a human homologous gene of cpr-1, and REG3G, a human gene that has similar functions to clec-60. We have found that EVs produced from L. plantarum WCFS1 up-regulate the expression of host defense genes and provide protective effects on hosts. Using probiotic-derived EVs instead of probiotic bacteria themselves, this study provides a new direction to treat antimicrobial resistant pathogens, such as VRE.

  5. Components of coated vesicles and nuclear pore complexes share a common molecular architecture.

    Directory of Open Access Journals (Sweden)

    Damien Devos

    2004-12-01

    Full Text Available Numerous features distinguish prokaryotes from eukaryotes, chief among which are the distinctive internal membrane systems of eukaryotic cells. These membrane systems form elaborate compartments and vesicular trafficking pathways, and sequester the chromatin within the nuclear envelope. The nuclear pore complex is the portal that specifically mediates macromolecular trafficking across the nuclear envelope. Although it is generally understood that these internal membrane systems evolved from specialized invaginations of the prokaryotic plasma membrane, it is not clear how the nuclear pore complex could have evolved from organisms with no analogous transport system. Here we use computational and biochemical methods to perform a structural analysis of the seven proteins comprising the yNup84/vNup107-160 subcomplex, a core building block of the nuclear pore complex. Our analysis indicates that all seven proteins contain either a beta-propeller fold, an alpha-solenoid fold, or a distinctive arrangement of both, revealing close similarities between the structures comprising the yNup84/vNup107-160 subcomplex and those comprising the major types of vesicle coating complexes that maintain vesicular trafficking pathways. These similarities suggest a common evolutionary origin for nuclear pore complexes and coated vesicles in an early membrane-curving module that led to the formation of the internal membrane systems in modern eukaryotes.

  6. Extracellular Vesicles As Modulators of Tumor Microenvironment and Disease Progression in Glioma

    Directory of Open Access Journals (Sweden)

    Abir Mondal

    2017-07-01

    Full Text Available Diffuse gliomas are lethal tumors of the central nervous system (CNS characterized by infiltrative growth, aggressive nature, and therapeutic resistance. The recent 2016 WHO classification for CNS tumors categorizes diffuse glioma into two major types that include IDH wild-type glioblastoma, which is the predominant type and IDH-mutant glioblastoma, which is less common and displays better prognosis. Recent studies suggest presence of a distinct cell population with stem cell features termed as glioma stem cells (GSCs to be causal in driving tumor growth in glioblastoma. The presence of a stem and progenitor population possibly makes glioblastoma highly heterogeneous. Significantly, tumor growth is driven by interaction of cells residing within the tumor with the surrounding milieu termed as the tumor microenvironment. It comprises of various cell types such as endothelial cells, secreted factors, and the surrounding extracellular matrix, which altogether help perpetuate the proliferation of GSCs. One of the important mediators critical to the cross talk is extracellular vesicles (EVs. These nano-sized vesicles play important roles in intercellular communication by transporting bioactive molecules into the surrounding milieu, thereby altering cellular functions and/or reprogramming recipient cells. With the growing information on the contribution of EVs in modulation of the tumor microenvironment, it is important to determine their role in both supporting as well as promoting tumor growth in glioma. In this review, we provide a comprehensive overview of the role of EVs in tumor progression and glioma pathogenesis.

  7. Magnetic Resonance Imaging of Seminal Vesicle Cyst Associated with Ipsilateral Urinary Anomalies

    Directory of Open Access Journals (Sweden)

    Huan-Wu Chen

    2006-01-01

    Conclusion: Most seminal vesicle cysts were asymptomatic and did not change during long-term follow-up. MRI is a powerful tool for detecting seminal vesicle cysts and in delineating associated congenital anomalies of the urogenital tract.

  8. 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.

  9. Matrix-dependent local retention of secretory vesicle cargo in cortical neurons

    NARCIS (Netherlands)

    de Wit, J.; Toonen, R.F.G.; Verhage, M.

    2009-01-01

    Neurons secrete many diffusible signals from synaptic and other secretory vesicles. We characterized secretion of guidance cues, neuropeptides, neurotrophins, and proteases from single secretory vesicles using pHluorin-tagged cargo in cortical neurons. Stimulation triggered transient and persistent

  10. 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.

  11. Commercial cow milk contains physically stable extracellular vesicles expressing immunoregulatory TGF-beta

    NARCIS (Netherlands)

    Pieters, B.C.; Arntz, O.J.; Bennink, M.B.; Broeren, M.G.; Caam, A.P.M. van; Koenders, M.I.; Lent, P.L. van; Berg, W.B. van den; Vries, M. de; Kraan, P.M. van der; Loo, F.A.J. van de

    2015-01-01

    SCOPE: 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

  12. Design and characterization of synthetic bilayer vesicles with a polymerized inner bilayer leaflet

    NARCIS (Netherlands)

    Ravoo, B.J; Weringa, W.D; Engberts, J.B.F.N.

    1996-01-01

    Four new phospholipid derivatives containing a beta-nitrostyrene unit linked to the phosphate headgroup have been synthesized and converted into unilamellar vesicles. The vesicles were characterized by freeze-fracture transmission electron microscopy (FFEM), cryo-scanning electron microscopy

  13. Proteomic characterization of macro-, micro- and nano-extracellular vesicles derived from the same first trimester placenta: relevance for feto-maternal communication.

    Science.gov (United States)

    Tong, Mancy; Kleffmann, Torsten; Pradhan, Shantanu; Johansson, Caroline L; DeSousa, Joana; Stone, Peter R; James, Joanna L; Chen, Qi; Chamley, Larry W

    2016-04-01

    What proteins are carried by extracellular vesicles (EVs) released from normal first trimester placentae? One thousand five hundred and eighty-five, 1656 and 1476 proteins were characterized in macro-, micro- and nano-vesicles, respectively, from first trimester placentae, with all EV fractions being enriched for proteins involved in vesicle transport and inflammation. Placental EVs are being increasingly recognized as important mediators of both healthy and pathological pregnancies. However, current research has focused on detecting changes in specific proteins in particular fractions of vesicles during disease. This is the first study to investigate the full proteome of different-sized fractions of EVs from the same first trimester placenta and highlights the differences/similarities between the vesicle fractions. A well-established ex vivo placental explant culture model was used to generate macro-, micro- and nano-vesicles from 56 first trimester placentae. Vesicle fractions were collected by differential ultracentrifugation, quantified and characterized. Placental macro-, micro- and nano-vesicles were characterized by microscopy, dynamic light scattering and nanoparticle tracking analysis. The proteome of each EV fraction was interrogated using liquid chromatography-coupled tandem mass spectrometry. Results were validated by semi-quantitative western blotting. A total of 1585, 1656 and 1476 proteins were identified in macro-, micro- and nano-vesicles, respectively. One thousand one hundred and twenty-five proteins were shared between all three fractions while up to 223 proteins were unique to each fraction. Gene Ontology pathway analysis showed an enrichment of proteins involved in vesicle transport and inflammation in all three fractions of EVs. The expression levels of proteins involved in internalization of vesicles (annexin V, calreticulin, CD31, CD47), the complement pathway [C3, decay-accelerating factor (DAF), membrane cofactor protein (MCP), protectin

  14. 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.

  15. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles. Volume I

    International Nuclear Information System (INIS)

    Campbell, K.P.

    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 Ca 2+ + Mg 2+ 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 Ca 2+ + Mg 2+ 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 Ca 2+ /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

  16. Single-step isolation of extracellular vesicles by size-exclusion chromatography

    OpenAIRE

    Böing, Anita N.; van der Pol, Edwin; Grootemaat, Anita E.; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk

    2014-01-01

    Background: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively.Aim: To develop a single-step protocol to isolate vesicles from human body fluids.Methods: Platelet-free supernatant, derived from platelet...

  17. Trafficking of Sendai virus nucleocapsids is mediated by intracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Raychel Chambers

    2010-06-01

    Full Text Available Paramyxoviruses are assembled at the plasma membrane budding sites after synthesis of all the structural components in the cytoplasm. Although viral ribonuclocapsid (vRNP is an essential component of infectious virions, the process of vRNP translocation to assembly sites is poorly understood.To analyze real-time trafficking of vRNPs in live infected cells, we created a recombinant Sendai virus (SeV, rSeVLeGFP, which expresses L protein fused to enhanced green fluorescent protein (eGFP. The rSeVLeGFP showed similar growth kinetics compared to wt SeV, and newly synthesized LeGFP could be detected as early as 8 h postinfection. The majority of LeGFP co-localized with other components of vRNPs, NP and P proteins, suggesting the fluorescent signals of LeGFP represent the locations of vRNPs. Analysis of LeGFP movement using time-lapse digital video microscopy revealed directional and saltatory movement of LeGFP along microtubules. Treatment of the cells with nocodazole restricted vRNP movement and reduced progeny virion production without affecting viral protein synthesis, suggesting the role of microtubules in vRNP trafficking and virus assembly. Further study with an electron microscope showed close association of vRNPs with intracellular vesicles present in infected cells. In addition, the vRNPs co-localized with Rab11a protein, which is known to regulate the recycling endocytosis pathway and Golgi-to-plasma membrane trafficking. Simultaneous movement between LeGFP and Rab11a was also observed in infected cells, which constitutively express mRFP-tagged Rab11a. Involvement of recycling endosomes in vRNP translocation was also suggested by the fact that vRNPs move concomitantly with recycling transferrin labeled with Alexa 594.Collectively, our results strongly suggest a previously unrecognized involvement of the intracellular vesicular trafficking pathway in vRNP translocation and provide new insights into the transport of viral structural

  18. Analysis of long-chain fatty acid binding activity in vesicles of the outer membrane generated from Escherchia coli

    International Nuclear Information System (INIS)

    Black, P.N.

    1987-01-01

    Escherichia coli transports long-chain fatty acids across the dual membrane by a high affinity, saturable, energy-dependent process. The fadL gene codes for an outer membrane protein which appears to act specifically as a long-chain fatty acid binding protein when fatty acid utilization is blocked by mutation. In an effort to understand the function of the fadL gene product, FLP, membranes have been isolated from fadL + and fadL - strains following osmotic lysis. Following isolation, total membranes were separated into inner and outer membrane fractions and assayed for long-chain fatty acid binding activity. Outer membrane vesicles were incubated 2-5 min at 37 0 C with 3 H oleate (C/sub 18:1/), cooled to 0 0 C, and centrifuged through a Lipidex 100 column for 3 min to remove the unbound fatty acid. The level of fatty acid binding was quantitated by scintillation counting of the eluate. Outer membrane vesicles generated from a fadL + strain bind 325 pmol fatty acid/mg protein whereas vesicles generated for a mutant strain bind 175 pmol fatty acid/mg protein. These data suggest that FLP acts at least as a long-chain fatty acid binding protein on the surface of the cell

  19. Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Kuehn Meta J

    2009-02-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF. To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells. Results Purified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER marker, TRAPα, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939, an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association. Conclusion These data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.

  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. Swinging of two-domains vesicles in shear flow

    Science.gov (United States)

    Viallat, Annie; Tusch, Simon; Khelloufi, Kamel; Leonetti, Marc

    2014-11-01

    Giant lipid vesicles and red blood cells in shear flow at low shear rates tank tread (TT) at small viscosity ratio between the inner particle volume and the external fluid, and flip or tumble (T) at large viscosity ratio. The phase diagram of motion of red blood cells is however much more complex. Swinging superimposes to TT, cells wobble and roll rather than tumble with increasing shear rate and present a shear-rate driven transition between TT to T. These features are attributed to the shear elasticity and the non spherical stress-free shape of the cell membrane, which stores shear elastic energy as a function of the relative position of its elements. We have created vesicles with a phase diagram of motion comparable to that of red blood cells by preparing membranes with two lipids and cholesterol. These membranes present two domains separated by a contact line. The line has a tension energy that depends on its relative position on the vesicle. Similarly to red blood cells, two-domains vesicles swing and wobble. An analytical model where line tension energy is added to the Keller and Skalak's model fits our experimental data without any adjustable parameter. Our experiments and model shed light on the motion of deformable particles in shear flow.

  2. 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.

  3. Interaction of cholesterol-crystallization-promoting proteins with vesicles

    NARCIS (Netherlands)

    de Bruijn, M. A.; Goldhoorn, B. G.; Zijlstra, A. I.; Tytgat, G. N.; Groen, A. K.

    1995-01-01

    In this study, the interaction of mucin and concanavalin A-binding proteins isolated from human bile with cholesterol/phospholipid vesicles was investigated. Using resonance energy transfer assays originally developed by Struck, Hoekstra and Pagano [(1981) Biochemistry 20, 4093-4099], no significant

  4. Packing states of multilamellar vesicles in a nonionic surfactant system

    DEFF Research Database (Denmark)

    Le, T.D.; Olsson, U.; Mortensen, K.

    2001-01-01

    under shear. Here, we focused only in the MLV region, L-alpha(*), of a temperature sensitive surfactant system (C12E4-water) to investigate the packing of multilamellar vesicles as a function of temperature under constant shear. Two sets of temperature scan experiments were performed in the L...

  5. Intermedin inhibits norepinephrine-induced contraction of rat seminal vesicle

    Directory of Open Access Journals (Sweden)

    P.F. Wong

    2014-09-01

    Conclusion: The results demonstrated that the inhibitory action of IMD on NE-induced seminal vesicle contraction was mediated via the ADM receptor(s and the nitric oxide production pathway, partially by the IMD receptor, but not by the CGRP receptor and the cAMP-PKA pathway.

  6. Glycosylation of extracellular vesicles : current knowledge, tools and clinical perspectives

    NARCIS (Netherlands)

    Williams, Charles; Royo, Felix; Aizpurua-Olaizola, Oier; Pazos, Raquel; Boons, Geert-Jan|info:eu-repo/dai/nl/088245489; Reichardt, Niels-Christian; Falcon-Perez, Juan M

    2018-01-01

    It is now acknowledged that extracellular vesicles (EVs) are important effectors in a vast number of biological processes through intercellular transfer of biomolecules. Increasing research efforts in the EV field have yielded an appreciation for the potential role of glycans in EV function. Indeed,

  7. The function of vesicles in the actinomycete Frankia

    NARCIS (Netherlands)

    Meesters, T.

    1988-01-01

    The actinomycete Frankia is a symbiotic nitrogen fixer, living in root nodules of many non-leguminous plants. A typical characteristic of this endophytic organism is the formation of specialized swollen cell structures, called vesicles. Frankia

  8. Removal of vesicle structures from transmission electron microscope images

    DEFF Research Database (Denmark)

    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 reconstruct...

  9. Transmembrane topology of the acetylcholine receptor examined in reconstituted vesicles

    International Nuclear Information System (INIS)

    McCrea, P.D.

    1987-01-01

    Each of the five acetylcholine receptor (AChR) subunits, α 2 β-γδ, is believed to have the same number of transmembrane crossing and to share the same general folding pattern. AChR isolated from the electric organ of electric fish is predominantly dimeric. We have used this bridge as a marker for the C-terminus of the δ subunit, and presumably that of the other subunits in addition. The disulfide's accessibility to hydrophilic reductants, principally glutathione (GSH), was tested in a reconstituted vesicle system. The reduction of the δ-δ desulfide, as evidenced by the transition of AChrR dimers to monomers, was quantitatively monitored on velocity sedimentation sucrose gradients. Alternatively, the reduction of δ 2 to δ was followed by employing non-reducing SDS-PAGE. Reductants such as GSH were able to access the bridge in intact right-side-out vesicles. No acceleration of this process was evident when the vesicles were disrupted by freeze-thaw or by detergents. Control experiments which determined the rate of reduction of entrapped diphtheria toxin, or that of 3 H-GSH efflux, demonstrated that intact reconstituted vesicles provide an adequate permeability barrier to GSH access of their intravesicular space

  10. Extracellular vesicles in human follicular fluid do not promote coagulation

    NARCIS (Netherlands)

    Franz, Cordula; Böing, Anita N.; Montag, Markus; Strowitzki, Thomas; Markert, Udo R.; Mastenbroek, Sebastiaan; Nieuwland, Rienk; Toth, Bettina

    2016-01-01

    Body fluids contain extracellular vesicles expressing tissue factor on their surface and serve as an additional trigger for coagulation. During the menstrual cycle ovarian tissue restoration is mandatory and it is unknown whether follicular fluid might provide procoagulant substances. Within an

  11. Penetration and fusion of phospholipid vesicles by lysozyme

    International Nuclear Information System (INIS)

    Kim, J.; Kim, H.

    1989-01-01

    The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-([ 125 I]iodophenyl)diazirine ([ 125 I]TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent [ 125 I]TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion

  12. The role of extracellular vesicles in neurodegenerative diseases.

    Science.gov (United States)

    Quek, Camelia; Hill, Andrew F

    2017-02-19

    Extracellular vesicles, including exosomes, are small membranous vesicles released from many biotypes, contributing to the disease progression and spreading. These extracellular vesicles provide an important mode of cell-to-cell communication by delivering proteins, lipids and RNA to target cells. Exosomes are found associated with neurodegenerative diseases, which are characterised by progressive degeneration of neurons and often associated with misfolded protein. The common diseases include Parkinson's disease (PD), Alzheimer's diseases (AD), amyotrophic lateral sclerosis (ALS), and the prion diseases. Of all neurodegenerative diseases, prion diseases are classified as the distinctive group owing to its transmissible and infectious nature of misfolded prion protein. The infectious prion particles have been demonstrated to be present in exosomes to spread prion infectivity within cells. Similarly, misfolded proteins involved in other neurodegenerative diseases such as Amyloid-β and tau in AD, α-synuclein in PD, and superoxide dismutase 1 in ALS have been demonstrated to exploit exosomes for induced spreading of misfolded proteins in a prion-like mechanism. Furthermore, RNA molecules can be taken up by the recipient cells as cargo in exosomes. These RNAs can module the expression of the target genes by repressing or inhibiting protein translation. Here we review the role of exosomes in prion diseases and other common neurodegenerative diseases, and discuss the potential of these vesicles for disease pathogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Dimensional characterization of extracellular vesicles using atomic force microscopy

    NARCIS (Netherlands)

    Sebaihi, N.; de Boeck, B.; Yuana, Y.; Nieuwland, R.; Petry, J.

    2017-01-01

    Extracellular vesicles (EV) are small biological entities released from cells into body fluids. EV are recognized as mediators in intercellular communication and influence important physiological processes. It has been shown that the concentration and composition of EV in body fluids may differ from

  14. Antibody-Based Assays for Phenotyping of Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Lotte Hatting Pugholm

    2015-01-01

    Full Text Available Extracellular vesicles (EVs are a heterogeneous population of membrane-enclosed vesicles. EVs are recognized as important players in cell-to-cell communication and are described to be involved in numerous biological and pathological processes. The fact that EVs are involved in the development and progression of several diseases has formed the basis for the use of EV analysis in a clinical setting. As the interest in EVs has increased immensely, multiple techniques have been developed aiming at characterizing these vesicles. These techniques characterize different features of EVs, like the size distribution, enumeration, protein composition, and the intravesicular cargo (e.g., RNA. This review focuses on techniques that exploit the specificity and sensitivity associated with antibody-based assays to characterize the protein phenotype of EVs. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. Thus, protein profiling of EVs holds great diagnostic and prognostic potential.

  15. Antibody-Based Assays for Phenotyping of Extracellular Vesicles.

    Science.gov (United States)

    Pugholm, Lotte Hatting; Revenfeld, Anne Louise Schacht; Søndergaard, Evo Kristina Lindersson; Jørgensen, Malene Møller

    2015-01-01

    Extracellular vesicles (EVs) are a heterogeneous population of membrane-enclosed vesicles. EVs are recognized as important players in cell-to-cell communication and are described to be involved in numerous biological and pathological processes. The fact that EVs are involved in the development and progression of several diseases has formed the basis for the use of EV analysis in a clinical setting. As the interest in EVs has increased immensely, multiple techniques have been developed aiming at characterizing these vesicles. These techniques characterize different features of EVs, like the size distribution, enumeration, protein composition, and the intravesicular cargo (e.g., RNA). This review focuses on techniques that exploit the specificity and sensitivity associated with antibody-based assays to characterize the protein phenotype of EVs. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. Thus, protein profiling of EVs holds great diagnostic and prognostic potential.

  16. Cdk5 is essential for synaptic vesicle endocytosis

    DEFF Research Database (Denmark)

    Tan, Timothy C; Valova, Valentina A; Malladi, Chandra S

    2003-01-01

    Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin...

  17. Transcutol containing vesicles for topical delivery of minoxidil.

    Science.gov (United States)

    Mura, Simona; Manconi, Maria; Valenti, Donatella; Sinico, Chiara; Vila, Amparo Ofelia; Fadda, Anna Maria

    2011-04-01

    The aim of this work was to evaluate the ability of Transcutol (Trc) to produce elastic vesicles with soy lecithin (SL) and study the influence of the obtained vesicles on in vitro (trans)dermal delivery of minoxidil. To this purpose, so-called penetration enhancer-containing vesicles (PEVs) were prepared using Trc aqueous solutions (5-10-20-30% v/v) as hydrophilic phase. SL liposomes, without Trc, were used as control. Prepared formulations were characterized in terms of size distribution, morphology, zeta potential, deformability, and rheological behavior. The influence of the obtained PEVs on (trans)dermal delivery of minoxidil was studied by in vitro diffusion experiments through pig skin. Results showed that all prepared PEVs were able to give good entrapment efficiency (E%≈67) similar to that of conventional liposomes. Trc-containing PEVs showed to be more deformable than liposomes only when minoxidil was loaded in 5 and 10% Trc-containing vesicles. Rheological studies showed that PEVs have higher fluidity than conventional liposomes. All PEVs showed a higher stability than liposomes as shown by studying zeta potential and size distribution during three months. Results of in vitro diffusion experiments showed that Trc-containing PEVs are able to deliver minoxidil to deep skin layers without any transdermal permeation.

  18. Ultrasound-guided seminal vesicle biopsies in prostate cancer

    NARCIS (Netherlands)

    Wymenga, LFA; Duisterwinkel, FJ; Groenier, K; Mensink, HJA

    2000-01-01

    Invasion of prostatic adenocarcinoma into the seminal vesicles (SV) is generally accepted as an index of poor prognosis. The pre-operative identification of SV invasion is an important element in staging since it may alter subsequent treatment decisions. We studied the possibility of diagnosing SV

  19. Expression of Vesicular Nucleotide Transporter in Rat Odontoblasts

    International Nuclear Information System (INIS)

    Ikeda, Erina; Goto, Tetsuya; Gunjigake, Kaori; Kuroishi, Kayoko; Ueda, Masae; Kataoka, Shinji; Toyono, Takashi; Nakatomi, Mitsushiro; Seta, Yuji; Kitamura, Chiaki; Nishihara, Tatsuji; Kawamoto, Tatsuo

    2016-01-01

    Several theories have been proposed regarding pain transmission mechanisms in tooth. However, the exact signaling mechanism from odontoblasts to pulp nerves remains to be clarified. Recently, ATP-associated pain transmission has been reported, but it is unclear whether ATP is involved in tooth pain transmission. In the present study, we focused on the vesicular nucleotide transporter (VNUT), a transporter of ATP into vesicles, and examined whether VNUT was involved in ATP release from odontoblasts. We examined the expression of VNUT in rat pulp by RT-PCR and immunostaining. ATP release from cultured odontoblast-like cells with heat stimulation was evaluated using ATP luciferase methods. VNUT was expressed in pulp tissue, and the distribution of VNUT-immunopositive vesicles was confirmed in odontoblasts. In odontoblasts, some VNUT-immunopositive vesicles were colocalized with membrane fusion proteins. Additionally P2X 3 , an ATP receptor, immunopositive axons were distributed between odontoblasts. The ATP release by thermal stimulation from odontoblast-like cells was inhibited by the addition of siRNA for VNUT. These findings suggest that cytosolic ATP is transported by VNUT and that the ATP in the vesicles is then released from odontoblasts to ATP receptors on axons. ATP vesicle transport in odontoblasts seems to be a key mechanism for signal transduction from odontoblasts to axons in the pulp

  20. Decreased intestinal calcium absorption in vivo and normal brush border membrane vesicle calcium uptake in cortisol-treated chickens: evidence for dissociation of calcium absorption from brush border vesicle uptake.

    OpenAIRE

    Shultz, T D; Bollman, S; Kumar, R

    1982-01-01

    The influence of cortisol on intestinal calcium transport was studied in isolated duodenal loops and brush border membrane (BBM) vesicles of vitamin D-deficient or replete chickens. Four- to five-week-old vitamin D-deficient cockerels were dosed intraperitoneally with 1 microgram of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] alone 15 hr before sacrifice or in combination with 1, 3, or 5 mg of cortisol 24 and 48 hr before sacrifice. After a 1-microgram dose of 1,25-)OH)2D3 the in situ intestinal ...

  1. The Molecular Chaperone Hsc70 Interacts with Tyrosine Hydroxylase to Regulate Enzyme Activity and Synaptic Vesicle Localization.

    Science.gov (United States)

    Parra, Leonardo A; Baust, Tracy B; Smith, Amanda D; Jaumotte, Juliann D; Zigmond, Michael J; Torres, Soledad; Leak, Rehana K; Pino, Jose A; Torres, Gonzalo E

    2016-08-19

    We previously reported that the vesicular monoamine transporter 2 (VMAT2) is physically and functionally coupled with Hsc70 as well as with the dopamine synthesis enzymes tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase, providing a novel mechanism for dopamine homeostasis regulation. Here we expand those findings to demonstrate that Hsc70 physically and functionally interacts with TH to regulate the enzyme activity and synaptic vesicle targeting. Co-immunoprecipitation assays performed in brain tissue and heterologous cells demonstrated that Hsc70 interacts with TH and aromatic amino acid decarboxylase. Furthermore, in vitro binding assays showed that TH directly binds the substrate binding and carboxyl-terminal domains of Hsc70. Immunocytochemical studies indicated that Hsc70 and TH co-localize in midbrain dopaminergic neurons. The functional significance of the Hsc70-TH interaction was then investigated using TH activity assays. In both dopaminergic MN9D cells and mouse brain synaptic vesicles, purified Hsc70 facilitated an increase in TH activity. Neither the closely related protein Hsp70 nor the unrelated Hsp60 altered TH activity, confirming the specificity of the Hsc70 effect. Overexpression of Hsc70 in dopaminergic MN9D cells consistently resulted in increased TH activity whereas knockdown of Hsc70 by short hairpin RNA resulted in decreased TH activity and dopamine levels. Finally, in cells with reduced levels of Hsc70, the amount of TH associated with synaptic vesicles was decreased. This effect was rescued by addition of purified Hsc70. Together, these data demonstrate a novel interaction between Hsc70 and TH that regulates the activity and localization of the enzyme to synaptic vesicles, suggesting an important role for Hsc70 in dopamine homeostasis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. NMR studies of cation transport across membranes

    Energy Technology Data Exchange (ETDEWEB)

    Shochet, N.R.

    1985-01-01

    /sup 23/Na NMR Studies of cation transport across membranes were conducted both on model and biological membranes. Two ionophores, the carrier monensin and the channel-former gramicidin, were chosen to induce cation transport in large unilamellar phosphatidylcholine vesicles. The distinction between the NMR signals arising from the two sides of the membrane was achieved by the addition of an anionic paramagnetic shift reagent to the outer solution. The kinetics of the cation transport across the membrane was observed simultaneously monitoring the changes in the /sup 23/Na NMR signals of both compartments. Two mathematical models were developed for the estimation of the transport parameters of the monensin- and gramicidin-induced cation transport. The models were able to fit the experimental data very well. A new method for the estimation of the volume trapped inside the vesicles was developed. The method uses the relative areas of the intra- and extravesicular NMR signals arising from a suspension of vesicles bathed in the same medium they contain, as a measure for the relative volumes of these compartments. Sodium transport across biological membranes was studied by /sup 23/ NMR, using suspensions of cultured nerve cells. The sodium influx through voltage-gated channels was studied using the channel modifier batrachotoxin in combination with scorpion toxin.

  3. Interactions of Phospholipid Vesicles with Cationic and Anionic Oligomeric Surfactants.

    Science.gov (United States)

    Chen, Yao; Qiao, Fulin; Fan, Yaxun; Han, Yuchun; Wang, Yilin

    2017-07-27

    This work studied the interactions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with cationic ammonium surfactants and anionic sulfate or sulfonate surfactants of different oligomeric degrees, including cationic monomeric DTAB, dimeric C 12 C 3 C 12 Br 2 , and trimeric DDAD as well as anionic monomeric SDS, dimeric C 12 C 3 C 12 (SO 3 ) 2 , and trimeric TED-(C 10 SO 3 Na) 3 . The partition coefficient P of these surfactants between the DOPC vesicles and water was determined with isothermal titration microcalorimetry (ITC) by titrating concentrated DOPC solution into the monomer solution of these surfactants. It was found that the P value increases with the increase of the surfactant oligomeric degree. Moreover, the enthalpy change and the Gibbs free energy for the transition of these surfactants from water into the DOPC bilayer become more negative with increasing the oligomeric degree. Meanwhile, the calcein release experiment proves that the surfactant with a higher oligomeric degree shows stronger ability of changing the permeability of the DOPC vesicles. Furthermore, the solubilization of the DOPC vesicles by these oligomeric surfactants was studied by ITC, turbidity, and dynamic light scattering, and thus the phase boundaries for the surfactant/lipid mixtures have been determined. The critical surfactant to lipid ratios for the onset and end of the solubilization for the DOPC vesicles derived from the phase boundaries decrease remarkably with increasing the oligomeric degree. Overall, the surfactant with a larger oligomerization degree shows stronger ability in incorporating into the lipid bilayer, altering the membrane permeability and solubilizing lipid vesicles, which provides comprehensive understanding about the effects of structure and shape of oligomeric surfactant molecules on lipid-surfactant interactions.

  4. Energetics and mechanism of drug transport mediated by the lactococcal multidrug transporter LmrP

    NARCIS (Netherlands)

    Bolhuis, H; van Veen, H.W.; Brands, J.R; Putman, M; Poolman, B.; Driessen, A.J.M.; Konings, W.N

    1996-01-01

    The gene encoding the secondary multidrug transporter LmrP of Lactococcus lactis was heterologously expressed in Escherichia coli. The energetics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E. coli, LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+)

  5. Emergent properties arising from the assembly of amphiphiles. Artificial vesicle membranes as reaction promoters and regulators.

    Science.gov (United States)

    Walde, Peter; Umakoshi, Hiroshi; Stano, Pasquale; Mavelli, Fabio

    2014-09-14

    This article deals with artificial vesicles and their membranes as reaction promoters and regulators. Among the various molecular assemblies which can form in an aqueous medium from amphiphilic molecules, vesicle systems are unique. Vesicles compartmentalize the aqueous solution in which they exist, independent on whether the vesicles are biological vesicles (existing in living systems) or whether they are artificial vesicles (formed in vitro from natural or synthetic amphiphiles). After the formation of artificial vesicles, their aqueous interior (the endovesicular volume) may become - or may be made - chemically different from the external medium (the exovesicular solution), depending on how the vesicles are prepared. The existence of differences between endo- and exovesicular composition is one of the features on the basis of which biological vesicles contribute to the complex functioning of living organisms. Furthermore, artificial vesicles can be formed from mixtures of amphiphiles in such a way that the vesicle membranes become molecularly, compositionally and organizationally highly complex, similarly to the lipidic matrix of biological membranes. All the various properties of artificial vesicles as membranous compartment systems emerge from molecular assembly as these properties are not present in the individual molecules the system is composed of. One particular emergent property of vesicle membranes is their possible functioning as promoters and regulators of chemical reactions caused by the localization of reaction components, and possibly catalysts, within or on the surface of the membranes. This specific feature is reviewed and highlighted with a few selected examples which range from the promotion of decarboxylation reactions, the selective binding of DNA or RNA to suitable vesicle membranes, and the reactivation of fragmented enzymes to the regulation of the enzymatic synthesis of polymers. Such type of emergent properties of vesicle membranes may

  6. Electron tomography of fusiform vesicles and their organization in urothelial cells.

    Directory of Open Access Journals (Sweden)

    Samo Hudoklin

    Full Text Available The formation of fusiform vesicles (FVs is one of the most distinctive features in the urothelium of the urinary bladder. FVs represent compartments for intracellular transport of urothelial plaques, which modulate the surface area of the superficial urothelial (umbrella cells during the distension-contraction cycle. We have analysed the three-dimensional (3D structure of FVs and their organization in umbrella cells of mouse urinary bladders. Compared to chemical fixation, high pressure freezing gave a new insight into the ultrastructure of urothelial cells. Electron tomography on serial sections revealed that mature FVs had a shape of flattened discs, with a diameter of up to 1.2 µm. The lumen between the two opposing asymmetrically thickened membranes was very narrow, ranging from 5 nm to 10 nm. Freeze-fracturing and immunolabelling confirmed that FVs contain two opposing urothelial plaques connected by a hinge region that made an omega shaped curvature. In the central cytoplasm, 4-15 FVs were often organized into stacks. In the subapical cytoplasm, FVs were mainly organized as individual vesicles. Distension-contraction cycles did not affect the shape of mature FVs; however, their orientation changed from parallel in distended to perpendicular in contracted bladder with respect to the apical plasma membrane. In the intermediate cells, shorter and more dilated immature FVs were present. The salient outcome from this research is the first comprehensive, high resolution 3D view of the ultrastructure of FVs and how they are organized differently depending on their location in the cytoplasm of umbrella cells. The shape of mature FVs and their organization into tightly packed stacks makes them a perfect storage compartment, which transports large amounts of urothelial plaques while occupying a small volume of umbrella cell cytoplasm.

  7. Genetically controlled fusion, exocytosis and fission of artificial vesicles-a roadmap

    DEFF Research Database (Denmark)

    Bönzli, Eva; Hadorn, Maik; de Lucrezia, Davide

    2011-01-01

    enclosed synthesized peptides in vesicles to induce in a next step fusion of adjacent vesicles, fission and exocytosis of nested vesicles. Second, we will replace the peptides by an enclosed cell-free expression system to internally synthesize fusion peptides. To control the gene expression, different...... of principle. Furthermore, we optimized the already established protocol (Hadorn et al. 2011) to produce nested vesicles in the presence of peptides. This project may present an important step towards an artificial cell. Especially vesicle division is discussed as one of the upcoming challenges in designing...

  8. 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.

  9. The 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

    2013-01-01

    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. PMID:23766262

  10. The pustule in palmoplantar psoriasis: transformed vesicle or mature microabscess? A three-dimensional study.

    Science.gov (United States)

    Krieg, P H; Bacharach-Buhles, M; el-Gammal, S; Altmeyer, P

    1992-01-01

    Psoriasis on the palms and soles is characterized by pustules, vesicles and hyperkeratotic plaques. Several studies have postulated that the pustules and vesicles are round or egg-shaped. This three-dimensional study reveals that neither pustules nor vesicles have a regular spherical morphology. The vesicles are located in the upper and middle layers of the epidermis and may exhibit dumb-bell- or banana-shaped lumina. The pustules consist of different compartments combining characteristics of a dyshidrotic vesicle with those of a microabscess of Munro.

  11. 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)

    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; 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.

  12. 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

  13. Fusion of Sendai virus with vesicles of oligomerizable lipids: a microcalorimetric analysis of membrane fusion.

    Science.gov (United States)

    Ravoo, B J; Weringa, W D; Engberts, J B

    2000-01-01

    Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se. Copyright 2000 Academic Press.

  14. Membrane vesicles in sea water: heterogeneous DNA content and implications for viral abundance estimates

    Science.gov (United States)

    Biller, Steven J; McDaniel, Lauren D; Breitbart, Mya; Rogers, Everett; Paul, John H; Chisholm, Sallie W

    2017-01-01

    Diverse microbes release membrane-bound extracellular vesicles from their outer surfaces into the surrounding environment. Vesicles are found in numerous habitats including the oceans, where they likely have a variety of functional roles in microbial ecosystems. Extracellular vesicles are known to contain a range of biomolecules including DNA, but the frequency with which DNA is packaged in vesicles is unknown. Here, we examine the quantity and distribution of DNA associated with vesicles released from five different bacteria. The average quantity of double-stranded DNA and size distribution of DNA fragments released within vesicles varies among different taxa. Although some vesicles contain sufficient DNA to be visible following staining with the SYBR fluorescent DNA dyes typically used to enumerate viruses, this represents only a small proportion (vesicles. Thus DNA is packaged heterogeneously within vesicle populations, and it appears that vesicles are likely to be a minor component of SYBR-visible particles in natural sea water compared with viruses. Consistent with this hypothesis, chloroform treatment of coastal and offshore seawater samples reveals that vesicles increase epifluorescence-based particle (viral) counts by less than an order of magnitude and their impact is variable in space and time. PMID:27824343

  15. 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.

  16. Dynamics of fatty acid vesicles in response to pH stimuli

    DEFF Research Database (Denmark)

    Ikari, Keita; Sakuma, Yuka; Jimbo, Takehiro

    2015-01-01

    We investigate the dynamics of decanoic acid/decanoate (DA) vesicles in response to pH stimuli. Two types of dynamic processes induced by the micro injection of NaOH solutions are sequentially observed: deformations and topological transitions. In the deformation stage, DA vesicles show a series...... of shape deformations, i.e., prolate–oblate–stomatocyte-sphere. In the topological transition stage, spherical DA vesicles follow either of the two pathways, pore formation and vesicle fusion. The pH stimuli modify a critical aggregation concentration of DA molecules, which causes the solubilization of DA...... molecules in the outer leaflet of the vesicle bilayers. This solubilization decreases the outer surface area of the vesicle, thereby increasing surface tension. A kinetic model based on area difference elasticity theory can accurately describe the dynamics of DA vesicles triggered by pH stimuli....

  17. Lipid vesicle shape analysis from populations using light video microscopy and computer vision.

    Directory of Open Access Journals (Sweden)

    Jernej Zupanc

    Full Text Available We present a method for giant lipid vesicle shape analysis that combines manually guided large-scale video microscopy and computer vision algorithms to enable analyzing vesicle populations. The method retains the benefits of light microscopy and enables non-destructive analysis of vesicles from suspensions containing up to several thousands of lipid vesicles (1-50 µm in diameter. For each sample, image analysis was employed to extract data on vesicle quantity and size distributions of their projected diameters and isoperimetric quotients (measure of contour roundness. This process enables a comparison of samples from the same population over time, or the comparison of a treated population to a control. Although vesicles in suspensions are heterogeneous in sizes and shapes and have distinctively non-homogeneous distribution throughout the suspension, this method allows for the capture and analysis of repeatable vesicle samples that are representative of the population inspected.

  18. Seminal vesicle intraepithelial involvement by prostate cancer: putative mechanism and clinicopathological significance.

    Science.gov (United States)

    Miyai, Kosuke; Kristiansen, Anna; Egevad, Lars; Pina-Oviedo, Sergio; Divatia, Mukul K; Shen, Steven S; Miles, Brian J; Ayala, Alberto G; Park, Yong Wook; Ro, Jae Y

    2014-09-01

    We have recently shown seminal vesicle intraepithelial involvement of prostate cancer in cases with seminal vesicle invasion (pT3b). Based on the manner of seminal vesicle invasion, there could be 2 possible mechanisms of seminal vesicle intraepithelial involvement: direct intraepithelial invasion from prostate carcinoma in the muscular wall of seminal vesicles or intraepithelial involvement of cancer from the invaginated extraprostatic space (IES)/ejaculatory duct system to extraprostatic seminal vesicle. We aimed to clarify the manner and clinicopathological significance of seminal vesicle intraepithelial involvement. Of 1629 consecutive radical prostatectomies, 109 cases (6.7%) showed seminal vesicle invasion in whole-mounted radical prostatectomy specimens. In these pT3b cases, 18 (17%) showed seminal vesicle intraepithelial involvement by prostate cancer. Stromal invasion of the IES/ejaculatory duct system and ejaculatory duct intraepithelial invasion by prostate cancer were identified in 62 and 5 of 109 pT3b cases, respectively. However, the presence/absence of IES/ejaculatory duct system involvement by prostate cancer does not predict seminal vesicle intraepithelial involvement. No statistically significant correlation was observed between all pathologic parameters/biochemical recurrence and the presence/absence of seminal vesicle intra-epithelial involvement in the pT3b cases. These findings suggest that seminal vesicle intraepithelial involvement is more likely due to direct invasion of carcinoma from the muscular wall of seminal vesicles rather than intraepithelial extension from the ejaculatory duct system in the IES. Further studies with a substantially greater case number are needed to clarify the clinicopathological significance of seminal vesicle intraepithelial involvement in a better manner. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Comparative Study of Extracellular Vesicles from the Urine of Healthy Individuals and Prostate Cancer Patients.

    Directory of Open Access Journals (Sweden)

    Olga E Bryzgunova

    Full Text Available Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90-95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.

  20. Comparative Study of Extracellular Vesicles from the Urine of Healthy Individuals and Prostate Cancer Patients.

    Science.gov (United States)

    Bryzgunova, Olga E; Zaripov, Marat M; Skvortsova, Tatyana E; Lekchnov, Evgeny A; Grigor'eva, Alina E; Zaporozhchenko, Ivan A; Morozkin, Evgeny S; Ryabchikova, Elena I; Yurchenko, Yuri B; Voitsitskiy, Vladimir E; Laktionov, Pavel P

    2016-01-01

    Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90-95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.

  1. Signal focusing through active transport

    Science.gov (United States)

    Godec, Aljaž; Metzler, Ralf

    2015-07-01

    The accuracy of molecular signaling in biological cells and novel diagnostic devices is ultimately limited by the counting noise floor imposed by the thermal diffusion. Motivated by the fact that messenger RNA and vesicle-engulfed signaling molecules transiently bind to molecular motors and are actively transported in biological cells, we show here that the random active delivery of signaling particles to within a typical diffusion distance to the receptor generically reduces the correlation time of the counting noise. Considering a variety of signaling particle sizes from mRNA to vesicles and cell sizes from prokaryotic to eukaryotic cells, we show that the conditions for active focusing—faster and more precise signaling—are indeed compatible with observations in living cells. Our results improve the understanding of molecular cellular signaling and novel diagnostic devices.

  2. Significance of Extracellular Vesicles: Pathobiological Roles in Disease.

    Science.gov (United States)

    Yamamoto, Seiji; Azuma, Erika; Muramatsu, Masashi; Hamashima, Takeru; Ishii, Yoko; Sasahara, Masakiyo

    2016-11-25

    Over the past decade, many studies have been conducted on extracellular vesicles (EVs) in the fields of basic and clinical research. EVs are small sized membranous vesicles generated from many type of cells upon activation by environmental stresses such as heat, hypoxia, and irradiation. EVs theoretically consist of microparticles/microvesicles, exosomes, and apoptotic bodies by different productive mechanisms. Clinically, EVs are observed in the blood stream of patients suffering from acute and chronic inflammation evoked by various diseases, and number of EVs in blood flow is often dependent on the inflammatory status and severity of the diseases. To date, it has been reported that small molecules such as RNAs and proteins are encapsulated in EVs; however, the functions of EVs are still unclear in the biological, pathological, and clinical aspects. In this review, we summarize and discuss the biogenesis-based classification, expected function, and pathobiological activities of EVs.

  3. Emerging roles of extracellular vesicles in cellular senescence and aging.

    Science.gov (United States)

    Takasugi, Masaki

    2018-02-01

    Cellular senescence is a cellular program that prevents the proliferation of cells at risk of neoplastic transformation. On the other hand, age-related accumulation of senescent cells promotes aging at least partially due to the senescence-associated secretory phenotype, whereby cells secrete high levels of inflammatory cytokines, chemokines, and matrix metalloproteinases. Emerging evidence, however, indicates that extracellular vesicles (EVs) are important mediators of the effects of senescent cells on their microenvironment. Senescent cells secrete more EphA2 and DNA via EVs, which can promote cancer cell proliferation and inflammation, respectively. Extracellular vesicles secreted from DNA-damaged cells can also affect telomere regulation. Furthermore, it has now become clear that EVs actually play important roles in many aspects of aging. This review is intended to summarize these recent progresses, with emphasis on relationships between cellular senescence and EVs. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  4. Extracellular Vesicles as Therapeutic Agents in Systemic Lupus Erythematosus.

    Science.gov (United States)

    Perez-Hernandez, Javier; Redon, Josep; Cortes, Raquel

    2017-03-28

    Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease that affects multiple organs. Currently, therapeutic molecules present adverse side effects and are only effective in some SLE patient subgroups. Extracellular vesicles (EV), including exosomes, microvesicles and apoptotic bodies, are released by most cell types, carry nucleic acids, proteins and lipids and play a crucial role in cell-to-cell communication. EVs can stimulate or suppress the immune responses depending on the context. In SLE, EVs can work as autoadjuvants, enhance immune complex formation and maintaining inflammation state. Over the last years, EVs derived from mesenchymal stem cells and antigen presenting cells have emerged as cell-free therapeutic agents to treat autoimmune and inflammatory diseases. In this review, we summarize the current therapeutic applications of extracellular vesicles to regulate immune responses and to ameliorate disease activity in SLE and other autoimmune disorders.

  5. Complex motions of vesicles and capsules in flow

    Science.gov (United States)

    Vlahovska, Petia; Young, Yuan-Nan; Misbah, Chaouqi

    2009-11-01

    Membrane-bound particles exhibit rich dynamics when placed in flow. For example, in simple shear flow, vesicles made of lipid bilayers tank-tread or tumble. Capsules and red blood cells also show oscillations in the tank-treading inclination angle, called swinging. This motion originates from membrane shear--elasticity and non--spherical unstressed shape. We develop an analytical theory that quantitatively describes the swinging dynamics. Our analysis takes into account that the membrane is deformable, incompressible, and resists bending and shearing. Analytical results for the shape evolution are derived by considering a nearly-spherical particle shape. The phase diagram is constructed and compared to previous models which assume fixed ellipsoidal shape. Dynamics in quadratic and time-dependent flows is also discussed. Floquet analysis is conducted to investigate the vesicle dynamics and conditions for chaotic shape and flow dynamics are established.

  6. Thermal and active fluctuations of a compressible bilayer vesicle

    Science.gov (United States)

    Sachin Krishnan, T. V.; Yasuda, Kento; Okamoto, Ryuichi; Komura, Shigeyuki

    2018-05-01

    We discuss thermal and active fluctuations of a compressible bilayer vesicle by using the results of hydrodynamic theory for vesicles. Coupled Langevin equations for the membrane deformation and the density fields are employed to calculate the power spectral density matrix of membrane fluctuations. Thermal contribution is obtained by means of the fluctuation dissipation theorem, whereas active contribution is calculated from exponentially decaying time correlation functions of active random forces. We obtain the total power spectral density as a sum of thermal and active contributions. An apparent response function is further calculated in order to compare with the recent microrheology experiment on red blood cells. An enhanced response is predicted in the low-frequency regime for non-thermal active fluctuations.

  7. Controlled release of hydrophilic guest molecules from photoresponsive nucleolipid vesicles.

    Science.gov (United States)

    Sun, Yawei; Yan, Yongfeng; Wang, Mingqing; Chen, Cuixia; Xu, Hai; Lu, Jian R

    2013-07-10

    Amphiphilic hybrid nucleolipids bear the structural and functional hallmarks of both lipids and nucleic acids and hold great potential for biotechnological applications. However, further tailoring of their structures and properties for specific applications represents a major challenge. We here report a novel design and synthesis of a light-responsive nucleolipid by introducing an o-nitrobenzyl group that acts as a linker between a nucleotide and a lipid. The nucleolipid was applied readily to preparing smart vesicles and encapsulating hydrophilic guest molecules 5(6)-carboxyfluorescein (CF) in their inner aqueous phase. Upon light irradiation, their vesicular structure was disrupted as a result of the photolytic degradation of the nucleotide, resulting in CF release. Furthermore, temporally controlled CF release from these vesicles could be readily realized by turning on and off light. By demonstrating the molecular assembly and photodisassembly cycle, this report aims to stimulate further research exploring practical applications of nucleolipids.

  8. Extracellular vesicles provide a means for tissue crosstalk during exercise

    DEFF Research Database (Denmark)

    Whitham, Martin; Parker, Benjamin L; Friedrichsen, Martin

    2018-01-01

    Exercise stimulates the release of molecules into the circulation, supporting the concept that inter-tissue signaling proteins are important mediators of adaptations to exercise. Recognizing that many circulating proteins are packaged in extracellular vesicles (EVs), we employed quantitative...... proteomic techniques to characterize the exercise-induced secretion of EV-contained proteins. Following a 1-hr bout of cycling exercise in healthy humans, we observed an increase in the circulation of over 300 proteins, with a notable enrichment of several classes of proteins that compose exosomes and small...... vesicles. Pulse-chase and intravital imaging experiments suggested EVs liberated by exercise have a propensity to localize in the liver and can transfer their protein cargo. Moreover, by employing arteriovenous balance studies across the contracting human limb, we identified several novel candidate...

  9. CAPS and Munc13: CATCHRs that SNARE vesicles

    Directory of Open Access Journals (Sweden)

    Declan J James

    2013-12-01

    Full Text Available Abstract. CAPS (Calcium-dependent Activator Protein for Secretion, aka CADPS and Munc13 (Mammalian Unc-13 proteins function to prime vesicles for Ca2+-triggered exocytosis in neurons and neuroendocrine cells. CAPS and Munc13 proteins contain conserved C-terminal domains that promote the assembly of SNARE complexes for vesicle priming. Similarities of the C-terminal domains of CAPS/Munc13 proteins with CATCHR (Complex Associated with Tethering Containing Helical Rods domains in multi-subunit tethering complexes have been reported. Multi-subunit tethering complexes coordinate multiple interactions for SNARE complex assembly at constitutive membrane fusion steps. We review aspects of these diverse tethering and priming factors to identify common operating principles.

  10. Imaging and Quantification of Extracellular Vesicles by Transmission Electron Microscopy.

    Science.gov (United States)

    Linares, Romain; Tan, Sisareuth; Gounou, Céline; Brisson, Alain R

    2017-01-01

    Extracellular vesicles (EVs) are cell-derived vesicles that are present in blood and other body fluids. EVs raise major interest for their diverse physiopathological roles and their potential biomedical applications. However, the characterization and quantification of EVs constitute major challenges, mainly due to their small size and the lack of methods adapted for their study. Electron microscopy has made significant contributions to the EV field since their initial discovery. Here, we describe the use of two transmission electron microscopy (TEM) techniques for imaging and quantifying EVs. Cryo-TEM combined with receptor-specific gold labeling is applied to reveal the morphology, size, and phenotype of EVs, while their enumeration is achieved after high-speed sedimentation on EM grids.

  11. Pressure exerted by a vesicle on a surface

    International Nuclear Information System (INIS)

    Owczarek, A L; Prellberg, T

    2014-01-01

    Several recent works have considered the pressure exerted on a wall by a model polymer. We extend this consideration to vesicles attached to a wall, and hence include osmotic pressure. We do this by considering a two-dimensional directed model, namely that of area-weighted Dyck paths. Not surprisingly, the pressure exerted by the vesicle on the wall depends on the osmotic pressure inside, especially its sign. Here, we discuss the scaling of this pressure in the different regimes, paying particular attention to the crossover between positive and negative osmotic pressure. In our directed model, there exists an underlying Airy function scaling form, from which we extract the dependence of the bulk pressure on small osmotic pressures. (paper)

  12. Lubrication synergy: Mixture of hyaluronan and dipalmitoylphosphatidylcholine (DPPC) vesicles

    DEFF Research Database (Denmark)

    Raj, Akanksha; Wang, Min; Zander, Thomas

    2017-01-01

    consisting of non-homogeneous phospholipid bilayer with hyaluronan/DPPC aggregates on top. The presence of these aggregates generates a long-range repulsive surface force as two such surfaces are brought together. However, the aggregates are easily deformed, partly rearranged into multilayer structures......Phospholipids and hyaluronan have been implied to fulfil important roles in synovial joint lubrication. Since both components are present in synovial fluid, self-assembly structures formed by them should also be present. We demonstrate by small angle X-ray scattering that hyaluronan associates...... with the outer shell of dipalmitoylphophatidylcholine (DPPC) vesicles in bulk solution. Further, we follow adsorption to silica from mixed hyaluronan/DPPC vesicle solution by Quartz Crystal Microbalance with Dissipation measurements. Atomic Force Microscope imaging visualises the adsorbed layer structure...

  13. Pulmonary Extracellular Vesicles as Mediators of Local and Systemic Inflammation

    OpenAIRE

    Wahlund, Casper J. E.; Eklund, Anders; Grunewald, Johan; Gabrielsson, Susanne

    2017-01-01

    Cells of the airways are constantly exposed to environmental hazards including cigarette smoke, irritants, pathogens, and mechanical insults. Maintaining barrier integrity is vital, and mounting responses to threats depends on intercellular communication. Extracellular vesicles (EVs), including exosomes and microvesicles, are major signal mediators between cells, shuttling cargo in health and disease. Depending on the state of the originating cells, EVs are capable of inducing proinflammatory...

  14. Magnetic field alignable domains in phospholipid vesicle membranes containing lanthanides.

    Science.gov (United States)

    Beck, Paul; Liebi, Marianne; Kohlbrecher, Joachim; Ishikawa, Takashi; Rüegger, Heinz; Zepik, Helmut; Fischer, Peter; Walde, Peter; Windhab, Erich

    2010-01-14

    Magnetic fields were applied as a structuring force on phospholipid-based vesicular systems, using paramagnetic lanthanide ions as magnetic handles anchored to the vesicle membrane. Different vesicle formulations were investigated using small angle neutron scattering (SANS) in a magnetic field of up to 8 T, cryo-transmission electron microscopy (cryo-TEM), (31)P NMR spectroscopy, dynamic light scattering (DLS), and permeability measurements with a fluorescent water-soluble marker (calcein). The investigated vesicle formulations consisted usually of 80 mol % of the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 20 mol % of a chelator lipid (DMPE-DTPA; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-diethylenetriaminepentaacetate) with complexed lanthanide ions (Tm(3+), Dy(3+), or La(3+)), and the total lipid concentration was 15 mM. Vesicles containing the paramagnetic lanthanide Tm(3+) or Dy(3+) exhibited a temperature-dependent response to magnetic fields, which can be explained by considering the formation of lipid domains, which upon reaching a critical size become alignable in a magnetic field. The features of this "magnetic field alignable domain model" are as follows: with decreasing temperature (from 30 to 2.5 degrees C) solid domains, consisting mainly of the higher melting phospholipid (DMPE-DTPA.lanthanide), begin to form and grow in size. The domains assemble the large magnetic moments conferred by the lanthanides and orient in magnetic fields. The direction of alignment depends on the type of lanthanide used. The domains orient with their normal parallel to the magnetic field with thulium (Tm(3+)) and perpendicular with dysprosium (Dy(3+)). No magnetic field alignable domains were observed if DMPE-DTPA is replaced either by POPE-DTPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine-diethylenetriamine-pentaacetate) or by DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine).

  15. Potential Roles of Fungal Extracellular Vesicles during Infection

    OpenAIRE

    Joffe, Luna S.; Nimrichter, Leonardo; Rodrigues, Marcio L.; Del Poeta, Maurizio

    2016-01-01

    ABSTRACT Extracellular vesicles (EVs) are produced by virtually all cell types. Within the past few years, work in this field has revealed more information about fungal EVs. Fungal EVs have been shown to carry proteins, lipids, pigments, polysaccharides, and RNA; these components are known virulence factors, a fact which supports the hypothesis that fungal EVs concentrate pathogenic determinants. Additionally, recent studies have demonstrated that fungal EVs stimulate the host immune system. ...

  16. 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

  17. Dimensional characterization of extracellular vesicles using atomic force microscopy

    International Nuclear Information System (INIS)

    Sebaihi, N; De Boeck, B; Pétry, J; Yuana, Y; Nieuwland, R

    2017-01-01

    Extracellular vesicles (EV) are small biological entities released from cells into body fluids. EV are recognized as mediators in intercellular communication and influence important physiological processes. It has been shown that the concentration and composition of EV in body fluids may differ from healthy subjects to patients suffering from particular disease. So, EV have gained a strong scientific and clinical interest as potential biomarkers for diagnosis and prognosis of disease. Due to their small size, accurate detection and characterization of EV remain challenging. The aim of the presented work is to propose a characterization method of erythrocyte-derived EV using atomic force microscopy (AFM). The vesicles are immobilized on anti-CD235a-modified mica and analyzed by AFM under buffer liquid and dry conditions. EV detected under both conditions show very similar sizes namely ∼30 nm high and ∼90 nm wide. The size of these vesicles remains stable over drying time as long as 7 d at room temperature. Since the detected vesicles are not spherical, EV are characterized by their height and diameter, and not only by the height as is usually done for spherical nanoparticles. In order to obtain an accurate measurement of EV diameters, the geometry of the AFM tip was evaluated to account for the lateral broadening artifact inherent to AFM measurements. To do so, spherical polystyrene (PS) nanobeads and EV were concomitantly deposited on the same mica substrate and simultaneously measured by AFM under dry conditions. By applying this procedure, direct calibration of the AFM tip could be performed together with EV characterization under identical experimental conditions minimizing external sources of uncertainty on the shape and size of the tip, thus allowing standardization of EV measurement. (paper)

  18. Dimensional characterization of extracellular vesicles using atomic force microscopy

    Science.gov (United States)

    Sebaihi, N.; De Boeck, B.; Yuana, Y.; Nieuwland, R.; Pétry, J.

    2017-03-01

    Extracellular vesicles (EV) are small biological entities released from cells into body fluids. EV are recognized as mediators in intercellular communication and influence important physiological processes. It has been shown that the concentration and composition of EV in body fluids may differ from healthy subjects to patients suffering from particular disease. So, EV have gained a strong scientific and clinical interest as potential biomarkers for diagnosis and prognosis of disease. Due to their small size, accurate detection and characterization of EV remain challenging. The aim of the presented work is to propose a characterization method of erythrocyte-derived EV using atomic force microscopy (AFM). The vesicles are immobilized on anti-CD235a-modified mica and analyzed by AFM under buffer liquid and dry conditions. EV detected under both conditions show very similar sizes namely ~30 nm high and ~90 nm wide. The size of these vesicles remains stable over drying time as long as 7 d at room temperature. Since the detected vesicles are not spherical, EV are characterized by their height and diameter, and not only by the height as is usually done for spherical nanoparticles. In order to obtain an accurate measurement of EV diameters, the geometry of the AFM tip was evaluated to account for the lateral broadening artifact inherent to AFM measurements. To do so, spherical polystyrene (PS) nanobeads and EV were concomitantly deposited on the same mica substrate and simultaneously measured by AFM under dry conditions. By applying this procedure, direct calibration of the AFM tip could be performed together with EV characterization under identical experimental conditions minimizing external sources of uncertainty on the shape and size of the tip, thus allowing standardization of EV measurement.

  19. Extracellular Vesicle Isolation and Analysis by Western Blotting.

    Science.gov (United States)

    Kowal, Emma J K; Ter-Ovanesyan, Dmitry; Regev, Aviv; Church, George M

    2017-01-01

    Extracellular vesicles (EVs) are released by mammalian cells and are thought to be important mediators of intercellular communication. There are many methods for isolating EVs from cell culture media, but the most popular methods involve purification based on ultracentrifugation . Here, we provide a detailed protocol for isolating EVs by differential ultracentrifugation and analyzing EV proteins (such as the tetraspanins CD9 , CD63 and CD81 ) by western blotting.

  20. Extracellular Vesicles Produced by the Gram-positive Bacterium Bacillus subtilis are Disrupted by the Lipopeptide Surfactin

    OpenAIRE

    Brown, Lisa; Kessler, Anne; Cabezas-Sanchez, Pablo; Luque-Garcia, Jose L.; Casadevall, Arturo

    2014-01-01

    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...

  1. Magnetic resonance imaging and clinical findings in seminal vesicle pathologies

    Science.gov (United States)

    Ozmen, Zafer; Aktas, Fatma; Uluocak, Nihat; Albayrak, Eda; Altunkaş, Ayşegül; Çelikyay, Fatih

    2018-01-01

    ABSTRACT Purpose Congenital and acquired pathologies of the seminal vesicles (SV) are rare diseases. The diagnosis of SV anomalies is frequently delayed or wrong due to the rarity of these diseases and the lack of adequate evaluation of SV pathology. For this reason, we aimed to comprehensively evaluate SV pathologies and accompanying genitourinary system abnormalities. Materials and Methods Between March 2012 and December 2015, 1455 male patients with different provisional diagnosis underwent MRI. Congenital and acquired pathology of the SV was identified in 42 of these patients. The patients were categorized according to their SV pathologies. The patients were analyzed in terms of genitourinary system findings associated with SV pathologies. Results SV pathologies were accompanied by other genitourinary system findings. Congenital SV pathologies were bilateral or predominantly in the left SV. Patients with bilateral SV hypoplasia were diagnosed at an earlier age compared to patients with unilateral SV agenesis. There was a significant association between abnormal signal intensity in the SV and benign prostate hypertrophy (BPH) and patient age. Conclusion SV pathologies are rare diseases of the genitourinary system. The association between seminal vesicle pathology and other genitourinary system diseases requires complete genitourinary system evaluation that includes the seminal vesicles. PMID:28853814

  2. Phenotyping of Leukocytes and Leukocyte-Derived Extracellular Vesicles.

    Science.gov (United States)

    Pugholm, Lotte Hatting; Bæk, Rikke; Søndergaard, Evo Kristina Lindersson; Revenfeld, Anne Louise Schacht; Jørgensen, Malene Møller; Varming, Kim

    2016-01-01

    Extracellular vesicles (EVs) have a demonstrated involvement in modulating the immune system. It has been proposed that EVs could be used as biomarkers for detection of inflammatory and immunological disorders. Consequently, it is of great interest to investigate EVs in more detail with focus on immunological markers. In this study, five major leukocyte subpopulations and the corresponding leukocyte-derived EVs were phenotyped with focus on selected immunological lineage-specific markers and selected vesicle-related markers. The leukocyte-derived EVs displayed phenotypic differences in the 34 markers investigated. The majority of the lineage-specific markers used for identification of the parent cell types could not be detected on EVs released from monocultures of the associated cell types. In contrast, the vesicular presentation of CD9, CD63, and CD81 correlated to the cell surface expression of these markers, however, with few exceptions. Furthermore, the cellular expression of CD9, CD63, and CD81 varied between leukocytes present in whole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-related markers may differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.

  3. 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.

  4. Myeloid extracellular vesicles: messengers from the demented brain

    Directory of Open Access Journals (Sweden)

    Annamaria eNigro

    2016-01-01

    Full Text Available Blood-borne monocyte derived cells play a pivotal, initially unrecognized, role in most central nervous system disorders, including diseases initially classified as purely neurodegenerative (i.e. AD, PD, and ALS. Their trafficking to the brain and spinal cord has been extensively studied in classical neuroinflammatory disorders such as multiple sclerosis. Central nervous system resident myeloid cells, namely microglia and perivascular macrophages, also are in the spotlight of investigations on neurological disorders. Myeloid cells, such as infiltrating macrophages and microglia, have been described as having both protective and destructive features in neurological disorders, thus identification of their functional phenotype during disease evolution would be of paramount importance. Extracellular vesicles, namely exosomes and shed vesicles, are released by virtually any cell type and can be detected and identified in terms of cell origin in biological fluids. They therefore constitute an ideal tool to access information on cells residing in an inaccessible site such as the brain. We will review here available information on extracellular vesicles detection in neurological disorders with special emphasis on neurodegenerative diseases.

  5. Outer membrane vesicles of Lysobacter sp. XL1: biogenesis, functions, and applied prospects.

    Science.gov (United States)

    Kudryakova, Irina V; Shishkova, Nina A; Vasilyeva, Natalia V

    2016-06-01

    Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have been intensively investigated in recent times. Vesicle formation models have been proposed, some factors affecting the process were established, and important roles vesicles play in vital activities of their producing cells were determined. Studies of pathogenic bacterial vesicles contribute to understanding the causes of acute infection and developing drugs on their basis. Despite intensive research, issues associated with the understanding of vesicle biogenesis, the mechanisms of bacterium-bacterium and pathogen-host interactions with participation of vesicles, still remain unresolved. This review discusses some results obtained in the research into OMVs of Lysobacter sp. XL1 VKM B-1576. This bacterium secretes into the environment a spectrum of bacteriolytic enzymes that hydrolyze peptidoglycan of competing bacteria, thus leading to their lysis. One of these enzymes, lytic endopeptidase L5, has been shown not only to be secreted by means of vesicles but also to be involved in their formation. As part of vesicles, the antimicrobial potential of L5 enzyme has been found to be considerably expanded. Vesicles have been shown to have a therapeutic effect in respect of anthrax infection and staphylococcal sepsis modelled in mice. The scientific basis for constructing liposomal antimicrobial preparations from vesicle phospholipids and recombinant bacteriolytic enzyme L5 has been formed.

  6. 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 pathophysiological roles. However, certain issues regarding diverse subtypes and the complex pathophysiological roles of extracellular vesicles are not yet clearly elucidated. In this review, we first briefly introduce the complexity of extracellular vesicles in terms of their vesicular cargos and protein-protein interaction networks, their diverse subtypes, and multifaceted pathophysiological functions. Then, we introduce the limitation of reductionist approaches in understanding the complexity of extracellular vesicles. We finally suggest that molecular systems biology approaches based on the concept of emergent properties are essential for a comprehensive understanding of the complex pathophysiological functions of heterogeneous extracellular vesicles, either at the single vesicle level or at a systems level as a whole.

  7. Cycling of dense core vesicles involved in somatic exocytosis of serotonin by leech neurons

    Directory of Open Access Journals (Sweden)

    Citlali eTrueta

    2012-06-01

    Full Text Available We studied the cycling of dense core vesicles producing somatic exocytosis of serotonin. Our experiments were made using electron microscopy and vesicle staining with fluorescent dye FM1-43 in Retzius neurons of the leech, which secrete serotonin from clusters of dense core vesicles in a frequency-dependent manner. Electron micrographs of neurons at rest or after 1 Hz stimulation showed two pools of dense core vesicles. A perinuclear pool near Golgi apparatuses, from which vesicles apparently form, and a peripheral pool with vesicle clusters at a distance from the plasma membrane. By contrast, after 20 Hz electrical stimulation 47% of the vesicle clusters were apposed to the plasma membrane, with some omega exocytosis structures. Dense core and small clear vesicles apparently originating from endocytosis were incorporated in multivesicular bodies. In another series of experiments, neurons were stimulated at 20 Hz while bathed in a solution containing peroxidase. Electron micrographs of these neurons contained gold particles coupled to anti-peroxidase antibodies in dense core vesicles and multivesicular bodies located near the plasma membrane. Cultured neurons depolarized with high potassium in the presence of FM1-43 displayed superficial fluorescent spots, each reflecting a vesicle cluster. A partial bleaching of the spots followed by another depolarization in the presence of FM1-43 produced restaining of some spots, other spots disappeared, some remained without restaining and new spots were formed. Several hours after electrical stimulation the FM1-43 spots accumulated at the center of the somata. This correlated with electron micrographs of multivesicular bodies releasing their contents near Golgi apparatuses. Our results suggest that dense core vesicle cycling related to somatic serotonin release involves two steps: the production of clear vesicles and multivesicular bodies after exocytosis, and the formation of new dense core vesicles in

  8. Effect of nephrotoxicants on renal membrane transport: In vitro studies

    International Nuclear Information System (INIS)

    Ansari, R.A.; Berndt, W.O.

    1990-01-01

    It is possible to study the effects of nephrotoxicants on membrane function free of other cellular influences. By the use of Percoll gradient centrifugation, highly purified preparations of right-side-out basolateral (BL) and brush border (BB) membrane vesicles can be obtained from rat (male, Sprague-Dawley) renal cortex. Membrane function can be monitored by evaluation of sodium driven transport: 14 C-p-aminohippurate (PAH) for BL and 14 C-glucose for BB. Transport was measured by the rapid filtration technique. Each vesicle preparation was preincubated with the nephrotoxicant for five minutes before initiation of transport. Control vesicles showed a prominant overshoot 1 to 2 minutes after start of transport. Mercuric ion (Hg) had no effect on transport by BB at concentrations as high as 10μM. Transport by BL was reduced significantly at Hg concentrations as low as 100 nM. Chromate (Cr) also reduced BL transport at 100 nM and had no effect on BB transport. Citrinin significantly reduced both BB and BL transport, but the sensitivity of the membrane preparations differed. These data are consistent with the hypothesis that some nephrotoxicants may act on either side of the renal tubular cell membrane

  9. Label-free tracking of single extracellular vesicles in a nano-fluidic optical fiber (Conference Presentation)

    Science.gov (United States)

    van der Pol, Edwin; Weidlich, Stefan; Lahini, Yoav; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk; Schmidt, Markus A.; Faez, Sanli; van Leeuwen, Ton G.

    2016-03-01

    Background: Extracellular vesicles, such as exosomes, are abundantly present in human body fluids. Since the size, concentration and composition of these vesicles change during disease, vesicles have promising clinical applications, including cancer diagnosis. However, since ~70% of the vesicles have a diameter vesicles remains challenging. Thus far, vesicles vesicles to be adhered to a surface. Consequently, the majority of vesicles have never been studied in their physiological environment. We present a novel label-free optical technique to track single vesicles vesicles were contained within a single-mode light-guiding silica fiber containing a 600 nm nano-fluidic channel. Light from a diode laser (660 nm wavelength) was coupled to the fiber, resulting in a strongly confined optical mode in the nano-fluidic channel, which continuously illuminated the freely diffusing vesicles inside the channel. The elastic light scattering from the vesicles, in the direction orthogonal to the fiber axis, was collected using a microscope objective (NA=0.95) and imaged with a home-built microscope. Results: We have tracked single urinary vesicles as small as 35 nm by elastic light scattering. Please note that vesicles are low-refractive index (nvesicles vesicle-based clinical applications.

  10. Transport in Halobacterium Halobium: Light-Induced Cation-Gradients, Amino Acid Transport Kinetics, and Properties of Transport Carriers

    Science.gov (United States)

    Lanyi, Janos K.

    1977-01-01

    Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na(+). Measurements of Na-22 flux, exterior pH change, and membrane potential, Delta(psi) (with the dye 3,3'-dipentyloxadicarbocyanine) indicate that the means of Na(+) transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H(+)/Na(++ greater than 1). The resulting large chemical gradient for Na(+) (outside much greater than inside), as well as the membrane potential, will drive the transport of 18 amino acids. The I9th, glutamate, is unique in that its accumulation is indifferent to Delta(psi): this amino acid is transported only when a chemical gradient for Na(+) is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H+() collapses within 1 min, while the large Na(+) gradient and glutamate transporting activity persists for 10- 15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na(+), arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V(sub max) and K(sub m) comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na(+), in an electrically neutral fashion, so that only the chemical component of the Na(+) gradient is a driving force.

  11. Extracellular Vesicles and Their Role in Urologic Malignancies.

    Science.gov (United States)

    Junker, Kerstin; Heinzelmann, Joana; Beckham, Carla; Ochiya, Takahiro; Jenster, Guido

    2016-08-01

    Research has increased significantly on small vesicles secreted by healthy and diseased cells. Recent discoveries have revealed their functional and biomarker roles in urologic diseases. Whether and how this knowledge of extracellular vesicles (EVs) affects translational research and clinical practices have become pertinent questions. To provide an overview of the currently available literature on the rising field of EVs, focusing on function and pathogenesis in urologic cancers and the usefulness of EVs as biomarkers. A systematic literature search was conducted using PubMed to identify original articles, review articles, and editorials regarding EVs in different types of urologic tumor diseases. Articles published between 2005 and 2015 were reviewed and selected with the consensus of all authors. Besides soluble factors, different types of EVs are involved in the complex cross talk between different cell types. EVs regulate normal physiologic processes like spermatogenesis and renal function, as well as disease-specific processes including bladder, kidney, and prostate cancer. The content of EVs is derived from the cytoplasm of the donor cell. The proteins and RNAs within these EVs can be isolated from body fluids (eg, urine and blood) and represent potential diagnostic and prognostic biomarkers. EVs are also candidate therapeutic targets and potentially useful as therapeutic vehicles. The current data suggest that EVs are important regulators of cell-cell communication. The growing knowledge about their roles in urologic malignancies provides the basis for novel therapeutic strategies. In addition, nucleic acid and the protein content of EVs holds promise for the discovery of urine- or serum-based biomarkers for kidney, bladder, and prostate cancer. Normal and cancer cells secrete small vesicles that contain proteins and RNAs from the cell of origin. Changes in the diseased cells can be detected by examining the altered content of these vesicles when secreted in

  12. Bacterial membrane vesicles, an overlooked environmental colloid: Biology, environmental perspectives and applications.

    Science.gov (United States)

    Toyofuku, Masanori; Tashiro, Yosuke; Hasegawa, Yusuke; Kurosawa, Masaharu; Nomura, Nobuhiko

    2015-12-01

    Phospholipid vesicles play important roles in biological systems. Bacteria are one of the most abundant organisms on Earth, and bacterial membrane vesicles (MVs) were first observed 50 years ago. Many bacteria release MVs to the environment that mainly consist of the cell membrane and typically range from 20 to 400 nm in size. Bacterial MVs are involved in several biological functions, such as delivery of cargo, virulence and gene transfer. MVs can be isolated from laboratory culture and directly from the environment, indicating their high abundance in and impact on ecosystems. Many colloidal particles in the environment ranging in size from 1 nm to 1 μm have been reported but not characterized at the molecular level, and MVs remain to be explored. Hence, MVs can be considered terra incognita in environmental colloid research. Although MV biogenesis and biological roles are yet to be fully understood, the accumulation of knowledge has opened new avenues for their applications. Via genetic engineering, the MV yield can be greatly increased, and the components of MVs can be tailored. Recent studies have demonstrated that MVs have promising potential for applications such as drug delivery systems and nanobiocatalysts. For instance, MV vaccines have been extensively studied and have already been approved in Europe. Recent MV studies have evoked great interest in the fields of biology and biotechnology, but fundamental questions, such as their transport in the environment or physicochemical features of MVs, remain to be addressed. In this review, we present the current understanding of bacterial MVs and environmental perspectives and further introduce their applications. Copyright © 2015. Published by Elsevier B.V.

  13. First in vivo detection and characterization of hyaluronan-coated extracellular vesicles in human synovial fluid.

    Science.gov (United States)

    Mustonen, Anne-Mari; Nieminen, Petteri; Joukainen, Antti; Jaroma, Antti; Kääriäinen, Tommi; Kröger, Heikki; Lázaro-Ibáñez, Elisa; Siljander, Pia R-M; Kärjä, Vesa; Härkönen, Kai; Koistinen, Arto; Rilla, Kirsi

    2016-11-01

    Extracellular vesicles (EVs) function in intercellular signaling by transporting different membrane and cytosolic molecules, including hyaluronan (HA) and its synthesis machinery. As both EVs and HA are abundant in synovial fluid, we hypothesized that HA synthesized in synovial membrane would be carried on the surface of EVs. Synovial fluid (n = 15) and membrane samples (n = 5) were obtained from knee surgery patients. HA concentrations were analyzed in synovial fluid and HA and its synthesis machinery were examined with histochemical stainings in synovial membrane. To assess the size distribution of EVs in synovial fluid and to visualize HA on EVs, nanoparticle tracking analysis (NTA), confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were utilized. The average HA concentration in synovial fluid was 2.0 ± 0.21 mg/ml without significant differences between the patients with trauma/diagnostic arthroscopy and primary or post-traumatic osteoarthritis. Positive stainings of HA synthases (HAS1-3), HA and its receptor CD44 in synovial cells indicated active HA secretion in synovial membrane. According to NTA, EVs were abundant in synovial fluid and their main populations were ≤300 nm in diameter after differential centrifugation. There were no significant differences in the EV counts between the patients with primary or post-traumatic osteoarthritis. TEM verified that HA-positive particles detected by CLSM were lipid membrane vesicles surrounded by a HA coat. Our results provide the first in vivo evidence that human synovial fluid contains HA-positive EVs, one source of which presumably is the long HAS-positive protrusions of synovial fibroblasts. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1960-1968, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  14. Mechanism of ochratoxin A transport in kidney

    Energy Technology Data Exchange (ETDEWEB)

    Sokol, P.P.; Ripich, G.; Holohan, P.D.; Ross, C.R.

    1988-08-01

    The effect of the fungal metabolite (mycotoxin) Ochratoxin A (OTA) on the transport of p-amino(/sup 3/H)hippurate (PAH), a prototypic organic anion, was examined in renal brush border (BBMV) and basolateral membrane vesicles (BLMV). OTA was as effective an inhibitor of PAH uptake in both membranes as probenecid. The dose response curves for OTA in BBMV and BLMV gave IC50 values of 20 +/- 6 and 32 +/- 7 microM, respectively. The effect was specific since the transport of the organic cation N1-methylnicotinamide was not affected. The phenomenon of counterflow was studied to establish that OTA is translocated. OTA produced trans stimulation of PAH transport in both BBMV and BLMV, demonstrating that OTA is transported across both these membranes. The data suggest that OTA interacts with the PAH transport system in both BBMV and BLMV. We conclude that OTA transport in the kidney is mediated via the renal organic anion transport system.

  15. 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...... is observed all over the gradient. The variety of the membrane vesicles is currently being investigated further by several means. Summary/conclusion: A new procedure for easy and gentle isolation of bovine milk membrane vesicles encompassing ultracentrifugation and size-exclusion chromatography has been...... established. The resulting vesicle isolate exhibits the general membrane vesicle characteristics and provides an appropriate start material from which the variety of milk vesicles can be investigated...

  16. Vesicles from Amphiphilic Dumbbells and Janus Dendrimers: Bioinspired Self-Assembled Structures for Biomedical Applications

    Directory of Open Access Journals (Sweden)

    Soraya Taabache

    2017-07-01

    Full Text Available The current review focuses on vesicles obtained from the self-assembly of two types of dendritic macromolecules, namely amphiphilic Janus dendrimers (forming dendrimersomes and amphiphilic dumbbells. In the first part, we will present some synthetic strategies and the various building blocks that can be used to obtain dendritic-based macromolecules, thereby showing their structural versatility. We put our focus on amphiphilic Janus dendrimers and amphiphilic dumbbells that form vesicles in water but we also encompass vesicles formed thereof in organic solvents. The second part of this review deals with the production methods of these vesicles at the nanoscale but also at the microscale. Furthermore, the influence of various parameters (intrinsic to the amphiphilic JD and extrinsic—from the environment on the type of vesicle formed will be discussed. In the third part, we will review the numerous biomedical applications of these vesicles of nano- or micron-size.

  17. The Role of Extracellular Vesicles: An Epigenetic View of the Cancer Microenvironment.

    Science.gov (United States)

    Qian, Zhongrun; Shen, Qi; Yang, Xi; Qiu, Yongming; Zhang, Wenbin

    2015-01-01

    Exosomes, microvesicles, and other extracellular vesicles are released by many cell types, including cancer cells and cancer-related immune cells. Extracellular vesicles can directly or indirectly facilitate the transfer of bioinformation to recipient cells or to the extracellular environment. In cancer, exosomes have been implicated in tumor initiation, proliferation, and metastasis. Extracellular vesicles can transmit proteins and nucleic acids that participate in DNA methylation, histone modification, and posttranscriptional regulation of RNA. Factors transmitted by extracellular vesicles reflect the donor cell status, and extracellular vesicles derived from tumor cells may be also responsible for altering expression of tumor promoting and tumor suppressing genes in recipient cells. Thus, circulating extracellular vesicles may act as biomarkers of cancer, and detection of these biomarkers may be applied to diagnosis or assessment of prognosis in patients with cancer.

  18. 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.

  19. 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.

  20. Inkjet formation of unilamellar lipid vesicles for cell-like encapsulation.

    Science.gov (United States)

    Stachowiak, Jeanne C; Richmond, David L; Li, Thomas H; Brochard-Wyart, Françoise; Fletcher, Daniel A

    2009-07-21

    Encapsulation of macromolecules within lipid vesicles has the potential to drive biological discovery and enable development of novel, cell-like therapeutics and sensors. However, rapid and reliable production of large numbers of unilamellar vesicles loaded with unrestricted and precisely-controlled contents requires new technologies that overcome size, uniformity, and throughput limitations of existing approaches. Here we present a high-throughput microfluidic method for vesicle formation and encapsulation using an inkjet printer at rates up to 200 Hz. We show how multiple high-frequency pulses of the inkjet's piezoelectric actuator create a microfluidic jet that deforms a bilayer lipid membrane, controlling formation of individual vesicles. Variations in pulse number, pulse voltage, and solution viscosity are used to control the vesicle size. As a first step toward cell-like reconstitution using this method, we encapsulate the cytoskeletal protein actin and use co-encapsulated microspheres to track its polymerization into a densely entangled cytoskeletal network upon vesicle formation.

  1. Lactate/H+ transport kinetics in rat skeletal muscle related to fibre type and changes in transport capacity

    DEFF Research Database (Denmark)

    Juel; Pilegaard

    1998-01-01

    and hypothyroidism was due to a decrease in Vmax. The denervation-induced decline in lactate/H+ transport capacity resulted from both an increased Km and a reduced Vmax. The present data show that muscle type differences and most changes in the lactate/H+ transport capacity are mediated by modifications in Vmax......Lactate/H+ transport kinetics were determined by means of the pH-sensitive probe BCECF in sarcolemmal giant vesicles, obtained from rat skeletal muscle, and related to variations in lactate/H+ transport capacity. Vesicle preparations were made from red and white muscles, mixed muscles, denervated...... muscles, muscles of old rats and rats that had been subjected to high-intensity training, endurance training, repeated exposure to hypoxia, and hypothyroid or hyperthyroid treatments. The lactate/H+ transport capacity of red muscles was greater than that of white muscles, and this difference...

  2. Lactate/H+ transport kinetics in rat skeletal muscle related to fibre type and changes in transport capacity

    DEFF Research Database (Denmark)

    Juel; Pilegaard

    1998-01-01

    muscles, muscles of old rats and rats that had been subjected to high-intensity training, endurance training, repeated exposure to hypoxia, and hypothyroid or hyperthyroid treatments. The lactate/H+ transport capacity of red muscles was greater than that of white muscles, and this difference......Lactate/H+ transport kinetics were determined by means of the pH-sensitive probe BCECF in sarcolemmal giant vesicles, obtained from rat skeletal muscle, and related to variations in lactate/H+ transport capacity. Vesicle preparations were made from red and white muscles, mixed muscles, denervated...... and hypothyroidism was due to a decrease in Vmax. The denervation-induced decline in lactate/H+ transport capacity resulted from both an increased Km and a reduced Vmax. The present data show that muscle type differences and most changes in the lactate/H+ transport capacity are mediated by modifications in Vmax...

  3. Na+-independent D-glucose transport in rabbit renal basolateral membranes

    International Nuclear Information System (INIS)

    Cheung, P.T.; Hammerman, M.R.

    1988-01-01

    To define the mechanism by which glucose is transported across the basolateral membrane of the renal proximal tubular cell, we measured D-[14C]glucose uptake in basolateral membrane vesicles from rabbit kidney. Na+-dependent D-glucose transport, demonstrable in brush-border vesicles, could not be demonstrated in basolateral membrane vesicles. In the absence of Na+, the uptake of D-[14C]glucose in basolateral vesicles was more rapid than that of L-[3H]glucose over a concentration range of 1-50 mM. Subtraction of the latter from the former uptakes revealed a saturable process with apparent Km of 9.9 mM and Vmax of 0.80 nmol.mg protein-1.s-1. To characterize the transport component of D-glucose uptake in basolateral vesicles, we measured trans stimulation of 2 mM D-[14C]glucose entry in the absence of Na+. Trans stimulation could be effected by preloading basolateral vesicles with D-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose, but not with L-glucose or alpha-methyl-D-glucoside. Trans-stimulated D-[14C]glucose uptake was inhibited by 0.1 mM phloretin or cytochalasin B but not phlorizin. In contrast, Na+-dependent D-[14C]glucose transport in brush-border vesicles was inhibited by phlorizin but not phloretin or cytochalasin B. Our findings are consistent with the presence of a Na+-independent D-glucose transporter in the proximal tubular basolateral membrane with characteristics similar to those of transporters present in nonepithelial cells

  4. Volume regulatory responses of basolateral membrane vesicles from Necturus enterocytes: Role of the cytoskeleton

    OpenAIRE

    Dubinsky, William P.; Mayorga-Wark, Otilia; Schultz, Stanley G.

    1999-01-01

    Previous studies from this laboratory have demonstrated that basolateral membrane vesicles isolated from Necturus maculosus small intestinal epithelial cells possess a K+ channel that is inhibited by ATP. In the present studies, we demonstrate that these vesicles, which are essentially devoid of soluble cytoplasmic contaminants, exhibit volume regulatory responses that parallel those of intact epithelial cells. Thus, suspension of these vesicles in a solution that is hypotonic to the intraves...

  5. 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 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....

  6. Plasma biomarker discovery in preeclampsia using a novel differential isolation technology for circulating extracellular vesicles.

    Science.gov (United States)

    Tan, Kok Hian; Tan, Soon Sim; Sze, Siu Kwan; Lee, Wai Kheong Ryan; Ng, Mor Jack; Lim, Sai Kiang

    2014-10-01

    To circumvent the complex protein milieu of plasma and discover robust predictive biomarkers for preeclampsia (PE), we investigate if phospholipid-binding ligands can reduce the milieu complexity by extracting plasma extracellular vesicles for biomarker discovery. Cholera toxin B chain (CTB) and annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine were used to isolate extracellular vesicles from plasma of PE patients and healthy pregnant women. The proteins in the vesicles were identified using enzyme-linked immunosorbent assay, antibody array, and mass spectrometry. CTB and AV were found to bind 2 distinct groups of extracellular vesicles. Antibody array and enzyme-linked immunosorbent assay revealed that PE patients had elevated levels of CD105, interleukin-6, placental growth factor, tissue inhibitor of metallopeptidase 1, and atrial natriuretic peptide in cholera toxin B- but not AV-vesicles, and elevated levels of plasminogen activator inhibitor-1, pro-calcitonin, S100b, tumor growth factor β, vascular endothelial growth factor receptor 1, brain natriuretic peptide, and placental growth factor in both cholera toxin B- and AV-vesicles. CD9 level was elevated in cholera toxin B-vesicles but reduced in AV vesicles of PE patients. Proteome analysis revealed that in cholera toxin B-vesicles, 87 and 222 proteins were present only in PE patients and healthy pregnant women respectively while in AV-vesicles, 104 and 157 proteins were present only in PE and healthy pregnant women, respectively. This study demonstrated for the first time that CTB and AV bind unique extracellular vesicles, and their protein cargo reflects the disease state of the patient. The successful use of these 2 ligands to isolate circulating plasma extracellular vesicles for biomarker discovery in PE represents a novel technology for biomarker discovery that can be applied to other specialties. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. 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 ...

  8. Evidence that membrane transduction of oligoarginine does not require vesicle formation

    International Nuclear Information System (INIS)

    Zaro, Jennica L.; Shen Weichiang

    2005-01-01

    The involvement of vesicular formation processes in the membrane transduction and nuclear transport of oligoarginine is currently a subject of controversy. In this report, a novel quantitative method which allows for the selective measurement of membrane transduction excluding concurrent endocytosis was used to determine the effects of temperature, endosomal acidification, endosomolysis, and several known inhibitors of endocytic pathways on the internalization of oligoarginine. The results show that, unlike endocytosis, transduction of oligoarginine was not affected by incubation at 16 deg. C as compared to the 37 deg. C control, and was only partially inhibited at 4 deg. C incubation. Additionally, membrane transduction was not inhibited to the same extent as endocytosis following treatment with ammonium chloride, hypertonic medium, amiloride, or filipin. The endosomolytic activity of oligoarginine was investigated by examining the leakage of FITC-dextran into the cytosolic compartment, which was not higher in the presence of oligoarginine. Furthermore, ammonium chloride showed no effect on the nuclear transport of oligoarginine. The data presented in this report indicate that membrane transduction is likely to occur at the plasma membrane without the formation of membrane vesicles, and the nuclear localization involves membrane transduction, rather than endocytosis of oligoarginine

  9. 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

  10. 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

    2014-07-01

    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 harbouring 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. © 2014 John Wiley & Sons Ltd.

  11. The Influence of Vesicle Shape and Medium Conductivity on Possible Electrofusion under a Pulsed Electric Field.

    Science.gov (United States)

    Liu, Linying; Mao, Zheng; Zhang, Jianhua; Liu, Na; Liu, Qing Huo

    2016-01-01

    The effects of electric field on lipid membrane and cells have been extensively studied in the last decades. The phenomena of electroporation and electrofusion are of particular interest due to their wide use in cell biology and biotechnology. However, numerical studies on the electrofusion of cells (or vesicles) with different deformed shapes are still rare. Vesicle, being of cell size, can be treated as a simple model of cell to investigate the behaviors of cell in electric field. Based on the finite element method, we investigate the effect of vesicle shape on electrofusion of contact vesicles in various medium conditions. The transmembrane voltage (TMV) and pore density induced by a pulsed field are examined to analyze the possibility of vesicle fusion. In two different medium conditions, the prolate shape is observed to have selective electroporation at the contact area of vesicles when the exterior conductivity is smaller than the interior one; selective electroporation is more inclined to be found at the poles of the oblate vesicles when the exterior conductivity is larger than the interior one. Furthermore, we find that when the exterior conductivity is lower than the internal conductivity, the pulse can induce a selective electroporation at the contact area between two vesicles regardless of the vesicle shape. Both of these two findings have important practical applications in guiding electrofusion experiments.

  12. Hydrodynamic interaction between two vesicles in a linear shear flow: asymptotic study.

    Science.gov (United States)

    Gires, P Y; Danker, G; Misbah, C

    2012-07-01

    Interactions between two vesicles in an imposed linear shear flow are studied theoretically, in the limit of almost spherical vesicles, with a large intervesicle distance, in a strong flow, with a large inner to outer viscosity ratio. This allows to derive a system of ordinary equations describing the dynamics of the two vesicles. We provide an analytic expression for the interaction law. We find that when the vesicles are in the same shear plane, the hydrodynamic interaction leads to a repulsion. When they are not, the interaction may turn into attraction instead. The interaction law is discussed and analyzed as a function of relevant parameters.

  13. A preliminary proteomic characterisation of extracellular vesicles released by the ovine parasitic nematode, Teladorsagia circumcincta.

    Science.gov (United States)

    Tzelos, Thomas; Matthews, Jacqueline B; Buck, Amy H; Simbari, Fabio; Frew, David; Inglis, Neil F; McLean, Kevin; Nisbet, Alasdair J; Whitelaw, C Bruce A; Knox, David P; McNeilly, Tom N

    2016-05-15

    Teladorsagia circumcincta is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The development of high levels of anthelmintic resistance in this nematode species challenges its future control. Recent research indicates that many parasite species release extracellular vesicles into their environment, many of which have been classified as endocytic in origin, termed exosomes. These vesicles are considered to play important roles in the intercellular communication between parasites and their hosts, and thus represent potentially useful targets for novel control strategies. Here, we demonstrate that exosome-like extracellular vesicles can be isolated from excretory-secretory (ES) products released by T. circumcincta fourth stage larvae (Tci-L4ES). Furthermore, we perform a comparative proteomic analysis of vesicle-enriched and vesicle-free Tci-L4ES. Approximately 73% of the proteins identified in the vesicle-enriched fraction were unique to this fraction, whilst the remaining 27% were present in both vesicle-enriched and vesicle-free fraction. These unique proteins included structural proteins, nuclear proteins, metabolic proteins, proteolytic enzymes and activation-associated secreted proteins. Finally, we demonstrate that molecules present within the vesicles-enriched material are targets of the IgA and IgG response in T. circumcincta infected sheep, and could potentially represent useful targets for future vaccine intervention studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. 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.

  15. Identification of EDIL3 on extracellular vesicles involved in breast cancer cell invasion.

    Science.gov (United States)

    Lee, Jeong-Eun; Moon, Pyong-Gon; Cho, Young-Eun; Kim, Young-Bum; Kim, In-San; Park, Hoyong; Baek, Moon-Chang

    2016-01-10

    Cancer cell-derived extracellular vesicles have been linked to the pathogenesis of various cancers; however, the role of extracellular vesicles in tumorigenesis remains unclear. To identify extracellular vesicle proteins involved in cancer metastasis, quantitative proteomic analyses were performed on extracellular vesicles derived from two representative breast cancer cell lines: the less invasive MCF-7 and the invasive MDA-MB-231. Proteomic analysis allowed for the identification of 270 proteins in the extracellular vesicles. Here we report a new function of EDIL3 on extracellular vesicles, which are sufficient for enhancement of cell invasion and for acceleration of lung metastasis in vivo. This invasion is most likely mediated via the integrin-FAK signaling cascade in breast cancer cells. However, these effects are suppressed when EDIL3 is inactivated, providing evidence for a critical role of EDIL3 in development of cancer. Consistently, in human patients with metastatic breast cancer, the levels of EDIL3 on circulating extracellular vesicles are significantly elevated. This information is a remarkable breakthrough in understanding of the molecular mechanism underlying metastasis of breast cancer as well as in the research for cancer biomarkers using circulating extracellular vesicles. Furthermore, targeting EDIL3 on extracellular vesicles may lead to a new therapeutic option for treatment of breast cancer. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Extracellular vesicles: An overview of biogenesis, function, and role in breast cancer.

    Science.gov (United States)

    Zha, Quan Bin; Yao, Yu Feng; Ren, Zhao Jun; Li, Xiu Juan; Tang, Jin Hai

    2017-02-01

    Extracellular vesicles have emerged as important mediators of intercellular communication and play an active role in cancer, including breast cancer. Despite limited studies, initial observations suggest that these vesicles are important in breast physiology and pathophysiology. We here, in brief, describe their potential use as future biomarkers and therapeutic agents in breast cancer. Extracellular vesicles in blood and breast fluid may have a great potential to detect and predict the presence of breast cancer, and extracellular vesicles modulation may emerge as a therapeutic approach in cancer therapy.

  17. Antibody Binding Alters the Characteristics and Contents of Extracellular Vesicles Released by Histoplasma capsulatum.

    Science.gov (United States)

    Matos Baltazar, Ludmila; Nakayasu, Ernesto S; Sobreira, Tiago J P; Choi, Hyungwon; Casadevall, Arturo; Nimrichter, Leonardo; Nosanchuk, Joshua D

    2016-01-01

    Histoplasma capsulatum produces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment of H. capsulatum cells with monoclonal antibodies (MAbs) can change the outcome of infection in mice. We evaluated the sizes, enzymatic contents, and proteomic profiles of the vesicles released by fungal cells treated with either protective MAb 6B7 (IgG1) or nonprotective MAb 7B6 (IgG2b), both of which bind H. capsulatum heat shock protein 60 (Hsp60). Our results showed that treatment with either MAb was associated with changes in size and vesicle loading. MAb treatments reduced vesicle phosphatase and catalase activities compared to those of vesicles from untreated controls. We identified 1,125 proteins in vesicles, and 250 of these manifested differences in abundance relative to that of proteins in vesicles isolated from yeast cells exposed to Hsp60-binding MAbs, indicating that surface binding of fungal cells by MAbs modified protein loading in the vesicles. The abundance of upregulated proteins in vesicles upon MAb 7B6 treatment was 44.8% of the protein quantities in vesicles from fungal cells treated with MAb 6B7. Analysis of orthologous proteins previously identified in vesicles from other fungi showed that different ascomycete fungi have similar proteins in their extracellular milieu, many of which are associated with virulence. Our results demonstrate that antibody binding can modulate fungal cell responses, resulting in differential loading of vesicles, which could alter fungal cell susceptibility to host defenses. This finding provides additional evidence that antibody binding modulates microbial physiology and suggests a new function for specific immunoglobulins through alterations of fungal secretion. IMPORTANCE Diverse fungal species release extracellular vesicles, indicating that this is a common pathway for the delivery of molecules to the extracellular space. However, there has

  18. Proteome of cell wall-extracts from pathogenic Paracoccidioides brasiliensis: Comparison among morphological phases, isolates, and reported fungal extracellular vesicle proteins

    Directory of Open Access Journals (Sweden)

    Larissa V.G. Longo

    2014-06-01

    Full Text Available We identified non-covalently linked cell wall proteins from Paracoccidioides brasiliensis yeasts and mycelia, with focus on the yeast pathogenic phase, and correlated them with reported fungal extracellular vesicle proteins. We studied isolates Pb3 and Pb18, which evoke distinct patterns of experimental paracoccidioidomycosis and represent two phylogenetic groups. Proteins were extracted mildly with dithiothreitol, trypsinized, and peptides analyzed by liquid chromatography coupled to high-resolution mass spectrometry. Among 132 yeast-exclusive sequences, 92 were Pb18-exclusive. About 80% of total proteins were classified as secretory, mostly showing non-conventional signals. Extracellular vesicular transportation could be involved, since 60% had orthologs reported in fungal extracellular vesicles.

  19. Basic mechanisms for recognition and transport of synaptic cargos

    NARCIS (Netherlands)

    M.A. Schlager (Max); C.C. Hoogenraad (Casper)

    2009-01-01

    textabstractSynaptic cargo trafficking is essential for synapse formation, function and plasticity. In order to transport synaptic cargo, such as synaptic vesicle precursors, mitochondria, neurotransmitter receptors and signaling proteins to their site of action, neurons make use of molecular motor

  20. Synaptic vesicle dynamic changes in a model of fragile X.

    Science.gov (United States)

    Broek, Jantine A C; Lin, Zhanmin; de Gruiter, H Martijn; van 't Spijker, Heleen; Haasdijk, Elize D; Cox, David; Ozcan, Sureyya; van Cappellen, Gert W A; Houtsmuller, Adriaan B; Willemsen, Rob; de Zeeuw, Chris I; Bahn, Sabine

    2016-01-01

    Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MS(E)) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS.