La(3+) and Gd(3+) induce shape change of giant unilamellar vesicles of phosphatidylcholine.

Science.gov (United States)

Tanaka, Tomoki; Tamba, Yukihiro; Masum, Shah Md; Yamashita, Yuko; Yamazaki, Masahito

2002-08-19

Lanthanides such as La(3+) and Gd(3+) are well known to have large effects on the function of membrane proteins such as mechanosensitive ionic channels and voltage-gated sodium channels, and also on the structure of phospholipid membranes. In this report, we have investigated effects of La(3+) and Gd(3+) on the shape of giant unilamellar vesicle (GUV) of dioleoylphosphatidylcholine (DOPC-GUV) and GUV of DOPC/cholesterol by the phase-contrast microscopy. The addition of 10-100 microM La(3+) (or Gd(3+)) through a 10-microm diameter micropipette near the DOPC-GUV (or DOPC/cholesterol-GUV) triggered several kinds of shape changes. We have found that a very low concentration (10 microM) of La(3+) (or Gd(3+)) induced a shape change of GUV such as the discocyte via stomatocyte to inside budded shape transformation, the two-spheres connected by a neck to prolate transformation, and the pearl on a string to cylinder (or tube) transformation. To understand the effect of these lanthanides on the shape of the GUV, we have also investigated phase transitions of 30 microM dipalmitoylphosphatidylcholine-multilamellar vesicle (DPPC-MLV) by the ultra-sensitive differential scanning calorimetry (DSC). The chain-melting phase transition temperature and the L(beta') to P(beta') phase transition temperature of DPPC-MLV increased with an increase in La(3+) concentration. This result indicates that the lateral compression pressure of the membrane increases with an increase in La(3+) concentration. Thereby, the interaction of La(3+) (or Gd(3+)) on the external monolayer membrane of the GUV induces a decrease in its area (A(ex)), whereas the area of the internal monolayer membrane (A(in)) keeps constant. Therefore, the shape changes of the GUV induced by these lanthanides can be explained reasonably by the decrease in the area difference between two monolayers (DeltaA=A(ex)-A(in)).

Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.

Directory of Open Access Journals (Sweden)

Akira C Saito

Full Text Available Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC field to induce a linear cell-GUV alignment, and then a direct current (DC pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.

Antimicrobial Peptide Lactoferricin B-Induced Rapid Leakage of Internal Contents from Single Giant Unilamellar Vesicles.

Science.gov (United States)

Moniruzzaman, Md; Alam, Jahangir Md; Dohra, Hideo; Yamazaki, Masahito

2015-09-29

Enzymatic digestion of bovine lactoferrin generates lactoferricin B (Lfcin B), a 25-mer peptide with strong antimicrobial activity of unknown mechanism. To elucidate the mechanistic basis of Lfcin B bactericidal activity, we investigated the interaction of Lfcin B with Escherichia coli and liposomes of lipid membranes. Lfcin B induced the influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli into its cytoplasm. Lfcin B induced gradual leakage of calcein from large unilamellar vesicles (LUVs) of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. To clarify the cause of Lfcin B-induced leakage of calcein from the LUVs, we used the single giant unilamellar vesicle (GUV) method to investigate the interaction of Lfcin B with calcein-containing DOPG/DOPC-GUVs. We observed that a rapid leakage of calcein from a GUV started stochastically; statistical analysis provided a rate constant for Lfcin B-induced pore formation, kp. On the other hand, phase-contrast microscopic images revealed that Lfcin B induced a rapid leakage of sucrose from the single GUVs with concomitant appearance of a spherical GUV of smaller diameter. Because of the very fast leakage, and at the present time resolution of the experiments (33 ms), we could not follow the evolution of pore nor the process of the structural changes of the GUV. Here we used the term "local rupture" to express the rapid leakage of sucrose and determined the rate constant of local rupture, kL. On the basis of the comparison between kp and kL, we concluded that the leakage of calcein from single GUVs occurred as a result of a local rupture in the GUVs and that smaller pores inducing leakage of calcein were not formed before the local rupture. The results of the effect of the surface charge density of lipid membranes and that of salt concentration in buffer on kp clearly show that kp increases with an increase in the extent of electrostatic interactions due to

Structure factor of dimyristoylphosphatidylcholine unilamellar vesicles: small-angle x-ray scattering study

International Nuclear Information System (INIS)

Kiselev, M.A.; Aksenov, V.L.; Lombardo, D.; Kisselev, A.M.; Lesieur, P.

2003-01-01

Small-angle X-ray scattering (SAXS) experiments have been performed on dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles in 40% aqueous sucrose solution. Model of separated form factors was applied for the evaluation of SAXS curves from large unilamellar vesicles. For the first time vesicle structure factor, polydispersity, average radius and membrane thickness were calculated simultaneously from the SAXS curves at T=30 deg C for DMPC concentrations in the range from 15 to 75 mM (1-5% w/w). Structure factor correction to the scattering curve was shown to be negligibly small for the lipid concentration of 15 mM (1% w/w). It was proved to be necessary to introduce structure factor correction to the scattering curves for lipid concentrations ≥ 30 mM (2% w/w)

Structure Factor of Dimyristoylphosphatidylcholine Unilamellar Vesicles Small-Angle X-Ray Scattering Study

CERN Document Server

Kiselev, M A; Kisselev, A M; Lesieur, P; Aksenov, V L

2003-01-01

Small-angle X-ray scattering (SAXS) experiments have been performed on dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles in 40 % aqueous sucrose solution. Model of separated form factors was applied for the evaluation of SAXS curves from large unilamellar vesicles. For the first time vesicle structure factor, polydispersity, average radius and membrane thickness were calculated simultaneously from the SAXS curves at T=306{\\circ}C for DMPC concentrations in the range from 15 to 75 mM (1-5 % w/w). Structure factor correction to the scattering curve was shown to be negligibly small for the lipid concentration of 15 mM (1 % w/w). It was proved to be necessary to introduce structure factor correction to the scattering curves for lipid concentrations {\\ge}30 mM (2 % w/w).

Dark-field microscopic study of the interactions between gold/silver nanoparticles and giant unilamellar vesicles

Science.gov (United States)

Bhat, Anupama; Zhao, Jian; Cooks, Tiana; Ren, Jun; Lu, Qi

2018-02-01

Giant unilamellar vesicles (GUVs) are well-established model systems for studying lipid packing and membrane dynamics. With sizes larger than 1 μm, GUVs are easily observable using optical microscopy. Gold nanoparticles (AuNPs) are well known for their biocompatibility and such biomedical applications in drug and gene delivery as well as medical diagnostics and therapeutics. On the other hand, silver nanoparticles (AgNPs) have long been known for their potent antimicrobial and anti-inflammatory effects for such applications as wound dressing and biomedical implants. In this work, we employed the dark-field microscopy (CytoViva Inc.) to study the interactions between AuNPs/AgNPs and GUVs, respectively. The GUVs used in this study were prepared with 1,2 dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as well as cholesterol (chol) at various mol% concentrations (0, 10, 20, 30, 40%). The electroformed GUVs were allowed to incubate with gold or silver nanoparticles of various sizes (between 10 and 100 nm) for 2 hrs before microscopic examination. The experiment has shown that the size of nanoparticles is a critical factor that determines the penetration rate. In addition, the membrane rigidity increases with the molar concentration of cholesterol hence making the NP penetration more difficult. Comparative studies have been made between AuNPs and AgNPs in regard to NP penetration and loading rate as well as the morphological changes induced in GUVs. This work aims to better understand the mechanisms of AuNP/AgNP and membrane interactions for their respective future applications in nanomedicine and nanotechnology.

Solid state deuterium nuclear magnetic resonance detection of transmembrane-potential-driven tetraphenylphosphonium redistribution across Giant Unilamellar Vesicle bilayers

International Nuclear Information System (INIS)

Franzin, Carla Maria Mirella

1995-01-01

It has been demonstrated that deuterium nuclear magnetic resonance ( 2 H NMR) of Giant Unilamellar Vesicles (GUVs) consisting of specifically choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), plus 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and cholesterol can be used to monitor the transbilayer redistribution of tetraphenylphosphonium (TPP + ) in response to a transmembrane potential (δψ tm ). The 2 H quadrupolar splittings (δν Q 's) measured reflect the level of TPP + bound at the membrane surface due to the latter's effect on the membrane surface electrostatic potential, ψ s . Results reveal the appearance of two distinct δν Q 's, due to differences in bound TPP + at the inner versus the outer monolayer in response to a δψ tm . The observed values of the δν Q 's agree with theoretical predictions based on a derived mathematical model that takes into account δψ tm , plus ψ s , plus the equilibrium binding of TPP + from solution onto the membrane surface, plus the sensitivity of δν Q to the amount of bound TPP + . This model identifies experimental factors that lead to improvements in spectral resolution. Henceforth, 2 H NMR is a valuable tool for quantifying transmembrane asymmetries of ψ s . (author)

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...... agreement with previously reported data obtained by other methods. For example, our computed tie lines were found to be nonhorizontal, indicating a difference in cholesterol content in the coexisting phases. This new, to our knowledge, analytical strategy offers a way to further exploit fluorescence...

Osmotic Gradients Induce Bio-reminiscent Morphological Transformations in Giant Unilamellar Vesicles

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Kamila eOglecka

2012-05-01

Full Text Available We report observations of large-scale, in-plane and out-of-plane membrane deformations in giant uni- and multilamellar vesicles composed of binary and ternary lipid mixtures in the presence of net transvesicular osmotic gradients. The lipid mixtures we examined consisted of binary mixtures of DOPC and DPPC lipids and ternary mixtures comprising POPC, sphingomyelin, and cholesterol over a range of compositions – both of which produce co-existing phases for selected ranges of compositions at room temperature under thermodynamic equilibrium. In the presence of net osmotic gradient, we find that the in-plane phase separation potential of these mixtures is non-trivially altered and a variety of out-of-plane morphological remodeling occurs. The repertoire of membrane deformations we observe display striking resemblance to their biological counterparts in live cells encompassing vesiculation, membrane fission and fusion, tubulation and pearling, as well as expulsion of entrapped vesicles from multicompartmental GUV architectures through large, self-healing transient pores. These observations suggest that the forces introduced by simple osmotic gradients across membrane boundaries could act as a trigger for shape-dependent membrane and vesicle trafficking activities. We speculate that such coupling of osmotic gradients with membrane properties might have provided lipid-mediated mechanisms during the early evolution of membrane compartmentalization in the absence of osmoregulatory protein machinery.

Small unilamellar vesicles as reagents: a chemically defined, quantitative assay for lectins

Energy Technology Data Exchange (ETDEWEB)

Rando, R.R.

1981-01-01

Samll unilamellar vesicles containing synthetic glycolipids can be prepared. These vesicles are aggregated by the appropriate lectin (Orr et al., 1979; Rando and Bangerter, 1979; Slama and Rando, 1980). It is shown here that extent of aggregation of these vesicles as measured by light scattering at 360 nm, is, under certain conditions, linear with amount of lectin added. This forms the basis of a rapid and simple quantitative assay for lectins using the modified vesicles as a defined chemical substrate. The assay is sensitive to lectin concentrations in the low ..mu..g range. The assay is applied here to studies on concanavalin A, Ricinus communis agglutinin and the ..cap alpha..-fucosyl binding lectin from Ulex europaeus (Type I).

Structure of unilamellar vesicles: Numerical analysis based on small-angle neutron scattering data

International Nuclear Information System (INIS)

Zemlyanaya, E. V.; Kiselev, M. A.; Zbytovska, J.; Almasy, L.; Aswal, V. K.; Strunz, P.; Wartewig, S.; Neubert, R.

2006-01-01

The structure of polydispersed populations of unilamellar vesicles is studied by small-angle neutron scattering for three types of lipid systems, namely, single-, two-and four-component vesicular systems. Results of the numerical analysis based on the separated-form-factor model are reported

Defined DNA-mediated assemblies of gene-expressing giant unilamellar vesicles

DEFF Research Database (Denmark)

Hadorn, M.; Boenzli, E.; Sørensen, Kristian T.

2013-01-01

The technological aspects of artificial vesicles as prominent cell mimics are evolving toward higher-order assemblies of functional vesicles with tissuelike architectures. Here, we demonstrate the spatially controlled DNA-directed bottom-up synthesis of complex microassemblies and macroassemblies...

SANS study of the unilamellar DMPC vesicles. The fluctuation model of lipid bilayer

International Nuclear Information System (INIS)

Kiselev, M.A.; Zemlyanaya, E.V.; Vinod, A.

2003-01-01

On the basis of the separated form-factors model, parameters of the polydispersed unilamellar DMPC vesicle population are analyzed. The neutron scattering length density across the membrane is simulated on the basis of fluctuated model of lipid bilayer. The hydration of vesicle is described by sigmoid distribution function of the water molecules. The results of fitting of the experimental data obtained at the small angle spectrometer SANS-I, PSI (Switzerland) are: average vesicle radius 272±0.4 Armstrong, polydispersity of the radius 27 %, membrane thickness 50.6± Armstrong, thickness of hydrocarbon chain region 21.4±2.8 Armstrong, number of water molecules located per lipid molecule 13±1, and DMPC surface area 59±2 Armstrong 2 . The calculated water distribution function across the bilayer directly explains why lipid membrane is easy penetrated by water molecules

Small angle neutron scattering and calorimetric studies of large unilamellar vesicles of the phospholipid dipalmitoylphosphatidylcholine

Energy Technology Data Exchange (ETDEWEB)

Mason, P.C.; Gaulin, B.D. [Department of Physics and Astronomy, McMaster University, Hamilton, Ontario, L8S 4M1 (CANADA); Epand, R.M. [Department of Biochemistry, McMaster University, Hamilton, Ontario, L8N 3Z5 (CANADA); Wignall, G.D.; Lin, J.S. [Center for Small-Angle Scattering Research, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States)

1999-03-01

High-resolution differential scanning calorimetry (DSC) and small angle neutron scattering (SANS) experiments have been conducted on large unilamellar vesicles (LUV{close_quote}s) of the phospholipid dipalmitoylphosphatidylcholine (DPPC) in excess water. The DSC results indicate a phase transition at temperatures corresponding to the gel (L{sub {beta}{sup {prime}}}) to ripple (P{sub {beta}{sup {prime}}}) phase transition seen in multilamellar vesicles of DPPC while the SANS experiments provide direct evidence for the formation of the P{sub {beta}{sup {prime}}} phase in these systems. In addition, it is shown that SANS is an effective technique for extracting structural parameters such as vesicle radius and thickness in LUV model membrane systems. {copyright} {ital 1999} {ital The American Physical Society}

Giant lipid vesicles under electric field pulses assessed by non invasive imaging.

Science.gov (United States)

Mauroy, Chloé; Portet, Thomas; Winterhalder, Martin; Bellard, Elisabeth; Blache, Marie-Claire; Teissié, Justin; Zumbusch, Andreas; Rols, Marie-Pierre

2012-10-01

We present experimental results regarding the effects of electric pulses on giant unilamellar vesicles (GUVs). We have used phase contrast and coherent anti-Stokes Raman scattering (CARS) microscopy as relevant optical approaches to gain insight into membrane changes under electropermeabilization. No addition of exogenous molecules (lipid analogue, fluorescent dye) was needed. Therefore, experiments were performed on pure lipid systems avoiding possible artefacts linked to their use. Structural membrane changes were assessed by loss of contrast inside the GUVs due to sucrose and glucose mixing. Our observations, performed at the single vesicle level, indicate these changes are under the control of the number of pulses and field intensity. Larger number of pulses enhances membrane alterations. A threshold value of the field intensity must be applied to allow exchange of molecules between GUVs and the external medium. This threshold depends on the size of the vesicles, the larger GUVs being affected at lower electric field strengths than the smaller ones. Our experimental data are well described by a simple model in which molecule entry is driven by direct exchange. The CARS microscopic study of the effect of pulse duration confirms that pulses, in the ms time range, induce loss of lipids and membrane deformations facing the electrodes. Copyright © 2012 Elsevier B.V. All rights reserved.

Quantitative Studies of Antimicrobial Peptide Pore Formation in Large Unilamellar Vesicles by Fluorescence Correlation Spectroscopy (FCS)

DEFF Research Database (Denmark)

Kristensen, Kasper; Henriksen, Jonas Rosager; Andresen, Thomas Lars

2013-01-01

In spite of intensive research efforts over the past decades, the mechanisms by which membrane-active antimicrobial peptides interact with phospholipid membranes are not yet fully elucidated. New tools that can be used to characterize antimicrobial peptide-lipid membrane interactions are therefore...... to quantify leakage from large unilamellar vesicles is associated with a number of experimental pitfalls. Based on theoretical and experimental considerations, we discuss how to properly design experiments to avoid these pitfalls. Subsequently, we apply fluorescence correlation spectroscopy to quantify...

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

DEFF Research Database (Denmark)

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

2015-01-01

The capability of membrane-active peptides to disrupt phospholipid membranes is often studied by investigating peptide-induced leakage of quenched fluorescent molecules from large unilamellar lipid vesicles. In this article, we explore two fluorescence microscopy-based single-vesicle detection...... methods as alternatives to the quenching-based assays for studying peptide-induced leakage from large unilamellar lipid vesicles. Specifically, we use fluorescence correlation spectroscopy (FCS) to study the leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles...... dispersed in aqueous solution, and we use confocal imaging of surface-immobilized large unilamellar lipid vesicles to investigate whether there are heterogeneities in leakage between individual vesicles. Of importance, we design an experimental protocol that allows us to quantitatively correlate the results...

The kinetics of the phospholipase A2-catalyzed hydrolysis of Egg phosphatidylcholine in unilamellar vesicles. Product inhibition and its relief by serum albumin.

Science.gov (United States)

Kupferberg, J P; Yokoyama, S; Kézdy, F J

1981-06-25

Only the lecithin in the outer leaflet (representing 70% of the total) of egg lecithin unilamellar vesicles is hydrolyzed by Crotalus atrox phospholipase A2. Hydrolyzed vesicles remain intact and impermeable to ionic solutes. The fatty acids produced in the hydrolysis remain on the vesicle and are only partially ionized at neutral pH due to electrostatic repulsions. About 40% of the lysolecithin product is desorbed from the vesicle. In the presence of a large excess of bovine serum albumin, the reaction is first order with respect to both the enzyme and the substrate. At 21 degrees C, pH 7.2, I = 0.16 M, and [Ca2+] = 7 mM, the second order rate constant is kex(2) = 1.5 X 10(6) M-1 s-1. In the absence of albumin, the reaction is inhibited competitively by both the monomeric (KIm = 4.5 X 10(-8) M) and micellar (nKIa = 3.7 X 10(-7) M) forms of lysolecithin ([critical micelle concentration] = 4.3 X 10(-6) M). Bovine serum albumin complexes two molecules of lysolecithin with a dissociation constant, Kb = 5 X 10(-8) M. With substoichiometric albumin, the reaction is biphasic, and, when the albumin is saturated with lysolecithin, the kinetics become similar to those observed in the absence of albumin. The action of phospholipase A2 shows that in unilamellar vesicles there is only one major lecithin conformation in the outer leaflet, or that all conformations are rapidly interconvertible.

Detection of association and fusion of giant vesicles using a fluorescence-activated cell sorter.

Science.gov (United States)

Sunami, Takeshi; Caschera, Filippo; Morita, Yuuki; Toyota, Taro; Nishimura, Kazuya; Matsuura, Tomoaki; Suzuki, Hiroaki; Hanczyc, Martin M; Yomo, Tetsuya

2010-10-05

We have developed a method to evaluate the fusion process of giant vesicles using a fluorescence-activated cell sorter (FACS). Three fluorescent markers and FACS technology were used to evaluate the extent of association and fusion of giant vesicles. Two fluorescent markers encapsulated in different vesicle populations were used as association markers; when these vesicles associate, the two independent markers should be observed simultaneously in a single detection event. The quenched fluorescent marker and the dequencher, which were encapsulated in separate vesicle populations, were used as the fusion marker. When the internal aqueous solutions mix, the quenched marker is liberated by the dequencher and emits the third fluorescent signal. Although populations of pure POPC vesicles showed no detectable association or fusion, the same populations, oppositely charged by the exogenous addition of charged amphiphiles, showed up to 50% association and 30% fusion upon population analysis of 100,000 giant vesicles. Although a substantial fraction of the vesicles associated in response to a small amount of the charged amphiphiles (5% mole fraction compared to POPC alone), a larger amount of the charged amphiphiles (25%) was needed to induce vesicle fusion. The present methodology also revealed that the association and fusion of giant vesicles was dependent on size, with larger giant vesicles associating and fusing more frequently.

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.

Spontaneous formation of small unilamellar vesicles by pH jump: A pH gradient across the bilayer membrane as the driving force

International Nuclear Information System (INIS)

Hauser, H.; Mantsch, H.H.; Casal, H.L.

1990-01-01

31 P NMR and infrared spectroscopic methods have been used to study the formation of small unilamellar vesicles by the pH-jump method. It is shown that increasing the pH of different lamellar phospholipid dispersions (phosphatidic acids and phosphatidylserines) induces a pH gradient. This pH gradient is estimated to be 4 ± 1 pH units, and its direction is such that the inner monolayer of the vesicles is at lower pH. There is spectroscopic evidence for tighter packing of the lipid hydrocarbon chains in the inner monolayer, probably due to the constraints imposed by the high curvature of the small vesicles formed. These results are discussed in terms of the driving force of the spontaneous vesiculation

Non-leaky vesiculation of large unilamellar vesicles (LUV) induced by plasma high density lipoproteins (HDL): Detection by HPLC

International Nuclear Information System (INIS)

Tischler, U.; Rueckert, D.S.; Schubert, R.; Jaroni, H.W.; Schmidt, K.H.

1989-01-01

Interaction of large unilamellar phosphatidylcholine vesicles (LUV, 75nm) and plasma high density lipoproteins (HDL) resulted in a non-leaky vesiculation of LUV. This vesiculation was detected by a HPLC-system consisting of a combination of three TSK-gel columns (6000PW, 5000PW, 3000SW). With increasing incubation time liposomal [ 14 C]PC, entrapped [ 3 H]inulin, and apoprotein of HDL origin decreased. The decrease was accompanied by a formation of new particles, consisting of liposomal PC and apoprotein. These particles also enclosed [3H]inulin, reflecting a hydrophilic inner space. The formation of the particles reached a maximum after one day of incubation. Retention time was 21 minutes for LUV, 28 minutes for the new particles, and 36 minutes for HDL. In vesicles with membranes consisting of phosphatidylcholine and 30% cholesterol no interactions were observed

Formation of Kinetically Trapped Nanoscopic Unilamellar Vesicles from Metastable Nanodiscs

Energy Technology Data Exchange (ETDEWEB)

Nieh, Mu-Ping [Univ. of Connecticut, Storrs, CT (United States). Inst. of Materials Science, Dept. of Chemical, Materials & Biomolecular Engineering; Dolinar, Paul [Univ. of Ottawa, ON (Canada); Kucerka, Norbert [National Research Council, Chalk River, ON (Canada). Chalk River Lab., Canadian Neutron Beam Centre; Comenius Univ., Bratislava (Slovakia). Dept. of Physical Chemistry of Drugs; Kline, Steven R. [National Inst. of Standards and Technology (NIST), Gaithersburg, MD (United States); Debeer-Schmitt, Lisa M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Neutron Scattering Science Division; Littrell, Kenneth C. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Neutron Scattering Science Division; Katsaras, John [National Research Council, Chalk River, ON (Canada). Chalk River Lab., Canadian Neutron Beam Centre; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Neutron Scattering Science Division; Brock Univ., St. Catharines, ON (Canada). Dept. of Physics; Univ. of Guelph, ON (Canada). Guelph-Waterloo Physics Inst.

2011-09-27

Zwitterionic long-chain lipids (e.g., dimyristoyl phosphatidylcholine, DMPC) spontaneously form onion-like, thermodynamically stable structures in aqueous solutions (commonly known as multilamellar vesicles, or MLVs). It has also been reported that the addition of zwitterionic short-chain (i.e., dihexanoyl phosphatidylcholine, DHPC) and charged long-chain (i.e., dimyristoyl phosphatidylglycerol, DMPG) lipids to zwitterionic long-chain lipid solutions results in the formation of unilamellar vesicles (ULVs). Here, we report a kinetic study on lipid mixtures composed of DMPC, DHPC, and DMPG. Two membrane charge densities (i.e., [DMPG]/[DMPC] = 0.01 and 0.001) and two solution salinities (i.e., [NaCl] = 0 and 0.2 M) are investigated. Upon dilution of the high-concentration samples at 50 °C, thermodynamically stable MLVs are formed, in the case of both weakly charged and high salinity solution mixtures, implying that the electrostatic interactions between bilayers are insufficient to cause MLVs to unbind. Importantly, in the case of these samples small angle neutron scattering (SANS) data show that, initially, nanodiscs (also known as bicelles) or bilayered ribbons form at low temperatures (i.e., 10 °C), but transform into uniform size, nanoscopic ULVs after incubation at 10 °C for 20 h, indicating that the nanodisc is a metastable structure. The instability of nanodiscs may be attributed to low membrane rigidity due to a reduced charge density and high salinity. Moreover, the uniform-sized ULVs persist even after being heated to 50 °C, where thermodynamically stable MLVs are observed. This result clearly demonstrates that these ULVs are kinetically trapped, and that the mechanical properties (e.g., bending rigidity) of 10 C nanodiscs favor the formation of nanoscopic ULVs over that of MLVs. From a practical point of view, this method of forming uniform-sized ULVs may lend itself to their mass production, thus making them economically feasible for medical

Formation of Kinetically Trapped Nanoscopic Unilamellar Vesicles from Metastable Nanodiscs

International Nuclear Information System (INIS)

Nieh, Mu-Ping; Dolinar, Paul; Kucerka, Norbert; Kline, Steven R.; Debeer-Schmitt, Lisa M.; Littrell, Ken; Katsaras, John

2011-01-01

Zwitterionic long-chain lipids (e.g., dimyristoyl phosphatidylcholine, DMPC) spontaneously form onion-like, thermodynamically stable structures in aqueous solutions (commonly known as multilamellar vesicles, or MLVs). It has also been reported that the addition of zwitterionic short-chain (i.e., dihexanoyl phosphatidylcholine, DHPC) and charged long-chain (i.e., dimyristoyl phosphatidylglycerol, DMPG) lipids to zwitterionic long-chain lipid solutions results in the formation of unilamellar vesicles (ULVs). Here, we report a kinetic study on lipid mixtures composed of DMPC, DHPC, and DMPG. Two membrane charge densities (i.e., (DMPG)/(DMPC) = 0.01 and 0.001) and two solution salinities (i.e., (NaCl) = 0 and 0.2 M) are investigated. Upon dilution of the high-concentration samples at 50 C, thermodynamically stable MLVs are formed, in the case of both weakly charged and high salinity solution mixtures, implying that the electrostatic interactions between bilayers are insufficient to cause MLVs to unbind. Importantly, in the case of these samples small angle neutron scattering (SANS) data show that, initially, nanodiscs (also known as bicelles) or bilayered ribbons form at low temperatures (i.e., 10 C), but transform into uniform size, nanoscopic ULVs after incubation at 10 C for 20 h, indicating that the nanodisc is a metastable structure. The instability of nanodiscs may be attributed to low membrane rigidity due to a reduced charge density and high salinity. Moreover, the uniform-sized ULVs persist even after being heated to 50 C, where thermodynamically stable MLVs are observed. This result clearly demonstrates that these ULVs are kinetically trapped, and that the mechanical properties (e.g., bending rigidity) of 10 C nanodiscs favor the formation of nanoscopic ULVs over that of MLVs. From a practical point of view, this method of forming uniform-sized ULVs may lend itself to their mass production, thus making them economically feasible for medical applications that

Incorporation of photosenzitizer hypericin into synthetic lipid-based nano-particles for drug delivery and large unilamellar vesicles with different content of cholesterol

Science.gov (United States)

Joniova, Jaroslava; Blascakova, Ludmila; Jancura, Daniel; Nadova, Zuzana; Sureau, Franck; Miskovsky, Pavol

2014-08-01

Low-density lipoproteins (LDL) and high-density lipoproteins (HDL) are attractive natural occurring vehicles for drug delivery and targeting to cancer tissues. The capacity of both types of the lipoproteins to bind hydrophobic drugs and their functionality as drug carriers have been examined in several studies and it has been also shown that mixing of anticancer drugs with LDL or HDL before administration led to an increase of cytotoxic effects of the drugs in the comparison when the drugs were administered alone. However, a difficult isolation of the lipoproteins in large quantity from a biological organism as well as a variability of the composition and size of these molecules makes practical application of LDL and HDL as drug delivery systems quite complicated. Synthetic LDL and HDL and large unilamellar vesicles (LUV) are potentially suitable candidates to substitute the native lipoproteins for targeted and effective drug delivery. In this work, we have studied process of an association of potent photosensitizer hypericin (Hyp) with synthetic lipid-based nano-particles (sLNP) and large unilamellar vesicles (LUV) containing various amount of cholesterol. Cholesterol is one of the main components of both LDL and HDL particles and its presence in biological membranes is known to be a determining factor for membrane properties. It was found that the behavior of Hyp incorporation into sLNP particles with diameter ca ~ 90 nm is qualitatively very similar to that of Hyp incorporation into LDL (diameter ca. 22 nm) and these particles are able to enter U-87 MG cells by endocytosis. The presence of cholesterol in LUV influences the capacity of these vesicles to incorporate Hyp into their structure.

Precise detection of pH inside large unilamellar vesicles using membrane-impermeable dendritic porphyrin-based nanoprobes.

Science.gov (United States)

Leiding, Thom; Górecki, Kamil; Kjellman, Tomas; Vinogradov, Sergei A; Hägerhäll, Cecilia; Arsköld, Sindra Peterson

2009-05-15

Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu(3), which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe's pK is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe's pK was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu(3) was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu(3) was found to be superior to the commercially available pH indicators.

Photoinduced bimolecular electron transfer kinetics in small unilamellar vesicles

International Nuclear Information System (INIS)

Choudhury, Sharmistha Dutta; Kumbhakar, Manoj; Nath, Sukhendu; Pal, Haridas

2007-01-01

Photoinduced electron transfer (ET) from N,N-dimethylaniline to some coumarin derivatives has been studied in small unilamellar vesicles (SUVs) of the phospholipid, DL-α-dimyristoyl-phosphatidylcholine, using steady-state and time-resolved fluorescence quenching, both below and above the phase transition temperature of the vesicles. The primary interest was to examine whether Marcus inversion [H. Sumi and R. A. Marcus, J. Chem. Phys. 84, 4894 (1986)] could be observed for the present ET systems in these organized assemblies. The influence of the topology of SUVs on the photophysical properties of the reactants and consequently on their ET kinetics has also been investigated. Absorption and fluorescence spectral data of the coumarins in SUVs and the variation of their fluorescence decays with temperature indicate that the dyes are localized in the bilayer of the SUVs. Time-resolved area normalized emission spectra analysis, however, reveals that the dyes are distributed in two different microenvironments in the SUVs, which we attribute to the two leaflets of the bilayer, one toward bulk water and the other toward the inner water pool. The microenvironments in the two leaflets are, however, not indicated to be that significantly different. Time-resolved anisotropy decays were biexponential for all the dyes in SUVs, and this has been interpreted in terms of the compound motion model according to which the dye molecules can experience a fast wobbling-in-cone type of motion as well as a slow overall rotating motion of the cone containing the molecule. The expected bimolecular diffusion-controlled rates in SUVs, as estimated by comparing the microviscosities in SUVs (determined from rotational correlation times) and that in acetonitrile solution, are much slower than the observed fluorescence quenching rates, suggesting that reactant diffusion (translational) does not play any role in the quenching kinetics in the present systems. Accordingly, clear inversions are

Phase separation in artificial vesicles driven by light and curvature

Science.gov (United States)

Rinaldin, Melissa; Pomp, Wim; Schmidt, Thomas; Giomi, Luca; Kraft, Daniela; Physics of Life Processes Team; Soft; Bio Mechanics Collaboration; Self-Assembly in Soft Matter Systems Collaboration

The role of phase-demixing in living cells, leading to the lipid-raft hypothesis, has been extensively studied. Lipid domains of higher lipid chain order are proposed to regulate protein spatial organization. Giant Unilamellar Vesicles provide an artificial model to study phase separation. So far temperature was used to initiate the process. Here we introduce a new methodology based on the induction of phase separation by light. To this aim, the composition of the lipid membrane is varied by photo-oxidation of lipids. The control of the process gained by using light allowed us to observe vesicle shape fluctuations during phase-demixing. The presence of fluctuations near the critical mixing point resembles features of a critical process. We quantitatively analyze these fluctuations using a 2d elastic model, from which we can estimate the material parameters such as bending rigidity and surface tension, demonstrating the non-equilibrium critical behaviour. Finally, I will describe recent attempts toward tuning the membrane composition by controlling the vesicle curvature.

Viscoelastic deformation of lipid bilayer vesicles.

Science.gov (United States)

Wu, Shao-Hua; Sankhagowit, Shalene; Biswas, Roshni; Wu, Shuyang; Povinelli, Michelle L; Malmstadt, Noah

2015-10-07

Lipid bilayers form the boundaries of the cell and its organelles. Many physiological processes, such as cell movement and division, involve bending and folding of the bilayer at high curvatures. Currently, bending of the bilayer is treated as an elastic deformation, such that its stress-strain response is independent of the rate at which bending strain is applied. We present here the first direct measurement of viscoelastic response in a lipid bilayer vesicle. We used a dual-beam optical trap (DBOT) to stretch 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) giant unilamellar vesicles (GUVs). Upon application of a step optical force, the vesicle membrane deforms in two regimes: a fast, instantaneous area increase, followed by a much slower stretching to an eventual plateau deformation. From measurements of dozens of GUVs, the average time constant of the slower stretching response was 0.225 ± 0.033 s (standard deviation, SD). Increasing the fluid viscosity did not affect the observed time constant. We performed a set of experiments to rule out heating by laser absorption as a cause of the transient behavior. Thus, we demonstrate here that the bending deformation of lipid bilayer membranes should be treated as viscoelastic.

Formation of cubic phases from large unilamellar vesicles of dioleoylphosphatidylglycerol/monoolein membranes induced by low concentrations of Ca2+.

Science.gov (United States)

Awad, Tarek S; Okamoto, Yoshihide; Masum, Shah Md; Yamazaki, Masahito

2005-12-06

We developed a new method for the transformation of large unilamellar vesicles (LUVs) into the cubic phase. We found that the addition of low concentrations of Ca(2+) to suspensions of multilamellar vesicles (MLVs) of membranes of monoolein (MO) and dioleoylphosphatidylglycerol (DOPG) mixtures (DOPG/MO) changed their L(alpha) phase to the cubic phases. For instance, the addition of 15-25 mM Ca(2+) to 30%-DOPG/70%-MO-MLVs induced the Q(229) phase, whereas the addition of > or =28 mM Ca(2+) induced the Q(224) phase. LUVs of DOPG/MO membranes containing > or =25 mol % DOPG were prepared easily. Low concentrations of Ca(2+) transformed these LUVs in excess buffer into the Q(224) or the Q(229) phase, depending on the Ca(2+) concentration. For example, 15 and 50 mM Ca(2+) induced the Q(224) and Q(229) phase in the 30%-DOPG/70%-MO-LUVs at 25 degrees C, respectively. This finding is the first demonstration of transformation of LUVs of lipid membranes into the cubic phase under excess water condition.

Ganglioside GM1 spontaneous transfer between phospholipid vesicles

International Nuclear Information System (INIS)

Brown, R.E.; Sugar, I.P.; Thompson, T.E.

1986-01-01

The transfer kinetics of the monosiaylated glycosphingolipid, GM 1 , between different size phospholipid vesicles was measured using molecular sieve chromatography. At desired time intervals, small unilamellar donor vesicles were separated from large unilamellar acceptor vesicles by elution from a Sephacryl S-500 column [ 3 H]-GM 1 net transfer was calculated relative to [ 14 C]-cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. The initial GM 1 transfer rate between 1-palmitoyl-2-oleoyl phosphatidylcholine vesicles at 45 0 C deviated slightly from first order kinetics and possessed a half time of 3.6 days. This transfer half time is an order of magnitude shorter than that observed from the desiaylated derivative of GM 1 . The transfer kinetics are consistent with the authors recent electron microscopic results suggesting a molecular distribution of GM 1 in liquid-crystalline phosphatidylcholine bilayers

Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state.

Science.gov (United States)

Gabriel, N E; Agman, N V; Roberts, M F

1987-11-17

Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.

Influence of molecular weight and pH on adsorption of chitosan at the surface of large and giant vesicles.

Science.gov (United States)

Quemeneur, Francois; Rinaudo, Marguerite; Pépin-Donat, Brigitte

2008-01-01

This paper describes the mechanisms of adsorption of chitosan, a positively charged polyelectrolyte, on the DOPC lipid membrane of large and giant unilamellar vesicles (respectively, LUVs and GUVs). We observe that the variation of the zeta potential of LUVs as a function of chitosan concentration is independent on the chitosan molecular weight (Mw). This result is interpreted in terms of electrostatic interactions, which induce a flat adsorption of the chitosan on the surface of the membrane. The role of electrostatic interactions is further studied by observing the variation of the zeta potential as a function of the chitosan concentration for two different charge densities tuned by the pH. Results show a stronger chitosan-membrane affinity at pH 6 (lipids are negatively charged, and 40% chitosan amino groups are protonated) than at pH 3.4 (100% of protonated amino groups but zwitterionic lipids are positively charged) which confirms that adsorption is of electrostatic origin. Then, we investigate the stability of decorated LUVs and GUVs in a large range of pH (6.0 pH variation of the zeta potential as a function of the pH (2.0 pH pH pH > 10.0, in the absence of chitosan, the vesicles present complex shapes, contrary to the chitosan-decorated vesicles which remain spherical, confirming thus that chitosan remains adsorbed on vesicles in basic conditions up to pH = 12.0. These results, in addition with our previous data, show that the chitosan-decorated vesicles are stable over a very broad range of pH (2.0 pH < 12.0), which holds promise for their in vivo applications. Finally, the quantification of the chitosan adsorption on a LUV membrane is performed by zeta potential and fluorescence measurements. The fraction of membrane surface covered by chitosan is estimated to be lower than 40 %, which corresponds to the formation of a flat layer of chitosan on the membrane surface on an electrostatic basis.

Frequency-dependent electrodeformation of giant phospholipid vesicles in AC electric field

Science.gov (United States)

2010-01-01

A model of vesicle electrodeformation is described which obtains a parametrized vesicle shape by minimizing the sum of the membrane bending energy and the energy due to the electric field. Both the vesicle membrane and the aqueous media inside and outside the vesicle are treated as leaky dielectrics, and the vesicle itself is modeled as a nearly spherical shape enclosed within a thin membrane. It is demonstrated (a) that the model achieves a good quantitative agreement with the experimentally determined prolate-to-oblate transition frequencies in the kilohertz range and (b) that the model can explain a phase diagram of shapes of giant phospholipid vesicles with respect to two parameters: the frequency of the applied alternating current electric field and the ratio of the electrical conductivities of the aqueous media inside and outside the vesicle, explored in a recent paper (S. Aranda et al., Biophys J 95:L19–L21, 2008). A possible use of the frequency-dependent shape transitions of phospholipid vesicles in conductometry of microliter samples is discussed. PMID:21886342

Oscillatory phase separation in giant lipid vesicles induced by transmembrane osmotic differentials

Science.gov (United States)

Oglęcka, Kamila; Rangamani, Padmini; Liedberg, Bo; Kraut, Rachel S; Parikh, Atul N

2014-01-01

Giant lipid vesicles are closed compartments consisting of semi-permeable shells, which isolate femto- to pico-liter quantities of aqueous core from the bulk. Although water permeates readily across vesicular walls, passive permeation of solutes is hindered. In this study, we show that, when subject to a hypotonic bath, giant vesicles consisting of phase separating lipid mixtures undergo osmotic relaxation exhibiting damped oscillations in phase behavior, which is synchronized with swell–burst lytic cycles: in the swelled state, osmotic pressure and elevated membrane tension due to the influx of water promote domain formation. During bursting, solute leakage through transient pores relaxes the pressure and tension, replacing the domain texture by a uniform one. This isothermal phase transition—resulting from a well-coordinated sequence of mechanochemical events—suggests a complex emergent behavior allowing synthetic vesicles produced from simple components, namely, water, osmolytes, and lipids to sense and regulate their micro-environment. DOI: http://dx.doi.org/10.7554/eLife.03695.001 PMID:25318069

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

of vanadate (NaVO3) on glucose transporter (GLUT4) intrinsic activity (V(max) = intrinsic activity x [GLUT4 protein]) was studied in muscle plasma membrane giant vesicles. Giant vesicles (average diameter 7.6 microns) were produced by collagenase treatment of rat skeletal muscle. The vesicles were incubated......) 55% and 60%, respectively, compared with control. The plasma membrane GLUT4 protein content was not changed in response to vanadate. It is concluded that vanadate decreased glucose transport per GLUT4 (intrinsic activity). This finding suggests that regulation of glucose transport in skeletal muscle...

Sugar-Decorated Sugar Vesicles : Lectin-Carbohydrate Recognition at the Surface of Cyclodextrin Vesicles

NARCIS (Netherlands)

Voskuhl, Jens; Stuart, Marc C. A.; Ravoo, Bart Jan

2010-01-01

An artificial glycocalix self-assembles when unilamellar bilayer vesicles of amphiphilic beta-cyclodextrins are decorated with maltose and lactose by host-guest interactions. To this end, maltose and lactose were conjugated with adamantane through a tetra(ethyleneglycol) spacer. Both

Spontaneous transfer of ganglioside GM1 between phospholipid vesicles

International Nuclear Information System (INIS)

Brown, R.E.; Thompson, T.E.

1987-01-01

The transfer kinetics of the negatively charged glycosphingolipid II 3 -N-acetylneuraminosyl-gangliotetraosylceramide (GM 1 ) were investigated by monitoring tritiated GM 1 movement between donor and acceptor vesicles. After appropriate incubation times at 45 0 C, donor and acceptor vesicles were separated by molecular sieve chromatography. Donors were small unilamellar vesicles produced by sonication, whereas acceptors were large unilamellar vesicles produced by either fusion or ethanol injection. Initial GM 1 transfer to acceptors followed first-order kinetics with a half-time of about 40 h assuming that GM 1 is present in equal mole fractions in the exterior and interior surfaces of the donor vesicle bilayer and that no glycolipid flip-flop occurs. GM 1 net transfer was calculated relative to that of [ 14 C]cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. Factors affecting the GM 1 interbilayer transfer rate included phospholipid matrix composition, initial GM 1 concentration in donor vesicles, and the GM 1 distribution in donor vesicles with respect to total lipid symmetry. The findings provide evidence that GM 1 is molecularly dispersed at low concentrations within liquid-crystalline phospholipid bilayers

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)

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

Asymmetrical Polyhedral Configuration of Giant Vesicles Induced by Orderly Array of Encapsulated Colloidal Particles.

Science.gov (United States)

Natsume, Yuno; Toyota, Taro

2016-01-01

Giant vesicles (GVs) encapsulating colloidal particles by a specific volume fraction show a characteristic configuration under a hypertonic condition. Several flat faces were formed in GV membrane with orderly array of inner particles. GV shape changed from the spherical to the asymmetrical polyhedral configuration. This shape deformation was derived by entropic interaction between inner particles and GV membrane. Because a part of inner particles became to form an ordered phase in the region neighboring the GV membrane, free volume for the other part of particles increased. Giant vesicles encapsulating colloidal particles were useful for the model of "crowding effect" which is the entropic interaction in the cell.

Spontaneous transfer of gangliotetraosylceramide between phospholipid vesicles

International Nuclear Information System (INIS)

Brown, R.E.; Sugar, I.P.; Thompson, T.E.

1985-01-01

The transfer kinetics of the neutral glycosphingolipid gangliotetraosylceramide (asialo-GM1) were investigated by monitoring tritiated asialo-GM1 movement from donor to acceptor vesicles. Two different methods were employed to separate donor and acceptor vesicles at desired time intervals. In one method, a negative charge was imparted to dipalmitoylphosphatidylcholine donor vesicles by including 10 mol% dipalmitoylphosphatidic acid. Donors were separated from neutral dipalmitoylphosphatidylcholine acceptor vesicles by ion-exchange chromatography. In the other method, small, unilamellar donor vesicles and large, unilamellar acceptor vesicles were coincubated at 45 degrees C and then separated at desired time intervals by molecular sieve chromatography. The majority of asialo-GM1 transfer to acceptor vesicles occurred as a slow first-order process with a half-time of about 24 days assuming that the relative concentration of asialo-GM1 in the phospholipid matrix was identical in each half of the donor bilayer and that no glycolipid flip-flop occurred. Asialo-GM1 net transfer was calculated relative to that of [ 14 C]cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. A nearly identical transfer half-time was obtained when the phospholipid matrix was changed from dipalmitoylphosphatidylcholine to palmitoyloleoylphosphatidylcholine. Varying the acceptor vesicle concentration did not significantly alter the asialo-GM1 transfer half-time. This result is consistent with a transfer mechanism involving diffusion of glycolipid through the aqueous phase rather than movement of glycolipid following formation of collisional complexes between donor and acceptor vesicles. (Abstract Truncated)

Heterogeneous vesicles in mucous epithelial cells of posterior esophagus of Chinese giant salamander (Andrias davidianus

Directory of Open Access Journals (Sweden)

H. Zhang

2015-08-01

Full Text Available The Chinese giant salamander belongs to an old lineage of salamanders and endangered species. Many studies of breeding and disease regarding this amphibian had been implemented. However, the studies on the ultrastructure of this amphibian are rare. In this work, we provide a histological and ultrastructural investigation on posterior esophagus of Chinese giant salamander. The sections of amphibian esophagus were stained by hematoxylin & eosin (H&E. Moreover, the esophageal epithelium was observed by transmission electron microscopy (TEM. The results showed that esophageal epithelium was a single layer epithelium, which consisted of mucous cells and columnar cells. The esophageal glands were present in submucosa. The columnar cells were ciliated. According to the diverging ultrastructure of mucous vesicles, three types of mucous cells could be identified in the esophageal mucosa: i electron-lucent vesicles mucous cell (ELV-MC; ii electron-dense vesicles mucous cell (EDV-MC; and iii mixed vesicles mucous cell (MV-MC.

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

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...

Artificial vesicles as an animal cell model for the study of biological application of non-thermal plasma

International Nuclear Information System (INIS)

Ki, S H; Park, J K; Sung, C; Lee, C B; Uhm, H; Choi, E H; Baik, K Y

2016-01-01

Artificial cell-like model systems can provide information which is hard to obtain with real biological cells. Giant unilamellar vesicles (GUV) containing intra-membrane DNA or OH radical-binding molecules are used to visualize the cytolytic activity of OH radicals. Changes in the GUV membrane are observed by microscopy or flow cytometry as performed for animal cells after non-thermal plasma treatment. The experimental data shows that OH radicals can be detected inside the membrane, although the biological effects are not as significant as for H 2 O 2 . This artificial model system can provide a systemic means to elucidate the complex interactions between biological materials and non-thermal plasma. (paper)

Partitioning of organophosphorus pesticides into phosphatidylcholine small unilamellar vesicles studied by second-derivative spectrophotometry.

Science.gov (United States)

Takegami, Shigehiko; Kitamura, Keisuke; Ohsugi, Mayuko; Ito, Aya; Kitade, Tatsuya

2015-06-15

In order to quantitatively examine the lipophilicity of the widely used organophosphorus pesticides (OPs) chlorfenvinphos (CFVP), chlorpyrifos-methyl (CPFM), diazinon (DZN), fenitrothion (FNT), fenthion (FT), isofenphos (IFP), profenofos (PFF) and pyraclofos (PCF), their partition coefficient (Kp) values between phosphatidylcholine (PC) small unilamellar vesicles (SUVs) and water (liposome-water system) were determined by second-derivative spectrophotometry. The second-derivative spectra of these OPs in the presence of PC SUV showed a bathochromic shift according to the increase in PC concentration and distinct derivative isosbestic points, demonstrating the complete elimination of the residual background signal effects that were observed in the absorption spectra. The Kp values were calculated from the second-derivative intensity change induced by addition of PC SUV and obtained with a good precision of R.S.D. below 10%. The Kp values were in the order of CPFM>FT>PFF>PCF>IFP>CFVP>FNT⩾DZN and did not show a linear correlation relationship with the reported partition coefficients obtained using an n-octanol-water system (R(2)=0.530). Also, the results quantitatively clarified the effect of chemical-group substitution in OPs on their lipophilicity. Since the partition coefficient for the liposome-water system is more effective for modeling the quantitative structure-activity relationship than that for the n-octanol-water system, the obtained results are toxicologically important for estimating the accumulation of these OPs in human cell membranes. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular dynamics simulation of the formation, structure, and dynamics of small phospholipid vesicles

NARCIS (Netherlands)

Marrink, SJ; Mark, AE

2003-01-01

Here, we use coarse grained molecular dynamics (MD) simulations to study the spontaneous aggregation of dipalmitoylphosphatidylcholine (DPPC) lipids into small unilamellar vesicles. We show that the aggregation process occurs on a nanosecond time scale, with bicelles and cuplike vesicles formed at

Origins of microstructural transformations in charged vesicle suspensions: the crowding hypothesis.

Science.gov (United States)

Seth, Mansi; Ramachandran, Arun; Murch, Bruce P; Leal, L Gary

2014-09-02

It is observed that charged unilamellar vesicles in a suspension can spontaneously deflate and subsequently transition to form bilamellar vesicles, even in the absence of externally applied triggers such as salt or temperature gradients. We provide strong evidence that the driving force for this deflation-induced transition is the repulsive electrostatic pressure between charged vesicles in concentrated suspensions, above a critical effective volume fraction. We use volume fraction measurements and cryogenic transmission electron microscopy imaging to quantitatively follow both the macroscopic and microstructural time-evolution of cationic diC18:1 DEEDMAC vesicle suspensions at different surfactant and salt concentrations. A simple model is developed to estimate the extent of deflation of unilamellar vesicles caused by electrostatic interactions with neighboring vesicles. It is determined that when the effective volume fraction of the suspension exceeds a critical value, charged vesicles in a suspension can experience "crowding" due to overlap of their electrical double layers, which can result in deflation and subsequent microstructural transformations to reduce the effective volume fraction of the suspension. Ordinarily in polydisperse colloidal suspensions, particles interacting via a repulsive potential transform into a glassy state above a critical volume fraction. The behavior of charged vesicle suspensions reported in this paper thus represents a new mechanism for the relaxation of repulsive interactions in crowded situations.

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.

Electroformation of Giant Vesicles on a Polymer Mesh

Directory of Open Access Journals (Sweden)

Yukihisa Okumura

2011-07-01

Full Text Available Electroformation of cell-sized lipid membrane vesicles (giant vesicles, GVs from egg yolk phosphatidylcholine under applied electric voltage was examined on a substrate of a polymer mesh placed between two planar indium tin oxide coated glass electrodes. Under appropriate conditions, GVs were formed in good yield on meshes of various polymer materials, namely, hydrophobic poly(propylene, poly(ethylene terephthalate, a carbon fiber/nylon composite, and relatively hydrophilic nylon. Arranging threads in a mesh structure with appropriate openings improved GV formation compared to simply increasing the number of threads. For optimal electroformation of GVs, the size and shape of a mesh opening were crucial. With a too large opening, GV formation deteriorated. When the sides of an opening were partially missing, GV formation did not occur efficiently. With an adequate opening, a deposited lipid solution could fill the opening, and a relatively uniform lipid deposit formed on the surface of threads after evaporation of the solvent. This could supply a sufficient amount of lipids to the opening and also prevent a lipid deposit from becoming too thick for electroformation. As a result, good GV formation was often observed in openings filled with swelled lipid.

Phase Partitioning of GM1 and Its Bodipy-Labeled Analog Determine Their Different Binding to Cholera Toxin

DEFF Research Database (Denmark)

Rissanen, Sami; Grzybek, Michal; Orłowski, Adam

2017-01-01

membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane...

Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles

Science.gov (United States)

Litschel, Thomas; Ganzinger, Kristina A.; Movinkel, Torgeir; Heymann, Michael; Robinson, Tom; Mutschler, Hannes; Schwille, Petra

2018-05-01

Early protocells are commonly assumed to consist of an amphiphilic membrane enclosing an RNA-based self-replicating genetic system and a primitive metabolism without protein enzymes. Thus, protocell evolution must have relied on simple physicochemical self-organization processes within and across such vesicular structures. We investigate freeze-thaw (FT) cycling as a potential environmental driver for the necessary content exchange between vesicles. To this end, we developed a conceptually simple yet statistically powerful high-throughput procedure based on nucleic acid-containing giant unilamellar vesicles (GUVs) as model protocells. GUVs are formed by emulsion transfer in glass bottom microtiter plates and hence can be manipulated and monitored by fluorescence microscopy without additional pipetting and sample handling steps. This new protocol greatly minimizes artefacts, such as unintended GUV rupture or fusion by shear forces. Using DNA-encapsulating phospholipid GUVs fabricated by this method, we quantified the extent of content mixing between GUVs under different FT conditions. We found evidence of nucleic acid exchange in all detected vesicles if fast freezing of GUVs at ‑80 °C is followed by slow thawing at room temperature. In contrast, slow freezing and fast thawing both adversely affected content mixing. Surprisingly, and in contrast to previous reports for FT-induced content mixing, we found that the content is not exchanged through vesicle fusion and fission, but that vesicles largely maintain their membrane identity and even large molecules are exchanged via diffusion across the membranes. Our approach supports efficient screening of prebiotically plausible molecules and environmental conditions, to yield universal mechanistic insights into how cellular life may have emerged.

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

Science.gov (United States)

Nishigami, Misako; Mori, Takaaki; Tomita, Masahiro; Takiguchi, Kingo; Tsumoto, Kanta

2017-07-01

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases. Copyright © 2017 Elsevier B.V. All rights reserved.

Asymmetric Hybrid Polymer-Lipid Giant Vesicles as Cell Membrane Mimics.

Science.gov (United States)

Peyret, Ariane; Ibarboure, Emmanuel; Le Meins, Jean-François; Lecommandoux, Sebastien

2018-01-01

Lipid membrane asymmetry plays an important role in cell function and activity, being for instance a relevant signal of its integrity. The development of artificial asymmetric membranes thus represents a key challenge. In this context, an emulsion-centrifugation method is developed to prepare giant vesicles with an asymmetric membrane composed of an inner monolayer of poly(butadiene)- b -poly(ethylene oxide) (PBut- b -PEO) and outer monolayer of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC). The formation of a complete membrane asymmetry is demonstrated and its stability with time is followed by measuring lipid transverse diffusion. From fluorescence spectroscopy measurements, the lipid half-life is estimated to be 7.5 h. Using fluorescence recovery after photobleaching technique, the diffusion coefficient of 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(lissamine rhodamine B sulfonyl) (DOPE-rhod, inserted into the POPC leaflet) is determined to be about D = 1.8 ± 0.50 μm 2 s -1 at 25 °C and D = 2.3 ± 0.7 μm 2 s -1 at 37 °C, between the characteristic values of pure POPC and pure polymer giant vesicles and in good agreement with the diffusion of lipids in a variety of biological membranes. These results demonstrate the ability to prepare a cell-like model system that displays an asymmetric membrane with transverse and translational diffusion properties similar to that of biological cells.

TEMPERATURE DEPENDENT PHASE BEHAVIOR AND PROTEIN PARTITIONING IN GIANT PLASMA MEMBRANE VESICLES

OpenAIRE

Johnson, SA; Stinson, BM; Go, M; Carmona, LM; Reminick, JI; Fang, X; Baumgart, T

2010-01-01

Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured...

Bending Elasticity Modulus of Giant Vesicles Composed of Aeropyrum Pernix K1 Archaeal Lipid

Directory of Open Access Journals (Sweden)

Julia Genova

2015-03-01

Full Text Available Thermally induced shape fluctuations were used to study elastic properties of giant vesicles composed of archaeal lipids C25,25-archetidyl (glucosyl inositol and C25,25-archetidylinositol isolated from lyophilised Aeropyrum pernix K1 cells. Giant vesicles were created by electroformation in pure water environment. Stroboscopic illumination using a xenon flash lamp was implemented to remove the blur effect due to the finite integration time of the camera and to obtain an instant picture of the fluctuating vesicle shape. The mean weighted value of the bending elasticity modulus kc of the archaeal membrane determined from the measurements meeting the entire set of qualification criteria was (1.89 ± 0.18 × 10−19 J, which is similar to the values obtained for a membrane composed of the eukaryotic phospholipids SOPC (1.88 ± 0.17 × 10−19 J and POPC (2.00 ± 0.21 ´ 10−19 J. We conclude that membranes composed of archaeal lipids isolated from Aeropyrum pernix K1 cells have similar elastic properties as membranes composed of eukaryotic lipids. This fact, together with the importance of the elastic properties for the normal circulation through blood system, provides further evidence in favor of expectations that archaeal lipids could be appropriate for the design of drug delivery systems.

Vesicles and vesicle gels - structure and dynamics of formation

International Nuclear Information System (INIS)

Gradzielski, M

2003-01-01

Vesicles constitute an interesting morphology formed by self-aggregating amphiphilic molecules. They exhibit a rich structural variety and are of interest both from a fundamental point of view (for studying closed bilayer systems) and from a practical point of view (whenever one is interested in the encapsulation of active molecules). In many circumstances vesicular structures have to be formed by external forces, but of great interest are amphiphilic systems, where they form spontaneously. Here the question arises of whether this means that they are also thermodynamically stable structures, which at least in some systems appears to be the case. If such vesicles are well defined in size, it is possible to pack them densely and thereby form vesicle gels that possess highly elastic properties even for relatively low volume fractions of amphiphile. Conditions for the formation and the microstructure of such vesicle gels have been studied in some detail for the case of unilamellar vesicles. Another important and topical issue is the dynamics of vesicle formation/breakdown, as the understanding of the transition process will open the way to a deeper understanding of their stability and also allow controlling of the structures formed, by means of their formation processes. Significant progress in the study of the transformation processes has been achieved, in particular by means of time-resolved scattering experiments. (topical review)

Fusion of Sendai Virus with Vesicles of Oligomerizable Lipids: A Micro Calorimetric Analysis of Membrane Fusion

OpenAIRE

Ravoo, Bart Jan; Weringa, Wilke D.; Engberts, Jan B.F.N.

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°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 Sen...

Giant plasma membrane vesicles: models for understanding membrane organization.

Science.gov (United States)

Levental, Kandice R; Levental, Ilya

2015-01-01

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

Gold nanoparticles covalently assembled onto vesicle structures as possible biosensing platform

Directory of Open Access Journals (Sweden)

M. Fátima Barroso

2016-05-01

Full Text Available In this contribution a strategy is shown to covalently immobilize gold nanoparticles (AuNPs onto vesicle bilayers with the aim of using this nanomaterial as platform for the future design of immunosensors. A novel methodology for the self-assembly of AuNPs onto large unilamellar vesicle structures is described. The vesicles were formed with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC and 1-undecanethiol (SH. After, the AuNPs photochemically synthesized in pure glycerol were mixed and anchored onto SH–DOPC vesicles. The data provided by voltammetry, spectrometry and microscopy techniques indicated that the AuNPs were successfully covalently anchored onto the vesicle bilayer and decorated vesicles exhibit a spherical shape with a size of 190 ± 10 nm. The developed procedure is easy, rapid and reproducible to start designing a possible immunosensor by using environmentally friendly procedures.

Intermolecular crosslinks mediate aggregation of phospholipid vesicles by pulmonary surfactant-associated protein SAP-35

International Nuclear Information System (INIS)

Ross, G.R.; Sawyer, J.; Whitsett, J.

1987-01-01

Pulmonary surfactant-associated protein, Mr=35,000 (SAP-35) is known to bind phospholipids and is hypothesized to function in the organization of surfactant lipid membranes. SAP-35 has been observed to accelerate the calcium-induced aggregation of phospholipid vesicles. In order to define the molecular domains of SAP-35 which function in phospholipid aggregation, they have measured the light scattering properties (400nm) of purified canine SAP-35-phospholipid vesicle suspensions. Accelerated aggregation of unilamellar vesicles, requires SAP-35 and at least 2mM free calcium. The initial rate of A 400 change is proportional to the amount of native SAP-35 added over lipid:protein molar ratios ranging from 100:1 to 5000:1. Removal of the SAP-35 collagen-like domain and a specific cysteine residue involved in intermolecular disulfide bonding by bacterial collagenase digestion destroys the protein's lipid aggregation activity. Pre-incubation of SAP-35 with dithiothreitol (DTT) under nondenaturing conditions also results in a time-dependent loss of aggregation activity. Sucrose density gradient floatation of SAP-35 with 14 C dipalmitoyl phosphatidycholine labelled vesicles in the absence or presence of DTT suggests retention of SAP-35 lipid binding capacity. These data demonstrate the importance of SAP-35 triple helix and disulfide crosslinking integrity for the aggregation of unilamellar phospholipid vesicles

Giant Gold Nanowire Vesicle-Based Colorimetric and SERS Dual-Mode Immunosensor for Ultrasensitive Detection of Vibrio parahemolyticus.

Science.gov (United States)

Guo, Zhiyong; Jia, Yaru; Song, Xinxin; Lu, Jing; Lu, Xuefei; Liu, Baoqing; Han, Jiaojiao; Huang, Youju; Zhang, Jiawei; Chen, Tao

2018-05-15

Conventional methods for the detection of Vibrio parahemolyticus (VP) usually need tedious, labor-intensive processes, and have low sensitivity, which further limits their practical applications. Herein, we developed a simple and efficient colorimetry and surface-enhanced Raman scattering (SERS) dual-mode immunosensor for sensitive detection of VP, by employing giant Au vesicles with anchored tiny gold nanowires (AuNW) as a smart probe. Due to the larger specific surface and special hollow structure of giant Au vesicles, silver staining would easily lead to vivid color change for colorimetric analysis and further amplify SERS signals. The t-test was further used to determine if two sets of data from colorimetry and SERS were significantly different from each other. The result shows that there was no significant difference between data from the two methods. Two sets of data can mutually validate each other and avoid false positive and negative detection. The designed colorimetry-SERS dual-mode sensor would be very promising in various applications such as food safety inspection, personal healthcare, and on-site environmental monitoring.

Lipid lateral organization on giant unilamellar vesicles containing lipopolysaccharides

DEFF Research Database (Denmark)

Kubiak, Jakub; Brewer, Jonathan R.; Hansen, Søren

2011-01-01

15 mol % for LPS-smooth and LPS-Ra, and up to 25 mol % for LPS-Rc and LPS-Rd (with respect to total lipids). We used the GUVs to evaluate the impact of different LPS species on the lateral structure of the host membrane (i.e., E. coli polar lipid extract). Rhodamine-DPPE-labeled GUVs show...... model membranes, and that the size of these domains depends on the chemical structure and concentration of the LPSs....

Membrane Protrusion Coarsening and Nanotubulation within Giant Unilamellar Vesicles

KAUST Repository

Węgrzyn, Ilona; Jeffries, Gavin D. M.; Nagel, Birgit; Katterle, Martin; Gerrard, Simon R.; Brown, Tom; Orwar, Owe; Jesorka, Aldo

2011-01-01

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

Membrane orientation and lateral diffusion of BODIPY-cholesterol as a function of probe structure

DEFF Research Database (Denmark)

Solanko, Lukasz Michal; Wüstner, Daniel; Lund, Frederik Wendelboe

2013-01-01

-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal...

Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy

DEFF Research Database (Denmark)

Hedin, E.M.K.; Høyrup, Lise Pernille Kristine; Patkar, S.A.

2005-01-01

fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TILL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through......The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal......) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 1418514196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2...

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.

Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

Czech Academy of Sciences Publication Activity Database

Javanainen, M.; Martinez-Seara, Hector; Metzler, R.; Vattulainen, I.

2017-01-01

Roč. 8, č. 17 (2017), s. 4308-4313 ISSN 1948-7185 R&D Projects: GA ČR(CZ) GBP208/12/G016 Institutional support: RVO:61388963 Keywords : giant unilamellar vesicles * single-molecule tracking * lipid bilayer membranes Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 9.353, year: 2016

Phospholipase A/sub 2/ activity towards vesicles of DPPC and DMPC-DSPC containing small amounts of SMPC

DEFF Research Database (Denmark)

Høyrup, Lise Pernille Kristine; Mouritsen, Ole G.; Jørgensen, Kent

2001-01-01

Phospholipase A/sub 2/ (PLA/sub 2/) is an interfacially active enzyme whose hydrolytic activity is known to be enhanced in one-component phospholipid bilayer substrates exhibiting dynamic micro-heterogeneity. In this study the activity of PLA/sub 2/ towards large unilamellar vesicles composed of ...

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate

DEFF Research Database (Denmark)

Stock, Roberto; Brewer, Jonathan R.; Wagner, Kerstin

2012-01-01

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model...... membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy...... and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing...

Emergence of DNA-encapsulating liposomes from a DNA-lipid blend film

Science.gov (United States)

Shimobayashi, Shunsuke; Hishida, Mafumi; Kurimura, Tomo; Ichikawa, Masatoshi

A Micro-scale giant unilamellar vesicle (GUV) densely encapsulating molecular systems is one of the simplest life-mimicking model systems. The dehydration-rehydration process proposed by Deamer et al. more than 30 years ago generates vesicles to satisfy the constraints of micro-scale size, unilamellarity and densely polymer-encapsulation. Nevertheless, the physico-chemical mechanism of a set of dehydration-rehydration process has been poorly understood. The present study reveals crucial factors on the process through fluorescent microscopic observation and small angle x-ray scattering. From the results, we propose a plausible physical mechanism for the process, making it possible to optimize the encapsulation of any agent This work was supported by Grant-in-Aid for JSPS Fellows Grant (No. 25-1270) and by KAKENHI (Nos. 26707020, 25103012, and 26115709).

Fusion of Sendai Virus with Vesicles of Oligomerizable Lipids : A Micro Calorimetric Analysis of Membrane Fusion

NARCIS (Netherlands)

Ravoo, Bart Jan; Weringa, Wilke D.; Engberts, Jan B.F.N.

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°C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

DEFF Research Database (Denmark)

Sezgin, Erdinc; Betul Can, Fatma; Schneider, Falk

2016-01-01

Cholesterol is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of cholesterol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently...... for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase separated giant unilamellar vesicles (GUVs) and giant plasma membrane vesicles (GPMVs); 2) cellular trafficking, specifically subcellular localization in Niemann-Pick C...... in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent cholesterol analogs in visualizing cellular cholesterol dynamics....

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.

Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy

Czech Academy of Sciences Publication Activity Database

Macháň, Radek; Hof, Martin

2010-01-01

Roč. 1798, č. 7 (2010), s. 1377-1391 ISSN 0005-2736 R&D Projects: GA ČR GA203/08/0114; GA AV ČR GEMEM/09/E006 Institutional research plan: CEZ:AV0Z40400503 Keywords : supported lipid bilayer * giant unilamellar vesicle * fluorescence recovery after photobleaching Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.647, year: 2010

Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells

Czech Academy of Sciences Publication Activity Database

Čujová, Sabína; Bednárová, Lucie; Slaninová, Jiřina; Straka, J.; Čeřovský, Václav

2014-01-01

Roč. 20, č. 11 (2014), s. 885-895 ISSN 1075-2617 Institutional support: RVO:61388963 Keywords : antimicrobial peptides * wild-bee venom * CD spectroscopy * large unilamellar vesicles * membrane permeabilization * electron microscopy Subject RIV: CE - Biochemistry Impact factor: 1.546, year: 2014

Thermodynamics and Structural Evolution during a Reversible Vesicle-Micelle Transition of a Vitamin-Derived Bolaamphiphile Induced by Sodium Cholate.

Science.gov (United States)

Tian, Jun-Nan; Ge, Bing-Qiang; Shen, Yun-Feng; He, Yu-Xuan; Chen, Zhong-Xiu

2016-03-09

Interaction of endogenous sodium cholate (SC) with dietary amphiphiles would induce structural evolution of the self-assembled aggregates, which inevitably affects the hydrolysis of fat in the gut. Current work mainly focused on the interaction of bile salts with classical double-layered phospholipid vesicles. In this paper, the thermodynamics and structural evolution during the interaction of SC with novel unilamellar vesicles formed from vitamin-derived zwitterionic bolaamphiphile (DDO) were characterized. It was revealed that an increased temperature and the presence of NaCl resulted in narrowed micelle-vesicle coexistence and enlarged the vesicle region. The coexistence of micelles and vesicles mainly came from the interaction of monomeric SC with DDO vesicles, whereas micellar SC contributed to the total solubilization of DDO vesicles. This research may enrich the thermodynamic mechanism behind the structure transition of the microaggregates formed by amphiphiles in the gut. It will also contribute to the design of food formulation and drug delivery system.

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.

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.

Attachment of 99mTc to lipid vesicles containing the lipophilic chelate dipalmitoylphosphatidylethanolamine-DTPA

International Nuclear Information System (INIS)

Ahkong, Q.F.; Tilcock, C.

1992-01-01

The binding of 99m Tc to negatively-charged and neutral unilamellar lipid vesicles was investigated in the absence and presence of the ligand diethylenetriaminepentaacetic acid (DTPA) covalently attached to the headgroup of phosphatidylethanolamine at the surface of the membrane. Even in the absence of DTPA on the membrane surface, 99m Tc reduced by Sn bound to the membrane surface but rapidly dissociated from the vesicles in the presence of plasma in vitro. When DTPA was present on the membrane surface, dissociation of 99m Tc from the vesicle surface in plasma was much reduced. The dissociation of 99m Tc from the surface of negatively-charged vesicles was less than for neutral vesicles in the absence of ligand but was markedly greater than for vesicles containing the ligand DTPA, suggesting that the binding of 99m Tc to vesicles with surface-attached DTPA could not be explained solely on the basis of the negative charge provided by the DTPA. In vitro experiments using 14 C-labeled lipids as well as in vivo imaging studies indicated that dissociation of 99m Tc from the surface of the vesicle did not arise predominantly because of lipid exchange with plasma components or due to cleavage of Tc-DTPA from the vesicle surface. For vesicles with surface-attached DTPA, 99m Tc dissociation from the vesicle surface in plasma was further reduced by addition of the antioxidant ascorbate. (author)

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...... of the phenomenological constants in a canonical continuum description of fluid lipid-bilayer membranes and shown the consequences of this new interpretation in terms of the characteristics of the dynamics of vesicle shape fluctuations. Moreover, we have used the systematic formulation of our theory as a framework...... against which we have discussed the previously existing theories and their discrepancies. Finally, we have made a systematic prediction about the system-dependent characteristics of the relaxation dynamics of shape fluctuations of quasi-spherical vesicles with a view of experimental studies...

Mesoporous silica for drug delivery: Interactions with model fluorescent lipid vesicles and live cells.

Science.gov (United States)

Bardhan, Munmun; Majumdar, Anupa; Jana, Sayantan; Ghosh, Tapas; Pal, Uttam; Swarnakar, Snehasikta; Senapati, Dulal

2018-01-01

Formulated mesoporous silica nanoparticle (MSN) systems offer the best possible drug delivery system through the release of drug molecules from the accessible pores. In the present investigation, steady state and time resolved fluorescence techniques along with the fluorescence imaging were applied to investigate the interactions of dye loaded MSN with fluorescent unilamellar vesicles and live cells. Here 1,2-dimyristoyl-sn-glycero-3-phospocholine (DMPC) was used to prepare Small Unilamellar Vesicles (SUVs) as the model membrane with fluorescent 1,6-diphenyl-1,3,5-hexatriene (DPH) molecule incorporated inside the lipid bilayer. The interaction of DPH incorporated DMPC membrane with Fluorescein loaded MSN lead to the release of Fluorescein (Fl) dye from the interior pores of MSN systems. The extent of release of Fl and spatial distribution of the DPH molecule has been explored by monitoring steady-state fluorescence intensity and fluorescence lifetime at physiological condition. To investigate the fate of drug molecule released from MSN, fluorescence anisotropy has been used. The drug delivery efficiency of the MSN as a carrier for doxorubicin (DOX), a fluorescent chemotherapeutic drug, has also been investigated at physiological conditions. The study gives a definite confirmation for high uptake and steady release of DOX in primary oral mucosal non-keratinized squamous cells in comparison to naked DOX treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Morphology transition of raft-model membrane induced by osmotic pressure: Formation of double-layered vesicle similar to an endo- and/or exocytosis

International Nuclear Information System (INIS)

Onai, Teruaki; Hirai, Mitsuhiro

2010-01-01

The effect of osmotic pressure on the structure of large uni-lamellar vesicle (LUV) of the lipid mixtures of monosialoganglioside (G M1 )-cholesterol-dioleoyl-phosphatidylcholine (DOPC) was studies by using wide-angle X-ray scattering (WAXS) method. The molar ratios of the mixtures were 0.1/0.1/1, 0/0.1/1, and 0/0/1. The ternary lipid mixture is a model of lipid rafts. The value of osmotic pressure was varied from 0 to 4.16x10 5 N/m 2 by adding the polyvinylpyrrolidone (PVP) in the range from 0 to 25 % w/v. In the case of the mixtures without G M1 , the rise of the osmotic pressure just enhances the multi-lamellar stacking with deceasing the inter-lamellar spacing. On the other hand, the mixture containing G M1 shows the structural transition from a uni-lamellar vesicle to a double-layered vesicle (a liposome including a smaller one inside) by the rise of osmotic pressure. In this morphology transition the total surface area of the double-layered vesicle is mostly as same as that of the LUV at the initial state. The polar head region of G M1 is bulky and highly hydrophilic due to the oligosaccharide chain containing a sialic acid residue. Then, the present results suggest that the existence of G M1 in the outer-leaflet of the LUV is essentially important for such a double-layered vesicle formation. Alternatively, a phenomenon similar to an endo- and/or exocytosis in cells can be caused simply by a variation of osmotic pressure.

Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

DEFF Research Database (Denmark)

Börner, Richard; Ehrlich, Nicky; Hohlbein, Johannes

2016-01-01

Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels...... interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination...

Orthogonal functionalization of nanoporous substrates: control of 3D surface functionality.

Science.gov (United States)

Lazzara, Thomas D; Kliesch, Torben-Tobias; Janshoff, Andreas; Steinem, Claudia

2011-04-01

Anodic aluminum oxide (AAO) membranes with aligned, cylindrical, nonintersecting pores were selectively functionalized in order to create dual-functionality substrates with different pore-rim and pore-interior surface functionalities, using silane chemistry. We used a two-step process involving an evaporated thin gold film to protect the underlying surface functionality of the pore rims. Subsequent treatment with oxygen plasma of the modified AAO membrane removed the unprotected organic functional groups, i.e., the pore-interior surface. After gold removal, the substrate became optically transparent, and displayed two distinct surface functionalities, one at the pore-rim surface and another at the pore-interior surface. We achieved a selective hydrophobic functionalization with dodecyl-trichlorosilane of either the pore rims or the pore interiors. The deposition of planar lipid membranes on the functionalized areas by addition of small unilamellar vesicles occurred in a predetermined fashion. Small unilamellar vesicles only ruptured upon contact with the hydrophobic substrate regions forming solid supported hybrid bilayers. In addition, pore-rim functionalization with dodecyl-trichlorosilane allowed the formation of pore-spanning hybrid lipid membranes as a result of giant unilamellar vesicle rupture. Confocal laser scanning microscopy was employed to identify the selective spatial localization of the adsorbed fluorescently labeled lipids. The corresponding increase in the AAO refractive index due to lipid adsorption on the hydrophobic regions was monitored by optical waveguide spectroscopy. This simple orthogonal functionalization route is a promising method to control the three-dimensional surface functionality of nanoporous films. © 2011 American Chemical Society

Interaction of charged amphiphilic drugs with phosphatidylcholine vesicles studied by NMR

International Nuclear Information System (INIS)

Eriksson, L.E.G.

1987-01-01

Small unilamellar vesicles from egg phosphatidylcholine in NaCl solutions were exposed to some amphiphilic pharmaca. The aromatic drugs (chlorpromazine, dibucaine, tetracaine, imipramine and propranolol) were in their cationic form of the amines. By 1 H- and 31 P-NMR the membrane signals were observed. In particular, the N-methyl choline proton signals were followed upon drug addition. The intrinsic chemical shift difference (0.02 ppm) between the inner (upfield) and outer choline signals was influenced by the drug concentration. Packing properties of the lipid head groups and ring current shift probably contributed. At very high drug concentration, the vesicles are destroyed. A transformation into a micellar state with a high sample viscosity took place in a narrow concentration range of drug. The anion effects of Cl - were studied from the 35 Cl-NMR linewidth at 9.8 and 39.1 MHz. A continuous increase in the signal linewidth followed upon drug addition to the vesicles. Only chlorpromazine produced a broadening in the absence of vesicles (NaCl blank). The linewidth reflected a critical micelle concentration of this drug around 7 mM in 0.1 M NaCl. The 35 Cl-NMR experiments demonstrated the existence of an anionic counterion effect. This phenomenon should be accounted for when quantitatively analysing drug-membrane interactions in electrostatic terms. (Auth.)

Preparation and evaluation of unilamellar liposomes incorporating boron-containing derivatives of cholesterol

International Nuclear Information System (INIS)

Feakes, D.A.; Tate, C.C.; Stefanutti, S.J.

2000-01-01

The application of boron neutron capture therapy is dependent on the identification and preparation of boron-containing compounds that can be delivered and retained by the tumor cells. Unilamellar liposomes have been investigated as potential tumor-specific delivery vehicles for boron-containing compounds that have no inherent tumor specificity. A series of carborane-containing derivatives of cholesterol have been prepared and incorporated into the bilayer of unilamellar liposomes. The cholesterol derivatives vary in the linker moiety (ester and ether), the chain length between the cholesterol and the carborane substituent, and the identity of the carborane group itself (closo- and nido-). The ability of the boron-containing derivatives of cholesterol to be incorporated into the bilayer of the unilamellar liposomes and the stability of the resulting liposome formulations will be presented. (author)

Transfer of oleic acid between albumin and phospholipid vesicles

International Nuclear Information System (INIS)

Hamilton, J.A.; Cistola, D.P.

1986-01-01

The net transfer of oleic acid between egg phosphatidylcholine unilamellar vesicles and bovine serum albumin has been monitored by 13 C NMR spectroscopy and 90% isotopically substituted [1- 13 C]oleic acid. The carboxyl chemical shifts of oleic acid bound to albumin were different from those for oleic acid in phospholipid vesicles. Therefore, in mixtures of donor particles, the equilibrium distribution of oleic acid was determined from chemical shift and peak intensity data without separation of donor and acceptor particles. In a system containing equal masses of albumin and phospholipid and a stoichiometry of 4-5 mol of oleic acid per mol of albumin, the oleic acid distribution was pH dependent, with ≥80% of the oleic acid associated with albumin at pH 7.4; association was ≥90% at pH 8.0. Decreasing the pH below 7.4 markedly decreased the proportion of fatty acid bound to albumin. The distribution was reversible with pH and was independent of whether vesicles or albumin acted as a donor. These data suggest that pH may strongly influence the partitioning of fatty acid between cellular membranes and albumin. The 13 C NMR method is also advantageous because it provides information about the structural environments of oleic acid bound to albumin or phospholipid, the ionization state of oleic acid in each environment, and the structural integrity of the vesicles. In addition, minimum and maximum limits for the exchange rates of oleic acid among different environments were obtained from the NMR data

Biomembrane Permeabilization: Statistics of Individual Leakage Events Harmonize the Interpretation of Vesicle Leakage.

Science.gov (United States)

Braun, Stefan; Pokorná, Šárka; Šachl, Radek; Hof, Martin; Heerklotz, Heiko; Hoernke, Maria

2018-01-23

The mode of action of membrane-active molecules, such as antimicrobial, anticancer, cell penetrating, and fusion peptides and their synthetic mimics, transfection agents, drug permeation enhancers, and biological signaling molecules (e.g., quorum sensing), involves either the general or local destabilization of the target membrane or the formation of defined, rather stable pores. Some effects aim at killing the cell, while others need to be limited in space and time to avoid serious damage. Biological tests reveal translocation of compounds and cell death but do not provide a detailed, mechanistic, and quantitative understanding of the modes of action and their molecular basis. Model membrane studies of membrane leakage have been used for decades to tackle this issue, but their interpretation in terms of biology has remained challenging and often quite limited. Here we compare two recent, powerful protocols to study model membrane leakage: the microscopic detection of dye influx into giant liposomes and time-correlated single photon counting experiments to characterize dye efflux from large unilamellar vesicles. A statistical treatment of both data sets does not only harmonize apparent discrepancies but also makes us aware of principal issues that have been confusing the interpretation of model membrane leakage data so far. Moreover, our study reveals a fundamental difference between nano- and microscale systems that needs to be taken into account when conclusions about microscale objects, such as cells, are drawn from nanoscale models.

Membrane Restructuring Events during the Enzymatic Generation of Ceramides with Very Long-Chain Polyunsaturated Fatty Acids.

Science.gov (United States)

Peñalva, Daniel A; Antollini, Silvia S; Ambroggio, Ernesto E; Aveldaño, Marta I; Fanani, María L

2018-04-10

In rat sperm heads, sphingomyelin (SM) species that contain very long-chain polyunsaturated fatty acid (V-SM) become ceramides (V-Cer) after inducing in vitro the acrosomal reaction. The reason for such a specific location of this conversion, catalyzed by a sphingomyelinase (SMase), has received little investigation so far. Here, the effects of SMase were compared in unilamellar vesicles (large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs)) containing phosphatidylcholine, and either V-SM or a palmitate-rich SM (P-SM). In uniformly sized LUVs at 37 °C, more V-Cer was generated and more rapidly than P-Cer. Nephelometry and dynamic light scattering showed that LUVs tended to form large lipid particles more intensely, and Förster resonance energy transfer (FRET) increases suggested that lateral lipid mixing was more marked when V-Cer rather than P-Cer was produced. As reported by 6-dodecanoyl-2-dimethyl-aminopnaphthalene (Laurdan) and 1,6-diphenyl-1,3,5,-hexatriene (DPH), the production of V-Cer resulted in higher and faster restriction in lipid mobility than that of P-Cer, implying a stronger increase in membrane dehydration and microviscosity. Moreover, DPH anisotropy suggested a higher solubility of V-Cer than that of P-Cer in the liquid-disordered phase. At room temperature, liquid-condensed lateral domains appeared in P-SM- but not in V-SM-containing GUVs. The former maintained their size while losing their contents gradually during SMase action, whereas the latter became permeable earlier and reduced their size in few minutes until suddenly collapsing. The fast and potent generation of V-Cer may contribute to the membrane restructuring events that occur on the acrosome-reacted sperm head.

A metal-ion NMR investigation of the antibiotic facilitated transport of monovalent cations through the walls of phospholipid vesicles. II. Sulfur-33 NMR

International Nuclear Information System (INIS)

Buster, D.C.

1988-01-01

A technique has been developed to investigate the antibiotic facilitated transmembrane transport of monovalent cations using 23 Na and 7 Li Nuclear Magnetic Resonance spectroscopy. The initial portion of this thesis outlines the production and characterization of a model lipid system amenable to the NMR detection of cation transport. Large unilamellar vesicles (LUV) have been prepared from a 4:1 mixture of phosphatidylcholine and phosphatidylglycerol. The presence of the anionic chemical shift reagent dysprosium (III) tripolyphosphate, either inside or outside of the vesicles, allows for the spectroscopic separation of the NMR resonances arising from the inter- and extravesicular cation pools. The cation transporting properties of the channel-forming pentadecapeptide, gramicidin D, have been studied using the NMR technique

A Quantitative Analytical Method to Test for Salt Effects on Giant Unilamellar Vesicles

DEFF Research Database (Denmark)

Hadorn, Maik; Bönzli, Eva; Eggenberger Hotz, Peter

2011-01-01

preparation method with automatic haemocytometry. We found that this new quantitative screening method is highly reliable and consistent with previously reported results. Thus, this method may provide a significant methodological advance in analysis of effects on free-standing model membranes....

Transformation from Multilamellar to Unilamellar Vesicles by Addition of a Cationic Lipid to PEGylated Liposomes Explored with Synchrotron Small Angle X-ray Scattering

International Nuclear Information System (INIS)

Sakuragi, Mina; Sakurai, Kazuo; Koiwai, Kazunori; Nakamura, Kouji; Masunaga, Hiroyasu; Ogawa, Hiroki

2011-01-01

PEGylated liposomes composed of a benzamidine derivative (TRX), hydrogenated soybean phosphatidylcholine (HSPC), and N-(monomethoxy-polyethyleneglycolcarbamyl) distearoyl phosphatidylethanolamine (PEG-PE) were examined in terms of how the addition of TRX affects their structures with small angle x-ray scattering (SAXS) as well as transmission electron microscopy (TEM). TEM images showed the presence of unilamella vesicles for both with and without TRX, though a small amount of multilamella vesicles were observed in absence of TRX. We analyzed SAXS profiles at contained TRX composition combined with contrast variation technique by adding PEG solution and unilamella vesicle model could be reproduced. Subsequently, we analyzed SAXS profiles at no TRX composition. The mixture model of unilamella and multilamella vesicle was reconstructed and we estimated about 10 % multilamella vesicles from a fitting parameter.

Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures.

Science.gov (United States)

Semple, S C; Klimuk, S K; Harasym, T O; Dos Santos, N; Ansell, S M; Wong, K F; Maurer, N; Stark, H; Cullis, P R; Hope, M J; Scherrer, P

2001-02-09

Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15--0.25 g ODN/g lipid) of polyanionic ODN in lipid vesicles. This process requires the presence of up to 40% ethanol (v/v) and initial formulation at acidic pH values where the DODAP is positively charged. In addition, the presence of a poly(ethylene glycol)-lipid was required during the formulation process to prevent aggregation. The 'stabilized antisense-lipid particles' (SALP) formed are stable on adjustment of the external pH to neutral pH values and the formulation process allows encapsulation efficiencies of up to 70%. ODN encapsulation was confirmed by nuclease protection assays and (31)P NMR measurements. Cryo-electron microscopy indicated that the final particles consisted of a mixed population of unilamellar and small multilamellar vesicles (80--140 nm diameter), the relative proportion of which was dependent on the initial ODN to lipid ratio. Finally, SALP exhibited significantly enhanced circulation lifetimes in mice relative to free antisense ODN, cationic lipid/ODN complexes and SALP prepared with quaternary aminolipids. Given the small particle sizes and improved encapsulation efficiency, ODN to lipid ratios, and circulation times of this formulation compared to others, we believe SALP represent a viable candidate for systemic applications involving nucleic acid therapeutics.

Hydrolysis of triolein in phospholipid vesicles and microemulsions by a purified rat liver acid lipase.

Science.gov (United States)

Burrier, R E; Brecher, P

1983-10-10

An acid lipase was purified from rat liver lysosomes. Lipase purification involved affinity chromatography, gel filtration, and stabilization of the purified preparation using ethylene glycol and Triton X-100. A molecular weight of 67,000-69,000 was determined independently using density gradient centrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel filtration. To study enzyme action, model substrates were prepared by incorporating radiolabeled triolein into either unilamellar vesicles or microemulsions. Substrates were prepared by cosonicating aqueous dispersions of lecithin and triolein. Formation of vesicles or emulsions depended on the relative amount of each lipid and on sonication conditions. Vesicles were prepared at molar ratios between 70:1 and 26:1 (lecithin:triolein) and the microemulsion preparation at a molar ratio of 1:1. The substrate particles were of similar size (220-250 A) as determined by Bio-Gel A-15m chromatography. Hydrolysis of triolein contained in vesicles or emulsions was similar with respect to pH, temperature, and reaction products. Kinetic studies on vesicles with increasing triolein content showed progressively greater Vmax values (0-0.6 mumol/min/mg), and Vmax for the emulsion was 3.1 mumol/min/mg. Addition of human very low or low density lipoprotein produced a dose-dependent inhibition with both substrates. The results show that synthetically prepared microemulsions are stable and effective substrates for the acid lipase and indicate that surface-oriented triolein is hydrolyzed in both preparations.

Giant Plasma Membrane Vesicles: An Experimental Tool for Probing the Effects of Drugs and Other Conditions on Membrane Domain Stability.

Science.gov (United States)

Gerstle, Zoe; Desai, Rohan; Veatch, Sarah L

2018-01-01

Giant plasma membrane vesicles (GPMVs) are isolated directly from living cells and provide an alternative to vesicles constructed of synthetic or purified lipids as an experimental model system for use in a wide range of assays. GPMVs capture much of the compositional protein and lipid complexity of intact cell plasma membranes, are filled with cytoplasm, and are free from contamination with membranes from internal organelles. GPMVs often exhibit a miscibility transition below the growth temperature of their parent cells. GPMVs labeled with a fluorescent protein or lipid analog appear uniform on the micron-scale when imaged above the miscibility transition temperature, and separate into coexisting liquid domains with differing membrane compositions and physical properties below this temperature. The presence of this miscibility transition in isolated GPMVs suggests that a similar phase-like heterogeneity occurs in intact plasma membranes under growth conditions, albeit on smaller length scales. In this context, GPMVs provide a simple and controlled experimental system to explore how drugs and other environmental conditions alter the composition and stability of phase-like domains in intact cell membranes. This chapter describes methods to generate and isolate GPMVs from adherent mammalian cells and to interrogate their miscibility transition temperatures using fluorescence microscopy. © 2018 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2017-11-09

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

Synaptic Vesicle Endocytosis in Different Model Systems

Directory of Open Access Journals (Sweden)

Quan Gan

2018-06-01

Full Text Available Neurotransmission in complex animals depends on a choir of functionally distinct synapses releasing neurotransmitters in a highly coordinated manner. During synaptic signaling, vesicles fuse with the plasma membrane to release their contents. The rate of vesicle fusion is high and can exceed the rate at which synaptic vesicles can be re-supplied by distant sources. Thus, local compensatory endocytosis is needed to replenish the synaptic vesicle pools. Over the last four decades, various experimental methods and model systems have been used to study the cellular and molecular mechanisms underlying synaptic vesicle cycle. Clathrin-mediated endocytosis is thought to be the predominant mechanism for synaptic vesicle recycling. However, recent studies suggest significant contribution from other modes of endocytosis, including fast compensatory endocytosis, activity-dependent bulk endocytosis, ultrafast endocytosis, as well as kiss-and-run. Currently, it is not clear whether a universal model of vesicle recycling exist for all types of synapses. It is possible that each synapse type employs a particular mode of endocytosis. Alternatively, multiple modes of endocytosis operate at the same synapse, and the synapse toggles between different modes depending on its activity level. Here we compile review and research articles based on well-characterized model systems: frog neuromuscular junctions, C. elegans neuromuscular junctions, Drosophila neuromuscular junctions, lamprey reticulospinal giant axons, goldfish retinal ribbon synapses, the calyx of Held, and rodent hippocampal synapses. We will compare these systems in terms of their known modes and kinetics of synaptic vesicle endocytosis, as well as the underlying molecular machineries. We will also provide the future development of this field.

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.

Vesicle Encapsulation Studies Reveal that Single Molecule Ribozyme Heterogeneities Are Intrinsic

Science.gov (United States)

Okumus, Burak; Wilson, Timothy J.; Lilley, David M. J.; Ha, Taekjip

2004-01-01

Single-molecule measurements have revealed that what were assumed to be identical molecules can differ significantly in their static and dynamic properties. One of the most striking examples is the hairpin ribozyme, which was shown to exhibit two to three orders of magnitude variation in folding kinetics between molecules. Although averaged behavior of single molecules matched the bulk solution data, it was not possible to exclude rigorously the possibility that the variations around the mean values arose from different ways of interacting with the surface environment. To test this, we minimized the molecules' interaction with the surface by encapsulating DNA or RNA molecules inside 100- to 200-nm diameter unilamellar vesicles, following the procedures described by Haran and coworkers. Vesicles were immobilized on a supported lipid bilayer via biotin-streptavidin linkages. We observed no direct binding of DNA or RNA on the supported bilayer even at concentrations exceeding 100 nM, indicating that these molecules do not bind stably on the membrane. Since the vesicle diameter is smaller than the resolution of optical microscopy, the lateral mobility of the molecules is severely constrained, allowing long observation periods. We used fluorescence correlation spectroscopy, nuclease digestion, and external buffer exchange to show that the molecules were indeed encapsulated within the vesicles. When contained within vesicles, the natural form of the hairpin ribozyme exhibited 50-fold variation in both folding and unfolding rates in 0.5 mM Mg2+, which is identical to what was observed from the molecules tethered directly on the surface. This strongly indicates that the observed heterogeneity in dynamic properties does not arise as an artifact of surface attachment, but is intrinsic to the nature of the molecules. PMID:15454471

A bio-synthetic interface for discovery of viral entry mechanisms.

Energy Technology Data Exchange (ETDEWEB)

Gutzler, Mike; Maar, Dianna; Negrete, Oscar; Hayden, Carl C.; Sasaki, Darryl Yoshio; Stachowiak, Jeanne C.; Wang, Julia

2010-09-01

Understanding and defending against pathogenic viruses is an important public health and biodefense challenge. The focus of our LDRD project has been to uncover the mechanisms enveloped viruses use to identify and invade host cells. We have constructed interfaces between viral particles and synthetic lipid bilayers. This approach provides a minimal setting for investigating the initial events of host-virus interaction - (i) recognition of, and (ii) entry into the host via membrane fusion. This understanding could enable rational design of therapeutics that block viral entry as well as future construction of synthetic, non-proliferating sensors that detect live virus in the environment. We have observed fusion between synthetic lipid vesicles and Vesicular Stomatitis virus particles, and we have observed interactions between Nipah virus-like particles and supported lipid bilayers and giant unilamellar vesicles.

Determination of the unilamellar dimyristoylphosphatidylcholine vesicle structure from the small-angle scattering data in the framework of a model of separated form-factors

International Nuclear Information System (INIS)

Zemlyanaya, E.V.; Kiselev, M.A.

2002-01-01

On the basis of the model of separated form-factors, a code for fitting of small-angle neutron scattering spectra of the polydispersed vesicle population has been developed with corrections to the resolution function of the YuMO spectrometer. Vesicle and membrane bilayer parameters have been analyzed for various hierarchical models of the neutron scattering length density across the membrane. It was shown that hydration of vesicle can be described by the linear distribution function of water molecules. For the first time from the small-angle experiment, without additional methods, the average radius and polydispersity of the vesicle population, thickness of the membrane bilayer, thickness of hydrophobic and hydrophilic parts of bilayer, water distribution function and number of water molecules in the hydrophilic part have been calculated. (author)

Calcium Directly Regulates Phosphatidylinositol 4,5-Bisphosphate Headgroup Conformation and Recognition

DEFF Research Database (Denmark)

Bilkova, Eva; Pleskot, Roman; Rissanen, Sami

2017-01-01

), is completely inhibited in the presence of Ca2+, while Mg2+ has no effect with 100 nm liposomes and modest effect with giant unilamellar vesicles. Consistent with biochemical data, vibrational sum frequency spectroscopy and atomistic molecular dynamics simulations reveal how Ca2+ binding to the PI(4,5)P2...... phosphoinositide clustering, little is known about the molecular basis for this or its significance in cellular signaling. Here, we study the direct interaction of Ca2+ with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), the main lipid marker of the plasma membrane. Electrokinetic potential measurements of PI...

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate.

Directory of Open Access Journals (Sweden)

Roberto P Stock

Full Text Available The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1 ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2 the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3 in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

Thermal structural evolutions of DMPC-water biomimetic systems investigated by Raman Spectroscopy.

Science.gov (United States)

Fasanella, A; Cosentino, K; Beneduci, A; Chidichimo, G; Cazzanelli, E; Barberi, R C; Castriota, M

2018-06-01

Many cell membranes of living organisms can be represented as phospholipid bilayers immersed into a water environment. The physical-chemical interactions at the membranes/water interface are responsible for the stabilization of the membranes. In addition, the drug efficiency, the pharmaceutical mechanism and the improvement of the drug design can be addressed to the interactions between the membranes-water interface with the drug and to the membrane-drug interface. In this framework, it is important to find membranes models able to simulate and simultaneously simplify the biological systems to better understand both physical and chemical interactions at the interface level. Dimyristoylphosphatidylcholine (DMPC) is a synthetic phospholipid used in order to make Multilamellar Vesicle (MLV), Large Unilamellar Vesicle (LUV) and Giant Unilamellar Vesicle (GUV). In order to understand the mechanisms of vesicle formation, we have analyzed mixtures of DMPC and water by micro-Raman spectroscopy at different temperatures in the range between 10 and 35 °C. Particularly, we analyzed the temperature dependence of the CN vibrational frequency, which appears well correlated to the order degree of the various phases. These investigations, beyond the determination of phospholipid hydrocarbon chains order, provide information about the conformation of the lipid membranes. We have identified the mixture of DMPC/water that is best suited for Raman studies and can be used as an in-vitro model for biological systems. A peculiar frequency shift across the transition gel-ripple-liquid crystalline phases has been proposed as a useful diagnostic marker to detect the "order degree" and subsequently the phases of biomimetic membranes made by DMPC. Copyright © 2018 Elsevier B.V. All rights reserved.

Lyophilization and rehydration of polymer-coated lipid vesicles containing a lipophilic chelator in the presence of sucrose: Labeling with99m Tc and biodistribution studies

International Nuclear Information System (INIS)

Szucs, Margaret; Tilcock, Colin

1995-01-01

In this paper we describe studies of the effect of lyophilization and rehydration of polymer-coated lipid vesicles bearing a lipophilic surface chelator upon subsequent labeling with technetium-99m and in vivo biodistribution behavior. Unilamellar vesicles of average diameter 100 nm were prepared containing 2 mol% of the lipophilic chelator phosphatidylethanolamine-diethylenetriaminetetraacetic acid (PE-DTTA) and either 0 or 3 mol% of the lipid-polymer conjugate, dipalmitoyl-phosphatidylethanolamine-monomethoxy polyethylene glycol 5000 (PE-MPEG) in 0.9% sodium chloride to which was added varying amounts of sucrose to a final concentration of 100-500 mM. The size of the vesicles in sucrose was determined before lyophilization and after rehydration and the effect of sucrose on the ability to label the vesicles with pertechnetate in the presence of stannous chloride was determined. Biodistribution studies were done in rabbits with vesicles before lyophilization and after rehydration in order to determine whether the rate of clearance from the blood pool was affected by the lyophilization/rehydration procedure. Results demonstrate that vesicles containing PE-DTTA and without PE-MPEG can be lyophilized and rehydrated with no change in average size or size distribution so long as the external sucrose concentration is greater than approx. 250 mM. When PE-MPEG is also present in the membrane the average vesicle size increases from approx. 140 to 200 nm, consistent with vesicle fusion. However, this small change in vesicle size makes no difference to the resulting circulation half-life. In no case does the presence of sucrose on the exterior of the vesicle interfere with technetium labeling of the vesicle surface

Exploring the raft-hypothesis by probing planar bilayer patches of free-standing giant vesicles at nanoscale resolution, with and without Na,K-ATPase

DEFF Research Database (Denmark)

Bhartia, Tripta; Cornelius, Flemming; Ipsen, J. H.

2016-01-01

The structure of functional lipid domains (rafts) in biological membranes has for long time been unresolved due to their small length scales and transient nature. These cooperative properties of the lipid bilayer matrix are modelled by free-standing giant unilammellar vesicles (GUVs) with well...... mixtures of DOPC/DPPC/cholesterol with and without Na,K-ATPase (NKA), a transmembrane protein known to be associated with rafts. Two mechanisms of domain formation are revealed: 1) close to lo/ld phase coexistence, domains in size up to 100 nm appear as thermally induced droplet fluctuations, 2) NKA shows...... interfacial activity and cluster in lo/ld micro-emulsion droplets. Some perspectives for the application of the techniques and the understanding of the nature of raft domains are outlined....

Stability of phospholipid vesicles studied by asymmetrical flow field-flow fractionation and capillary electrophoresis

Energy Technology Data Exchange (ETDEWEB)

Yohannes, Gebrenegus [Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, FIN-00014 University of Helsinki (Finland); Pystynen, Kati-Henna [Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, FIN-00014 University of Helsinki (Finland); Riekkola, Marja-Liisa [Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, FIN-00014 University of Helsinki (Finland); Wiedmer, Susanne K. [Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, FIN-00014 University of Helsinki (Finland)]. E-mail: susanne.wiedmer@helsinki.fi

2006-02-23

The stability of zwitterionic phosphatidylcholine vesicles in the presence of 20 mol% phosphatidyl serine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI), and diacylphosphatidyl glycerol (PG) phospholipid vesicles, and cholesterol or calcium chloride was investigated by asymmetrical flow field-flow fractionation (AsFlFFF). Large unilamellar vesicles (LUV, diameter 100 nm) prepared by extrusion at 25 deg. C were used. Phospholipid vesicles (liposomes) were stored at +4 and -18 deg. C over an extended period of time. Extruded egg yolk phosphatidylcholine (EPC) particle diameters at peak maximum and mean measured by AsFlFFF were 101 {+-} 3 nm and 122 {+-} 5 nm, respectively. No significant change in diameter was observed after storage at +4 deg. C for about 5 months. When the storage period was extended to about 8 months (250 days) larger destabilized aggregates were formed (172 and 215 nm at peak maximum and mean diameters, respectively). When EPC was stored at -18 deg. C, large particles with diameters of 700-800 nm were formed as a result of dehydration, aggregation, and fusion processes. In the presence of calcium chloride, EPC alone did not form large aggregates. Addition of 20 mol% of negatively charged phospholipids (PS, PA, PI, or PG) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles increased the electrostatic interactions between calcium ion and the vesicles and large aggregates were formed. In the presence of cholesterol, large aggregates of about 250-350 nm appeared during storage at +4 and -18 deg. C for more than 1 day. The effect of liposome storage temperature on phospholipid coatings applied in capillary electrophoresis (CE) was studied by measuring the electroosmotic flow (EOF). EPC coatings with and without cholesterol, PS, or calcium chloride, prepared from liposomes stored at +25, +4, and -18 deg. C, were studied at 25 deg. C. The performances of the coatings were further evaluated with three uncharged compounds

Development and characterization of nanopore system for nano-vesicle analysis

Science.gov (United States)

Goyal, Gaurav

Nano-vesicles have recently attracted a lot of attention in research and medical communities and are very promising next-generation drug delivery vehicles. This is due to their biocompatibility, biodegradability and their ability to protect drug cargo and deliver it to site-specific locations, while maintaining the desired pharmacokinetic profile. The interaction of these drug loaded vesicles with the recipient cells via adsorption, endocytosis or receptor mediated internalization involve significant bending and deformation and is governed by mechanical properties of the nano-vesicles. Currently, the mechanical characteristics of nano-vesicles are left unexplored because of the difficulties associated with vesicle analysis at sub-100 nm length scale. The need for a complete understanding of nano-vesicle interaction with each other and the recipient cells warrants development of an analytical tool capable of mechanical investigation of individual vesicles at sub-100 nm scale. This dissertation presents investigation of nano-vesicle deformability using resistive pulse sensing and solid-state nanopore devices. The dissertation is divided into four chapters. Chapter 1 discusses the motivation, specific aims and presents an overview of nanoparticle characterization techniques, resistive pulse sensing background and principles, techniques for fabricating solid-state nanopores, as well the deformation behavior of giant vesicles when placed in electric field. Chapter 2 is dedicated to understanding of the scientific principles governing transport of sub-100 nm particles in dilute solutions. We investigated the translocation of rigid nanoparticles through nanopores at salt concentrations exosomes derived from human breast cancer cell line. Exosomes also exhibit co-translocational deformation behavior; however, they appear to be less affected by the deforming force inside the nanopore compared to the DOPC liposomes. We believe, the results of this research will bring about a

Capturing suboptical dynamic structures in lipid bilayer patches formed from free-standing giant unilamellar vesicles

DEFF Research Database (Denmark)

Bhatia, Tripta; Cornelius, Flemming; Ipsen, John H.

2017-01-01

. The method has been applied to classical lipid raft mixtures in which suboptical domain fluctuations have been imaged in both the liquid-ordered and liquid-disordered membrane phases. High-resolution scanning by atomic force microscopy (AFM) of membranes composed of binary and ternary lipid mixtures...

Peptide and nucleic acid-directed self-assembly of cationic nanovehicles through giant unilamellar vesicle modification

DEFF Research Database (Denmark)

Tagalakis, A. D.; Maeshima, R.; Yu-Wai-Man, C.

2017-01-01

into a neuroblastoma xenograft mouse model, nanovesicle complexes were found to distribute throughout the tumour interstitium, thus providing an alternative safer approach for future development of tumour-specific therapeutic nucleic acid interventions. On oropharyngeal instillation, nanovesicle complexes displayed...

Membrane fusion of pH-sensitive liposomes – a quantitative study using giant unilamellar vesicles

DEFF Research Database (Denmark)

Trier, Sofie; Henriksen, Jonas Rosager; Andresen, Thomas Lars

2011-01-01

This article presents a methodology for developing small-signal behavioral electromagnetic (EM) models of p-i-n photodiodes (PDs) for high-speed applications. The EM model includes RC bandwidth limitation effect and transit-time effect. The model is capable of accurately modeling arbitrary comple...

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

DEFF Research Database (Denmark)

Bagatolli, Luis; Needham, David

2014-01-01

to study composition-structure-property materials relationships of free-standing lipid bilayer membranes. Because their size (~5 to 100 m diameter) that is well above the resolution limit of regular light microscopes, GUVs are suitable membrane models for optical microscopy and micromanipulation...

Exploring the raft-hypothesis by probing planar bilayer patches of free-standing giant vesicles at nanoscale resolution, with and without Na,K-ATPase.

Science.gov (United States)

Bhatia, T; Cornelius, F; Ipsen, J H

2016-12-01

The structure of functional lipid domains (rafts) in biological membranes has for long time been unresolved due to their small length scales and transient nature. These cooperative properties of the lipid bilayer matrix are modelled by free-standing giant unilammellar vesicles (GUVs) with well-characterized lipid composition. We review a series of recent advances in preparation and analysis of GUVs, which allows for characterization of small domains by high-resolution imaging techniques. These includes a new GUV preparation method with a desired overall lipid composition achieved by mixing small unilammellar vesicles (SUVs), test of the lipids compositional uniformity in GUVs and swift adsorption of GUVs to solid support by kinetically arresting the lateral structure of membrane prior to collapse for subsequent imaging. The techniques are applied to the analysis of membrane domains in GUVs formed from mixtures of DOPC/DPPC/cholesterol with and without Na,K-ATPase (NKA), a transmembrane protein known to be associated with rafts. Two mechanisms of domain formation are revealed: 1) close to l o /l d phase coexistence, domains in size up to 100nm appear as thermally induced droplet fluctuations, 2) NKA shows interfacial activity and cluster in l o /l d micro-emulsion droplets. Some perspectives for the application of the techniques and the understanding of the nature of raft domains are outlined. Copyright © 2016 Elsevier B.V. All rights reserved.

Controlled drug release under a low frequency magnetic field: effect of the citrate coating on magnetoliposomes stability

KAUST Repository

Nappini, Silvia; Bonini, Massimo; Bombelli, Francesca Baldelli; Pineider, Francesco; Sangregorio, Claudio; Baglioni, Piero; Nordè n, Bengt

2011-01-01

The paper describes the effect of a low-frequency alternating magnetic field (LF-AMF) on the permeability and release properties of large (LUVs) and giant (GUVs) unilamellar vesicles loaded with citrate coated cobalt ferrite nanoparticles (NPs). The citrate shell allows a high loading of NPs in lipid vesicles without modifying their magnetic properties. The increase of magnetic LUVs permeability upon exposure to LF-AMF has been evaluated as the fluorescence self-quenching of carboxyfluorescein (CF) entrapped inside the liposome aqueous pool. Liposome leakage has been monitored as a function of field frequency, time exposure and concentration of the citrate coated NPs. Confocal Laser Scanning Microscopy (CLSM) experiments performed on magnetic GUVs labeled with the fluorescent probe DiIC18 and loaded with Alexa 488-C5-maleimide fluorescent dye provided insights on the release mechanism induced by LF-AMF. The results show that LF-AMF strongly affects vesicles permeability, suggesting the formation of pores in the lipid bilayer due to both hyperthermic effects and nanoparticle oscillations in the vesicles pool at the applied frequency. The behaviour of these magnetic vesicles in the presence of LF-AMF makes this system a good candidate for controlled drug delivery. © 2011 The Royal Society of Chemistry.

What can we learn about the lipid vesicle structure from the small angle neutron scattering experiment?

International Nuclear Information System (INIS)

Kiselev, M.A.; Zemlyanaya, E.V.; Aswal, V.K.; Neubert, R.H.H.

2005-01-01

Small angle neutron scattering (SANS) on the unilamellar vesicle populations (diameter of 500 and 1000 Armstrong) was used to characterize lipid vesicles from dimyristoylphosphatidylcholine (DMPC) in three phases (gel, ripple, and liquid). Parameters of vesicle populations and internal structure of the DMPC bilayer were characterized on the basis of the Separated Form Factor (SFF) model. Vesicle shape changes from about spherical in liquid phase to elliptical in ripple and gel phases for vesicles prepared via extrusion through pores with the diameter of 500 Armstrong. Parameters of the internal bilayer structure (membrane thickness, thickness of the hydrophobic core, hydration, and surface area of lipid molecule) were determined on the basis of the Hydrophobic-Hydrophilic (HH) approximation of neutron scattering length density across the bilayer ρ(x) and on the basis of the Step Function (SF) approximation of ρ(x). It was demonstrated in the framework of HH approximation that DMPC membrane thickness in the liquid phase (T = 30 deg C) depends on the membrane curvature. Vesicle population prepared via extrusion through pores with the diameter of 500 Armstrong is characterized by an average radius of 275.6 ± 0.5 Armstrong, polydispersity of 27%, membrane thickness of 47.8 ± 0.2 Armstrong, thickness of hydrophobic core of 20.5 ± 0.3 Armstrong, surface area per DMPC molecule of 61.0 ± 0.4 A 2 Armstrong, and the number of water molecules per DMPC molecule of 11.9 ± 0.3. Vesicles prepared via extrusion through pores with the diameter of 1000 Armstrong have a polydispersity of 48%, and a membrane thickness of 45.6 ± 0.2 Armstrong. SF approximation was used to describe the DMPC membrane structure in gel (T 10 deg C) and ripple (T = 20 deg C) phases. DMPC vesicles prepared via extrusion through 1000- Armstrong pores have a membrane thickness of 49.6 ± 0.5 Armstrong in the gel phase and 48.3 ± 0.6 Armstrong in the ripple phase. The dependence of the DMPC membrane

Antifungal Effect of (+-Pinoresinol Isolated from Sambucus williamsii

Directory of Open Access Journals (Sweden)

Bomi Hwang

2010-05-01

Full Text Available In this study, we investigated the antifungal activity and mechanism of action of (+-pinoresinol, a biphenolic compound isolated from the herb Sambucus williamsii,used in traditional medicine. (+-Pinoresinol displays potent antifungal properties without hemolytic effects on human erythrocytes. To understand the antifungal mechanism of (+-pinoresinol, we conducted fluorescence experiments on the human pathogen Candida albicans. Fluorescence analysis using 1,6-diphenyl-1,3,5-hexatriene (DPH indicated that the (+-pinoresinol caused damage to the fungal plasma membrane. This result was confirmed by using rhodamine-labeled giant unilamellar vesicle (GUV experiments. Therefore, the present study indicates that (+-pinoresinol possesses fungicidal activities and therapeutic potential as an antifungal agent for the treatment of fungal infectious diseases in humans.

Optical stretching as a tool to investigate the mechanical properties of lipid bilayers.

Science.gov (United States)

Solmaz, Mehmet E; Sankhagowit, Shalene; Biswas, Roshni; Mejia, Camilo A; Povinelli, Michelle L; Malmstadt, Noah

2013-10-07

Measurements of lipid bilayer bending modulus by various techniques produce widely divergent results. We attempt to resolve some of this ambiguity by measuring bending modulus in a system that can rapidly process large numbers of samples, yielding population statistics. This system is based on optical stretching of giant unilamellar vesicles (GUVs) in a microfluidic dual-beam optical trap (DBOT). The microfluidic DBOT system is used here to measure three populations of GUVs with distinct lipid compositions. We find that gel-phase membranes are significantly stiffer than liquid-phase membranes, consistent with previous reports. We also find that the addition of cholesterol does not alter the bending modulus of membranes composed of a monounsaturated phospholipid.

Optical stretching as a tool to investigate the mechanical properties of lipid bilayers†

Science.gov (United States)

Solmaz, Mehmet E.; Sankhagowit, Shalene; Biswas, Roshni; Mejia, Camilo A.; Povinelli, Michelle L.; Malmstadt, Noah

2013-01-01

Measurements of lipid bilayer bending modulus by various techniques produce widely divergent results. We attempt to resolve some of this ambiguity by measuring bending modulus in a system that can rapidly process large numbers of samples, yielding population statistics. This system is based on optical stretching of giant unilamellar vesicles (GUVs) in a microfluidic dual-beam optical trap (DBOT). The microfluidic DBOT system is used here to measure three populations of GUVs with distinct lipid compositions. We find that gel-phase membranes are significantly stiffer than liquid-phase membranes, consistent with previous reports. We also find that the addition of cholesterol does not alter the bending modulus of membranes composed of a monounsaturated phospholipid. PMID:24244843

Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered

Czech Academy of Sciences Publication Activity Database

Koukalová, Alena; Amaro, Mariana; Aydogan, Gokcan; Gröbner, G.; Williamson, P. T. F.; Mikhalyov, I.; Hof, Martin; Šachl, Radek

2017-01-01

Roč. 7, JUL 2017 (2017), č. článku 5460. ISSN 2045-2322 R&D Projects: GA ČR GA17-03160S Institutional support: RVO:61388955 Keywords : FLUORESCENCE CORRELATION SPECTROSCOPY * PLASMA-MEMBRANE VESICLES * RESONANCE ENERGY-TRANSFER Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 4.259, year: 2016

Morphological study of lipid vesicles in presence of amphotericin B via modification of the microfluidic CellASIC platform and LED illumination microscopy

International Nuclear Information System (INIS)

Genova, J; Decheva-Zarkova, M; Pavlič, J I

2016-01-01

Giant lipid vesicles (liposomes) are the simplest model of the biological cell and can be easily formed from natural or synthetic lipid species with controlled composition and properties. This is the reason why they are the preferred objects for various scientific investigations. Amphotericin B (AmB) is a membrane active drug, used for treatment of systemic fungal infections. In this work we studied the morphological behavior of giant SOPC vesicles in asymmetrical presence of amphotericin B antibiotic in the vicinity of the lipid membrane. The visualization of the vesicles was carried out via inverted phase contrast microscopy. The illumination source was modified in a way that tungsten light bulb was replaced by 10 W white LED chip. All the experiments were performed using CellASIC ONIX Microfluidic Platform. The setup has been modified thus opening new opportunities for a variety of experimental realizations. The performed morphological studies showed strong and irreversible effect on the vesicle shape at the presence of amphotericin B in concentration 10 -5 g/l in the outer for the liposome's membrane solution. At concentration 10 -3 g/l AmB the effect was less visible and in 15-20 minutes the vesicles regained its initial spherical shape. (paper)

Fusion of Selected Cells and Vesicles Mediated by Optically Trapped Plasmonic Nanoparticles

DEFF Research Database (Denmark)

Bahadori, Azra

. In this work, we introduce a novel and extremely flexible physical method which can trigger membrane fusion in a highly selective manner not only between synthetic GUVs of different compositions, but also between live cells which remain viable after fusion. Optical tweezers’ laser (1064 nm) is used to position....... The concept of cellular delivery is also known as targeted drug delivery and is quite a hot research topic internationally. Therefore, there have been efforts to develop various chemical molecules, proteins/peptides and physical approaches to trigger membrane fusion between synthetic giant unilamellar...... and merging of the two membranes results in merging the two membranes thereby completes the fusion. Complete fusion is associated with lipid mixing and lumen mixing which are both imaged by a high resolution confocal microscope. The confocal imaging enables quantification of the associated lipid mixing...

The effect of peptides and ions interacting with an electrically neutral membrane interface on the structure and stability of lipid membranes in the liquid-crystalline phase and in the liquid-ordered phase

Science.gov (United States)

Sano, Ryoko; Masum, Shah Md; Tanaka, Tomoki; Yamashita, Yuko; Levadny, Victor; Yamazaki, Masahito

2005-08-01

We investigated the effects of a de novo designed peptide, WLFLLKKK (peptide-1) and La3+, which can bind with the electrically neutral lipid membrane interface, on the stability of the phosphatidylcholine (PC) membrane in the Lα phase and that of the liquid-ordered (lo) phase membranes. The results of spacing of the multilamellar vesicle and shape changes of the giant unilamellar vesicle (GUV) indicate that the peptide-1 can be partitioned into the membrane interface in the Lα phase but not into that in the lo phase. La3+ induced shape changes of GUVs of the lo phase membrane, which are the same as those of GUVs in the Lα phase. This indicates that the binding of La3+ induced an increase in the lateral compression pressure of the membrane, which decreased the surface area of the membrane in the lo phase. The difference of the membrane interface between the Lα phase and the lo phase is discussed.

The effect of peptides and ions interacting with an electrically neutral membrane interface on the structure and stability of lipid membranes in the liquid-crystalline phase and in the liquid-ordered phase

International Nuclear Information System (INIS)

Sano, Ryoko; Masum, Shah Md; Tanaka, Tomoki; Yamashita, Yuko; Levadny, Victor; Yamazaki, Masahito

2005-01-01

We investigated the effects of a de novo designed peptide, WLFLLKKK (peptide-1) and La 3+ , which can bind with the electrically neutral lipid membrane interface, on the stability of the phosphatidylcholine (PC) membrane in the L α phase and that of the liquid-ordered (lo) phase membranes. The results of spacing of the multilamellar vesicle and shape changes of the giant unilamellar vesicle (GUV) indicate that the peptide-1 can be partitioned into the membrane interface in the L α phase but not into that in the lo phase. La 3+ induced shape changes of GUVs of the lo phase membrane, which are the same as those of GUVs in the L α phase. This indicates that the binding of La 3+ induced an increase in the lateral compression pressure of the membrane, which decreased the surface area of the membrane in the lo phase. The difference of the membrane interface between the L α phase and the lo phase is discussed

Centerband-only-detection-of-exchange (31)P nuclear magnetic resonance and phospholipid lateral diffusion: theory, simulation and experiment.

Science.gov (United States)

Lai, Angel; Saleem, Qasim; Macdonald, Peter M

2015-10-14

Centerband-only-detection-of-exchange (CODEX) (31)P NMR lateral diffusion measurements were performed on dimyristoylphosphatidylcholine (DMPC) assembled into large unilamellar spherical vesicles. Optimization of sample and NMR acquisition conditions provided significant sensitivity enhancements relative to an earlier first report (Q. Saleem, A. Lai, H. Morales, and P. M. Macdonald, Chem. Phys. Lipids, 2012, 165, 721). An analytical description was developed that permitted the extraction of lateral diffusion coefficients from CODEX data, based on a Gaussian-diffusion-on-a-sphere model (A. Ghosh, J. Samuel, and S. Sinha, Europhys. Lett., 2012, 98, 30003-p1) as relevant to CODEX (31)P NMR measurements on a population of spherical unilamellar phospholipid bilayer vesicles displaying a distribution of vesicle radii.

Repeated intraperitoneal injections of liposomes containing phosphatidic acid and cardiolipin reduce amyloid-β levels in APP/PS1 transgenic mice

DEFF Research Database (Denmark)

Ordóñez-Gutiérrez, Lara; Re, Francesca; Bereczki, Erika

2015-01-01

, it was hypothesized that shifting this equilibrium towards the blood by enhancing peripheral clearance might reduce Aβ levels in the brain: the 'sink effect'. We tested this hypothesis by intraperitoneally injecting APP/PS1 transgenic mice with small unilamellar vesicles containing either phosphatidic acid...... Aβ may be therapeutically relevant in AD. FROM THE CLINICAL EDITOR: Intraperitoneal injection of small unilamellar vesicles containing phosphatidic acid or cardiolipin significantly reduced the amount of amyloid-beta (Aß) peptide in the plasma in a rodent model. Brain levels of Aß were also affected...

Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

DEFF Research Database (Denmark)

Jalmar, Olivier; Franc¸ois-Moutal, Liberty; García-Sáez, Ana-Jesus

2013-01-01

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activa...

NH125 kills methicillin-resistant Staphylococcus aureus persisters by lipid bilayer disruption.

Science.gov (United States)

Kim, Wooseong; Fricke, Nico; Conery, Annie L; Fuchs, Beth Burgwyn; Rajamuthiah, Rajmohan; Jayamani, Elamparithi; Vlahovska, Petia M; Ausubel, Frederick M; Mylonakis, Eleftherios

2016-01-01

NH125, a known WalK inhibitor kills MRSA persisters. However, its precise mode of action is still unknown. The mode of action of NH125 was investigated by comparing its spectrum of antimicrobial activity and its effects on membrane permeability and giant unilamellar vesicles (GUVs) with walrycin B, a WalR inhibitor and benzyldimethylhexadecylammonium chloride (16-BAC), a cationic surfactant. NH125 killed persister cells of a variety of Staphylococcus aureus strains. Similar to 16-BAC, NH125 killed MRSA persisters by inducing rapid membrane permeabilization and caused the rupture of GUVs, whereas walrycin B did not kill MRSA persisters or induce membrane permeabilization and did not affect GUVs. NH125 kills MRSA persisters by interacting with and disrupting membranes in a detergent-like manner.

The Role of Membrane Fluidization in the Gel-Assisted Formation of Giant Polymersomes.

Directory of Open Access Journals (Sweden)

Adrienne C Greene

Full Text Available Polymersomes are being widely explored as synthetic analogs of lipid vesicles based on their enhanced stability and potential uses in a wide variety of applications in (e.g., drug delivery, cell analogs, etc.. Controlled formation of giant polymersomes for use in membrane studies and cell mimetic systems, however, is currently limited by low-yield production methodologies. Here, we describe for the first time, how the size distribution of giant poly(ethylene glycol-poly(butadiene (PEO-PBD polymersomes formed by gel-assisted rehydration may be controlled based on membrane fluidization. We first show that the average diameter and size distribution of PEO-PBD polymersomes may be readily increased by increasing the temperature of the rehydration solution. Further, we describe a correlative relationship between polymersome size and membrane fluidization through the addition of sucrose during rehydration, enabling the formation of PEO-PBD polymersomes with a range of diameters, including giant-sized vesicles (>100 μm. This correlative relationship suggests that sucrose may function as a small molecule fluidizer during rehydration, enhancing polymer diffusivity during formation and increasing polymersome size. Overall the ability to easily regulate the size of PEO-PBD polymersomes based on membrane fluidity, either through temperature or fluidizers, has broadly applicability in areas including targeted therapeutic delivery and synthetic biology.

Applying fluorescence correlation spectroscopy to investigate peptide-induced membrane disruption

DEFF Research Database (Denmark)

Kristensen, Kasper; Henriksen, Jonas Rosager; Andresen, Thomas Lars

2017-01-01

to quantify leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles, thereby providing a tool for estimating the size of peptide-induced membrane disruptions. If fluorescently labeled lipids are incorporated into the membranes of the vesicles, FCS can also be used to obtain...

The effect of peptides and ions interacting with an electrically neutral membrane interface on the structure and stability of lipid membranes in the liquid-crystalline phase and in the liquid-ordered phase

Energy Technology Data Exchange (ETDEWEB)

Sano, Ryoko [Department of Physics, Faculty of Science, Shizuoka University, Shizuoka, 422-8529 (Japan); Masum, Shah Md [Material Science, Graduate School of Science and Engineering, Shizuoka University, 422-8529 (Japan); Tanaka, Tomoki [Material Science, Graduate School of Science and Engineering, Shizuoka University, 422-8529 (Japan); Yamashita, Yuko [Department of Physics, Faculty of Science, Shizuoka University, Shizuoka, 422-8529 (Japan); Levadny, Victor [Department of Physics, Faculty of Science, Shizuoka University, Shizuoka, 422-8529 (Japan); Scientific Council for Cybernetics, Russian Academy of Sciences, Vavilov street 34, 333117, Moscow (Russian Federation); Yamazaki, Masahito [Department of Physics, Faculty of Science, Shizuoka University, Shizuoka, 422-8529 (Japan); Material Science, Graduate School of Science and Engineering, Shizuoka University, 422-8529 (Japan)

2005-08-10

We investigated the effects of a de novo designed peptide, WLFLLKKK (peptide-1) and La{sup 3+}, which can bind with the electrically neutral lipid membrane interface, on the stability of the phosphatidylcholine (PC) membrane in the L{sub {alpha}} phase and that of the liquid-ordered (lo) phase membranes. The results of spacing of the multilamellar vesicle and shape changes of the giant unilamellar vesicle (GUV) indicate that the peptide-1 can be partitioned into the membrane interface in the L{sub {alpha}} phase but not into that in the lo phase. La{sup 3+} induced shape changes of GUVs of the lo phase membrane, which are the same as those of GUVs in the L{sub {alpha}} phase. This indicates that the binding of La{sup 3+} induced an increase in the lateral compression pressure of the membrane, which decreased the surface area of the membrane in the lo phase. The difference of the membrane interface between the L{sub {alpha}} phase and the lo phase is discussed.

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity.

Science.gov (United States)

Morigaki, Kenichi; Tanimoto, Yasushi

2018-03-14

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates. Copyright © 2018 Elsevier B.V. All rights reserved.

Effective protection of biological membranes against photo-oxidative damage: Polymeric antioxidant forming a protecting shield over the membrane.

Science.gov (United States)

Mertins, Omar; Mathews, Patrick D; Gomide, Andreza B; Baptista, Mauricio S; Itri, Rosangela

2015-10-01

We have prepared a chitosan polymer modified with gallic acid in order to develop an efficient protection strategy biological membranes against photodamage. Lipid bilayers were challenged with photoinduced damage by photosensitization with methylene blue, which usually causes formation of hydroperoxides, increasing area per lipid, and afterwards allowing leakage of internal materials. The damage was delayed by a solution of gallic acid in a concentration dependent manner, but further suppressed by the polymer at very low concentrations. The membrane of giant unilamellar vesicles was covered with this modified macromolecule leading to a powerful shield against singlet oxygen and thus effectively protecting the lipid membrane from oxidative stress. The results have proven the discovery of a promising strategy for photo protection of biological membranes. Copyright © 2015 Elsevier B.V. All rights reserved.

Viscoelastic deformation of lipid bilayer vesicles†

Science.gov (United States)

Wu, Shao-Hua; Sankhagowit, Shalene; Biswas, Roshni; Wu, Shuyang; Povinelli, Michelle L.

2015-01-01

Lipid bilayers form the boundaries of the cell and its organelles. Many physiological processes, such as cell movement and division, involve bending and folding of the bilayer at high curvatures. Currently, bending of the bilayer is treated as an elastic deformation, such that its stress-strain response is independent of the rate at which bending strain is applied. We present here the first direct measurement of viscoelastic response in a lipid bilayer vesicle. We used a dual-beam optical trap (DBOT) to stretch 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) giant unilamellar vesicles (GUVs). Upon application of a step optical force, the vesicle membrane deforms in two regimes: a fast, instantaneous area increase, followed by a much slower stretching to an eventual plateau deformation. From measurements of dozens of GUVs, the average time constant of the slower stretching response was 0.225 ± 0.033 s (standard deviation, SD). Increasing the fluid viscosity did not affect the observed time constant. We performed a set of experiments to rule out heating by laser absorption as a cause of the transient behavior. Thus, we demonstrate here that the bending deformation of lipid bilayer membranes should be treated as viscoelastic. PMID:26268612

Phase behavior, rheological property, and transmutation of vesicles in fluorocarbon and hydrocarbon surfactant mixtures.

Science.gov (United States)

Yuan, Zaiwu; Qin, Menghua; Chen, Xiushan; Liu, Changcheng; Li, Hongguang; Hao, Jingcheng

2012-06-26

We present a detailed study of a salt-free cationic/anionic (catanionic) surfactant system where a strongly alkaline cationic surfactant (tetradecyltrimethylammonium hydroxide, TTAOH) was mixed with a single-chain fluorocarbon acid (nonadecafluorodecanoic acid, NFDA) and a hyperbranched hydrocarbon acid [di-(2-ethylhexyl)phosphoric acid, DEHPA] in water. Typically the concentration of TTAOH is fixed while the total concentration and mixing molar ratio of NFDA and DEHPA is varied. In the absence of DEHPA and at a TTAOH concentration of 80 mmol·L(-1), an isotropic L(1) phase, an L(1)/L(α) two-phase region, and a single L(α) phase were observed successively with increasing mixing molar ratio of NFDA to TTAOH (n(NFDA)/n(TTAOH)). In the NFDA-rich region (n(NFDA)/n(TTAOH) > 1), a small amount of excess NFDA can be solubilized into the L(α) phase while a large excess of NFDA eventually leads to phase separation. When NFDA is replaced gradually by DEHPA, the mixed system of TTAOH/NFDA/DEHPA/H(2)O follows the same phase sequence as that of the TTAOH/NFDA/H(2)O system and the phase boundaries remain almost unchanged. However, the viscoelasticity of the samples in the single L(α) phase region becomes higher at the same total surfactant concentration as characterized by rheological measurements. Cryo-transmission electron microscopic (cryo-TEM) observations revealed a microstructural evolution from unilamellar vesicles to multilamellar ones and finally to gaint onions. The size of the vesicle and number of lamella can be controlled by adjusting the molar ratio of NFDA to DEHPA. The dynamic properties of the vesicular solutions have also been investigated. It is found that the yield stress and the storage modulus are time-dependent after a static mixing process between the two different types of vesicle solutions, indicating the occurrence of a dynamic fusion between the two types of vesicles. The microenvironmental changes induced by aggregate transitions were probed by

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......Biological cells are highly dynamic, and continually move material around their own volume and between their interior and exterior. Much of this transport encapsulates the material inside phospholipid vesicles that shuttle to and fro, fusing with, and budding from, other membranes. A feature...

Emergence and stability of intermediate open vesicles in disk-to-vesicle transitions.

Science.gov (United States)

Li, Jianfeng; Zhang, Hongdong; Qiu, Feng; Shi, An-Chang

2013-07-01

The transition between two basic structures, a disk and an enclosed vesicle, of a finite membrane is studied by examining the minimum energy path (MEP) connecting these two states. The MEP is constructed using the string method applied to continuum elastic membrane models. The results reveal that, besides the commonly observed disk and vesicle, open vesicles (bowl-shaped vesicles or vesicles with a pore) can become stable or metastable shapes. The emergence, stability, and probability distribution of these open vesicles are analyzed. It is demonstrated that open vesicles can be stabilized by higher-order elastic energies. The estimated probability distribution of the different structures is in good agreement with available experiments.

Ripple formation in unilamellar-supported lipid bilayer revealed by FRAPP.

Science.gov (United States)

Harb, Frédéric; Simon, Anne; Tinland, Bernard

2013-12-01

The mechanisms of formation and conditions of the existence of the ripple phase are fundamental thermodynamic questions with practical implications for medicine and pharmaceuticals. We reveal a new case of ripple formation occurring in unilamellar-supported bilayers in water, which results solely from the bilayer/support interaction, without using lipid mixtures or specific ions. This ripple phase is detected by FRAPP using diffusion coefficient measurements as a function of temperature: a diffusivity plateau is observed. It occurs in the same temperature range where ripple phase existence has been observed using other methods. When AFM experiments are performed in the appropriate temperature range the ripple phase is confirmed.

Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes.

Science.gov (United States)

Moniruzzaman, Md; Islam, Md Zahidul; Sharmin, Sabrina; Dohra, Hideo; Yamazaki, Masahito

2017-08-22

Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

Layer-by-layer cell membrane assembly

Science.gov (United States)

Matosevic, Sandro; Paegel, Brian M.

2013-11-01

Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

Induction of Highly Curved Structures in Relation to Membrane Permeabilization and Budding by the Triterpenoid Saponins, α- and δ-Hederin

Science.gov (United States)

Lorent, Joseph; Le Duff, Cécile S.; Quetin-Leclercq, Joelle; Mingeot-Leclercq, Marie-Paule

2013-01-01

The interactions of triterpenoid monodesmosidic saponins, α-hederin and δ-hederin, with lipid membranes are involved in their permeabilizing effect. Unfortunately, the interactions of these saponins with lipid membranes are largely unknown, as are the roles of cholesterol or the branched sugar moieties (two for α-hederin and one for δ-hederin) on the aglycone backbone, hederagenin. The differences in sugar moieties are responsible for differences in the molecular shape of the saponins and the effects on membrane curvature that should be the most positive for α-hederin in a transbilayer direction. In large unilamellar vesicles and monocyte cells, we showed that membrane permeabilization was dependent on the presence of membrane cholesterol and saponin sugar chains, being largest for α-hederin and smallest for hederagenin. In the presence of cholesterol, α-hederin induced the formation of nonbilayer phases with a higher rate of Brownian tumbling or lateral diffusion. A reduction of Laurdan's generalized polarization in relation to change in order of the polar heads of phospholipids was observed. Using giant unilamellar vesicles, we visualized the formation of wrinkled borders, the decrease in liposome size, budding, and the formation of macroscopic pores. All these processes are highly dependent on the sugars linked to the aglycone, with α-hederin showing a greater ability to induce pore formation and δ-hederin being more efficient in inducing budding. Hederagenin induced intravesicular budding but no pore formation. Based on these results, a curvature-driven permeabilization mechanism dependent on the interaction between saponin and sterols and on the molecular shape of the saponin and its ability to induce local spontaneous curvature is proposed. PMID:23530040

Differential dynamic and structural behavior of lipid-cholesterol domains in model membranes.

Directory of Open Access Journals (Sweden)

Luis F Aguilar

Full Text Available Changes in the cholesterol (Chol content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs for cuvette and giant unilamellar vesicles (GUVs for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC and dioctadecyl phosphatidylcholine (DOPC in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo.

Effect of biocompatible polymers on the structural integrity of lipid bilayers under external stimuli

Science.gov (United States)

Wang, Jia-Yu; Kausik, Ravinath; Chen, Chi-Yuan; Han, Song-I.; Marks, Jeremy; Lee, Ka Yee

2010-03-01

Cell membrane dysfunction due to loss of structural integrity is the pathology of tissue death in trauma and common diseases. It is now established that certain biocompatible polymers, such as Poloxamer 188, Poloxamine 1107 and polyethylene glycol (PEG), are effective in sealing of injured cell membranes, and able to prevent acute necrosis. Despite these broad applications of these polymers for human health, the fundamental mechanisms by which these polymers interact with cell membranes are still under debate. Here, the effects of a group of biocompatible polymers on phospholipid membrane integrity under osmotic and oxidative stress were explored using giant unilamellar vesicles as model cell membranes. Our results suggest that the adsorption of the polymers on the membrane surface is responsible for the cell membrane resealing process due to its capability of slowing down the surface hydration dynamics.

Vesicle electrohydrodynamics.

Science.gov (United States)

Schwalbe, Jonathan T; Vlahovska, Petia M; Miksis, Michael J

2011-04-01

A small amplitude perturbation analysis is developed to describe the effect of a uniform electric field on the dynamics of a lipid bilayer vesicle in a simple shear flow. All media are treated as leaky dielectrics and fluid motion is described by the Stokes equations. The instantaneous vesicle shape is obtained by balancing electric, hydrodynamic, bending, and tension stresses exerted on the membrane. We find that in the absence of ambient shear flow, it is possible that an applied stepwise uniform dc electric field could cause the vesicle shape to evolve from oblate to prolate over time if the encapsulated fluid is less conducting than the suspending fluid. For a vesicle in ambient shear flow, the electric field damps the tumbling motion, leading to a stable tank-treading state.

Functional assay of Salmonella typhi OmpC using reconstituted large unilamellar vesicles: a general method for characterization of outer membrane proteins.

Science.gov (United States)

Sundara Baalaji, N; Mathew, M K; Krishnaswamy, S

2006-10-01

The immunodominant trimeric beta-barrel outer membrane protein OmpC from Salmonella typhi, the causative agent of typhoid, has been functionally characterized here. The activity in the vesicle environment was studied in vitro using OmpC reconstituted into proteoliposomes. Passage of polysaccharides and polyethyleneglycols through OmpC has been examined to determine the permeability properties. The relative rate of neutral solute flux yields a radius of 1.1 nm for the S. typhi OmpC pore. This is almost double the pore size of Escherichia coli. This provides an example of large pore size present in the porins that form trimers as in the general bacterial porin family. The method used in this study provides a good membrane model for functional studies of porins.

Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics

Science.gov (United States)

Weiss, Marian; Frohnmayer, Johannes Patrick; Benk, Lucia Theresa; Haller, Barbara; Janiesch, Jan-Willi; Heitkamp, Thomas; Börsch, Michael; Lira, Rafael B.; Dimova, Rumiana; Lipowsky, Reinhard; Bodenschatz, Eberhard; Baret, Jean-Christophe; Vidakovic-Koch, Tanja; Sundmacher, Kai; Platzman, Ilia; Spatz, Joachim P.

2018-01-01

Compartments for the spatially and temporally controlled assembly of biological processes are essential towards cellular life. Synthetic mimics of cellular compartments based on lipid-based protocells lack the mechanical and chemical stability to allow their manipulation into a complex and fully functional synthetic cell. Here, we present a high-throughput microfluidic method to generate stable, defined sized liposomes termed `droplet-stabilized giant unilamellar vesicles (dsGUVs)’. The enhanced stability of dsGUVs enables the sequential loading of these compartments with biomolecules, namely purified transmembrane and cytoskeleton proteins by microfluidic pico-injection technology. This constitutes an experimental demonstration of a successful bottom-up assembly of a compartment with contents that would not self-assemble to full functionality when simply mixed together. Following assembly, the stabilizing oil phase and droplet shells are removed to release functional self-supporting protocells to an aqueous phase, enabling them to interact with physiologically relevant matrices.

DIFFERENTIAL HEPATIC PROCESSING AND BILIARY-SECRETION OF HEADGROUP AND ACYL CHAINS OF LIPOSOMAL PHOSPHATIDYLCHOLINES

NARCIS (Netherlands)

VERKADE, HJ; DERKSEN, JTP; GERDING, A; SCHERPHOF, GL; VONK, RJ; KUIPERS, F

1991-01-01

To investigate the contribution of plasma-derived phosphatidylcholine (PC) to bile PC, the hepatic processing and biliary secretion of liposome-associated PC was studied in rats. For this purpose, small unilamellar vesicles (SUV), containing trace amounts of

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

Energy Technology Data Exchange (ETDEWEB)

Beschiaschvili, G.; Seelig, J. (Univ. of Basel (Switzerland))

1990-01-09

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

International Nuclear Information System (INIS)

Beschiaschvili, G.; Seelig, J.

1990-01-01

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart

The effect of reticuloendothelial blockade on the blood clearance and tissue distribution of liposomes

International Nuclear Information System (INIS)

Souhami, R.L.; Patel, H.M.; Ryman, B.E.

1981-01-01

The blood clearance and tissue distribution of liposomes have been studied in mice subjected to reticuloendothelial blockade with dextran sulphate or carbon. The liposomes have been labelled in the lipid membranes with [ 3 H]-cholesterol, [ 14 C]phosphatidylcholine and/or 99 sup(m)Tc and the content with [ 14 C]inulin. Reticuloendothelial blockade has been shown to slow the rate of clearance of neutral, positively and negatively charged liposomes and of both small unilamellar vesicles and large multilamellar vesicles. In normal animals, the liver uptake accounted for only 20-55% of the total injected radioactivity, the amount varying with the charge and size of the liposomes. Following blockade, the liver uptake of charged and neutral multilamellar liposomes was depressed. This was also true for negatively charged small unilamellar vesicles. The degree of depression of hepatic uptake was between 25-50%, which contrasts with the 80-90% reduction in uptake of a wholly phagocytosed particle (sheep red cells). This difference suggets that mechanisms other than Kupffer cell phagocytosis are also responsible for the normal uptake of liposomes into the liver. In the case of neutral and positively charged small unilamellar vesicles, delayed clearance due to blockade was not associated with depressed hepatic uptake. The site of action of blockading agents for these preparations is not clear. With all preparations of liposomes, blockade produced a slight and variable increase in uptake in the lung and spleen. The alteration of distribution of liposomes by reticuloendothelial blockade is therefore not great and the value of the technique in modifying the tissue distribution of substances within liposomes may be limited. (orig.)

Effect of vesicle size on the prodan fluorescence in diheptadecanoylphosphatidylcholine bilayer membrane under atmospheric and high pressures.

Science.gov (United States)

Goto, Masaki; Sawaguchi, Hiroshi; Tamai, Nobutake; Matsuki, Hitoshi; Kaneshina, Shoji

2010-08-17

The bilayer phase behavior of diheptadecanoylphosphatidylcholine (C17PC) with different vesicle sizes (large multilamellar vesicle (LMV) and giant multilamellar vesicle (GMV)) was investigated by fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan under atmospheric and high pressures. The difference in phase transitions and thermodynamic quantities of the transition was hardly observed between LMV and GMV used here. On the contrary, the Prodan fluorescence in the bilayer membranes changed depending on the size of vesicles as well as on the phase states. From the second derivative of fluorescence spectra, the three-dimensional image plots in which we can see the location of Prodan in the bilayer membrane as blue valleys were constructed for LMV and GMV under atmospheric pressure. The following characteristic behavior was found: (1) the Prodan molecules in GMV can be distributed to not only adjacent glycerol backbone region, but also near bulk-water region in the lamellar gel or ripple gel phase; (2) the blue valleys of GMV became deeper than those of LMV because of the greater surface density of the Prodan molecules per unit area of GMV than LMV; (3) the liquid crystalline phase of the bilayer excludes the Prodan molecules to a more hydrophilic region at the membrane surface with an increase in vesicle size; (4) the accurate information as to the phase transitions is gradually lost with increasing vesicle size. Under the high-pressure condition, the difference in Prodan fluorescence between LMV and GMV was essentially the same as the difference under atmospheric pressure except for the existence of the pressure-induced interdigitated gel phase. Further, we found that Prodan fluorescence spectra in the interdigitated gel phase were especially affected by the size of vesicles. This study revealed that the Prodan molecules can move around the headgroup region by responding not only to the phase state but also to the vesicle size, and they

Drug Delivery by an Enzyme-Mediated Cyclization of a Lipid Prodrug with Unique Bilayer-Formation Properties

DEFF Research Database (Denmark)

Linderoth, Lars; Peters, Günther H.j.; Madsen, Robert

2009-01-01

Special delivery: Liposomal drug-delivery systems in which prodrugs are activated specifically by disease-associated enzymes have great potential for the treatment of severe diseases, such as cancer. A new type of phospholipid-based prodrug has the ability to form stable small unilamellar vesicle...... (see picture). Activation of the prodrug vesicles by the enzyme sPLA2 initiates a cyclization reaction, which leads to the release of the drug....

Interaction between sodium dodecyl sulfate and membrane reconstituted aquaporins: A comparative study of spinach SoPIP2;1 and E. coli AqpZ

DEFF Research Database (Denmark)

Hansen, Jesper Schmidt; Vararattanavech, Ardcharaporn; Plasencia, Inés

2011-01-01

This study describes the interaction between sodium dodecyl sulfate (SDS) and membrane proteins reconstituted into large unilamellar lipid vesicles and detergent micelles studied by circular dichroism (CD) and polarity sensitive probe labeling. Specifically, we carried out a comparative study of ...

Vesicle-based rechargeable batteries

Energy Technology Data Exchange (ETDEWEB)

Stanish, I.; Singh, A. [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave., S.W., Washington, DC 20375 (United States); Lowy, D.A. [Nova Research, Inc., 1900 Elkin St., Alexandria, VA 22308 (United States); Hung, C.W. [Department of Chemical Engineering, University of Maryland, College Park, MD 20742 (United States)

2005-05-02

Vesicle-based rechargeable batteries can be fabricated by mounting polymerized vesicles filled with ferrocyanide or ferricyanide to a conductive surface. The potential can be adjusted by changing the concentration ratio of hydroquinone and benzoquinone bound to the vesicle membranes. These batteries show promise as a means of supplying portable power for future autonomous nanosystems. (Abstract Copyright [2005], Wiley Periodicals, Inc.)

Magnetic liposomes based on nickel ferrite nanoparticles for biomedical applications.

Science.gov (United States)

Rodrigues, Ana Rita O; Gomes, I T; Almeida, Bernardo G; Araújo, J P; Castanheira, Elisabete M S; Coutinho, Paulo J G

2015-07-21

Nickel ferrite nanoparticles with superparamagnetic behavior at room temperature were synthesized using a coprecipitation method. These magnetic nanoparticles were either covered with a lipid bilayer, forming dry magnetic liposomes (DMLs), or entrapped in liposomes, originating aqueous magnetoliposomes (AMLs). A new and promising method for the synthesis of DMLs is described. The presence of the lipid bilayer in DMLs was confirmed by FRET (Förster Resonance Energy Transfer) measurements between the fluorescent-labeled lipids NBD-C12-HPC (NBD acting as a donor) included in the second lipid layer and rhodamine B-DOPE (acceptor) in the first lipid layer. An average donor-acceptor distance of 3 nm was estimated. Assays of the non-specific interactions of magnetoliposomes with biological membranes (modeled using giant unilamellar vesicles, GUVs) were performed. Membrane fusion between both aqueous and dry magnetoliposomes and GUVs was confirmed by FRET, which is an important result regarding applications of these systems both as hyperthermia agents and antitumor drug nanocarriers.

How synthetic membrane systems contribute to the understanding of lipid-driven endocytosis.

Science.gov (United States)

Schubert, Thomas; Römer, Winfried

2015-11-01

Synthetic membrane systems, such as giant unilamellar vesicles and solid supported lipid bilayers, have widened our understanding of biological processes occurring at or through membranes. Artificial systems are particularly suited to study the inherent properties of membranes with regard to their components and characteristics. This review critically reflects the emerging molecular mechanism of lipid-driven endocytosis and the impact of model membrane systems in elucidating the complex interplay of biomolecules within this process. Lipid receptor clustering induced by binding of several toxins, viruses and bacteria to the plasma membrane leads to local membrane bending and formation of tubular membrane invaginations. Here, lipid shape, and protein structure and valency are the essential parameters in membrane deformation. Combining observations of complex cellular processes and their reconstitution on minimal systems seems to be a promising future approach to resolve basic underlying mechanisms. This article is part of a Special Issue entitled: Mechanobiology. Copyright © 2015 Elsevier B.V. All rights reserved.

A balance between membrane elasticity and polymerization energy sets the shape of spherical clathrin coats

Science.gov (United States)

Saleem, Mohammed; Morlot, Sandrine; Hohendahl, Annika; Manzi, John; Lenz, Martin; Roux, Aurélien

2015-02-01

In endocytosis, scaffolding is one of the mechanisms to create membrane curvature by moulding the membrane into the spherical shape of the clathrin cage. However, the impact of membrane elastic parameters on the assembly and shape of clathrin lattices has never been experimentally evaluated. Here, we show that membrane tension opposes clathrin polymerization. We reconstitute clathrin budding in vitro with giant unilamellar vesicles (GUVs), purified adaptors and clathrin. By changing the osmotic conditions, we find that clathrin coats cause extensive budding of GUVs under low membrane tension while polymerizing into shallow pits under moderate tension. High tension fully inhibits polymerization. Theoretically, we predict the tension values for which transitions between different clathrin coat shapes occur. We measure the changes in membrane tension during clathrin polymerization, and use our theoretical framework to estimate the polymerization energy from these data. Our results show that membrane tension controls clathrin-mediated budding by varying the membrane budding energy.

Membrane shape modulates transmembrane protein distribution.

Science.gov (United States)

Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E S; Bassereau, Patricia

2014-01-27

Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Hydrogel Micro-/Nanosphere Coated by a Lipid Bilayer: Preparation and Microscopic Probing

Directory of Open Access Journals (Sweden)

Sarah Rahni

2017-02-01

Full Text Available The result of polymeric nanogels and lipid vesicles interaction—lipobeads—can be considered as multipurpose containers for future therapeutic applications, such as targeted anticancer chemotherapy with superior tumor response and minimum side effects. In this work, micrometer sized lipobeads were synthesized by two methods: (i mixing separately prepared microgels made of poly(N-isopropylacrylamide (PNIPA and phospholipid vesicles of micrometer or nanometer size and (ii polymerization within the lipid vesicles. For the first time, a high vacuum scanning electron microscopy was shown to be suitable for a quick validation of the structural organization of wet lipobeads and their constituents without special sample preparation. In particular, the structural difference of microgels prepared by thermal and UV-polymerization in different solvents was revealed and three types of giant liposomes were recognized under high vacuum in conjunction with their size, composition, and method of preparation. Importantly, the substructure of the hydrogel core and multi- and unilamellar constructions of the peripheral lipid part were explicitly distinguished on the SEM images of lipobeads, justifying the spontaneous formation of a lipid bilayer on the surface of microgels and evidencing an energetically favorable structural organization of the hydrogel/lipid bilayer assembly. This key property can facilitate lipobeads’ preparation and decrease technological expenses on their scaled production. The comparison of the SEM imaging with the scanning confocal and atomic force microscopies data are also presented in the discussion.

Shear-Induced Membrane Fusion in Viscous Solutions

KAUST Repository

Kogan, Maxim; Feng, Bobo; Nordé n, Bengt; Rocha, Sandra; Beke-Somfai, Tamá s

2014-01-01

Large unilamellar lipid vesicles do not normally fuse under fluid shear stress. They might deform and open pores to relax the tension to which they are exposed, but membrane fusion occurring solely due to shear stress has not yet been reported. We

Single-vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles

NARCIS (Netherlands)

Farsi, Z.; Preobraschenski, J.; Bogaart, G. van den; Riedel, D.; Jahn, R.; Woehler, A.

2016-01-01

Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided

Single-vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles.

Science.gov (United States)

Farsi, Zohreh; Preobraschenski, Julia; van den Bogaart, Geert; Riedel, Dietmar; Jahn, Reinhard; Woehler, Andrew

2016-02-26

Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided by an electrochemical proton gradient. However, it is unclear how transmitter molecules carrying different net charges can be efficiently sequestered while maintaining charge neutrality and osmotic balance. We used single-vesicle imaging to monitor pH and electrical gradients and directly showed different uptake mechanisms for glutamate and γ-aminobutyric acid (GABA) operating in parallel. In contrast to glutamate, GABA was exchanged for protons, with no other ions participating in the transport cycle. Thus, only a few components are needed to guarantee reliable vesicle filling with different neurotransmitters. Copyright © 2016, American Association for the Advancement of Science.

Fusion of Nonionic Vesicles

DEFF Research Database (Denmark)

Bulut, Sanja; Oskolkova, M. Z.; Schweins, R.

2010-01-01

We present an experimental study of vesicle fusion using light and neutron scattering to monitor fusion events. Vesicles are reproducibly formed with an extrusion procedure using an single amphiphile triethylene glycol mono-n-decyl ether in water. They show long-term stability for temperatures ar...... a barrier to fusion changing from 15 k(B)T at T = 26 degrees C to 10k(H) T at T = 35 degrees C. These results are compatible with the theoretical predictions using the stalk model of vesicle fusion....

How to Characterize Individual Nano-Size Liposomes with Simple Self-Calibrating Fluorescence Microscopy

DEFF Research Database (Denmark)

Mortensen, Kim I.; Tassone, Chiara; Ehrlich, Nicky

2018-01-01

and structural properties. Consequently, vesicles must be characterized individually to ensure correct interpretation of experimental results. Here we do that using dual-color fluorescence labeling of vesicles-of their lipid bilayers and lumens, respectively. A vesicle then images as two spots, one in each color....... They in turn enable calibration of the dual-color fluorescence microscopy images they appear in. Since this calibration is not a separate experiment but an analysis of images of vesicles to be characterized, it eliminates the potential source of error that a separate calibration experiment would have been. Non...... channel. A simple image analysis determines the total intensity and width of each spot. These four data all depend on the vesicle radius in a simple manner for vesicles that are spherical, unilamellar, and optimal encapsulators of molecular cargo. This permits identification of such ideal vesicles...

Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis.

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Débora L Oliveira

2010-06-01

Full Text Available Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.We characterized extracellular vesicle production in wild type (WT and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex or MVB functionality (vps23, late endosomal trafficking revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the

Phase Partitioning of GM1 and Its Bodipy-Labeled Analog Determine Their Different Binding to Cholera Toxin

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Sami Rissanen

2017-05-01

Full Text Available Driven by interactions between lipids and proteins, biological membranes display lateral heterogeneity that manifests itself in a mosaic of liquid-ordered (Lo or raft, and liquid-disordered (Ld or non-raft domains with a wide range of different properties and compositions. In giant plasma membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1, whose headgroup accessibility and membrane order or phase partitioning may differ from those of GM1, rendering the interpretation of CTxB binding data quite problematic. To unravel the molecular basis of CTxB binding to GM1 and bdGM1, we explored the partitioning and the headgroup presentation of these gangliosides in the Lo and Ld phases using atomistic molecular dynamics simulations complemented by CTxB binding experiments. The conformation of both GM1 and bdGM1 was shown to be largely similar in the Lo and Ld phases. However, bdGM1 showed reduction in receptor availability when reconstituted into synthetic bilayer mixtures, highlighting that membrane phase partitioning of the gangliosides plays a considerable role in CTxB binding. Our results suggest that the CTxB binding is predominately modulated by the partitioning of the receptor to an appropriate membrane phase. Further, given that the Lo and Ld partitioning of bdGM1 differs from those of GM1, usage of bdGM1 for studying GM1 behavior in cells can lead to invalid interpretation of experimental data.

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...

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

Impermeability effects in three-dimensional vesicles

International Nuclear Information System (INIS)

Biscari, P; Canevese, S M; Napoli, G

2004-01-01

We analyse the effects of the impermeability constraint on the equilibrium shapes of a three-dimensional vesicle hosting a rigid inclusion. A given alteration of the inclusion and/or vesicle parameters leads to shape modifications of different orders of magnitude, when applied to permeable or impermeable vesicles. Moreover, the enclosed-volume constraint wrecks the uniqueness of stationary equilibrium shapes, and gives rise to pear-shaped or stomatocyte-like vesicles

INVESTIGATION ON THE MORPHOLOGY AND PROPERTIES OF AGGREGATE STRUCTURES OF NATURAL PHOSPHOLIPIDS IN AQUEOUS SYSTEM USING CRYO-TEM

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Dwi Hudiyanti

2012-02-01

Full Text Available Cryogenic transmission electron microscopy (Cryo-TEM was used to investigate the aggregates morphology and properties of candle tree (Aleurites moluccana endosperm, sesame (Sesamum indicum L. syn. seeds, and coconut (Cocos nucifera endosperm phospholipids in dilute aqueous system. The micrographs showed that candle tree phospholipids formed planar bilayer and cluster of vesicles with lipid droplets, while coconut and sesame phospholipids formed well-defined unilamellar vesicles. The vesicles size could be as small as 50 nm in diameter. Coconut phospholipids also showed a good bending ability. Formation of clusters of vesicles was also found in coconut phospholipids dispersion, but this cluster was easily broken by extrusion through a small pore membrane.

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

Spontaneous charged lipid transfer between lipid vesicles.

Science.gov (United States)

Richens, Joanna L; Tyler, Arwen I I; Barriga, Hanna M G; Bramble, Jonathan P; Law, Robert V; Brooks, Nicholas J; Seddon, John M; Ces, Oscar; O'Shea, Paul

2017-10-03

An assay to study the spontaneous charged lipid transfer between lipid vesicles is described. A donor/acceptor vesicle system is employed, where neutrally charged acceptor vesicles are fluorescently labelled with the electrostatic membrane probe Fluoresceinphosphatidylethanolamine (FPE). Upon addition of charged donor vesicles, transfer of negatively charged lipid occurs, resulting in a fluorescently detectable change in the membrane potential of the acceptor vesicles. Using this approach we have studied the transfer properties of a range of lipids, varying both the headgroup and the chain length. At the low vesicle concentrations chosen, the transfer follows a first-order process where lipid monomers are transferred presumably through the aqueous solution phase from donor to acceptor vesicle. The rate of transfer decreases with increasing chain length which is consistent with energy models previously reported for lipid monomer vesicle interactions. Our assay improves on existing methods allowing the study of a range of unmodified lipids, continuous monitoring of transfer and simplified experimental procedures.

Exosomal microRNAs in giant panda (Ailuropoda melanoleuca) breast milk: potential maternal regulators for the development of newborn cubs.

Science.gov (United States)

Ma, Jideng; Wang, Chengdong; Long, Keren; Zhang, Hemin; Zhang, Jinwei; Jin, Long; Tang, Qianzi; Jiang, Anan; Wang, Xun; Tian, Shilin; Chen, Li; He, Dafang; Li, Desheng; Huang, Shan; Jiang, Zhi; Li, Mingzhou

2017-06-14

The physiological role of miRNAs is widely understood to include fine-tuning the post-transcriptional regulation of a wide array of biological processes. Extensive studies have indicated that exosomal miRNAs in the bodily fluids of various organisms can be transferred between living cells for the delivery of gene silencing signals. Here, we illustrated the expression characteristics of exosomal miRNAs in giant panda breast milk during distinct lactation periods and highlighted the enrichment of immune- and development-related endogenous miRNAs in colostral and mature giant panda milk. These miRNAs are stable, even under certain harsh conditions, via the protection of extracellular vesicles. These findings indicate that breast milk may facilitate the dietary intake of maternal miRNAs by infants for the regulation of postnatal development. We also detected exogenous plant miRNAs from the primary food source of the giant panda (bamboo) in the exosomes of giant panda breast milk that were associated with regulatory roles in basic metabolism and neuron development. This result suggested that dietary plant miRNAs are absorbed by host cells and subsequently secreted into bodily fluids as potential cross-kingdom regulators. In conclusion, exosomal miRNAs in giant panda breast milk may be crucial maternal regulators for the development of intrinsic 'slink' newborn cubs.

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

Responsive Polydiacetylene Vesicles for Biosensing Microorganisms

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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.

Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (Hymenoptera: Andrenidae)

Czech Academy of Sciences Publication Activity Database

Čujová, Sabína; Slaninová, Jiřina; Monincová, Lenka; Fučík, Vladimír; Bednárová, Lucie; Štokrová, Jitka; Hovorka, Oldřich; Voburka, Zdeněk; Straka, J.; Čeřovský, Václav

2013-01-01

Roč. 45, č. 1 (2013), s. 143-157 ISSN 0939-4451 R&D Projects: GA ČR GA203/08/0536 Institutional support: RVO:61388963 Keywords : antimicrobial peptides * wild bee venom * CD spectroscopy * large unilamellar vesicles * electron microscopy Subject RIV: CE - Biochemistry Impact factor: 3.653, year: 2013

Statistical Analysis of Bending Rigidity Coefficient Determined Using Fluorescence-Based Flicker-Noise Spectroscopy.

Science.gov (United States)

Doskocz, Joanna; Drabik, Dominik; Chodaczek, Grzegorz; Przybyło, Magdalena; Langner, Marek

2018-06-01

Bending rigidity coefficient describes propensity of a lipid bilayer to deform. In order to measure the parameter experimentally using flickering noise spectroscopy, the microscopic imaging is required, which necessitates the application of giant unilamellar vesicles (GUV) lipid bilayer model. The major difficulty associated with the application of the model is the statistical character of GUV population with respect to their size and the homogeneity of lipid bilayer composition, if a mixture of lipids is used. In the paper, the bending rigidity coefficient was measured using the fluorescence-enhanced flicker-noise spectroscopy. In the paper, the bending rigidity coefficient was determined for large populations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. The quantity of obtained experimental data allows to perform statistical analysis aiming at the identification of the distribution, which is the most appropriate for the calculation of the value of the membrane bending rigidity coefficient. It has been demonstrated that the bending rigidity coefficient is characterized by an asymmetrical distribution, which is well approximated with the gamma distribution. Since there are no biophysical reasons for that we propose to use the difference between normal and gamma fits as a measure of the homogeneity of vesicle population. In addition, the effect of a fluorescent label and types of instrumental setups on determined values has been tested. Obtained results show that the value of the bending rigidity coefficient does not depend on the type of a fluorescent label nor on the type of microscope used.

Transcriptome of extracellular vesicles released by hepatocytes.

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Felix Royo

Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

Accumulation of Vesicle-Associated Human Tau in Distal Dendrites Drives Degeneration and Tau Secretion in an In Situ Cellular Tauopathy Model

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Sangmook Lee

2012-01-01

Full Text Available We used a nontransgenic cellular tauopathy model in which individual giant neurons in the lamprey CNS (ABCs overexpress human tau isoforms cell autonomously to characterize the still poorly understood consequences of disease-associated tau processing in situ. In this model, tau colocalizes with endogenous microtubules and is nontoxic when expressed at low levels, but is misprocessed by a toxicity-associated alternative pathway when expressed above levels that saturate dendritic microtubules, causing abnormally phosphorylated, vesicle-associated tau to accumulate in ABC distal dendrites. This causes localized microtubule loss and eventually dendritic degeneration, which is preceded by tau secretion to the extracellular space. This sequence is reiterated at successively more proximal dendritic locations over time, suggesting that tau-induced dendritic degeneration is driven by distal dendritic accumulation of hyperphosphorylated, vesicle-associated tau perpetuated by localized microtubule loss. The implications for the diagnosis and treatment of human disease are discussed.

Spontaneous Vesicle Recycling in the Synaptic Bouton

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Sven eTruckenbrodt

2014-12-01

Full Text Available The trigger for synaptic vesicle exocytosis is Ca2+, which enters the synaptic bouton following action potential stimulation. However, spontaneous release of neurotransmitter also occurs in the absence of stimulation in virtually all synaptic boutons. It has long been thought that this represents exocytosis driven by fluctuations in local Ca2+ levels. The vesicles responding to these fluctuations are thought to be the same ones that release upon stimulation, albeit potentially triggered by different Ca2+ sensors. This view has been challenged by several recent works, which have suggested that spontaneous release is driven by a separate pool of synaptic vesicles. Numerous articles appeared during the last few years in support of each of these hypotheses, and it has been challenging to bring them into accord. We speculate here on the origins of this controversy, and propose a solution that is related to developmental effects. Constitutive membrane traffic, needed for the biogenesis of vesicles and synapses, is responsible for high levels of spontaneous membrane fusion in young neurons, probably independent of Ca2+. The vesicles releasing spontaneously in such neurons are not related to other synaptic vesicle pools and may represent constitutively releasing vesicles (CRVs rather than bona fide synaptic vesicles. In mature neurons, constitutive traffic is much dampened, and the few remaining spontaneous release events probably represent bona fide spontaneously releasing synaptic vesicles (SRSVs responding to Ca2+ fluctuations, along with a handful of CRVs that participate in synaptic vesicle turnover.

Enhanced Synaptic Transmission at the Squid Giant Synapse by Artificial Seawater Based on Physically Modified Saline

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Soonwook eChoi

2014-02-01

Full Text Available Superfusion of the squid giant synapse with artificial seawater (ASW based on isotonic saline containing oxygen nanobubbles (RNS60 ASW generates an enhancement of synaptic transmission. This was determined by examining the postsynaptic response to single and repetitive presynaptic spike activation, spontaneous transmitter release, and presynaptic voltage clamp studies. In the presence of RNS60 ASW single presynaptic stimulation elicited larger postsynaptic potentials (PSP and more robust recovery from high frequency stimulation than in control ASW. Analysis of postsynaptic noise revealed an increase in spontaneous transmitter release with modified noise kinetics in RNS60 ASW. Presynaptic voltage clamp demonstrated an increased EPSP, without an increase in presynaptic ICa⁺⁺ amplitude during RNS60 ASW superfusion. Synaptic release enhancement reached stable maxima within 5 to 10 minutes of RNS60 ASW superfusion and was maintained for the entire recording time, up to one hour. Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles and large endosome-like vesicles near junctional sites. Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW. After ATP block the number of vesicles at the active zone and clathrin-coated vesicles was reduced, with an increase in large vesicles. The possibility that RNS60 ASW acts by increasing mitochondrial ATP synthesis was tested by direct determination of ATP levels in both presynaptic and postsynaptic structures. This was implemented using luciferin/luciferase photon emission, which demonstrated a marked increase in ATP synthesis following RNS60 administration. It is concluded that RNS60 positively modulates synaptic transmission by up-regulating ATP synthesis, thus leading to synaptic

Giant seminal vesicle cyst with ipsilateral hypoplastic kidney: Report of a case with review of literature

Directory of Open Access Journals (Sweden)

Dilip Kumar Pal

2006-01-01

Full Text Available We report a case of a congenital seminal vesicle cyst with ipsilateral hypoplastic kidney in a 51 year-old Indian man presenting with features of bladder outlet obstruction. Abdominal and pelvic Ultrasonography (USG, computed tomography revealed a retrovesical cystic mass measuring 10cm x 9cm with indentations over the left infero-lateral wall of the urinary bladder. On USG and radionuclide renal scanning the ipsilateral kidney was not found, which was located only on the CT scan. The cyst and the hypoplastic kidney was excised with an uneventful recovery.

Membrane-bound conformation of M13 major coat protein : a structure validation through FRET-derived constraints

NARCIS (Netherlands)

Vos, W.L.; Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

2005-01-01

M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein

Optogenetic acidification of synaptic vesicles and lysosomes.

Science.gov (United States)

Rost, Benjamin R; Schneider, Franziska; Grauel, M Katharina; Wozny, Christian; Bentz, Claudia; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

2015-12-01

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.

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.

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.

Novel chimeric peptide with enhanced cell specificity and anti-inflammatory activity.

Science.gov (United States)

Kim, Young-Min; Kim, Nam-Hong; Lee, Jong-Wan; Jang, Jin-Sun; Park, Yung-Hoon; Park, Seong-Cheol; Jang, Mi-Kyeong

2015-07-31

An antimicrobial peptide (AMP), Hn-Mc, was designed by combining the N-terminus of HPA3NT3 and the C-terminus of melittin. This chimeric AMP exhibited potent antibacterial activity with low minimal inhibitory concentrations (MICs), ranging from 1 to 2 μM against four drug-susceptible bacteria and ten drug-resistant bacteria. Moreover, the hemolysis and cytotoxicity was reduced significantly compared to those of the parent peptides, highlighting its high cell selectivity. The morphological changes in the giant unilamellar vesicles and bacterial cell surfaces caused by the Hn-Mc peptide suggested that it killed the microbial cells by damaging the membrane envelope. An in vivo study also demonstrated the antibacterial activity of the Hn-Mc peptide in a mouse model infected with drug-resistant bacteria. In addition, the chimeric peptide inhibited the expression of lipopolysaccharide (LPS)-induced cytokines in RAW 264.7 cells by preventing the interaction between LPS and Toll-like receptors. These results suggest that this chimeric peptide is an antimicrobial and anti-inflammatory candidate as a pharmaceutic agent. Copyright © 2015 Elsevier Inc. All rights reserved.

Separating attoliter-sized compartments using fluid pore-spanning lipid bilayers.

Science.gov (United States)

Lazzara, Thomas D; Carnarius, Christian; Kocun, Marta; Janshoff, Andreas; Steinem, Claudia

2011-09-27

Anodic aluminum oxide (AAO) is a porous material having aligned cylindrical compartments with 55-60 nm diameter pores, and being several micrometers deep. A protocol was developed to generate pore-spanning fluid lipid bilayers separating the attoliter-sized compartments of the nanoporous material from the bulk solution, while preserving the optical transparency of the AAO. The AAO was selectively functionalized by silane chemistry to spread giant unilamellar vesicles (GUVs) resulting in large continuous membrane patches covering the pores. Formation of fluid single lipid bilayers through GUV rupture could be readily observed by fluorescence microscopy and further supported by conservation of membrane surface area, before and after GUV rupture. Fluorescence recovery after photobleaching gave low immobile fractions (5-15%) and lipid diffusion coefficients similar to those found for bilayers on silica. The entrapment of molecules within the porous underlying cylindrical compartments, as well as the exclusion of macromolecules from the nanopores, demonstrate the barrier function of the pore-spanning membranes and could be investigated in three-dimensions using confocal laser scanning fluorescence imaging. © 2011 American Chemical Society

Localization and Orientation of Xanthophylls in a Lipid Bilayer.

Science.gov (United States)

Grudzinski, Wojciech; Nierzwicki, Lukasz; Welc, Renata; Reszczynska, Emilia; Luchowski, Rafal; Czub, Jacek; Gruszecki, Wieslaw I

2017-08-29

Xanthophylls (polar carotenoids) play diverse biological roles, among which are modulation of the physical properties of lipid membranes and protection of biomembranes against oxidative damage. Molecular mechanisms underlying these functions are intimately related to the localization and orientation of xanthophyll molecules in lipid membranes. In the present work, we address the problem of localization and orientation of two xanthophylls present in the photosynthetic apparatus of plants and in the retina of the human eye, zeaxanthin and lutein, in a single lipid bilayer membrane formed with dimyristoylphosphatidylcholine. By using fluorescence microscopic analysis and Raman imaging of giant unilamellar vesicles, as well as molecular dynamics simulations, we show that lutein and zeaxanthin adopt a very similar transmembrane orientation within a lipid membrane. In experimental and computational approach, the average tilt angle of xanthophylls relative to the membrane normal is independently found to be ~40 deg, and results from hydrophobic mismatch between the membrane thickness and the distance between the terminal hydroxyl groups of the xanthophylls. Consequences of such a localization and orientation for biological activity of xanthophylls are discussed.

Thermodynamics and kinetics of vesicles formation processes.

Science.gov (United States)

Guida, Vincenzo

2010-12-15

Vesicles are hollow aggregates, composed of bilayers of amphiphilic molecules, dispersed into and filled with a liquid solvent. These aggregates can be formed either as equilibrium or as out of equilibrium meta-stable structures and they exhibit a rich variety of different morphologies. The surprising richness of structures, the vast range of industrial applications and the presence of vesicles in a number of biological systems have attracted the interest of numerous researchers and scientists. In this article, we review both the thermodynamics and the kinetics aspects of the phenomena of formation of vesicles. We start presenting the thermodynamics of bilayer membranes formation and deformation, with the aim of deriving the conditions for the existence of equilibrium vesicles. Specifically, we use the results from continuum thermodynamics to discuss the possibility of formation of stable equilibrium vesicles, from both mixed amphiphiles and single component systems. We also link the bilayer membrane properties to the molecular structure of the starting amphiphiles. In the second part of this article, we focus on the dynamics and kinetics of vesiculation. We review the process of vesicles formation both from planar lamellar phase under shear and from isotropic micelles. In order to clarify the physical mechanisms of vesicles formation, we continuously draw a parallel between emulsification and vesiculation processes. Specifically, we compare the experimental results, the driving forces and the relative scaling laws identified for the two processes. Describing the dynamics of vesicles formation, we also discuss why non equilibrium vesicles can be formed by kinetics control and why they are meta-stable. Understanding how to control the properties, the stability and the formation process of vesicles is of fundamental importance for a vast number of industrial applications. Copyright © 2009. Published by Elsevier B.V.

Cargo Release from Polymeric Vesicles under Shear

Directory of Open Access Journals (Sweden)

Yingying Guo

2018-03-01

Full Text Available In this paper we study the release of cargo from polymeric nano-carriers under shear. Vesicles formed by two star block polymers— A 12 B 6 C 2 ( A B C and A 12 B 6 A 2 ( A B A —and one linear block copolymer— A 14 B 6 ( A B , are investigated using dissipative particle dynamics (DPD simulations. A - and C -blocks are solvophobic and B -block is solvophilic. The three polymers form vesicles of different structures. The vesicles are subjected to shear both in bulk and between solvophobic walls. In bulk shear, the mechanisms of cargo release are similar for all vesicles, with cargo travelling through vesicle membrane with no preferential release location. When sheared between walls, high cargo release rate is only observed with A B C vesicle after it touches the wall. For A B C vesicle, the critical condition for high cargo release rate is the formation of wall-polymersome interface after which the effect of shear rate in promoting cargo release is secondary. High release rate is achieved by the formation of solvophilic pathway allowing cargo to travel from the vesicle cavity to the vesicle exterior. The results in this paper show that well controlled target cargo release using polymersomes can be achieved with polymers of suitable design and can potentially be very useful for engineering applications. As an example, polymersomes can be used as carriers for surface active friction reducing additives which are only released at rubbing surfaces where the additives are needed most.

Lipid Directed Intrinsic Membrane Protein Segregation

DEFF Research Database (Denmark)

Hansen, Jesper S.; Thompson, James R.; Helix Nielsen, Claus

2013-01-01

We demonstrate a new approach for direct reconstitution of membrane proteins during giant vesicle formation. We show that it is straightforward to create a tissue-like giant vesicle film swelled with membrane protein using aquaporin SoPIP2;1 as an illustration. These vesicles can also be easily h...

Liposomes loaded with contrast material for image enhancement in computed tomography: work in progress

International Nuclear Information System (INIS)

Ryan, P.J.; Davis, M.A.; DeGaeta, L.R.; Woda, B.; Melchior, D.L.

1984-01-01

Large unilamellar phospholipid vesicles were prepared and loaded with various radiographic contrast media. Body CT following in vivo adminstration of these vesicles in the rat demonstrated opacification of organs associated with the reticuloendothelial system. Image enhancement in the spleen and liver was dose dependent and was linearly related within the dose range investigated. Clearance of the radiographic contrast material was complete within 24 hours. Diffuse splenic lymphoma following intraperitoneal or intrasplenic injection of lymphoma cells, and solitary lymphoma nodules following intrahepatic injection were readily detected as nonenhanced areas following injection of liposomes

Are calcifying matrix vesicles in atherosclerotic lesions of cellular origin?

Science.gov (United States)

Bobryshev, Yuri V; Killingsworth, Murray C; Huynh, Thuan G; Lord, Reginald S A; Grabs, Anthony J; Valenzuela, Stella M

2007-03-01

Over recent years, the role of matrix vesicles in the initial stages of arterial calcification has been recognized. Matrix calcifying vesicles have been isolated from atherosclerotic arteries and the biochemical composition of calcified vesicles has been studied. No studies have yet been carried out to examine the fine structure of matrix vesicles in order to visualize the features of the consequent stages of their calcification in arteries. In the present work, a high resolution ultrastructural analysis has been employed and the study revealed that matrix vesicles in human atherosclerotic lesions are heterogeneous with two main types which we classified. Type I calcified vesicles were presented by vesicles surrounded by two electron-dense layers and these vesicles were found to be resistant to the calcification process in atherosclerotic lesions in situ. Type II matrix vesicles were presented by vesicles surrounded by several electron-dense layers and these vesicles were found to represent calcifying vesicles in atherosclerotic lesions. To test the hypothesis that calcification of matrix vesicles surrounded by multilayer sheets may occur simply as a physicochemical process, independently from the cell regulation, we produced multilamellar liposomes and induced their calcification in vitro in a manner similar to that occurring in matrix vesicles in atherosclerotic lesions in situ.

Vesicles Are Persistent Features of Different Plastids.

Science.gov (United States)

Lindquist, Emelie; Solymosi, Katalin; Aronsson, Henrik

2016-10-01

Peripheral vesicles in plastids have been observed repeatedly, primarily in proplastids and developing chloroplasts, in which they are suggested to function in thylakoid biogenesis. Previous observations of vesicles in mature chloroplasts have mainly concerned low temperature pretreated plants occasionally treated with inhibitors blocking vesicle fusion. Here, we show that such vesicle-like structures occur not only in chloroplasts and proplastids, but also in etioplasts, etio-chloroplasts, leucoplasts, chromoplasts and even transforming desiccoplasts without any specific pretreatment. Observations are made both in C3 and C4 species, in different cell types (meristematic, epidermis, mesophyll, bundle sheath and secretory cells) and different organs (roots, stems, leaves, floral parts and fruits). Until recently not much focus has been given to the idea that vesicle transport in chloroplasts could be mediated by proteins, but recent data suggest that the vesicle system of chloroplasts has similarities with the cytosolic coat protein complex II system. All current data taken together support the idea of an ongoing, active and protein-mediated vesicle transport not only in chloroplasts but also in other plastids, obviously occurring regardless of chemical modifications, temperature and plastid developmental stage. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Enhanced synaptic transmission at the squid giant synapse by artificial seawater based on physically modified saline

Science.gov (United States)

Choi, Soonwook; Yu, Eunah; Rabello, Guilherme; Merlo, Suelen; Zemmar, Ajmal; Walton, Kerry D.; Moreno, Herman; Moreira, Jorge E.; Sugimori, Mutsuyuki; Llinás, Rodolfo R.

2014-01-01

Superfusion of the squid giant synapse with artificial seawater (ASW) based on isotonic saline containing oxygen nanobubbles (RNS60 ASW) generates an enhancement of synaptic transmission. This was determined by examining the postsynaptic response to single and repetitive presynaptic spike activation, spontaneous transmitter release, and presynaptic voltage clamp studies. In the presence of RNS60 ASW single presynaptic stimulation elicited larger postsynaptic potentials (PSP) and more robust recovery from high frequency stimulation than in control ASW. Analysis of postsynaptic noise revealed an increase in spontaneous transmitter release with modified noise kinetics in RNS60 ASW. Presynaptic voltage clamp demonstrated an increased EPSP, without an increase in presynaptic ICa++ amplitude during RNS60 ASW superfusion. Synaptic release enhancement reached stable maxima within 5–10 min of RNS60 ASW superfusion and was maintained for the entire recording time, up to 1 h. Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles (CCV) and large endosome-like vesicles near junctional sites. Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW. After ATP block the number of vesicles at the active zone and CCV was reduced, with an increase in large vesicles. The possibility that RNS60 ASW acts by increasing mitochondrial ATP synthesis was tested by direct determination of ATP levels in both presynaptic and postsynaptic structures. This was implemented using luciferin/luciferase photon emission, which demonstrated a marked increase in ATP synthesis following RNS60 administration. It is concluded that RNS60 positively modulates synaptic transmission by up-regulating ATP synthesis, thus leading to synaptic transmission enhancement. PMID:24575037

Telocytes in pancreas of the Chinese giant salamander (Andrias davidianus).

Science.gov (United States)

Zhang, Hui; Yu, Pengcheng; Zhong, Shengwei; Ge, Tingting; Peng, Shasha; Guo, Xiaoquan; Zhou, Zuohong

2016-11-01

Telocytes (TCs), novel interstitial cells, have been identified in various organs of many mammals. However, information about TCs of lower animals remains rare. Herein, pancreatic TCs of the Chinese giant salamanders (Andrias davidianus) were identified by CD34 immunohistochemistry (IHC) and transmission electron microscopy (TEM). The IHC micrographs revealed CD34 + TCs with long telopodes (Tps) that were located in the interstitium of the pancreas. CD34 + TCs/Tps were frequently observed between exocrine acinar cells and were close to blood vessels. The TEM micrographs also showed the existence of TCs in the interstitium of the pancreas. TCs had distinctive ultrastructural features, such as one to three very long and thin Tps with podoms and podomers, caveolae, dichotomous branching, neighbouring exosomes and vesicles. The Tps and exosomes were found in close proximity to exocrine acinar cells and α cells. It is suggested that TCs may play a role in the regeneration of acinar cells and α cells. In conclusion, our results demonstrated the presence of TCs in the pancreas of the Chinese giant salamander. This finding will assist us in a better understanding of TCs functions in the amphibian pancreas. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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....

Extracellular vesicles: Exosomes, microvesicles, and friends

NARCIS (Netherlands)

Raposo, G.; Stoorvogel, W.|info:eu-repo/dai/nl/074352385

2013-01-01

Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for

Slow sedimentation and deformability of charged lipid vesicles.

Directory of Open Access Journals (Sweden)

Iván Rey Suárez

Full Text Available The study of vesicles in suspension is important to understand the complicated dynamics exhibited by cells in in vivo and in vitro. We developed a computer simulation based on the boundary-integral method to model the three dimensional gravity-driven sedimentation of charged vesicles towards a flat surface. The membrane mechanical behavior was modeled using the Helfrich Hamiltonian and near incompressibility of the membrane was enforced via a model which accounts for the thermal fluctuations of the membrane. The simulations were verified and compared to experimental data obtained using suspended vesicles labelled with a fluorescent probe, which allows visualization using fluorescence microscopy and confers the membrane with a negative surface charge. The electrostatic interaction between the vesicle and the surface was modeled using the linear Derjaguin approximation for a low ionic concentration solution. The sedimentation rate as a function of the distance of the vesicle to the surface was determined both experimentally and from the computer simulations. The gap between the vesicle and the surface, as well as the shape of the vesicle at equilibrium were also studied. It was determined that inclusion of the electrostatic interaction is fundamental to accurately predict the sedimentation rate as the vesicle approaches the surface and the size of the gap at equilibrium, we also observed that the presence of charge in the membrane increases its rigidity.

Bubble-induced microstreaming: guiding and destroying lipid vesicles

Science.gov (United States)

Marmottant, Philippe; Hilgenfeldt, Sascha

2002-11-01

Micron-sized bubbles respond with strong oscillations when submitted to ultrasound. This has led to their use as echographic contrast enhancers. The large energy and force densities generated by the collapsing bubbles also make them non-invasive mechanical tools: Recently, it has been reported that the interaction of cavitating bubbles with nearby cells can render the latter permeable to large molecules (sonoporation), suggesting prospects for drug delivery and gene transfection. We have developed a laboratory setup that allows for a controlled study of the interaction of single microbubbles with single lipid bilayer vesicles. Substituting vesicles for cell membranes is advantageous because the mechanical properties of vesicles are well-known. Microscopic observations reveal that vesicles near a bubble follow the vivid streaming motion set up by the bubble. The vesicles "bounce" off the bubble, being periodically accelerated towards and away from it, and undergo well-defined shape deformations along their trajectory in accordance with fluid-dynamical theory. Break-up of vesicles could also be observed.

High-yield nontoxic gene transfer through conjugation of the CM₁₈-Tat₁₁ chimeric peptide with nanosecond electric pulses.

Science.gov (United States)

Salomone, Fabrizio; Breton, Marie; Leray, Isabelle; Cardarelli, Francesco; Boccardi, Claudia; Bonhenry, Daniel; Tarek, Mounir; Mir, Lluis M; Beltram, Fabio

2014-07-07

We report a novel nontoxic, high-yield, gene delivery system based on the synergistic use of nanosecond electric pulses (NPs) and nanomolar doses of the recently introduced CM18-Tat11 chimeric peptide (sequence of KWKLFKKIGAVLKVLTTGYGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein). This combined use makes it possible to drastically reduce the required CM18-Tat11 concentration and confines stable nanopore formation to vesicle membranes followed by DNA release, while no detectable perturbation of the plasma membrane is observed. Two different experimental assays are exploited to quantitatively evaluate the details of NPs and CM18-Tat11 cooperation: (i) cytofluorimetric analysis of the integrity of synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles exposed to CM18-Tat11 and NPs and (ii) the in vitro transfection efficiency of a green fluorescent protein-encoding plasmid conjugated to CM18-Tat11 in the presence of NPs. Data support a model in which NPs induce membrane perturbation in the form of transient pores on all cellular membranes, while the peptide stabilizes membrane defects selectively within endosomes. Interestingly, atomistic molecular dynamics simulations show that the latter activity can be specifically attributed to the CM18 module, while Tat11 remains essential for cargo binding and vector subcellular localization. We argue that this result represents a paradigmatic example that can open the way to other targeted delivery protocols.

The use of Trametes versicolor laccase for the polymerization of aniline in the presence of vesicles as templates.

Science.gov (United States)

Junker, Katja; Kissner, Reinhard; Rakvin, Boris; Guo, Zengwei; Willeke, Martin; Busato, Stephan; Weber, Thomas; Walde, Peter

2014-02-05

The enzymatic polymerization of aniline to polyaniline (PANI) with Trametes versicolor laccase (TvL) as catalyst and dioxygen (O₂) as oxidant was investigated in an aqueous medium containing unilamellar vesicles with an average diameter of about 80 nm formed from AOT (=sodium bis(2-ethylhexyl) sulfosuccinate). Compared to the same reaction carried out with horseradish peroxidase isoenzyme C (HRPC) as catalyst and hydrogen peroxide (H₂O₂) as oxidant, notable differences were found in the kinetics of the reaction, as well as in the characteristics of the PANI obtained. Under comparable optimal conditions, which are pH 3.5 for TvL/O₂ and pH 4.3 for HRPC/H₂O₂, the reaction with TvL/O₂ was much slower than with HRPC/H₂O₂, i.e. ≈27 days vs. 1 day reaction time to reach equilibrium with >90% yield at 25 °C. Although in both cases, aniline monomer coupling occurred mainly via the carbon atom in para position of aniline, UV-vis-NIR absorption and EPR measurements indicate that the reaction with TvL/O₂ yielded mainly overoxidized products (with λ(max)=730 nm). These products had a lower amount of unpaired electrons if compared with the products obtained with HRPC/H₂O₂ (with λ(max)≈1000 nm, which is characteristic for the polaron state of PANI-ES, the emeraldine salt form of PANI). Similarly to previous findings with HRPC/H₂O₂, enzyme inactivation occurred during the polymerization also in the case of TvL/O₂. Since the aqueous PANI-vesicle suspensions obtained are of high colloidal stability, they can be used directly as ink in a conventional thermal inkjet printer for printing on paper or on surface treated polyimide films. Printed PANI-ES patterns on paper changed colour from green (emeraldine salt) to blue (emeraldine base) upon exposure to ammonia gas, demonstrating the expected ammonia sensing properties. Copyright © 2013 Elsevier Inc. All rights reserved.

Membrane fusion

DEFF Research Database (Denmark)

Bendix, Pól Martin

2015-01-01

At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

ABC Triblock Copolymer Vesicles with Mesh-like Morphology

Science.gov (United States)

Zhao, Wei; Russell, Thomas; Grason, Gregory

2010-03-01

Polymer vesicles can be made from poly(isoprene-b-styrene-b-2-vinylpyridene) (PI-b-PS-b-P2VP) triblock copolymer under the confinement of anodic aluminum oxide (AAO) membrane. It was found that these vesicles have well-defined, nanoscopic size and a microphase-separated hydrophobic core, comprised of PS and PI blocks. Vesicle formation was tracked using both transmission and scanning electron microscopy. A mesh-like morphology formed in the core at a well-defined composition of three blocks. Confinement played an important role in generating these vesicles with such an unusual morphology.

Genetically Controlled Fusion, Exocytosis and Fission of Artificial Vesicles

DEFF Research Database (Denmark)

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

if a special class of viral proteins, termed fusogenic peptides, were added to the external medium. In the present work, we intend to develop genetically controlled fusion, fission and exocytosis of vesicles by the synthesis of peptides within vesicles. First, we 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 mechanisms are available, e.g. addition...... fusion, fission and exocytosis....

Primary vesicles, vesicle-rich segregation structures and recognition of primary and secondary porosities in lava flows from the Paraná igneous province, southern Brazil

Science.gov (United States)

Barreto, Carla Joana S.; de Lima, Evandro F.; Goldberg, Karin

2017-04-01

This study focuses on a volcanic succession of pāhoehoe to rubbly lavas of the Paraná-Etendeka Province exposed in a single road profile in southernmost Brazil. This work provides an integrated approach for examining primary vesicles and vesicle-rich segregation structures at the mesoscopic scale. In addition, this study provides a quantitative analysis of pore types in thin section. We documented distinct distribution patterns of vesicle and vesicle-rich segregation structures according to lava thickness. In compound pāhoehoe lavas, the cooling allows only vesicles (pipe vesicles to be frozen into place. In inflated pāhoehoe lavas, vesicles of different sizes are common, including pipe vesicles, and also segregation structures such as proto-cylinders, cylinders, cylinder sheets, vesicle sheets, and pods. In rubbly lavas, only vesicles of varying sizes occur. Gas release from melt caused the formation of primary porosity, while hydrothermal alteration and tectonic fracturing are the main processes that generated secondary porosity. Although several forms of porosity were created in the basaltic lava flows, the precipitation of secondary minerals within the pores has tended to reduce the original porosities. Late-stage fractures could create efficient channel networks for possible hydrocarbon/groundwater migration and entrapment owing to their ability to connect single pores. Quantitative permeability data should be gathered in future studies to confirm the potential of these lavas for store hydrocarbons or groundwater.

Giant grains

International Nuclear Information System (INIS)

Leitch-Devlin, M.A.; Millar, T.J.; Williams, D.A.

1976-01-01

Infrared observations of the Orion nebula have been interpreted by Rowan-Robinson (1975) to imply the existence of 'giant' grains, radius approximately 10 -2 cm, throughout a volume about a parsec in diameter. Although Rowan-Robinson's model of the nebula has been criticized and the presence of such grains in Orion is disputed, the proposition is accepted, that they exist, and in this paper situations in which giant grains could arise are examined. It is found that, while a giant-grain component to the interstellar grain density may exist, it is difficult to understand how giant grains arise to the extent apparently required by the Orion nebula model. (Auth.)

Synaptic vesicle distribution by conveyor belt.

Science.gov (United States)

Moughamian, Armen J; Holzbaur, Erika L F

2012-03-02

The equal distribution of synaptic vesicles among synapses along the axon is critical for robust neurotransmission. Wong et al. show that the continuous circulation of synaptic vesicles throughout the axon driven by molecular motors ultimately yields this even distribution. Copyright © 2012 Elsevier Inc. All rights reserved.

A scenario for a genetically controlled fission of artificial vesicles

DEFF Research Database (Denmark)

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

2011-01-01

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...... be incorporated into vesicles, and therefore allow the synthesis of a large number of proteins (Noireaux et al. 2005). However, vesicle fission remains one of the upcoming challenges in the artificial cell project (Noireaux et al. 2011). So far, vesicle fission is implemented by applying mechanical stress...

Molecular characterization of exosome-like vesicles from breast cancer cells

International Nuclear Information System (INIS)

Kruger, Stefan; Elmageed, Zakaria Y Abd; Hawke, David H; Wörner, Philipp M; Jansen, David A; Abdel-Mageed, Asim B; Alt, Eckhard U; Izadpanah, Reza

2014-01-01

Membrane vesicles released by neoplastic cells into extracellular medium contain potential of carrying arrays of oncogenic molecules including proteins and microRNAs (miRNA). Extracellular (exosome-like) vesicles play a major role in cell-to-cell communication. Thus, the characterization of proteins and miRNAs of exosome-like vesicles is imperative in clarifying intercellular signaling as well as identifying disease markers. Exosome-like vesicles were isolated using gradient centrifugation from MCF-7 and MDA-MB 231 cultures. Proteomic profiling of vesicles using liquid chromatography-mass spectrometry (LC-MS/MS) revealed different protein profiles of exosome-like vesicles derived from MCF-7 cells (MCF-Exo) than those from MDA-MB 231 cells (MDA-Exo). The protein database search has identified 88 proteins in MDA-Exo and 59 proteins from MCF-Exo. Analysis showed that among all, 27 proteins were common between the two exosome-like vesicle types. Additionally, MDA-Exo contains a higher amount of matrix-metalloproteinases, which might be linked to the enhanced metastatic property of MDA-MB 231 cells. In addition, microarray analysis identified several oncogenic miRNA between the two types vesicles. Identification of the oncogenic factors in exosome-like vesicles is important since such vesicles could convey signals to non-malignant cells and could have an implication in tumor progression and metastasis

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.

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.

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.

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

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.

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

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.

Calmodulin stimulation of calcium transport in carrot microsomal vesicles

International Nuclear Information System (INIS)

Pierce, W.S.; Sze, H.

1987-01-01

ATP-dependent 45 Ca 2+ uptake into microsomal vesicles isolated from cultured carrot cells (Daucus carota Danvers) was stimulated 2-3 fold by 5 ug/ml calmodulin (CaM). Microsomal vesicles separated with a linear sucrose gradient showed two peaks with CaM-stimulated Ca 2+ uptake activities. One peak (at 1.12 g/cc) comigrated with the activity of the antimycin A-insensitive NADH-dependent cytochrome c reductase. This transport activity was enhanced 10-20 fold by 10 mM oxalate and appeared to be associates with vesicles derived primarily from the ER. The other peak of CaM-stimulated Ca 2+ uptake (at 1.17 g/cc) was not affected by oxalate. These vesicles are probably derived from the plasma membrane. Preliminary experiments with the low-density vesicles (ER) vesicles, indicate that inositol-1,4,5-trisphosphate caused a transient reduction in intravesicular Ca 2+ . These results are consistent with the ER being an important site of intracellular Ca 2+ regulation

Motion mechanics of non-adherent giant liposomes with a combined optical and atomic force microscope

Science.gov (United States)

Moreno-Flores, Susana; Ortíz, Rocío

2017-11-01

Herein we present an investigation of the motional dynamics of single mesoscopic bodies of biological relevance with an AFM-based macromanipulation tool and an optical microscope. Giant liposomes are prominent case examples as minimal cell models; studying their mechanics provides a means to address the influence of structural components in the mechanical behaviour of living cells. However, they also pose an experimental challenge due to their lightness, fragility, and high mobility. Their entrapment in wells in a fluid of lower density allows their study under conditions of constrained motion, which enables the synchronous measurement of nanoforces with motion tracking. The procedure enables to estimate sliding friction coefficients and masses of vesicles, and sheds light upon the region between the vesicle and the underlying substrate. The present study paves the way for the investigation of motion and deformation mechanics with one combined technique and a single type of experiment traditionally vetoed to objects that can move as well as deform. Such an approach can be directly applied to cells in suspension, adherent cells or cellular 3D-assemblies so as to assess substrate biocompatibility, monitor adhesion, detachment, motility as well as deformability.

Motion mechanics of non-adherent giant liposomes with a combined optical and atomic force microscope

International Nuclear Information System (INIS)

Moreno-Flores, Susana; Ortíz, Rocío

2017-01-01

Herein we present an investigation of the motional dynamics of single mesoscopic bodies of biological relevance with an AFM-based macromanipulation tool and an optical microscope. Giant liposomes are prominent case examples as minimal cell models; studying their mechanics provides a means to address the influence of structural components in the mechanical behaviour of living cells. However, they also pose an experimental challenge due to their lightness, fragility, and high mobility. Their entrapment in wells in a fluid of lower density allows their study under conditions of constrained motion, which enables the synchronous measurement of nanoforces with motion tracking. The procedure enables to estimate sliding friction coefficients and masses of vesicles, and sheds light upon the region between the vesicle and the underlying substrate. The present study paves the way for the investigation of motion and deformation mechanics with one combined technique and a single type of experiment traditionally vetoed to objects that can move as well as deform. Such an approach can be directly applied to cells in suspension, adherent cells or cellular 3D-assemblies so as to assess substrate biocompatibility, monitor adhesion, detachment, motility as well as deformability. (paper)

Allometry indicates giant eyes of giant squid are not exceptional.

Science.gov (United States)

Schmitz, Lars; Motani, Ryosuke; Oufiero, Christopher E; Martin, Christopher H; McGee, Matthew D; Gamarra, Ashlee R; Lee, Johanna J; Wainwright, Peter C

2013-02-18

The eyes of giant and colossal squid are among the largest eyes in the history of life. It was recently proposed that sperm whale predation is the main driver of eye size evolution in giant squid, on the basis of an optical model that suggested optimal performance in detecting large luminous visual targets such as whales in the deep sea. However, it is poorly understood how the eye size of giant and colossal squid compares to that of other aquatic organisms when scaling effects are considered. We performed a large-scale comparative study that included 87 squid species and 237 species of acanthomorph fish. While squid have larger eyes than most acanthomorphs, a comparison of relative eye size among squid suggests that giant and colossal squid do not have unusually large eyes. After revising constants used in a previous model we found that large eyes perform equally well in detecting point targets and large luminous targets in the deep sea. The eyes of giant and colossal squid do not appear exceptionally large when allometric effects are considered. It is probable that the giant eyes of giant squid result from a phylogenetically conserved developmental pattern manifested in very large animals. Whatever the cause of large eyes, they appear to have several advantages for vision in the reduced light of the deep mesopelagic zone.

Magnetoliposomes based on nickel/silica core/shell nanoparticles: Synthesis and characterization

Energy Technology Data Exchange (ETDEWEB)

Rodrigues, Ana Rita O.; Gomes, I.T.; Almeida, Bernardo G. [Centro de Física (CFUM), Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal); Araújo, J.P. [IFIMUP/IN – Instituto de Nanociência e Nanotecnologia, R. Campo Alegre, 4169-007 Porto (Portugal); Castanheira, Elisabete M.S. [Centro de Física (CFUM), Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal); Coutinho, Paulo J.G., E-mail: pcoutinho@fisica.uminho.pt [Centro de Física (CFUM), Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal)

2014-12-15

In the present work, nickel magnetic nanoparticles with diameters lower than 100 nm, with and without silica shell, were synthesized by microheterogeneous templating. The magnetic properties of the nanoparticles show a typical ferromagnetic behavior with a coercive field of 80 Oe. Dry magnetoliposomes (DMLs) with diameter between 58 nm and 76 nm were obtained from the synthesis of nanoparticles in the presence of a lipid or surfactant layer, and aqueous magnetoliposomes (AMLs) were obtained by encapsulation of the nanoparticles in liposomes. FRET (Förster resonance energy transfer) experiments were performed to study the non-specific interactions between aqueous magnetoliposomes and giant unilamellar vesicles (GUVs), as models of cell membranes. It was possible to detect membrane fusion between GUVs and AMLs containing both NBD-C{sub 6}-HPC (donor) and the dye Nile Red (acceptor). - Highlights: • Magnetic nickel nanoparticles were synthesized in microheterogeneous media. • The nanoparticles were covered with a silica shell to improve biocompatibility. • Aqueous and dry magnetoliposomes were prepared, the latter with diameter around 70 nm. • Membrane fusion between magnetoliposomes and models of cell membranes was detected by FRET.

Magnetoliposomes based on nickel/silica core/shell nanoparticles: Synthesis and characterization

International Nuclear Information System (INIS)

Rodrigues, Ana Rita O.; Gomes, I.T.; Almeida, Bernardo G.; Araújo, J.P.; Castanheira, Elisabete M.S.; Coutinho, Paulo J.G.

2014-01-01

In the present work, nickel magnetic nanoparticles with diameters lower than 100 nm, with and without silica shell, were synthesized by microheterogeneous templating. The magnetic properties of the nanoparticles show a typical ferromagnetic behavior with a coercive field of 80 Oe. Dry magnetoliposomes (DMLs) with diameter between 58 nm and 76 nm were obtained from the synthesis of nanoparticles in the presence of a lipid or surfactant layer, and aqueous magnetoliposomes (AMLs) were obtained by encapsulation of the nanoparticles in liposomes. FRET (Förster resonance energy transfer) experiments were performed to study the non-specific interactions between aqueous magnetoliposomes and giant unilamellar vesicles (GUVs), as models of cell membranes. It was possible to detect membrane fusion between GUVs and AMLs containing both NBD-C 6 -HPC (donor) and the dye Nile Red (acceptor). - Highlights: • Magnetic nickel nanoparticles were synthesized in microheterogeneous media. • The nanoparticles were covered with a silica shell to improve biocompatibility. • Aqueous and dry magnetoliposomes were prepared, the latter with diameter around 70 nm. • Membrane fusion between magnetoliposomes and models of cell membranes was detected by FRET

Characterization of lipid films by an angle-interrogation surface plasmon resonance imaging device.

Science.gov (United States)

Liu, Linlin; Wang, Qiong; Yang, Zhong; Wang, Wangang; Hu, Ning; Luo, Hongyan; Liao, Yanjian; Zheng, Xiaolin; Yang, Jun

2015-04-01

Surface topographies of lipid films have an important significance in the analysis of the preparation of giant unilamellar vesicles (GUVs). In order to achieve accurately high-throughput and rapidly analysis of surface topographies of lipid films, a homemade SPR imaging device is constructed based on the classical Kretschmann configuration and an angle interrogation manner. A mathematical model is developed to accurately describe the shift including the light path in different conditions and the change of the illumination point on the CCD camera, and thus a SPR curve for each sampling point can also be achieved, based on this calculation method. The experiment results show that the topographies of lipid films formed in distinct experimental conditions can be accurately characterized, and the measuring resolution of the thickness lipid film may reach 0.05 nm. Compared with existing SPRi devices, which realize detection by monitoring the change of the reflective-light intensity, this new SPRi system can achieve the change of the resonance angle on the entire sensing surface. Thus, it has higher detection accuracy as the traditional angle-interrogation SPR sensor, with much wider detectable range of refractive index. Copyright © 2015 Elsevier B.V. All rights reserved.

High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope.

Science.gov (United States)

Li, Yongxiao; Montague, Samantha J; Brüstle, Anne; He, Xuefei; Gillespie, Cathy; Gaus, Katharina; Gardiner, Elizabeth E; Lee, Woei Ming

2018-02-28

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The influence of NBD fluorescent probe on model membranes containing POPC and DPPC.

Science.gov (United States)

Weng, Chi-Jung; Wu, Ju-Ping; Kuo, Ming-Yen; Hsueh, Ya-Wei

2016-03-01

To investigate the effect of fluorescent probe on the properties of membranes, we studied model membranes composed of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence and absence of fluorescent probe. The morphology of giant unilamellar vesicles (GUVs) has been observed as a function of temperature and composition by fluorescence microscopy using NBD-DOPE or C 6 -NBD-PC as the probe. The phase behavior of model membranes containing no fluorescent probe was investigated by 2 H-NMR spectroscopy. We found that the bright phase observed on GUVs was the fluid phase enriched in POPC and the dark phase was the gel phase enriched in DPPC. NBD-DOPE and C 6 -NBD-PC preferentially participated in the fluid-phase domains when GUVs were in the gel + fluid phase coexistence. Inclusion of both fluorescent probes (1 mol%) lowered the transition temperature of POPC/DPPC membranes. In addition, C 6 -NBD-PC exhibited a stronger effect than NBD-DOPE, which was considered to be associated with the structures of fluorescent molecules.

Deformation of phospholipid vesicles in an optical stretcher

OpenAIRE

Delabre , Ulysse; Feld , Kasper; Crespo , Eleonore; Whyte , Graeme; Sykes , Cecile; Seifert , Udo; Guck , Jochen

2015-01-01

International audience; Phospholipid vesicles are common model systems for cell membranes. Important aspects of the membrane function relate to its mechanical properties. Here we have investigated the deformation behaviour of phospholipid vesicles in a dual-beam laser trap, also called an optical stretcher. This study explicitly makes use of the inherent heating present in such traps to investigate the dependence of vesicle deformation on temperature. By using lasers with different wavelength...

Plasma membrane aquaporins mediates vesicle stability in broccoli.

Directory of Open Access Journals (Sweden)

Maria Del Carmen Martínez-Ballesta

Full Text Available 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.

Active elastohydrodynamics of vesicles in narrow blind constrictions

Science.gov (United States)

Fai, T. G.; Kusters, R.; Harting, J.; Rycroft, C. H.; Mahadevan, L.

2017-11-01

Fluid-resistance limited transport of vesicles through narrow constrictions is a recurring theme in many biological and engineering applications. Inspired by the motor-driven movement of soft membrane-bound vesicles into closed neuronal dendritic spines, here we study this problem using a combination of passive three-dimensional simulations and a simplified semianalytical theory for the active transport of vesicles forced through constrictions by molecular motors. We show that the motion of these objects is characterized by two dimensionless quantities related to the geometry and to the strength of forcing relative to the vesicle elasticity. We use numerical simulations to characterize the transit time for a vesicle forced by fluid pressure through a constriction in a channel and find that relative to an open channel, transport into a blind end leads to the formation of a smaller forward-flowing lubrication layer that strongly impedes motion. When the fluid pressure forcing is complemented by forces due to molecular motors that are responsible for vesicle trafficking into dendritic spines, we find that the competition between motor forcing and fluid drag results in multistable dynamics reminiscent of the real system. Our study highlights the role of nonlocal hydrodynamic effects in determining the kinetics of vesicular transport in constricted geometries.

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

DEFF Research Database (Denmark)

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

2017-01-01

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

Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles

Directory of Open Access Journals (Sweden)

Jan Lötvall

2014-12-01

Full Text Available 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.

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.

ABC triblock copolymer vesicles with mesh-like morphology.

Science.gov (United States)

Zhao, Wei; Chen, Dian; Hu, Yunxia; Grason, Gregory M; Russell, Thomas P

2011-01-25

Polymer vesicles made from poly(isoprene-b-styrene-b-2-vinyl pyridine) (PI-b-PS-b-P2VP) triblock copolymer confined within the nanopores of an anodic aluminum oxide (AAO) membrane are studied. It was found that these vesicles have well-defined, nanoscopic size, and complex microphase-separated hydrophobic membranes, comprised of the PS and PI blocks, while the coronas are formed by the P2VP block. Vesicle formation was tracked using both transmission and scanning electron microscopy. A mesh-like morphology formed in the membrane at a well-defined composition of the three blocks that can be tuned by changing the copolymer composition. The nanoscale confinement, copolymer composition, and subtle molecular interactions contribute to the generation of these vesicles with such unusual morphologies.

Vesicle interactions with polyamino acids and antibody: in vitro and in vivo studies

International Nuclear Information System (INIS)

Dunnick, J.K.; McDougall, I.R.; Aragon, S.; Goris, M.L.; Kriss, J.P.

1975-01-01

Artificial spherules or vesicles of 900 A in diameter formed from phosphatidylcholine and gangliosides and enclosing /sup 99m/TcO 4 - (standard preparation) survive intact in the circulation of the mouse. Polyamino acids and protein have been incorporated into and onto the vesicles; such vesicles remain intact as determined by diffusion dialysis studies and by electron paramagnetic resonance studies of vesicles enclosing spin label. In studying the distribution of polyamino acid-vesicles and protein vesicles in vivo, it was found that the latter distribute differently from standard vesicles or free protein alone whereas aromatic polyamino acid-vesicles concentrate in the liver and spleen to a greater extent than standard vesicles. The permeability and stability characteristics of vesicles may be preserved when they are modified by the addition of protein or polyamino acids and that such modification of vesicles may be associated with an alteration of their fate in vivo. The potential exists to use vesicles as carriers of radiopharmaceuticals and other drugs and to direct the vesicles preferentially to tissue targets in vivo. (U.S.)

Vesicle dynamics in shear and capillary flows

International Nuclear Information System (INIS)

Noguchi, Hiroshi; Gompper, Gerhard

2005-01-01

The deformation of vesicles in flow is studied by a mesoscopic simulation technique, which combines multi-particle collision dynamics for the solvent with a dynamically triangulated surface model for the membrane. Shape transitions are investigated both in simple shear flows and in cylindrical capillary flows. We focus on reduced volumes, where the discocyte shape of fluid vesicles is stable, and the prolate shape is metastable. In simple shear flow at low membrane viscosity, the shear induces a transformation from discocyte to prolate with increasing shear rate, while at high membrane viscosity, the shear induces a transformation from prolate to discocyte, or tumbling motion accompanied by oscillations between these two morphologies. In capillary flow, at small flow velocities the symmetry axis of the discocyte is found not to be oriented perpendicular to the cylinder axis. With increasing flow velocity, a transition to a prolate shape occurs for fluid vesicles, while vesicles with shear-elastic membranes (like red blood cells) transform into a coaxial parachute-like shape

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

Pulsed-laser polymerization in compartmentalized liquids. 1. Polymerization in vesicles

NARCIS (Netherlands)

Jung, M.; Casteren, van I.A.; Monteiro, M.J.; Herk, van A.M.; German, A.L.

2000-01-01

Polymerization in vesicles is a novel type of polymerization in heterogeneous media, leading to parachute-like vesicle-polymer hybrid morphologies. To explore the kinetics of vesicle polymerizations and to learn more about the actual locus of polymerization we applied the pulsed-laser polymerization

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.

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.

Extracellular vesicles: fundamentals and clinical relevance

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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.

The Bretherton Problem for a Vesicle

Science.gov (United States)

Barakat, Joseph; Spann, Andrew; Shaqfeh, Eric

2016-11-01

The motion of a lipid bilayer vesicle through a circular tube is investigated by singular perturbation theory in the limit of vanishing clearance. The vesicle is treated as a sac of fluid enclosed by a thin, elastic sheet that admits a bending stiffness. It is assumed that the vesicle is axisymmetric and swollen to a near-critical volume such that the clearance "e" between the membrane and the tube wall is very small. In this limit, bending resistance is of negligible importance compared to the isotropic tension, allowing the vesicle to be treated as a "no-slip bubble." The effective membrane tension is found to scale inversely with "e" raised to the 3/2 power with a comparatively weak Marangoni gradient. The extra pressure drop is found to have a leading contribution due to the cylindrical midsection, which scales inversely with "e," as well as a correction due to the end caps, which scales inversely with the square root of "e." The apparent viscosity is predicted as a unique function of the geometry. The theory exhibits excellent agreement with a simplified, "quasi-parallel" theory and with direct numerical simulations using the boundary element method. The results of this work are compared to those for bubbles, rigid particles, and red blood cells in confined flows.

Functionalization of Block Copolymer Vesicle Surfaces

Directory of Open Access Journals (Sweden)

Wolfgang Meier

2011-01-01

Full Text Available In dilute aqueous solutions certain amphiphilic block copolymers self-assemble into vesicles that enclose a small pool of water with a membrane. Such polymersomes have promising applications ranging from targeted drug-delivery devices, to biosensors, and nanoreactors. Interactions between block copolymer membranes and their surroundings are important factors that determine their potential biomedical applications. Such interactions are influenced predominantly by the membrane surface. We review methods to functionalize block copolymer vesicle surfaces by chemical means with ligands such as antibodies, adhesion moieties, enzymes, carbohydrates and fluorophores. Furthermore, surface-functionalization can be achieved by self-assembly of polymers that carry ligands at their chain ends or in their hydrophilic blocks. While this review focuses on the strategies to functionalize vesicle surfaces, the applications realized by, and envisioned for, such functional polymersomes are also highlighted.

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

were shown to fuse if a special class of viral proteins, termed fusogenic peptides, were added to the external medium (Nomura et al. 2004). In the present work, we intend to develop genetically controlled fusion, fission and exocytosis of vesicles by the synthesis of peptides within vesicles. First, we...... 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...

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.

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.

Assembly of cells and vesicles for organ engineering

International Nuclear Information System (INIS)

Taguchi, Tetsushi

2011-01-01

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. (topical review)

Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

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 using a combination of differential centrifugation and isopycnic zonal ultracentrifugation. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes whereas the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The sucrose HSR vesicles have an additional morphological feature which appears as membrane projections that resemble the SR feet. The freeze-fracture morphology of either type of SR reveals an asymmetric distribution of intramembraneous particles in the same orientation and distribution as the sarcoplasmic reticulum in vivo. Biochemical studies were made on the content of Ca, Mg, ATPase, and protein of the vesicles and phosphorylation of the vesicles. The biochemical and morphological data indicate that the LSR is derived from the longitudinal sarcoplasmic reticulum and the HSR is derived from the terminal cisternae of the sarcoplasmic reticulum, contains junctional SR membrane and has three unique proteins (calsequestrin, an intrinsic 30,000 dalton protein and a 9000 dalton proteolipid).

Interaction of a potyviral VPg with anionic phospholipid vesicles

International Nuclear Information System (INIS)

Rantalainen, Kimmo I.; Christensen, Peter A.; Hafren, Anders; Otzen, Daniel E.; Kalkkinen, Nisse; Maekinen, Kristiina

2009-01-01

The viral genome-linked protein (VPg) of Potato virus A (PVA) is a multifunctional protein that belongs to a class of intrinsically disordered proteins. Typically, this type of protein gains a more stable structure upon interactions or posttranslational modifications. In a membrane lipid strip overlay binding assay, PVA VPg was found to bind phosphatidylserine (PS), but not phosphatidylcholine (PC). According to circular dichroism spectroscopy, the secondary structure of PVA VPg was stabilized upon interactions with PS and phosphatidylglycerol (PG), but not with PC vesicles. It is possible that this stabilization favored the formation of α-helical structures. Limited tryptic digestion showed that the interaction with anionic vesicles protected certain, otherwise accessible, trypsin cleavage sites. An electron microscopy study revealed that interaction with VPg substantially increased the vesicle diameter and caused the formation of pore or plaque-like electron dense spots on the vesicle surface, which gradually led to disruption of the vesicles.

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.

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

THE REDSHIFT DISTRIBUTION OF GIANT ARCS IN THE SLOAN GIANT ARCS SURVEY

International Nuclear Information System (INIS)

Bayliss, Matthew B.; Gladders, Michael D.; Koester, Benjamin P.; Oguri, Masamune; Hennawi, Joseph F.; Sharon, Keren; Dahle, Haakon

2011-01-01

We measure the redshift distribution of a sample of 28 giant arcs discovered as a part of the Sloan Giant Arcs Survey. Gemini/GMOS-North spectroscopy provides precise redshifts for 24 arcs, and 'redshift desert' constrains for the remaining 4 arcs. This is a direct measurement of the redshift distribution of a uniformly selected sample of bright giant arcs, which is an observable that can be used to inform efforts to predict giant arc statistics. Our primary giant arc sample has a median redshift z = 1.821 and nearly two-thirds of the arcs, 64%, are sources at z ∼> 1.4, indicating that the population of background sources that are strongly lensed into bright giant arcs resides primarily at high redshift. We also analyze the distribution of redshifts for 19 secondary strongly lensed background sources that are not visually apparent in Sloan Digital Sky Survey imaging, but were identified in deeper follow-up imaging of the lensing cluster fields. Our redshift sample for the secondary sources is not spectroscopically complete, but combining it with our primary giant arc sample suggests that a large fraction of all background galaxies that are strongly lensed by foreground clusters reside at z ∼> 1.4. Kolmogorov-Smirnov tests indicate that our well-selected, spectroscopically complete primary giant arc redshift sample can be reproduced with a model distribution that is constructed from a combination of results from studies of strong-lensing clusters in numerical simulations and observational constraints on the galaxy luminosity function.

Giant CP stars

International Nuclear Information System (INIS)

Loden, L.O.; Sundman, A.

1989-01-01

This study is part of an investigation of the possibility of using chemically peculiar (CP) stars to map local galactic structure. Correct luminosities of these stars are therefore crucial. CP stars are generally regarded as main-sequence or near-main-sequence objects. However, some CP stars have been classified as giants. A selection of stars, classified in literature as CP giants, are compared to normal stars in the same effective temperature interval and to ordinary 'non giant' CP stars. There is no clear confirmation of a higher luminosity for 'CP giants', than for CP stars in general. In addition, CP characteristics seem to be individual properties not repeated in a component star or other cluster members. (author). 50 refs., 5 tabs., 3 figs

High acidity unilamellar zeolite MCM-56 and its pillared and delaminated derivatives.

Science.gov (United States)

Gil, Barbara; Makowski, Wacław; Marszalek, Bartosz; Roth, Wieslaw J; Kubu, Martin; Čejka, Jiři; Olejniczak, Zbigniew

2014-07-21

The unilamellar form of zeolite MWW, MCM-56, which is obtained by direct hydrothermal synthesis has been studied with regard to acidity and porosity in its original and post-synthesis modified pillared and delaminated forms. The acidity measured by FTIR was found to be only slightly lower than the highly active 3-D MWW forms, MCM-22 and MCM-49. Pivalonitrile adsorption, which is a measure of spatial openness, showed 50% accessibility vs. MCM-22/49. It highlights the potential of MCM-56 as a layered material with increased access to acid sites because it does not entail laborious post-synthesis modification. Swelling, pillaring and delamination of MCM-56 are facile but result in a reduction in the number of Brønsted acid sites (BAS) while increasing accessibility to pivalonitrile. The delamination procedure involving sonication and acidification of the highly basic mother liquor produces the most visible increase in surface area and access to all BAS. The accompanying doubling of the solid yield and the decrease in absolute number of BAS suggest significant precipitation of dissolved silica generated during swelling and sonication in high pH medium. The viability of separating surfactant covered layers upon sonication with the consequence of exposing hydrophobic hydrocarbon tails to aqueous environment is addressed.

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.

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.

Compartmentalization and Transport in Synthetic Vesicles

Directory of Open Access Journals (Sweden)

Christine eSchmitt

2016-02-01

Full Text Available Nano-scale vesicles have become a popular tool in life sciences. Besides liposomes that are generated from phospholipids of natural origin, polymersomes fabricated of synthetic block copolymers enjoy increasing popularity, as they represent more versatile membrane building blocks that can be selected based on their specific physicochemical properties, like permeability, stability or chemical reactivity.In this review, we focus on the application of simple and nested artificial vesicles in synthetic biology. First, we provide an introduction into the utilization of multi-compartmented vesosomes as compartmentalized nano-scale bioreactors. In the bottom-up development of protocells from vesicular nano-reactors, the specific exchange of pathway intermediates across compartment boundaries represents a bottleneck for future studies. To date, most compartmented bioreactors rely on unspecific exchange of substrates and products. This is either based on changes in permeability of the coblock polymer shell by physicochemical triggers or by the incorporation of unspecific porin proteins into the vesicle membrane. Since the incorporation of membrane transport proteins into simple and nested artificial vesicles offers the potential for specific exchange of substances between subcompartments, it opens new vistas in the design of protocells. Therefore we devote the main part of the review to summarize the technical advances in the use of phospholipids and block copolymers for the reconstitution of membrane proteins.

Chromatic response of polydiacetylene vesicle induced by the permeation of methotrexate.

Science.gov (United States)

Shin, Min Jae; Kim, Ye Jin; Kim, Jong-Duk

2015-07-07

The noble vesicular system of polydiacetylene showed a red shift using two types of detecting systems. One of the systems involves the absorption of target materials from the outer side of the vesicle, and the other system involves the permeation through the vesicular layers from within the vesicle. The chromatic mixed vesicles of N-(2-aminoethyl)pentacosa-10,12-diynamide (AEPCDA) and dimethyldioctadecylammonium chloride (DODAC) were fabricated by sonication, followed by polymerization by UV irradiation. The stability of monomeric vesicles was observed to increase with the polymerization of the vesicles. Methotrexate was used as a target material. The polymerized mixed vesicles having a blue color were exposed to a concentration gradient of methotrexate, and a red shift was observed indicating the adsorption of methotrexate on the polydiacetylene bilayer. In order to check the chromatic change by the permeation of methotrexate, we separated the vesicle portion, which contained methotrexate inside the vesicle, and checked chromatic change during the permeation of methotrexate through the vesicle. The red shift apparently indicates the disturbance in the bilayer induced by the permeation of methotrexate. The maximum contrast of color appeared at the equal molar ratio of AEPCDA and DODAC, indicating that the formation of flexible and deformable vesicular layers is important for red shift. Therefore, it is hypothesized that the system can be applicable for the chromatic detection of the permeation of methotrexate through the polydiacetylene layer.

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)

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.

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.

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

Adhesion signals of phospholipid vesicles at an electrified interface.

Science.gov (United States)

DeNardis, Nadica Ivošević; Žutić, Vera; Svetličić, Vesna; Frkanec, Ruža

2012-09-01

General adhesion behavior of phospholipid vesicles was examined in a wide range of potentials at the mercury electrode by recording time-resolved adhesion signals. It was demonstrated that adhesion-based detection is sensitive to polar headgroups in phospholipid vesicles. We identified a narrow potential window around the point of zero charge of the electrode where the interaction of polar headgroups of phosphatidylcholine vesicles with the substrate is manifested in the form of bidirectional signals. The bidirectional signal is composed of the charge flow due to the nonspecific interaction of vesicle adhesion and spreading and of the charge flow due to a specific interaction of the negatively charged electrode and the most exposed positively charged choline headgroups. These signals are expected to appear only when the electrode surface charge density is less than the surface charge density of the choline groups at the contact interface. In comparison, for the negatively charged phosphatidylserine vesicles, we identified the potential window at the mercury electrode where charge compensation takes place, and bidirectional signals were not detected.

v-SNAREs control exocytosis of vesicles from priming to fusion.

Science.gov (United States)

Borisovska, Maria; Zhao, Ying; Tsytsyura, Yaroslav; Glyvuk, Nataliya; Takamori, Shigeo; Matti, Ulf; Rettig, Jens; Südhof, Thomas; Bruns, Dieter

2005-06-15

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.

Intact deposition of cationic vesicles on anionic cellulose fibers: Role of vesicle size, polydispersity, and substrate roughness studied via streaming potential measurements.

Science.gov (United States)

Kumar, Abhijeet; Gilson, Laurent; Henrich, Franziska; Dahl, Verena; Kleinen, Jochen; Gambaryan-Roisman, Tatiana; Venzmer, Joachim

2016-07-01

Understanding the mechanism of intact vesicle deposition on solid surfaces is important for effective utilization of vesicles as active ingredient carriers in applications such as drug delivery and fabric softening. In this study, the deposition of large (davg=12μm) and small (davg=0.27μm) cationic vesicles of ditallowethylester dimethylammonium chloride (DEEDMAC) on smooth and rough anionic cellulose fibers is investigated. The deposition process is studied quantitatively using streaming potential measurements and spectrophotometric determination of DEEDMAC concentrations. Natural and regenerated cellulose fibers, namely cotton and viscose, having rough and smooth surfaces, respectively, are used as adsorbents. Equilibrium deposition data and profiles of substrate streaming potential variation with deposition are used to gain insights into the fate of vesicles upon deposition and the deposition mechanism. Intact deposition of DEEDMAC vesicles is ascertained based on streaming potential variation with deposition in the form of characteristic saturating profiles which symbolize particle-like deposition. The same is also confirmed by confocal fluorescence microscopy. Substrate roughness is found to considerably influence the deposition mechanism which, in a novel application of electrokinetic methods, is elucidated via streaming potential measurements. Copyright © 2016 Elsevier Inc. All rights reserved.

Dynamics of coarsening in multicomponent lipid vesicles with non-uniform mechanical properties

Science.gov (United States)

Funkhouser, Chloe M.; Solis, Francisco J.; Thornton, K.

2014-04-01

Multicomponent lipid vesicles are commonly used as a model system for the complex plasma membrane. One phenomenon that is studied using such model systems is phase separation. Vesicles composed of simple lipid mixtures can phase-separate into liquid-ordered and liquid-disordered phases, and since these phases can have different mechanical properties, this separation can lead to changes in the shape of the vesicle. In this work, we investigate the dynamics of phase separation in multicomponent lipid vesicles, using a model that couples composition to mechanical properties such as bending rigidity and spontaneous curvature. The model allows the vesicle surface to deform while conserving surface area and composition. For vesicles initialized as spheres, we study the effects of phase fraction and spontaneous curvature. We additionally initialize two systems with elongated, spheroidal shapes. Dynamic behavior is contrasted in systems where only one phase has a spontaneous curvature similar to the overall vesicle surface curvature and systems where the spontaneous curvatures of both phases are similar to the overall curvature. The bending energy contribution is typically found to slow the dynamics by stabilizing configurations with multiple domains. Such multiple-domain configurations are found more often in vesicles with spheroidal shapes than in nearly spherical vesicles.

Rapid formation of gas giants, ice giants and super-Earths

Energy Technology Data Exchange (ETDEWEB)

Boss, A P [DTM, Carnegie Institution of Washington, 5241 Broad Branch Road, NW, Washington, DC 20015 (United States)], E-mail: boss@dtm.ciw.edu

2008-08-15

Giant planets might have been formed by either of the two basic mechanisms, top-down (disk instability) or bottom-up (core accretion). The latter mechanism is the most generally accepted mechanism and it begins with the collisional accumulation of solid cores that may then accrete sufficient gas to become gas giants. The former mechanism is more heretical and begins with the gravitational instability of the protoplanetary disk gas, leading to the formation of self-gravitating protoplanets, within which the dust settles to form a solid core. The disk instability mechanism has been thought of primarily as a mechanism for the formation of gas giants, but if it occurs in a disk that is being photoevaporated by the ultraviolet radiation from nearby massive stars, then the outer gaseous protoplanets can be photoevaporated as well and stripped of their gaseous envelopes. The result would then be ice giants (cold super-Earths), such as the objects discovered recently by microlensing orbiting two presumed M dwarf stars. M dwarfs that form in regions of future high-mass star formation would be expected to produce cold super-Earths orbiting at distances of several astronomical units (AU) and beyond, while M dwarfs that form in regions of low-mass star formation would be expected to have gas giants at those distances. Given that most stars are born in the former rather than in the latter regions, M dwarfs should have significantly more super-Earths than gas giants on orbits of several AU or more.

Rapid formation of gas giants, ice giants and super-Earths

International Nuclear Information System (INIS)

Boss, A P

2008-01-01

Giant planets might have been formed by either of the two basic mechanisms, top-down (disk instability) or bottom-up (core accretion). The latter mechanism is the most generally accepted mechanism and it begins with the collisional accumulation of solid cores that may then accrete sufficient gas to become gas giants. The former mechanism is more heretical and begins with the gravitational instability of the protoplanetary disk gas, leading to the formation of self-gravitating protoplanets, within which the dust settles to form a solid core. The disk instability mechanism has been thought of primarily as a mechanism for the formation of gas giants, but if it occurs in a disk that is being photoevaporated by the ultraviolet radiation from nearby massive stars, then the outer gaseous protoplanets can be photoevaporated as well and stripped of their gaseous envelopes. The result would then be ice giants (cold super-Earths), such as the objects discovered recently by microlensing orbiting two presumed M dwarf stars. M dwarfs that form in regions of future high-mass star formation would be expected to produce cold super-Earths orbiting at distances of several astronomical units (AU) and beyond, while M dwarfs that form in regions of low-mass star formation would be expected to have gas giants at those distances. Given that most stars are born in the former rather than in the latter regions, M dwarfs should have significantly more super-Earths than gas giants on orbits of several AU or more

Getting there: vesicles en route for plant cytokinesis

NARCIS (Netherlands)

Ozdoba, A.

2007-01-01

In dividing plant cells, membranous vesicles (60-80 nm in diameter) are transported to the site where a new cell wall that separates the daughter cells is formed. In this thesis the physical parameters size and stiffness that vesicles require to reach the forming cell plate were studied. Synthetic

Giant Cell Arteritis

Science.gov (United States)

Giant cell arteritis is a disorder that causes inflammation of your arteries, usually in the scalp, neck, and arms. ... arteries, which keeps blood from flowing well. Giant cell arteritis often occurs with another disorder called polymyalgia ...

Integral equation methods for vesicle electrohydrodynamics in three dimensions

Science.gov (United States)

Veerapaneni, Shravan

2016-12-01

In this paper, we develop a new boundary integral equation formulation that describes the coupled electro- and hydro-dynamics of a vesicle suspended in a viscous fluid and subjected to external flow and electric fields. The dynamics of the vesicle are characterized by a competition between the elastic, electric and viscous forces on its membrane. The classical Taylor-Melcher leaky-dielectric model is employed for the electric response of the vesicle and the Helfrich energy model combined with local inextensibility is employed for its elastic response. The coupled governing equations for the vesicle position and its transmembrane electric potential are solved using a numerical method that is spectrally accurate in space and first-order in time. The method uses a semi-implicit time-stepping scheme to overcome the numerical stiffness associated with the governing equations.

Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

Energy Technology Data Exchange (ETDEWEB)

Campbell, K.P.

1978-01-01

Light and heavy sarcoplasmic reticulum vesicles isolated from rabbit leg muscle have been used in a study of chloride-induced calcium release. The biochemical and morphological data indicate that light sarcoplasmic reticulum vesicles are derived from the longitudinal reticulum and heavy sarcoplasmic reticulum vesicles are derived from the terminal cisternae of the sarcoplasmic reticulum. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP to amounts greater than 100 nmoles Ca/sup + +/ per mg of protein in less than one minute. Light and heavy sarcoplasmic reticulum vesicles each had a biphasic time course of calcium uptake. The initial uptake was followed by a rapid release after approximately one minute, of 30 to 40% of the accumulated calcium, which was then followed by a slower phase of calcium accumulation. Results indicate that the chloride induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization. The release of calcium from the light SR vesicles is probably due to osmotic swelling and the release of calcium from the heavy SR vesicles is probably due to depolarization.

Theory of Disk-to-Vesicle Transformation

Science.gov (United States)

Li, Jianfeng; Shi, An-Chang

2009-03-01

Self-assembled membranes from amphiphilic molecules, such as lipids and block copolymers, can assume a variety of morphologies dictated by energy minimization of system. The membrane energy is characterized by a bending modulus (κ), a Gaussian modulus (κG), and the line tension (γ) of the edge. Two basic morphologies of membranes are flat disks that minimize the bending energy at the cost of the edge energy, and enclosed vesicles that minimize the edge energy at the cost of bending energy. In our work, the transition from disk to vesicle is studied theoretically using the string method, which is designed to find the minimum energy path (MEP) or the most probable transition path between two local minima of an energy landscape. Previous studies of disk-to-vesicle transition usually approximate the transitional states by a series of spherical cups, and found that the spherical cups do not correspond to stable or meta-stable states of the system. Our calculation demonstrates that the intermediate shapes along the MEP are very different from spherical cups. Furthermore, some of these transitional states can be meta-stable. The disk-to-vesicle transition pathways are governed by two scaled parameters, κG/κ and γR0/4κ, where R0 is the radius of the disk. In particular, a meta-stable intermediate state is predicted, which may correspond to the open morphologies observed in experiments and simulations.

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.

Bifunctional viscous nanovesicles co-loaded with resveratrol and gallic acid for skin protection against microbial and oxidative injuries.

Science.gov (United States)

Vitonyte, Justina; Manca, Maria Letizia; Caddeo, Carla; Valenti, Donatella; Peris, Josè Esteban; Usach, Iris; Nacher, Amparo; Matos, Maria; Gutiérrez, Gemma; Orrù, Germano; Fernàndez-Busquets, Xavier; Fadda, Anna Maria; Manconi, Maria

2017-05-01

Resveratrol and gallic acid were co-loaded in phospholipid vesicles aiming at protecting the skin from external injuries, such as oxidative stress and microbial infections. Liposomes were prepared using biocompatible phospholipids dispersed in water. To improve vesicle stability and applicability, the phospholipids and the phenols were dispersed in water/propylene glycol or water/glycerol, thus obtaining PEVs and glycerosomes, respectively. The vesicles were characterized by size, morphology, physical stability, and their therapeutic efficacy was investigated in vitro. The vesicles were spherical, unilamellar and small in size: liposomes and glycerosomes were around 70nm in diameter, while PEVs were larger (∼170nm). The presence of propylene glycol or glycerol increased the viscosity of the vesicle systems, positively affecting their stability. The ability of the vesicles to promote the accumulation of the phenols (especially gallic acid) in the skin was demonstrated, as well as their low toxicity and great ability to protect keratinocytes and fibroblasts from oxidative damage. Additionally, an improvement of the antimicrobial activity of the phenols was shown against different skin pathogens. The co-loading of resveratrol and gallic acid in modified phospholipid vesicles represents an innovative, bifunctional tool for preventing and treating skin affections. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Two Novel Rab2 Interactors Regulate Dense-core Vesicle Maturation

Science.gov (United States)

Ailion, Michael; Hannemann, Mandy; Dalton, Susan; Pappas, Andrea; Watanabe, Shigeki; Hegermann, Jan; Liu, Qiang; Han, Hsiao-Fen; Gu, Mingyu; Goulding, Morgan Q.; Sasidharan, Nikhil; Schuske, Kim; Hullett, Patrick; Eimer, Stefan; Jorgensen, Erik M.

2014-01-01

Summary Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi, and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1 and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a new pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network. PMID:24698274

Vesicle biomechanics in a time-varying magnetic field.

Science.gov (United States)

Ye, Hui; Curcuru, Austen

2015-01-01

Cells exhibit distortion when exposed to a strong electric field, suggesting that the field imposes control over cellular biomechanics. Closed pure lipid bilayer membranes (vesicles) have been widely used for the experimental and theoretical studies of cellular biomechanics under this electrodeformation. An alternative method used to generate an electric field is by electromagnetic induction with a time-varying magnetic field. References reporting the magnetic control of cellular mechanics have recently emerged. However, theoretical analysis of the cellular mechanics under a time-varying magnetic field is inadequate. We developed an analytical theory to investigate the biomechanics of a modeled vesicle under a time-varying magnetic field. Following previous publications and to simplify the calculation, this model treated the inner and suspending media as lossy dielectrics, the membrane thickness set at zero, and the electric resistance of the membrane assumed to be negligible. This work provided the first analytical solutions for the surface charges, electric field, radial pressure, overall translational forces, and rotational torques introduced on a vesicle by the time-varying magnetic field. Frequency responses of these measures were analyzed, particularly the frequency used clinically by transcranial magnetic stimulation (TMS). The induced surface charges interacted with the electric field to produce a biomechanical impact upon the vesicle. The distribution of the induced surface charges depended on the orientation of the coil and field frequency. The densities of these charges were trivial at low frequency ranges, but significant at high frequency ranges. The direction of the radial force on the vesicle was dependent on the conductivity ratio between the vesicle and the medium. At relatively low frequencies (biomechanics under a time-varying magnetic field. Biological effects of clinical TMS are not likely to occur via alteration of the biomechanics of brain

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.

An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles

International Nuclear Information System (INIS)

Zhang, Xiaojun; Chen, Yuan; Chen, Yong

2014-01-01

Highlights: • Air drying induced the transformation of cell-surface membrane vesicles into pits. • An AFM-based pit-measuring method was developed to measure cell-surface vesicles. • Our method detected at least two populations of cell-surface membrane vesicles. - Abstract: Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM) has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release

Loading of Vesicles into Soft Amphiphilic Nanotubes using Osmosis

NARCIS (Netherlands)

Erne, Petra M.; van Bezouwen, Laura S.; Stacko, Peter; van Dtjken, Derk Jan; Chen, Jiawen; Stuart, Marc C. A.; Boekema, Eghert J.; Feringa, Ben L.

2015-01-01

The facile assembly of higher-order nanoarchitectures from simple building blocks is demonstrated by the loading of vesicles into soft amphiphilic nanotubes using osmosis. The nanotubes are constructed from rigid interdigitated bilayers which are capped with vesicles comprising phospholipid-based

Extracellular Membrane Vesicles and Phytopathogenicity of Acholeplasma laidlawii PG8

Directory of Open Access Journals (Sweden)

Vladislav M. Chernov

2012-01-01

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

Elastic vesicles for transdermal drug delivery of hydrophilic drugs: a comparison of important physicochemical characteristics of different vesicle types.

Science.gov (United States)

Ntimenou, Vassiliki; Fahr, Alfred; Antimisiaris, Sophia G

2012-08-01

The aim of this study is to evaluate the influence of different lipid vesicular systems on the skin permeation ability of hydrophilic molecules, and understand if and which vesicle physicochemical properties may be used as predictive tools. Calcein and carboxyfluorescein were used as hydrophilic drug models. All vesicles (conventional liposomes [CLs], transfersomes [TRs] and invasomes [INVs]), were characterized for particle size distribution, zeta-potential, vesicular shape and morphology, encapsulation efficiency, integrity, colloidal stability, elasticity and finally in vitro human skin permeation. Dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM) defined that almost all vesicles had spherical structure, low polydispersity (PI Elasticity values (measured by extrusion through membranes) were in the order INVs > TRs > CLs. Three vesicle types were selected (having different elasticity) and in vitro skin permeation experiments demonstrated that calcein permeation was minimal from an aqueous solution, slightly enhanced from CLs, and enhanced by 1.8 and 7.2 times from TRs and INVs, respectively. Permeation and elasticity values were correlated by rank order but not linearly, indicating that elasticity can be used as a crude predictive tool for enhancement of skin transport. Drug encapsulation efficiency was not found to be an important factor in the current study.

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 <70 nm, detection of single vesicles remains challenging. Thus far, vesicles <70 nm have only be studied by techniques that require the 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 <70 nm in suspension. Method: Urinary 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 (n<1.4) particles, which we confirmed by combining data on thermal diffusion and light scattering cross section. Conclusions: For the first time, we have studied vesicles <70 nm freely diffusing in suspension. The ease-of-use and performance of this technique support its potential for vesicle-based clinical applications.

Methods for the physical characterization and quantification of extracellular vesicles in biological samples.

Science.gov (United States)

Rupert, Déborah L M; Claudio, Virginia; Lässer, Cecilia; Bally, Marta

2017-01-01

Our body fluids contain a multitude of cell-derived vesicles, secreted by most cell types, commonly referred to as extracellular vesicles. They have attracted considerable attention for their function as intercellular communication vehicles in a broad range of physiological processes and pathological conditions. Extracellular vesicles and especially the smallest type, exosomes, have also generated a lot of excitement in view of their potential as disease biomarkers or as carriers for drug delivery. In this context, state-of-the-art techniques capable of comprehensively characterizing vesicles in biological fluids are urgently needed. This review presents the arsenal of techniques available for quantification and characterization of physical properties of extracellular vesicles, summarizes their working principles, discusses their advantages and limitations and further illustrates their implementation in extracellular vesicle research. The small size and physicochemical heterogeneity of extracellular vesicles make their physical characterization and quantification an extremely challenging task. Currently, structure, size, buoyant density, optical properties and zeta potential have most commonly been studied. The concentration of vesicles in suspension can be expressed in terms of biomolecular or particle content depending on the method at hand. In addition, common quantification methods may either provide a direct quantitative measurement of vesicle concentration or solely allow for relative comparison between samples. The combination of complementary methods capable of detecting, characterizing and quantifying extracellular vesicles at a single particle level promises to provide new exciting insights into their modes of action and to reveal the existence of vesicle subpopulations fulfilling key biological tasks. Copyright © 2016 Elsevier B.V. All rights reserved.

Understanding crumpling lipid vesicles at the gel phase transition

Science.gov (United States)

Hirst, Linda; Ossowski, Adam; Fraser, Matthew

2011-03-01

Wrinkling and crumpling transitions in different membrane types have been studied extensively in recent years both theoretically and computationally. There has also been very interesting recent work on defects in liquid crystalline shells. Lipid bilayer vesicles, widely used in biophysical research can be considered as a single layer smectic shell in the liquid crystalline phase. On cooling the lipid vesicle a transition to the gel phase may take place in which the lipid chains tilt and assume a more ordered packing arrangement. We observe large scale morphological changes in vesicles close to this transition point using fluorescence microscopy and investigate the possible mechanisms for this transition. Confocal microscopy is used to map 3D vesicle shape and crumpling length-scales. We also employ the molecular tilt sensitive dye, Laurdan to investigate the role of tilt domain formation on macroscopic structure. Funded by NSF CAREER award (DMR - BMAT #0852791).

Green fluorescent protein changes the conductance of connexin 43 (Cx43) hemichannels reconstituted in planar lipid bilayers.

Science.gov (United States)

Carnarius, Christian; Kreir, Mohamed; Krick, Marcel; Methfessel, Christoph; Moehrle, Volker; Valerius, Oliver; Brüggemann, Andrea; Steinem, Claudia; Fertig, Niels

2012-01-20

In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.

Dynamics of membrane nanotubes coated with I-BAR

Science.gov (United States)

Barooji, Younes F.; Rørvig-Lund, Andreas; Semsey, Szabolcs; Reihani, S. Nader S.; Bendix, Poul M.

2016-07-01

Membrane deformation is a necessary step in a number of cellular processes such as filopodia and invadopodia formation and has been shown to involve membrane shaping proteins containing membrane binding domains from the IRSp53-MIM protein family. In reconstituted membranes the membrane shaping domains can efficiently deform negatively charged membranes into tubules without any other proteins present. Here, we show that the IM domain (also called I-BAR domain) from the protein ABBA, forms semi-flexible nanotubes protruding into Giant Unilamellar lipid Vesicles (GUVs). By simultaneous quantification of tube intensity and tubular shape we find both the diameter and stiffness of the nanotubes. I-BAR decorated tubes were quantified to have a diameter of ~50 nm and exhibit no stiffening relative to protein free tubes of the same diameter. At high protein density the tubes are immobile whereas at lower density the tubes diffuse freely on the surface of the GUV. Bleaching experiments of the fluorescently tagged I-BAR confirmed that the mobility of the tubes correlates with the mobility of the I-BAR on the GUV membrane. Finally, at low density of I-BAR the protein upconcentrates within tubes protruding into the GUVs. This implies that I-BAR exhibits strong preference for negatively curved membranes.

Investigation of the chemical mechanisms involved in the electropulsation of membranes at the molecular level.

Science.gov (United States)

Breton, Marie; Mir, Lluis M

2018-02-01

The chemical consequences of electropulsation on giant unilamellar vesicles (GUVs), in particular the possible oxidation of unsaturated phospholipids, have been investigated by mass spectrometry, flow cytometry and absorbance methods. Pulse application induced oxidation of the GUV phospholipids and the oxidation level depended on the duration of the pulse. Light and O 2 increased the level of pulse-induced lipid peroxidation whereas the presence of antioxidants either in the membrane or in the solution completely suppressed peroxidation. Importantly, pulse application did not create additional reactive oxygen species (ROS) in GUV-free solution. Lipid peroxidation seems to result from a facilitation of the lipid peroxidation by the ROS already present in the solution before pulsing, not from a direct pulse-induced peroxidation. The pulse would facilitate the entrance of ROS in the core of the membrane, allowing the contact between ROS and lipid chains and provoking the oxidation. Our findings demonstrate that the application of electric pulses on cells could induce the oxidation of the membrane phospholipids since cell membranes contain unsaturated lipids. The chemical consequences of electropulsation will therefore have to be taken into account in future biomedical applications of electropulsation since oxidized phospholipids play a key role in many signaling pathways and diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes.

Science.gov (United States)

Niko, Yosuke; Didier, Pascal; Mely, Yves; Konishi, Gen-ichi; Klymchenko, Andrey S

2016-01-11

Imaging lipid organization in cell membranes requires advanced fluorescent probes. Here, we show that a recently synthesized push-pull pyrene (PA), similarly to popular probe Laurdan, changes the emission maximum as a function of lipid order, but outperforms it by spectroscopic properties. In addition to red-shifted absorption compatible with common 405 nm diode laser, PA shows higher brightness and much higher photostability than Laurdan in apolar membrane environments. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting liquid ordered and disordered phases in giant unilamellar vesicles. Fluorescence confocal microscopy in Hela cells revealed that PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging using PA reveals variation of lipid order within different cellular compartments: plasma membranes are close to liquid ordered phase of model membranes composed of sphingomyelin and cholesterol, while intracellular membranes are much less ordered, matching well membranes composed of unsaturated phospholipids without cholesterol. These differences in the lipid order were confirmed by fluorescence lifetime imaging (FLIM) at the blue edge of PA emission band. PA probe constitutes thus a new powerful tool for biomembrane research.

Quantification of leakage from large unilamellar lipid vesicles by fluorescence correlation spectroscopy

DEFF Research Database (Denmark)

Kristensen, Kasper; Henriksen, Jonas Rosager; Andresen, Thomas Lars

2014-01-01

Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that in recent years has found numerous applications for studying biological phenomena. In this article, we scrutinize one of these applications, namely, FCS as a technique for studying leakage of fluorescent molecul...

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...

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.

Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells.

Science.gov (United States)

Čujová, Sabína; Bednárová, Lucie; Slaninová, Jiřina; Straka, Jakub; Čeřovský, Václav

2014-11-01

The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan-containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detecting cytoplasmic β-galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Replication of simulated prebiotic amphiphile vesicles controlled by experimental lipid physicochemical properties

International Nuclear Information System (INIS)

Armstrong, Don L; Zidovetzki, Raphael; Markovitch, Omer; Lancet, Doron

2011-01-01

We present a new embodiment of the graded autocatalysis replication domain (GARD) for the growth, replication and evolution of lipid vesicles based on a semi-empirical foundation using experimentally measured kinetic values of selected extant lipid species. Extensive simulations using this formalism elucidated the details of the dependence of the replication and properties of the vesicles on the physicochemical properties and concentrations of the lipids, both in the environment and in the vesicle. As expected, the overall concentration and number of amphiphilic components strongly affect average replication time. Furthermore, variations in acyl chain length and unsaturation of vesicles also influence replication rate, as do the relative concentrations of individual lipid types. Understanding of the dependence of replication rates on physicochemical parameters opens a new direction in the study of prebiotic vesicles and lays the groundwork for future studies involving the competition between lipid vesicles for available amphiphilic monomers

The effect of spontaneous curvature on a two-phase vesicle

International Nuclear Information System (INIS)

Cox, Geoffrey; Lowengrub, John

2015-01-01

Vesicles are membrane-bound structures commonly known for their roles in cellular transport and the shape of a vesicle is determined by its surrounding membrane (lipid bilayer). When the membrane is composed of different lipids, it is natural for the lipids of similar molecular structure to migrate towards one another (via spinodal decomposition), creating a multi-phase vesicle. In this article, we consider a two-phase vesicle model which is driven by nature's propensity to maintain a minimal state of elastic energy. The model assumes a continuum limit, thereby treating the membrane as a closed three-dimensional surface. The main purpose of this study is to reveal the complexity of the Helfrich two-phase vesicle model with non-zero spontaneous curvature and provide further evidence to support the relevance of spontaneous curvature as a modelling parameter. In this paper, we illustrate the complexity of the Helfrich two-phase model by providing multiple examples of undocumented solutions and energy hysteresis. We also investigate the influence of spontaneous curvature on morphological effects and membrane phenomena such as budding and fusion. (paper)

Endocytic vesicle rupture is a conserved mechanism of cellular invasion by amyloid proteins.

Science.gov (United States)

Flavin, William P; Bousset, Luc; Green, Zachary C; Chu, Yaping; Skarpathiotis, Stratos; Chaney, Michael J; Kordower, Jeffrey H; Melki, Ronald; Campbell, Edward M

2017-10-01

Numerous pathological amyloid proteins spread from cell to cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression. Understanding the mechanism by which disease-associated amyloid protein assemblies enter target cells and induce cellular dysfunction is, therefore, key to understanding the progressive nature of such neurodegenerative diseases. In this study, we utilized an imaging-based assay to monitor the ability of disease-associated amyloid assemblies to rupture intracellular vesicles following endocytosis. We observe that the ability to induce vesicle rupture is a common feature of α-synuclein (α-syn) assemblies, as assemblies derived from WT or familial disease-associated mutant α-syn all exhibited the ability to induce vesicle rupture. Similarly, different conformational strains of WT α-syn assemblies, but not monomeric or oligomeric forms, efficiently induced vesicle rupture following endocytosis. The ability to induce vesicle rupture was not specific to α-syn, as amyloid assemblies of tau and huntingtin Exon1 with pathologic polyglutamine repeats also exhibited the ability to induce vesicle rupture. We also observe that vesicles ruptured by α-syn are positive for the autophagic marker LC3 and can accumulate and fuse into large, intracellular structures resembling Lewy bodies in vitro. Finally, we show that the same markers of vesicle rupture surround Lewy bodies in brain sections from PD patients. These data underscore the importance of this conserved endocytic vesicle rupture event as a damaging mechanism of cellular invasion by amyloid assemblies of multiple neurodegenerative disease-associated proteins, and suggest that proteinaceous inclusions such as Lewy bodies form as a consequence of continued fusion of autophagic vesicles in cells unable to degrade ruptured vesicles and their amyloid contents.

Ready-made chromatography columns for extracellular vesicle isolation from plasma

Directory of Open Access Journals (Sweden)

Joanne Louise Welton

2015-03-01

Full Text Available Proteomic studies of circulating vesicles are hampered by difficulties in purifying vesicles from plasma and serum. Isolations are contaminated with high-abundance blood proteins that may mask genuine vesicular-associated proteins and/or simply provide misleading data. In this brief report, we explored the potential utility of a commercially available size exclusion chromatography column for rapid vesicle purification. We evaluated the performance of the column, with cancer cell line conditioned medium or healthy donor plasma, in terms of removing non-vesicular protein and enriching for vesicles exhibiting exosome characteristics. Serial fractions revealed a peak for typical exosomal proteins (CD9, CD81 etc. that preceded the peak for highly abundant proteins, including albumin, for either sample type, and harvesting only this peak would represent elimination of >95% of protein from the sample. The columns showed good reproducibility, and streamlining the workflow would allow the exosome-relevant material to be collected in less than 10 minutes. Surprisingly, however, subsequent post-column vesicle concentration steps whilst resulting in some protein loss also lead to low vesicle recoveries, with a net effect of reducing sample purity (assessed by the particle-to-protein ratio. The columns provide a convenient, reproducible and highly effective means of eliminating >95% of non-vesicular protein from biological fluid samples such as plasma.

Brivaracetam augments short-term depression and slows vesicle recycling.

Science.gov (United States)

Yang, Xiaofeng; Bognar, Joseph; He, Tianyu; Mohammed, Mouhari; Niespodziany, Isabelle; Wolff, Christian; Esguerra, Manuel; Rothman, Steven M; Dubinsky, Janet M

2015-12-01

Brivaracetam (BRV) decreases seizure activity in a number of epilepsy models and binds to the synaptic vesicle glycoprotein 2A (SV2A) with a higher affinity than the antiepileptic drug levetiracetam (LEV). Experiments were performed to determine if BRV acted similarly to LEV to induce or augment short-term depression (STD) under high-frequency neuronal stimulation and slow synaptic vesicle recycling. Electrophysiologic field excitatory postsynaptic potential (fEPSP) recordings were made from CA1 synapses in rat hippocampal slices loaded with BRV or LEV during intrinsic activity or with BRV actively loaded during hypertonic stimulation. STD was examined in response to 5 or 40 Hz stimulus trains. Presynaptic release of FM1-43 was visualized using two-photon microscopy to assess drug effects upon synaptic vesicle mobilization. When hippocampal slices were incubated in 0.1-30 μm BRV or 30 μm-1 mm LEV for 3 h, the relative CA1 field EPSPs decreased over the course of a high-frequency train of stimuli more than for control slices. This STD was frequency- and concentration-dependent, with BRV being 100-fold more potent than LEV. The extent of STD depended on the length of the incubation time for both drugs. Pretreatment with LEV occluded the effects of BRV. Repeated hypertonic sucrose treatments and train stimulation successfully unloaded BRV from recycling vesicles and reversed BRVs effects on STD, as previously reported for LEV. At their maximal concentrations, BRV slowed FM1-43 release to a greater extent than in slices loaded with LEV during prolonged stimulation. BRV, similar to LEV, entered into recycling synaptic vesicles and produced a frequency-dependent decrement of synaptic transmission at 100-fold lower concentrations than LEV. In addition, BRV slowed synaptic vesicle mobilization more effectively than LEV, suggesting that these drugs may modify multiple functions of the synaptic vesicle protein SV2A to curb synaptic transmission and limit epileptic activity

Novel contrast agent for liver and spleen

International Nuclear Information System (INIS)

Seltzer, S.E.; Blau, M.; Adams, D.F.; Janoff, A.; Minchey, S.

1991-01-01

This paper determines whether the biodistribution and imaging characteristics of a liposome-encapsulated contrast agent, iotrolan-carrying interdigitation fusion (IF) vesicles, were acceptable for a liver-spleen CT contrast agent. IF vesicles with iotrolan in their aqueous phase were prepared by fusing small unilamellar liposomes into larger vesicles. The iodine-to-lipid ratio was 4.7. Biodistribution was measured with I-125 iotrolan-labeled IF vesicles in rats. CT imaging (Somatom Plus, Siemens Medical Systems) was performed in dogs. At the lowest dose (10 mg of iodine and 2.1 mg of lipid per kilogram) 72% of the ID was in the liver, 5% in spleen, and 1% in lungs at 1 hour. At the highest dose, (1,000 mg of iodine and 212 mg of lipid per kilogram), liver values were 68% ID, while spleen rose to 18%, lung 5%. Liver and spleen values stayed at peak for 24 hours then fell; the half-life was 6 days. In dogs, liver and spleen enhancement at 1 hour averaged 652 and 256 HU above baseline per gram of iodine per kilogram, respectively

Measurement of the membrane dipole electric field in DMPC vesicles using vibrational shifts of p-cyanophenylalanine and molecular dynamics simulations.

Science.gov (United States)

Shrestha, Rebika; Cardenas, Alfredo E; Elber, Ron; Webb, Lauren J

2015-02-19

The magnitude of the membrane dipole field was measured using vibrational Stark effect (VSE) shifts of nitrile oscillators placed on the unnatural amino acid p-cyanophenylalanine (p-CN-Phe) added to a peptide sequence at four unique positions. These peptides, which were based on a repeating alanine-leucine motif, intercalated into small unilamellar DMPC vesicles which formed an α-helix as confirmed by circular dichroic (CD) spectroscopy. Molecular dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the headgroup region of the lipid (αLAX(25)) and one buried in the interior of the bilayer (αLAX(16)), were used to examine the structure of the nitrile with respect to the membrane normal, the assumed direction of the dipole field, by quantifying both a small tilt of the helix in the bilayer and conformational rotation of the p-CN-Phe side chain at steady state. Vibrational absorption energies of the nitrile oscillator at each position showed a systematic blue shift as the nitrile was stepped toward the membrane interior; for several different concentrations of peptide, the absorption energy of the nitrile located in the middle of the bilayer was ∼3 cm(-1) greater than that of the nitrile closest to the surface of the membrane. Taken together, the measured VSE shifts and nitrile orientations within the membrane resulted in an absolute magnitude of 8-11 MV/cm for the dipole field, at the high end of the range of possible values that have been accumulated from a variety of indirect measurements. Implications for this are discussed.

Interaction of abscisic acid with phospholipid membranes

International Nuclear Information System (INIS)

Stillwell, W.; Brengle, B.; Hester, P.; Wassall, S.T.

1989-01-01

The plant hormone abscisic acid (ABA) is shown, under certain conditions, to greatly enhance the permeability of phospholipid bilayer membranes to the nonelectrolyte erythritol (followed spectrophotometrically by osmotic swelling) and the anion carboxyfluorescein (followed by fluorescence). The hormone is ineffective with single- and mixed-component phosphatidylcholine membranes in the liquid-crystalline or gel states. In contrast, substantial ABA-induced permeability is measured for two-component membranes containing lipids with different polar head groups or containing phosphatidylcholines with different acyl chains at temperatures where gel and liquid-crystalline phases coexist. Despite the large ABA-induced enhancement in bilayer permeability, no evidence for a substantial change at the molecular level was seen in the membranes by magnetic resonance techniques. 13 C NMR spin-lattice relaxation times, T 1 , in sonicated unilamellar vesicles and ESR of spin-labeled fatty acids intercalated into membranes showed negligible effect on acyl chain order and dynamics within the bilayer, while 31 P NMR of sonicated unilamellar vesicles indicated negligible effect on molecular motion and conformation in the head-group region. The authors propose that, instead of causing a general nonspecific perturbation to the membrane, the hormone acts at membrane defects formed due to mismatch in molecular packing where two different head groups or acyl chain states interface. Increased membrane disruption by ABA at these points of membrane instability could then produce an enhancement in permeability

Solubilization and localization of weakly polar lipids in unsonicated egg phosphatidylcholine: A 13C MAS NMR study

International Nuclear Information System (INIS)

Hamilton, J.A.; Fujito, D.T.; Hammer, C.F.

1991-01-01

The weakly polar lipids cholesteryl ester, triacylglycerol, and diacylglycerol incorporate to a limited extent into the lamellar structure of small unilamellar vesicles. The localization of the carbonyl group(s) at the aqueous interface was detected by [ 13 C]carbonyl chemical shift changes relative to the neat unhydrated lipid. This study uses 13 C NMR to investigate the interactions of thes lipids with unsonicated (multilamellar) phosphatidylcholine, a model system for cellular membranes and surfaces of emulsion particles with low curvature. Magic angle spinning reduced the broad lines of the unsonicated dispersions to narrow lines comparable to those from sonicated dispersions. [ 13 C]Carbonyl chemical shifts revealed incorporation of the three lipids into the lamellar structure of the unsonicated phospholipids and a partial hydration of the carbonyl groups similar to that observed in small vesicles. Other properties of interfacial weakly polar lipids in multilayers were similar to those in small unilamellar bilayers. There is thus a general tendency of weakly polar lipids to incorparate at least to a small extent into the lamellar structure of phospholipids and take on interfacial properties that are distinct from their bulk-phase properties. This pool of surface-located lipid is likely to be directly involved in enzymatyic transformations and protein-mediated transport. The 13 C magic angle spinning NMR method may be generally useful for determining the orientation of molecules in model membranes

Direct interaction between EgFABP1, a fatty acid binding protein from Echinococcus granulosus, and phospholipid membranes.

Directory of Open Access Journals (Sweden)

Jorge L Porfido

Full Text Available Growth and maintenance of hydatid cysts produced by Echinococcus granulosus have a high requirement for host lipids for biosynthetic processes, membrane building and possibly cellular and developmental signalling. This requires a high degree of lipid trafficking facilitated by lipid transporter proteins. Members of the fatty acid binding protein (FABP family have been identified in Echinococcus granulosus, one of which, EgFABP1 is expressed at the tegumental level in the protoscoleces, but it has also been described in both hydatid cyst fluid and secretions of protoscoleces. In spite of a considerable amount of structural and biophysical information on the FABPs in general, their specific functions remain mysterious.We have investigated the way in which EgFABP1 may interact with membranes using a variety of fluorescence-based techniques and artificial small unilamellar vesicles. We first found that bacterial recombinant EgFABP1 is loaded with fatty acids from the synthesising bacteria, and that fatty acid binding increases its resistance to proteinases, possibly due to subtle conformational changes induced on EgFABP1. By manipulating the composition of lipid vesicles and the ionic environment, we found that EgFABP1 interacts with membranes in a direct contact, collisional, manner to exchange ligand, involving both ionic and hydrophobic interactions. Moreover, we observed that the protein can compete with cytochrome c for association with the surface of small unilamellar vesicles (SUVs.This work constitutes a first approach to the understanding of protein-membrane interactions of EgFABP1. The results suggest that this protein may be actively involved in the exchange and transport of fatty acids between different membranes and cellular compartments within the parasite.

Quantal basis of vesicle growth and information content, a unified approach.

Science.gov (United States)

Nitzany, Eyal; Hammel, Ilan; Meilijson, Isaac

2010-09-07

Secretory vesicles express a periodic multimodal size distribution. The successive modes are integral multiples of the smallest mode (G(1)). The vesicle content ranges from macromolecules (proteins, mucopolysaccharides and hormones) to low molecular weight molecules (neurotransmitters). A steady-state model has been developed to emulate a mechanism for the introduction of vesicles of monomer size, which grow by a unit addition mechanism, G(1)+G(n)-->G(n+1) which, at a later stage are eliminated from the system. We describe a model of growth and elimination transition rates which adequately illustrates the distributions of vesicle population size at steady-state and upon elimination. Consequently, prediction of normal behavior and pathological perturbations is feasible. Careful analysis of spontaneous secretion, as compared to short burst-induced secretion, suggests that the basic character-code for reliable communication should be within a range of only 8-10 vesicles' burst which may serve as a yes/no message. Copyright 2010 Elsevier Ltd. All rights reserved.

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....

Packing states of multilamellar vesicles in a nonionic surfactant system

DEFF Research Database (Denmark)

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

2001-01-01

-alpha(*) phase using the noninvasive small-angle neutron scattering (SANS) technique, one while heating and the other while cooling the sample. Data from the heating and cooling cycles were used to demonstrate reversibility of the system. Three states of packing can be identified from the scattering profiles......Lyotropic lamellar phases under shear flow have been shown to form multilamellar vesicles (MLVs), an onion-like structure. The size of the vesicles is governed by the shear imposed on the sample. Previously, we studied the structural transformation from multilamellar vesicles to lamellae to sponge...... 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...

Vesicle fusion observed by content transfer across a tethered lipid bilayer.

Science.gov (United States)

Rawle, Robert J; van Lengerich, Bettina; Chung, Minsub; Bendix, Poul Martin; Boxer, Steven G

2011-10-19

Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

Previous work has showed that ergosterol and sphingolipids become sorted to secretory vesicles immunoisolated using a chimeric, artificial raft membrane protein as bait. In this study, we have extended this analysis to three populations of secretory vesicles isolated using natural yeast plasma...... 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...

Structure formation in binary mixtures of lipids and detergents: self-assembly and vesicle division.

Science.gov (United States)

Noguchi, Hiroshi

2013-01-14

Self-assembly dynamics in binary surfactant mixtures and structure changes of lipid vesicles induced by detergent solution are studied using coarse-grained molecular simulations. Disk-shaped micelles, the bicelles, are stabilized by detergents surrounding the rim of a bilayer disk of lipids. The self-assembled bicelles are considerably smaller than bicelles formed from vesicle rupture, and their size is determined by the concentrations of lipids and detergents and the interactions between the two species. The detergent-adsorption induces spontaneous curvature of the vesicle bilayer and results in vesicle division into two vesicles or vesicle rupture into worm-like micelles. The division occurs mainly via the inverse pathway of the modified stalk model. For large spontaneous curvature of the monolayers of the detergents, a pore is often opened, thereby leading to vesicle division or worm-like micelle formation.

Composition Effect of the Outer Layer on the Vesicle Fusion Catalyzed by Phospholipase D

Energy Technology Data Exchange (ETDEWEB)

Park, Jin Won [Seoul National University, Seoul (Korea, Republic of)

2014-09-15

Phospholipase D (PLD) catalyzed the generation of phosphatidic acid (PA) from phosphatidylcholine (PC) at the outer layer of the vesicles prepared through layer by layer via a double emulsion technique. The generation induced a curvature change in the vesicles, which eventually led them to fuse each other. The ratio of two-fattyacid-tail ethanolamine (PE) to one-fatty-acid-tail ethanolamine (PE) was found to acquire the condition where the mixed-phospholipid vesicles were stable identically with pure two-fatty-acid-tail PC. The effect of the outer-layer mixture on the PLD-induced vesicle fusion was investigated using the fluorescence intensity change. 8-Aminonaph- thalene-1,3,6-trisulfonic acid disodium salt (ANTS) and p-Xylene-bis(N-pyridinium bromide) (DPX) were encapsulated in the vesicles, respectively, for the quantification of the fusion. The fluorescence scale was calibrated with the fluorescence of a 1/1 mixture of ANTS and DPX vesicles in NaCl buffer taken as 100% fluorescence (0% fusion) and the vesicles containing both ANTS and DPX as 0% fluorescence (100% fusion), considering the leakage into the medium studied directly in a separate experiment using vesicles containing both ANTS and DPX. The fusion data for each composition were acquired with the subtraction of the leakage from the quenching. From the monitoring, the vesicle fusion caused by the PLD reaction seems dominantly to occur rather than the vesicle lysis, because the composition effect on the fusion was observed identically with that on the change in the vesicle structure. Furthermore, the diameter measurements also support the fusion dominancy.

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

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.

Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport across Phospholipid Membranes Induced by a Proton Gradient

Directory of Open Access Journals (Sweden)

Fatemeh Madani

2011-01-01

Full Text Available Detergent-mediated reconstitution of bacteriorhodopsin (BR into large unilamellar vesicles (LUVs was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane.

Water distribution function across the curved lipid bilayer: SANS study

International Nuclear Information System (INIS)

Kiselev, M.A.; Zemlyanaya, E.V.; Ryabova, N.Y.; Hauss, T.; Dante, S.; Lombardo, D.

2008-01-01

The neutron scattering length density across the membrane is simulated on the basis of fluctuated model of lipid bilayer. The use of a separated form factors method has been applied for the identification of the structural features of the polydispersed unilamellar DMPC vesicle system. The hydration of vesicle is described by sigmoid distribution function of the water molecules. The application of the model to the obtained SANS spectra allow the determination of the main parameters of the system, such as the average vesicle radius (and its polydispersity), the membrane thickness, the thickness of hydrocarbon chain region, the number of water molecules located per lipid molecule, and the phospholipid surface area. Moreover the approach allow the calculation of some relevant parameters connected with the water distribution function across the bilayer system. The main features of the obtained results furnish an explanation of why lipid membrane is easily penetrated by the water molecules of the solution

Structure of clathrin-coated vesicles from small-angle scattering experiments

DEFF Research Database (Denmark)

Pedersen, J.S.

1993-01-01

Previously published small-angle neutron and X-ray scattering data from coated vesicles, reassembled coats, and stripped vesicles have been analyzed in terms of one common model. The neutron data sets include contrast variation measurements at three different D2O solvent concentrations. The model...... used for interpreting the data has spherical symmetry and explicitly takes into account polydispersity, which is described by a Gaussian distribution. A constant thickness of the clathrin coats is assumed. The fitting of the model shows that the coated vesicles consist of a low-density outer protein....... Thus, the membrane and the high-density protein shell overlap in space, which shows that the lipid membrane contains protein. The molecular mass of the average particle is 27 x 10(6) Da. The coated vesicles consist, on average, of approximately 85% protein and 15% lipids. About 40% of the protein mass...

Models for randomly distributed nanoscopic domains on spherical vesicles

Science.gov (United States)

Anghel, Vinicius N. P.; Bolmatov, Dima; Katsaras, John

2018-06-01

The existence of lipid domains in the plasma membrane of biological systems has proven controversial, primarily due to their nanoscopic size—a length scale difficult to interrogate with most commonly used experimental techniques. Scattering techniques have recently proven capable of studying nanoscopic lipid domains populating spherical vesicles. However, the development of analytical methods able of predicting and analyzing domain pair correlations from such experiments has not kept pace. Here, we developed models for the random distribution of monodisperse, circular nanoscopic domains averaged on the surface of a spherical vesicle. Specifically, the models take into account (i) intradomain correlations corresponding to form factors and interdomain correlations corresponding to pair distribution functions, and (ii) the analytical computation of interdomain correlations for cases of two and three domains on a spherical vesicle. In the case of more than three domains, these correlations are treated either by Monte Carlo simulations or by spherical analogs of the Ornstein-Zernike and Percus-Yevick (PY) equations. Importantly, the spherical analog of the PY equation works best in the case of nanoscopic size domains, a length scale that is mostly inaccessible by experimental approaches such as, for example, fluorescent techniques and optical microscopies. The analytical form factors and structure factors of nanoscopic domains populating a spherical vesicle provide a new and important framework for the quantitative analysis of experimental data from commonly studied phase-separated vesicles used in a wide range of biophysical studies.

Molecular Recognition of Vesicles : Host-Guest Interactions Combined with Specific Dimerization of Zwitterions

NARCIS (Netherlands)

Voskuhl, Jens; Fenske, Tassilo; Stuart, Marc C. A.; Wibbeling, Birgit; Schmuck, Carsten; Ravoo, Bart Jan

2010-01-01

The aggregation of beta-cyclodextrin vesicles can be induced by an adamantyl-substituted zwitterionic guanidiniocarbonylpyrrole carboxylate guest molecule (1). Upon addition of 1 to the cyclodextrin vesicles at neutral pH, the vesicles aggregate (but do not fuse), as shown by using UV/Vis and

Visualizing the effect of dynamin inhibition on annular gap vesicle formation and fission.

Science.gov (United States)

Nickel, Beth; Boller, Marie; Schneider, Kimberly; Shakespeare, Teresa; Gay, Vernon; Murray, Sandra A

2013-06-15

Although gap junction plaque assembly has been extensively studied, mechanisms involved in plaque disassembly are not well understood. Disassembly involves an internalization process in which annular gap junction vesicles are formed. These vesicles undergo fission, but the molecular machinery needed for these fissions has not been described. The mechanoenzyme dynamin has been previously demonstrated to play a role in gap junction plaque internalization. To investigate the role of dynamin in annular gap junction vesicle fission, immunocytochemical, time-lapse and transmission electron microscopy were used to analyze SW-13 adrenocortical cells in culture. Dynamin was demonstrated to colocalize with gap junction plaques and vesicles. Dynamin inhibition, by siRNA knockdown or treatment with the dynamin GTPase inhibitor dynasore, increased the number and size of gap junction 'buds' suspended from the gap junction plaques. Buds, in control populations, were frequently released to form annular gap junction vesicles. In dynamin-inhibited populations, the buds were larger and infrequently released and thus fewer annular gap junction vesicles were formed. In addition, the number of annular gap junction vesicle fissions per hour was reduced in the dynamin-inhibited populations. We believe this to be the first report addressing the details of annular gap junction vesicle fissions and demonstrating a role of dynamin in this process. This information is crucial for elucidating the relationship between gap junctions, membrane regulation and cell behavior.

Giant pulses of pulsar radio emission

OpenAIRE

Kuzmin, A. D.

2007-01-01

Review report of giant pulses of pulsar radio emission, based on our detections of four new pulsars with giant pulses, and the comparative analysis of the previously known pulsars with giant pulses, including the Crab pulsar and millisecond pulsar PSR B1937+21.

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

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.

Kadar Prostaglandin F2? pada Cairan Vesikula Seminalis dan Produk Sel Monolayer Vesikula Seminalis Sapi Bali (CONCENTRATIONS OF PROSTAGLANDIN F2? IN SEMINAL VESICLE FLUID AND PRODUCT OF SEMINAL VESICLE MONOLAYER CELLS OF BALI CATTLE

Directory of Open Access Journals (Sweden)

Tjok Gde Oka Pemayun

2007-12-01

Full Text Available In this study, the concentration of prostaglandin F2 ? (PGF2? in seminal vesicle fluid and seminal vesicle monolayer cell cultures of Bali cattle was determined. The seminal vesicle fluid was aspirated and the epithelial cells of the seminal vesicles were cultured in tissue culture medium (TCM 199 growth medium containing 10% fetal calf serum (FCS and 10% oestrus mares serum (EMS with a density of 1.9 x 106 cells / ml medium. Following an incubation at 38.50 C in 5% CO2 atmosphere for 6 days and the level of PGF2 ? in the original seminal vesicle fluid and in the cell culture medium were determined by radioimmunoassay techniques (RIA. The results showed that the level of PGF2 ? in the non-extracted monolayer culture of seminal vesicle (1287,50 ± 3,39 pg/ml was significantly higher than that of detected in non-extracted seminal vesicle fluid (1,23 ± 0,79 pg/ml. In contrast, after extraction the level of PGF2 ? in seminal vesicle monolayer cell cultures (218,33 ± 2,87 pg/ml significantly decreased as compared to seminal vesicle fluid (1750,83 ± 2,71 pg/ml. In conclusion the highest level of PGF2 ? was found in the extract of seminal vesicle fluid.

Penetration enhancer-containing vesicles (PEVs) as carriers for cutaneous delivery of minoxidil.

Science.gov (United States)

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

2009-10-01

The aim of this work was to evaluate the ability of a few different penetration enhancers to produce elastic vesicles with soy lecithin and 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 as dehydrated-rehydrated vesicles by using soy lecithin and different amounts of three penetration enhancers, 2-(2-ethoxyethoxy)ethanol (Transcutol), capryl-caproyl macrogol 8-glyceride (Labrasol), and cineole. Soy lecithin liposomes, without penetration enhancers, were used as control. Prepared formulations were characterized in terms of size distribution, morphology, zeta potential, and vesicle deformability. The influence of PEVs on (trans)dermal delivery of minoxidil was studied by in vitro diffusion experiments through newborn pig skin in comparison with traditional liposomes and ethanolic solutions of the drug also containing each penetration enhancer. A skin pre-treatment study using empty PEVs and conventional liposomes was also carried out. Results showed that all the used penetration enhancers were able to give more deformable vesicles than conventional liposomes with a good drug entrapment efficiency and stability. In vitro skin penetration data showed that PEVs were able to give a statistically significant improvement of minoxidil deposition in the skin in comparison with classic liposomes and penetration enhancer-containing drug ethanolic solutions without any transdermal delivery. Moreover, the most deformable PEVs, prepared with Labrasol and cineole, were also able to deliver to the skin a higher total amount of minoxidil than the PE alcoholic solutions thus suggesting that minoxidil delivery to the skin was strictly correlated to vesicle deformability, and therefore to vesicle composition.

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.

Differential Regulation of Synaptic Vesicle Tethering and Docking by UNC-18 and TOM-1.

Science.gov (United States)

Gracheva, Elena O; Maryon, Ed B; Berthelot-Grosjean, Martine; Richmond, Janet E

2010-01-01

The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18), unc-64(syntaxin) and tom-1(tomosyn). We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25 nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin.

Differential regulation of synaptic vesicle tethering and docking by UNC-18 and TOM-1

Directory of Open Access Journals (Sweden)

Elena O Gracheva

2010-10-01

Full Text Available The assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin is required to prime synaptic vesicles for fusion. Since Munc18 and tomosyn compete for syntaxin interactions, the interplay between these proteins is predicted to be important in regulating synaptic transmission. We explored this possibility, by examining genetic interactions between C. elegans unc-18(Munc18, unc-64(syntaxin and tom-1(tomosyn. We have previously demonstrated that unc-18 mutants have reduced synaptic transmission, whereas tom-1 mutants exhibit enhanced release. Here we show that the unc-18 mutant release defect is associated with loss of two morphologically distinct vesicle pools; those tethered within 25nm of the plasma membrane and those docked with the plasma membrane. In contrast, priming defective unc-13 mutants accumulate tethered vesicles, while docked vesicles are greatly reduced, indicating tethering is UNC-18-dependent and occurs in the absence of priming. C. elegans unc-64 mutants phenocopy unc-18 mutants, losing both tethered and docked vesicles, whereas overexpression of open syntaxin preferentially increases vesicle docking, suggesting UNC-18/closed syntaxin interactions are responsible for vesicle tethering. Given the competition between vertebrate tomosyn and Munc18, for syntaxin binding, we hypothesized that C. elegans TOM-1 may inhibit both UNC-18-dependent vesicle targeting steps. Consistent with this hypothesis, tom-1 mutants exhibit enhanced UNC-18 plasma membrane localization and a concomitant increase in both tethered and docked synaptic vesicles. Furthermore, in tom-1;unc-18 double mutants the docked, primed vesicle pool is preferentially rescued relative to unc-18 single mutants. Together these data provide evidence for the differential regulation of two vesicle targeting steps by UNC-18 and TOM-1 through competitive interactions with syntaxin

Giant Oil Fields - The Highway to Oil: Giant Oil Fields and their Importance for Future Oil Production

International Nuclear Information System (INIS)

Robelius, Fredrik

2007-01-01

Since the 1950s, oil has been the dominant source of energy in the world. The cheap supply of oil has been the engine for economic growth in the western world. Since future oil demand is expected to increase, the question to what extent future production will be available is important. The belief in a soon peak production of oil is fueled by increasing oil prices. However, the reliability of the oil price as a single parameter can be questioned, as earlier times of high prices have occurred without having anything to do with a lack of oil. Instead, giant oil fields, the largest oil fields in the world, can be used as a parameter. A giant oil field contains at least 500 million barrels of recoverable oil. Only 507, or 1 % of the total number of fields, are giants. Their contribution is striking: over 60 % of the 2005 production and about 65 % of the global ultimate recoverable reserve (URR). However, giant fields are something of the past since a majority of the largest giant fields are over 50 years old and the discovery trend of less giant fields with smaller volumes is clear. A large number of the largest giant fields are found in the countries surrounding the Persian Gulf. The domination of giant fields in global oil production confirms a concept where they govern future production. A model, based on past annual production and URR, has been developed to forecast future production from giant fields. The results, in combination with forecasts on new field developments, heavy oil and oil sand, are used to predict future oil production. In all scenarios, peak oil occurs at about the same time as the giant fields peak. The worst-case scenario sees a peak in 2008 and the best-case scenario, following a 1.4 % demand growth, peaks in 2018

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

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

Science.gov (United States)

Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E

2015-01-01

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 also show that

Placental Nano-vesicles Target to Specific Organs and Modulate Vascular Tone In Vivo.

Science.gov (United States)

Tong, Mancy; Stanley, Joanna L; Chen, Q; James, Joanna L; Stone, Peter R; Chamley, Larry W

2017-11-01

How do nano-vesicles extruded from normal first trimester human placentae affect maternal vascular function? Placental nano-vesicles affect the ability of systemic mesenteric arteries to undergo endothelium- and nitric oxide- (NO-) dependent vasodilation in vivo in pregnant mice. Dramatic cardiovascular adaptations occur during human pregnancy, including a substantial decrease in total peripheral resistance in the first trimester. The human placenta constantly extrudes extracellular vesicles that can enter the maternal circulation and these vesicles may play an important role in feto-maternal communication. Human placental nano-vesicles were administered into CD1 mice via a tail vein and their localization and vascular effects at 30 min and 24 h post-injection were investigated. Nano-vesicles from normal first trimester human placentae were collected and administered into pregnant (D12.5) or non-pregnant female mice. After either 30 min or 24 h of exposure, all major organs were dissected for imaging (n = 7 at each time point) while uterine and mesenteric arteries were dissected for wire myography (n = 6 at each time point). Additional in vitro studies using HMEC-1 endothelial cells were also conducted to investigate the kinetics of interaction between placental nano-vesicles and endothelial cells. Nano-vesicles from first trimester human placentae localized to the lungs, liver and kidneys 24 h after injection into pregnant mice (n = 7). Exposure of pregnant mice to placental nano-vesicles for 30 min in vivo increased the vasodilatory response of mesenteric arteries to acetylcholine, while exposure for 24 h had the opposite effect (P nano-vesicles did not affect the function of uterine arteries or mesenteric arteries from non-pregnant mice. Placental nano-vesicles rapidly interacted with endothelial cells via a combination of phagocytosis, endocytosis and cell surface binding in vitro. N/A. As it is not ethical to administer labelled placental nano-vesicles to

Statistical Modelling of Synaptic Vesicles Distribution and Analysing their Physical Characteristics

DEFF Research Database (Denmark)

Khanmohammadi, Mahdieh

transmission electron microscopy is used to acquire images from two experimental groups of rats: 1) rats subjected to a behavioral model of stress and 2) rats subjected to sham stress as the control group. The synaptic vesicle distribution and interactions are modeled by employing a point process approach......This Ph.D. thesis deals with mathematical and statistical modeling of synaptic vesicle distribution, shape, orientation and interactions. The first major part of this thesis treats the problem of determining the effect of stress on synaptic vesicle distribution and interactions. Serial section...... on differences of statistical measures in section and the same measures in between sections. Three-dimensional (3D) datasets are reconstructed by using image registration techniques and estimated thicknesses. We distinguish the effect of stress by estimating the synaptic vesicle densities and modeling...

Thermally assisted acoustofluidic separation of extracellular vesicles from cells

Science.gov (United States)

Mirtaheri, Elnaz; Dolatmoradi, Ata; Pimentel, Krystine; Bhansali, Shekhar; El-Zahab, Bilal

2018-02-01

Extracellular vesicles (EVs) have been gaining increasing attention given their role in communicating information between cells. Composition-based isolation of EVs is particularly of high significance as the proteomic and lipidomic characterization of their cargo could provide valuable clues to the role of EVs in mediating the biology of various conditions. This has, however, proved to be challenging as EVs, despite their abundance, are very small and difficult to be differentiated from the other constituents of host media. In addition, currently available methods like ultracentrifugation and filtration are cumbersome and capable of achieving mostly size-based separations. In this work, we demonstrate the possibility of separating submicron EV-like vesicles from cancer cells using a thermally-assisted acoustophoretic device. In a system composed of MCF-7 breast cancer cells spiked with two different types of same-size vesicles, composition-based isolation of vesicles was shown to be realizable through opposite focusing of the system's components at the node and antinodes of the overlaid ultrasonic standing wave. By proper choice of temperature in the microchannel, we were able to achieve separations with purities exceeding 93%. Furthermore, cells recovered from the channel were shown to be viable after the separation.

Thin film drainage between pre-inflated capsules or vesicles

Science.gov (United States)

Keh, Martin; Walter, Johann; Leal, Gary

2013-11-01

Capsules and vesicles are often used as vehicles to carry active ingredients or fragrance in drug delivery and consumer products and oftentimes in these applications the particles may be pre-inflated due to the existence of a small osmotic pressure difference between the interior and exterior fluid. We study the dynamics of thin film drainage between capsules and vesicles in flow as it is crucial to fusion and deposition of the particles and, therefore, the stability and effectiveness of the products. Simulations are conducted using a numerical model coupling the boundary integral method for the motion of the fluids and a finite element method for the membrane mechanics. For low capillary numbers, the drainage behavior of vesicles and capsules are approximately the same, and also similar to that of drops as the flow-independent and uniform tension due to pre-inflation dominates. The tension due to deformation caused by flow will become more important as the strength of the external flow (i.e. the capillary number) increases. In this case, the shapes of the thin film region are fundamentally different for capsules and vesicles, and the drainage behavior in both cases differs from a drop. Funded by P&G.

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 ...

Excitation of giant resonances in heavy ion collisions

International Nuclear Information System (INIS)

Kuehn, W.

1991-01-01

Introduction: What are Giant Resonances? General Features of Giant Resonances, Macroscopic Description and Classification, Basic Excitation Mechanisms, Decay Modes, Giant Resonances Built on Excited States, Relativistic Coulomb Excitation of Giant Resonances, Experimental Situation. (orig.)

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

DEFF Research Database (Denmark)

Shillcock, J. C.

2012-01-01

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

Floret-like multinucleated giant cells in neurofibroma

Directory of Open Access Journals (Sweden)

Golka Dariusz

2007-12-01

Full Text Available Abstract This short report discusses a case of neurofibroma containing floret-like multinucleated giant cells. This being the second such case in the literature. Floret-like multinucleated giant cells have been reported in gynaecomastia and neurofibroma in neurofibromatosis type 1. These cells have been reported in uncommon soft tissue tumours including pleomorphic lipoma, giant cell collagenoma, giant cell fibroblastoma and giant cell angiofibroma. We recommend these cells to be interpreted carefully keeping in mind the rare malignant change in neurofibromas. Immunohistochemistry would help in defining the nature of such cells.

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

Science.gov (United States)

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

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

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

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.

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.

Folding Up of Gold Nanoparticle Strings into Plasmonic Vesicles for Enhanced Photoacoustic Imaging

KAUST Repository

Liu, Yijing

2015-11-11

The stepwise self-assembly of hollow plasmonic vesicles with vesicular membranes containing strings of gold nanoparticles (NPs) is reported. The formation of chain vesicles can be controlled by tuning the density of the polymer ligands on the surface of the gold NPs. The strong absorption of the chain vesicles in the near-infrared (NIR) region leads to a much higher efficiency in photoacoustic (PA) imaging than for non-chain vesicles. The chain vesicles were further employed for the encapsulation of drugs and the NIR light triggered release of payloads. This work not only offers a new platform for controlling the hierarchical self-assembly of NPs, but also demonstrates that the physical properties of the materials can be tailored by controlling the spatial arrangement of NPs within assemblies to achieve a better performance in biomedical applications.

Extracellular membrane vesicles in blood products-biology and clinical relevance

Directory of Open Access Journals (Sweden)

Emilija Krstova Krajnc

2016-01-01

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

Reactions of the hydrated electron with pyrene in lipid bilayer vesicles

International Nuclear Information System (INIS)

Schnecke, W.; Graetzel, M.; Henglein, A.

1977-01-01

Pyrene and some pyrene derivatives were solubilized in bilayer vesicles of lecithin and the rates of lecithin and the rates of reaction with the hydrated electron investigated. The concentration of the vesicles was 1.3 x 10 -7 M, that of pyrene 10 -6 - 10 -4 M. The rate constant decreases with increasing pyrene concentration. The effect is explained by the highly inhomogeneous distribution of pyrene molecules in the solutions. Only those pyrene molicules are reactive that reside close to the outer surface of the vesicles. The anions of pyrene formed disappear in a second order process. It is concluded that the anions are rapidly detached from their vesicular carriers and react with each other in the aqueous phase. Fluorescence, light scattering and electron microscopic investigations were also carried out to obtain information about the properties of the vesicles used. (orig.) [de

Release of canine parvovirus from endocytic vesicles

International Nuclear Information System (INIS)

Suikkanen, Sanna; Antila, Mia; Jaatinen, Anne; Vihinen-Ranta, Maija; Vuento, Matti

2003-01-01

Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A 2 like domain in N-terminus of VP1. In this study we characterized the role of PLA 2 activity on CPV entry process. PLA 2 activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA 2 inhibitors inhibited the viral proliferation suggesting that PLA 2 activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA 2 activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A 1 , brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A 1 , brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA 2 activity of the virus. These results suggest that parvoviral PLA 2 activity is essential for productive infection and

Secretory Vesicle Priming by CAPS Is Independent of Its SNARE-Binding MUN Domain

Directory of Open Access Journals (Sweden)

Cuc Quynh Nguyen Truong

2014-11-01

Full Text Available Priming of secretory vesicles is a prerequisite for their Ca2+-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca2+-dependent activator protein for secretion (CAPS also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin by MUN domains of CAPSs, and is therefore not redundant with Munc13 action.

Nanodielectrics with giant permittivity

Indian Academy of Sciences (India)

Following the prediction, during the last couple of years we have investigated the effect of giant permittivity in one-dimensional systems of conventional metals and conjugated polymer chains. In this article, we have tried to summarize the works on giant permittivity and finally the fabrication of nanocapacitor using metal ...

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

Science.gov (United States)

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

2015-08-01

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

A study of the enhanced sensitizing capacity of a contact allergen in lipid vesicle formulations

DEFF Research Database (Denmark)

Simonsson, Carl; Madsen, Jakob Torp; Graneli, Annette

2011-01-01

, an indicator of a compounds sensitizing capacity, increased when RBITC was applied in lipid vesicles as compared to an ethanol:water (Et:W) solution. Micro-scale vesicles showed a slightly higher cell proliferative response compared to nano-scale vesicles. TPM imaging revealed that the vesicle formulations...... improved the skin penetration of RBITC compared to the Et:W solution. A strong fluorescent region in the stratum corneum and upper epidermis implies elevated association of RBITC to these skin layers when formulated in lipid vesicles. In conclusion, the results indicate that there could be an elevated risk...... of sensitization when haptens are delivered in vehicles containing lipid vesicles. Although the size of the vesicles seems to be of minor importance, further studies are needed before a more generalized conclusion can be drawn. It is likely that the enhanced sensitizing capacity is a consequence of the improved...

From red giants to planetary nebulae

International Nuclear Information System (INIS)

Kwok, S.

1982-01-01

The transition from red giants to planetary nebulae is studied by comparing the spectral characteristics of red giant envelopes and planetary nebulae. Observational and theoretical evidence both suggest that remnants of red giant envelopes may still be present in planetary nebula systems and should have significant effects on their formation. The dynamical effects of the interaction of stellar winds from central stars of planetary nebulae with the remnant red giant envelopes are evaluated and the mechanism found to be capable of producing the observed masses and momenta of planetary nebulae. The observed mass-radii relation of planetary nebulae may also be best explained by the interacting winds model. The possibility that red giant mass loss, and therefore the production of planetary nebulae, is different between Population I and II systems is also discussed

Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery

NARCIS (Netherlands)

Merchant, M.L.; Rood, I.M.; Deegens, J.K.J.; Klein, J.B.

2017-01-01

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.

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

Penetration and fusion of phospholipid vesicles by lysozyme

Energy Technology Data Exchange (ETDEWEB)

Kim, J.; Kim, H. (Korea Advanced Institute of Science and Technology, Seoul)

1989-10-01

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-(({sup 125}I)iodophenyl)diazirine (({sup 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 ({sup 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.

Selective Metal-Ion-Mediated Vesicle Adhesion Based on Dynamic Self-Organization of a Pyrene-Appended Glutamic Acid.

Science.gov (United States)

Xing, Pengyao; Wang, Yajie; Yang, Minmin; Zhang, Yimeng; Wang, Bo; Hao, Aiyou

2016-07-13

Vesicles with dynamic membranes provide an ideal model system for investigating biological membrane activities, whereby vesicle aggregation behaviors including adhesion, fusion, fission, and membrane contraction/extension have attracted much attention. In this work we utilize an aromatic amino acid (pyrene-appended glutamic acid, PGlu) to prepare nanovesicles that aggregate to form vesicle clusters selectively induced by Fe(3+) or Cu(2+), and the vesicles transform into irregular nano-objects when interacting with Al(3+). Vesicle clusters have better stability than pristine vesicles, which hinders the spontaneous morphological transformation from vesicles into lamellar nanosheets with long incubation period. The difference between complexation of Fe(3+) and Al(3+) with vesicles was studied by various techniques. On the basis of metal ion-vesicle interactions, this self-assembled nanovesicle system also behaves as an effective fluorescent sensor for Fe(3+) and Al(3+), which cause fluorescence quenching and enhanced excimer emission, respectively.

Giant multipole resonances: an experimental review

International Nuclear Information System (INIS)

Bertrand, F.E.

1979-01-01

During the past several years experimental evidence has been published for the existance of nondipole giant resonances. These giant multipole resonances, the so-called new giant resonances were first observed through inelastic hadron and electron scattering and such measurements have continued to provide most of the information in this field. A summary is provided of the experimental evidence for these new resonances. The discussion deals only with results from inelastic scattering and only with the electric multipoles. Emphasis is placed on the recent observations of the giant monopole resonance. Results from recent heavy-ion and pion inelastic scattering are discussed. 38 references

Giant dipole resonance in hot nuclei

International Nuclear Information System (INIS)

Mau, N.V.

1993-01-01

Giant resonances built on an excited state of the nucleus at a finite temperature T are studied. The following questions are investigated: how long such collective effects occur in a nucleus when T increases. How the properties of the giant resonances vary when the temperature increases. How the study of giant resonances in hot nuclei can give information on the structure of the nucleus in a highly excited state. The special case of the giant dipole resonance is studied. Some of the experimental results are reviewed and in their theoretical interpretation is discussed. (K.A.). 56 refs., 20 figs., 4 tabs

Irregular bilayer structure in vesicles prepared from Halobacterium cutirubrum lipids

Science.gov (United States)

Lanyi, J. K.

1974-01-01

Fluorescent probes were used to study the structure of the cell envelope of Halobacterium cutirubrum, and, in particular, to explore the effect of the heterogeneity of the lipids in this organism on the structure of the bilayers. The fluorescence polarization of perylene was followed in vesicles of unfractionated lipids and polar lipids as a function of temperature in 3.4 M solutions of NaCl, NaNO3, and KSCN, and it was found that vesicles of unfractionated lipids were more perturbed by chaotropic agents than polar lipids. The dependence of the relaxation times of perylene on temperature was studied in cell envelopes and in vesicles prepared from polar lipids, unfractionated lipids, and mixtures of polar and neutral lipids.

Seminal vesicle metastasis after partial hepatectomy for hepatocellular carcinoma

International Nuclear Information System (INIS)

Gong, Li; Zheng, Minwen; Li, Yanhong; Zhang, Wendong; Bu, Wangjun; Shi, Lifang; Zhang, Wei; Yan, Hong

2011-01-01

Metastasis to the seminal vesicle is extremely rare for hepatocellular carcinoma (HCC). To our knowledge, it has been not reported in literature. The purpose of the present paper was to report a case of metastasis to the seminal vesicle after HCC resection, along with its histological features and immunohistochemical characteristics. A 46-year-old Chinese man was admitted to our hospital due to abdominal distension. He had a history of HCC related to hepatitis B virus infection. Moreover, left partial hepatectomy was performed in another hospital 28 months ago, and right partial hepatectomy for HCC recurrence in our hospital 4 months ago. After resection, radiofrequency ablation therapy had been performed. About 27 months after the initial operation, contrast-enhanced computed tomography (CT) of the pelvic cavity revealed a mass with homogeneous enhancement in the seminal vesicle. Transrectal needle biopsy revealed a poorly differentiated adenocarcinoma. Therefore, seminal vesiculectomy was resected. The histological diagnosis of the removed tumor was compatible with the original HCC. Immunohistochemical examination demonstrated that the tumor cells were positive for glypican-3 (GPC3), alpha-fetoprotein (AFP), hepatocyte paraffin-1 (Hep Par 1), cytokeratin 18 (CK 18), and hepatocyte antigen, which confirmed that the seminal vesicle tumor was a metastatic tumor of HCC. However, CT subsequently revealed multiple metastatic foci in the abdominal and pelvic cavities in May 2009 and August 2009, respectively. The seminal vesicle is an extremely rare metastatic site for HCC, and the prognosis is very poor. A combination of clinical and pathological features is necessary for a correct diagnosis, and primary tumor should be excluded before diagnosing metastatic foci

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

Science.gov (United States)

Pi, Fengmei; Binzel, Daniel W.; Lee, Tae Jin; Li, Zhefeng; Sun, Meiyan; Rychahou, Piotr; Li, Hui; Haque, Farzin; Wang, Shaoying; Croce, Carlo M.; Guo, Bin; Evers, B. Mark; Guo, Peixuan

2018-01-01

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

Electrified BPS giants: BPS configurations on giant gravitons with static electric field

International Nuclear Information System (INIS)

Ali-Akbari, Mohammad; Sheikh-Jabbari, Mohammad Mahdi

2007-01-01

We consider D3-brane action in the maximally supersymmetric type IIB plane-wave background. Upon fixing the light-cone gauge, we obtain the light-cone Hamiltonian which is manifestly supersymmetric. The 1/2 BPS solutions of this theory (solutions which preserve 16 supercharges) are either of the form of spherical three branes, the giant gravitons, or zero size point like branes. We then construct specific classes of 1/4 BPS solutions of this theory in which static electric field on the brane is turned on. These solutions are deformations about either of the two 1/2 BPS solutions. In particular, we study in some detail 1/4 BPS configurations with electric dipole on the three sphere giant, i.e. BIons on the giant gravitons, which we hence call BIGGons. We also study BPS configurations corresponding to turning on a background uniform constant electric field. As a result of this background electric field the three sphere giant is deformed to squashed sphere, while the zero size point like branes turn into circular or straight fundamental strings in the plane-wave background, with their tension equal to the background electric field

Small round blue cell tumor of seminal vesicle in a young patient

Directory of Open Access Journals (Sweden)

Adriano A. De Paula

2006-10-01

Full Text Available Seminal vesicle tumor is a rare disease with unclear origin. Generally, it is presented as a pelvic mass that can be detected by sonography and digital rectal exam. The authors report a 25-year-old patient with a pelvic mass which the magnetic resonance and surgical specimen reveal a seminal vesicle tumor. Immunohistochemical findings favored a primitive neuroectodermal tumor of the seminal vesicle. Herein, the treatment, histological and histochemical findings of this entity are discussed.

Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses

Science.gov (United States)

Moon, James J.; Suh, Heikyung; Bershteyn, Anna; Stephan, Matthias T.; Liu, Haipeng; Huang, Bonnie; Sohail, Mashaal; Luo, Samantha; Ho Um, Soong; Khant, Htet; Goodwin, Jessica T.; Ramos, Jenelyn; Chiu, Wah; Irvine, Darrell J.

2011-03-01

Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8+ T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.

Giant cells around bone biomaterials: Osteoclasts or multi-nucleated giant cells?

Science.gov (United States)

Miron, Richard J; Zohdi, Hamoon; Fujioka-Kobayashi, Masako; Bosshardt, Dieter D

2016-12-01

Recently accumulating evidence has put into question the role of large multinucleated giant cells (MNGCs) around bone biomaterials. While cells derived from the monocyte/macrophage lineage are one of the first cell types in contact with implanted biomaterials, it was originally thought that specifically in bone tissues, all giant cells were bone-resorbing osteoclasts whereas foreign body giant cells (FBGCs) were found associated with a connective tissue foreign body reaction resulting in fibrous encapsulation and/or material rejection. Despite the great majority of bone grafting materials routinely found with large osteoclasts, a special subclass of bone biomaterials has more recently been found surrounded by large giant cells virtually incapable of resorbing bone grafts even years after their implantation. While original hypotheses believed that a 'foreign body reaction' may be taking place, histological data retrieved from human samples years after their implantation have put these original hypotheses into question by demonstrating better and more stable long-term bone volume around certain bone grafts. Exactly how or why this 'special' subclass of giant cells is capable of maintaining long-term bone volume, or methods to scientifically distinguish them from osteoclasts remains extremely poorly studied. The aim of this review article was to gather the current available literature on giant cell markers and differences in expression patterns between osteoclasts and MNGCs utilizing 19 specific markers including an array of CD-cell surface markers. Furthermore, the concept of now distinguishing between pro-inflammatory M1-MNGCs (previously referred to as FBGCs) as well as wound-healing M2-MNGCs is introduced and discussed. This review article presents 19 specific cell-surface markers to distinguish between osteoclasts and MNGCs including an array of CD-cell surface markers. Furthermore, the concept of now distinguishing between pro-inflammatory M1-MNGCs (often

Structure and organization of phospholipid/polysaccharide nanoparticles

International Nuclear Information System (INIS)

Gerelli, Y; Bari, M T Di; Deriu, A; Cantu, L; Colombo, P; Como, C; Motta, S; Sonvico, F; May, R

2008-01-01

In recent years nanoparticles and microparticles composed of polymeric or lipid material have been proposed as drug carriers for improving the efficacy of encapsulated drugs. For the production of these systems different materials have been proposed, among them phospholipids and polysaccharides due to their biocompatibility, biodegradability, low cost and safety. We report here a morphological and structural investigation, performed using cryo-TEM, static light scattering and small angle neutron and x-ray scattering, on phospholipid/saccharide nanoparticles loaded with a lipophilic positively charged drug (tamoxifen citrate) used in breast cancer therapy. The lipid component was soybean lecithin; the saccharide one was chitosan that usually acts as an outer coating increasing vesicle stability. The microscopy and scattering data indicate the presence of two distinct nanoparticle families: uni-lamellar vesicles with average radius 90 A and multi-lamellar vesicles with average radius 440 A. In both families the inner core is occupied by the solvent. The presence of tamoxifen gives rise to a multi-lamellar structure of the lipid outer shell. It also induces a positive surface charge into the vesicles, repelling the positively charged chitosan molecules which therefore do not take part in nanoparticle formation

Structure and organization of phospholipid/polysaccharide nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Gerelli, Y; Bari, M T Di; Deriu, A [Dipartimento di Fisica and CNISM, Universita degli Studi di Parma and CRS SOFT, INFM-CNR (Italy); Cantu, L [Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina-LITA, Universita di Milano (Italy); Colombo, P; Como, C; Motta, S; Sonvico, F [Dipartimento Farmaceutico, Universita degli Studi di Parma (Italy); May, R [Institut Laue-Langevin, Grenoble (France)], E-mail: Antonio.Deriu@fis.unipr.it

2008-03-12

In recent years nanoparticles and microparticles composed of polymeric or lipid material have been proposed as drug carriers for improving the efficacy of encapsulated drugs. For the production of these systems different materials have been proposed, among them phospholipids and polysaccharides due to their biocompatibility, biodegradability, low cost and safety. We report here a morphological and structural investigation, performed using cryo-TEM, static light scattering and small angle neutron and x-ray scattering, on phospholipid/saccharide nanoparticles loaded with a lipophilic positively charged drug (tamoxifen citrate) used in breast cancer therapy. The lipid component was soybean lecithin; the saccharide one was chitosan that usually acts as an outer coating increasing vesicle stability. The microscopy and scattering data indicate the presence of two distinct nanoparticle families: uni-lamellar vesicles with average radius 90 A and multi-lamellar vesicles with average radius 440 A. In both families the inner core is occupied by the solvent. The presence of tamoxifen gives rise to a multi-lamellar structure of the lipid outer shell. It also induces a positive surface charge into the vesicles, repelling the positively charged chitosan molecules which therefore do not take part in nanoparticle formation.

Structure and organization of phospholipid/polysaccharide nanoparticles

Science.gov (United States)

Gerelli, Y.; Di Bari, M. T.; Deriu, A.; Cantù, L.; Colombo, P.; Como, C.; Motta, S.; Sonvico, F.; May, R.

2008-03-01

In recent years nanoparticles and microparticles composed of polymeric or lipid material have been proposed as drug carriers for improving the efficacy of encapsulated drugs. For the production of these systems different materials have been proposed, among them phospholipids and polysaccharides due to their biocompatibility, biodegradability, low cost and safety. We report here a morphological and structural investigation, performed using cryo-TEM, static light scattering and small angle neutron and x-ray scattering, on phospholipid/saccharide nanoparticles loaded with a lipophilic positively charged drug (tamoxifen citrate) used in breast cancer therapy. The lipid component was soybean lecithin; the saccharide one was chitosan that usually acts as an outer coating increasing vesicle stability. The microscopy and scattering data indicate the presence of two distinct nanoparticle families: uni-lamellar vesicles with average radius 90 Å and multi-lamellar vesicles with average radius 440 Å. In both families the inner core is occupied by the solvent. The presence of tamoxifen gives rise to a multi-lamellar structure of the lipid outer shell. It also induces a positive surface charge into the vesicles, repelling the positively charged chitosan molecules which therefore do not take part in nanoparticle formation.

Phycocyanin-encapsulating hyalurosomes as carrier for skin delivery and protection from oxidative stress damage.

Science.gov (United States)

Castangia, Ines; Manca, Maria Letizia; Catalán-Latorre, Ana; Maccioni, Anna Maria; Fadda, Anna Maria; Manconi, Maria

2016-04-01

The phycobiliprotein phycocyanin, extracted from Klamath algae, possesses important biological properties but it is characterized by a low bioavailability due to its high molecular weight. To overcome the bioavailability problems, phycocyanin was successfully encapsulated, using an environmentally-friendly method, into hyalurosomes, a new kind of phospholipid vesicles immobilised with hyaluronan sodium salt by the simple addition of drug/sodium hyaluronate water dispersion to phospholipids. Liposomes were used as a comparison. Vesicles were small in size and homogeneously dispersed, being the mean size always smaller than 150 nm and PI never higher than 0.31. Liposomes were unilamellar and spherical, the addition of the polymer slightly modify the vesicular shape which remain spherical, while the addition of PEG improve the lamellarity of vesicles being multilamellar vesicles. In all cases phycocyanin was encapsulated in good amount especially using hyalurosomes and PEG hyalurosomes (65 and 61% respectively). In vitro penetration studies suggested that hyalurosomes favoured the phycocyanin deposition in the deeper skin layers probably thanks to their peculiar hyaluronan-phospholipid structure. Moreover, hyalurosomes were highly biocompatible and improved phycocyanin antioxidant activity on stressed human keratinocytes respect to the drug solution.

Structural modification of unilamellar and multilamellar vesicles in the presence of vitamin D

Science.gov (United States)

Devarajan, A.; Raouf, Y. A.; Rashid, S.; Law, R. L.; Stojanoff, V.; Isakovic, A. F.; Gater, D. L.

Chronic vitamin D deficiency is increasingly associated with a range of health conditions, such as cardiovascular disease, diabetes and certain cancers. Our report contributes to a mechanistic understanding of these associations. Vitamin D is a lipophilic compound that is synthesized in the skin by the action of UV light on 7-dehydrocholesterol and obtained from dietary sources. We look at how vitamin D could be extracted from either skin membranes or therapeutic liposomes and transported through the body by its associated proteins. A variety of physical techniques (FTIR, DLS, UV-Vis spectroscopy, NMR, XRD) are brought to investigate: (a) the behavior of vitamin D in model membranes, and (b) the effect of vitamin D-associated proteins on membrane structure. Our results include: (1) vitamin D can be incorporated into DOPC membranes up to 40mol% with only minor changes in the dynamics of the lipid acyl chains; (2) liposomes containing larger quantities of vitamin D may have reduced stability over time; (3) the vitamin D binding protein and the vitamin D receptor do associate with and alter the behavior of model membranes, including systems that do not contain vitamin D. We acknowledge the support from KU-KAIST collaborative Grant program, and support from BNL staff.

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

Science.gov (United States)

Guillén-González, F.; Tierra, G.

2018-02-01

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

Self-assembly of star micelle into vesicle in solvents of variable quality: the star micelle retains its core-shell nanostructure in the vesicle.

Science.gov (United States)

Liu, Nijuan; He, Qun; Bu, Weifeng

2015-03-03

Intra- and intermolecular interactions of star polymers in dilute solutions are of fundamental importance for both theoretical interest and hierarchical self-assembly into functional nanostructures. Here, star micelles with a polystyrene corona and a small ionic core bearing platinum(II) complexes have been regarded as a model of star polymers to mimic their intra- and interstar interactions and self-assembled behaviors in solvents of weakening quality. In the chloroform/methanol mixture solvents, the star micelles can self-assemble to form vesicles, in which the star micelles shrink significantly and are homogeneously distributed on the vesicle surface. Unlike the morphological evolution of conventional amphiphiles from micellar to vesicular, during which the amphiphilic molecules are commonly reorganized, the star micelles still retain their core-shell nanostructures in the vesicles and the coronal chains of the star micelle between the ionic cores are fully interpenetrated.

Two types of mineral-related matrix vesicles in the bone mineralization of zebrafish

International Nuclear Information System (INIS)

Yang, L; Zhang, Y; Cui, F Z

2007-01-01

Two types of mineral-related matrix vesicle, multivesicular body (MVB) and monovesicle, were detected in the skeletal bone of zebrafish. Transmission electron microscopy and energy dispersive spectroscopy (EDS) analyses of the vesicular inclusions reveal that both types of vesicles contain calcium and phosphorus, suggesting that these vesicles may be involved in mineral ion delivery for the bone mineralization of zebrafish. However, their size and substructure are quite different. Monovesicles, whose diameter ranges from 100 nm to 550 nm, are similar to the previously reported normal matrix vesicles, while MVBs have a larger size of 700-1000 nm in nominal diameter and possess a substructure that is composed of smaller vesicles with their average size around 100 nm. The presence of mineral-related MVBs, which is first identified in zebrafish bone, indicates that the mineralization-associated transportation process of mineral ions is more complicated than is ordinarily imagined

Calcium transport in sealed vesicles from red beet (Beta vulgaris L.) storage tissue. II. Characterization of 45Ca2+ uptake into plasma membrane vesicles

International Nuclear Information System (INIS)

Giannini, J.L.; Ruiz-Cristin, J.; Briskin, D.P.

1987-01-01

Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using 45 Ca 2+ . Uptake of 45 Ca 2+ by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of 45 Ca 2+ uptake to ATP utilization via ΔμH + , no evidence for a secondary H + /Ca 2+ antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca 2+ and an imposed pH gradient could not drive 45 Ca 2+ uptake. Optimal uptake of 45 Ca 2+ occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of 45 Ca 2+ uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K/sub m/ values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving 45 Ca 2+ uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell

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...

Giant Planets: Good Neighbors for Habitable Worlds?

Science.gov (United States)

Georgakarakos, Nikolaos; Eggl, Siegfried; Dobbs-Dixon, Ian

2018-04-01

The presence of giant planets influences potentially habitable worlds in numerous ways. Massive celestial neighbors can facilitate the formation of planetary cores and modify the influx of asteroids and comets toward Earth analogs later on. Furthermore, giant planets can indirectly change the climate of terrestrial worlds by gravitationally altering their orbits. Investigating 147 well-characterized exoplanetary systems known to date that host a main-sequence star and a giant planet, we show that the presence of “giant neighbors” can reduce a terrestrial planet’s chances to remain habitable, even if both planets have stable orbits. In a small fraction of systems, however, giant planets slightly increase the extent of habitable zones provided that the terrestrial world has a high climate inertia. In providing constraints on where giant planets cease to affect the habitable zone size in a detrimental fashion, we identify prime targets in the search for habitable worlds.

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.

Regulatory Multidimensionality of Gas Vesicle Biogenesis in Halobacterium salinarum NRC-1

Directory of Open Access Journals (Sweden)

Andrew I. Yao

2011-01-01

Full Text Available It is becoming clear that the regulation of gas vesicle biogenesis in Halobacterium salinarum NRC-1 is multifaceted and appears to integrate environmental and metabolic cues at both the transcriptional and posttranscriptional levels. The mechanistic details underlying this process, however, remain unclear. In this manuscript, we quantify the contribution of light scattering made by both intracellular and released gas vesicles isolated from Halobacterium salinarum NRC-1, demonstrating that each form can lead to distinct features in growth curves determined by optical density measured at 600 nm (OD600. In the course of the study, we also demonstrate the sensitivity of gas vesicle accumulation in Halobacterium salinarum NRC-1 on small differences in growth conditions and reevaluate published works in the context of our results to present a hypothesis regarding the roles of the general transcription factor tbpD and the TCA cycle enzyme aconitase on the regulation of gas vesicle biogenesis.

Therapeutic application of extracellular vesicles in acute and chronic renal injury.

Science.gov (United States)

Rovira, Jordi; Diekmann, Fritz; Campistol, Josep M; Ramírez-Bajo, María José

A new cell-to-cell communication system was discovered in the 1990s, which involves the release of vesicles into the extracellular space. These vesicles shuttle bioactive particles, including proteins, mRNA, miRNA, metabolites, etc. This particular communication has been conserved throughout evolution, which explains why most cell types are capable of producing vesicles. Extracellular vesicles (EVs) are involved in the regulation of different physiological processes, as well as in the development and progression of several diseases. EVs have been widely studied over recent years, especially those produced by embryonic and adult stem cells, blood cells, immune system and nervous system cells, as well as tumour cells. EV analysis from bodily fluids has been used as a diagnostic tool for cancer and recently for different renal diseases. However, this review analyses the importance of EVs generated by stem cells, their function and possible clinical application in renal diseases and kidney transplantation. Copyright © 2016. Published by Elsevier España, S.L.U.

Adrenomedullin increases the short-circuit current in the mouse seminal vesicle: actions on chloride secretion.

Science.gov (United States)

Liao, S B; Cheung, K H; O, W S; Tang, Fai

2014-08-01

Adrenomedullin (ADM) may regulate seminal vesicle fluid secretion, and this may affect sperm quality. In this study, we investigated the effect of ADM on chloride secretion in the mouse seminal vesicle. The presence of ADM in mouse seminal vesicle was confirmed using immunostaining, and the molecular species was determined using gel filtration chromatography coupled with enzyme-linked assay for ADM. The effects of ADM on chloride secretion were studied by short-circuit current technique in a whole-mount preparation of mouse seminal vesicle in an Ussing chamber. The effects of specific ADM and calcitonin gene-related peptide (CGRP) receptor antagonists were investigated. Whether the ADM effect depended on the cAMP- and/or calcium-activated chloride channel was also studied using specific chloride channel blockers. The results showed that ADM was present in seminal vesicle epithelial cells. The major molecular species was precursor in the mouse seminal vesicle. ADM increased short-circuit current through the calcium-activated chloride channel in mouse seminal vesicle, and CGRP receptor was involved. We conclude that ADM may regulate chloride and fluid secretion from the seminal vesicle, which may affect the composition of the seminal plasma bathing the sperm and, hence, fertility. © 2014 by the Society for the Study of Reproduction, Inc.

Polymer Vesicles as Robust Scaffolds for the Directed Assembly of Highly Crystalline Nanocrystals †

KAUST Repository

Wang, Mingfeng

2009-12-15

We report the incorporation of various inorganic nanoparticles (NPs) (PbS, LaOF, LaF3, and TiO2, each capped by oleic acid, and CdSe/ZnS core/shell QDs capped by trioctylphosphine oxide) into vesicles (d = 70-150 nm) formed by a sample of poly(styrene-b-acrylic acid) (PS4o4-b-PAA 62, where the subscripts refer to the degree of polymerization) in mixtures of tetrahydrofuran (THF), dioxane, and water. The block copolymer formed mixtures of crew-cut micelles and vesicles with some enhancement of the vesicle population when the NPs were present. The vesicle fraction could be isolated by selective sedimentation via centrifugation, followed by redispersion in water. The NPs appeared to be incorporated into the PAA layers on the internal and external walls of the vesicles (strongly favoring the former). NPs on the exterior surface of the vesicles could be removed completely by treating the samples with a solution of ethylenediaminetetraacetate (EDTA) in water. The triangular nanoplatelets of LaF3 behaved differently. Stacks of these platelets were incorporated into solid colloidal entities, similar in size to the empty vesicles that accompanied them, during the coassembly as water was added to the polymer/LaF3/THF/ dioxane mixture. © 2009 American Chemical Society.

EVpedia: an integrated database of high-throughput data for systemic analyses of extracellular vesicles

Directory of Open Access Journals (Sweden)

Dae-Kyum Kim

2013-03-01

Full Text Available Secretion of extracellular vesicles is a general cellular activity that spans the range from simple unicellular organisms (e.g. archaea; Gram-positive and Gram-negative bacteria to complex multicellular ones, suggesting that this extracellular vesicle-mediated communication is evolutionarily conserved. Extracellular vesicles are spherical bilayered proteolipids with a mean diameter of 20–1,000 nm, which are known to contain various bioactive molecules including proteins, lipids, and nucleic acids. Here, we present EVpedia, which is an integrated database of high-throughput datasets from prokaryotic and eukaryotic extracellular vesicles. EVpedia provides high-throughput datasets of vesicular components (proteins, mRNAs, miRNAs, and lipids present on prokaryotic, non-mammalian eukaryotic, and mammalian extracellular vesicles. In addition, EVpedia also provides an array of tools, such as the search and browse of vesicular components, Gene Ontology enrichment analysis, network analysis of vesicular proteins and mRNAs, and a comparison of vesicular datasets by ortholog identification. Moreover, publications on extracellular vesicle studies are listed in the database. This free web-based database of EVpedia (http://evpedia.info might serve as a fundamental repository to stimulate the advancement of extracellular vesicle studies and to elucidate the novel functions of these complex extracellular organelles.

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

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers*

Science.gov (United States)

Carnarius, Christian; Kreir, Mohamed; Krick, Marcel; Methfessel, Christoph; Moehrle, Volker; Valerius, Oliver; Brüggemann, Andrea; Steinem, Claudia; Fertig, Niels

2012-01-01

In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein. PMID:22139870

Model of SNARE-mediated membrane adhesion kinetics.

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Jason M Warner

Full Text Available SNARE proteins are conserved components of the core fusion machinery driving diverse membrane adhesion and fusion processes in the cell. In many cases micron-sized membranes adhere over large areas before fusion. Reconstituted in vitro assays have helped isolate SNARE mechanisms in small membrane adhesion-fusion and are emerging as powerful tools to study large membrane systems by use of giant unilamellar vesicles (GUVs. Here we model SNARE-mediated adhesion kinetics in SNARE-reconstituted GUV-GUV or GUV-supported bilayer experiments. Adhesion involves many SNAREs whose complexation pulls apposing membranes into contact. The contact region is a tightly bound rapidly expanding patch whose growth velocity v(patch increases with SNARE density Gamma(snare. We find three patch expansion regimes: slow, intermediate, fast. Typical experiments belong to the fast regime where v(patch ~ (Gamma(snare(2/3 depends on SNARE diffusivities and complexation binding constant. The model predicts growth velocities ~10 - 300 microm/s. The patch may provide a close contact region where SNAREs can trigger fusion. Extending the model to a simple description of fusion, a broad distribution of fusion times is predicted. Increasing SNARE density accelerates fusion by boosting the patch growth velocity, thereby providing more complexes to participate in fusion. This quantifies the notion of SNAREs as dual adhesion-fusion agents.

β-CD-dextran polymer for efficient sequestration of cholesterol from phospholipid bilayers: Mechanistic and safe-toxicity investigations.

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Stelzl, Dominik; Nielsen, Thorbjørn Terndrup; Hansen, Terkel; di Cagno, Massimiliano

2015-12-30

The aim of this work was to investigate the suitability of β-cyclodextrin-dextran (BCD-dextran) polymer as cholesterol sequestering agent in vitro. For this purpose, BCD-dextran-cholesterol complexation was studied by phase solubility studies as well as with a specifically designed in vitro model based on giant unilamellar vesicles (GUVs) to evaluate the ability of this polymer to sequestrate cholesterol from phospholipid bilayers. Cholesterol-sequestering ability of BCD-dextran was also investigated on different cell lines relevant for the hematopoietic system and results were correlated to cells toxicity. BCD-dextran polymer was capable of extracting significant amount of cholesterol from phospholipid bilayers and to a higher extent in comparison to available β-cyclodextrins (BCDs). The ability of BCD-dextran in sequestering cholesterol resulted also very high on cell lines relevant for the hematopoietic system. Moreover, BCD-dextran resulted less toxic on cell cultures due to higher selectivity in sequestering cholesterol in comparison to MBCD (that sequestrated also significant amounts of cholesteryl esters). In conclusion, BCD-dextran resulted an extremely efficient cholesterol-sequestering agent and BCD-dextran resulted more selective to cholesterol extraction in comparison to other BCDs (therefore of lower cytotoxicity). This phenomenon might play a key role to develop an efficient treatment for hypercholesterolemia based on cholesterol segregation. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of Lateral and Terminal Chains of X-Shaped Bolapolyphiles with Oligo(phenylene ethynylene Cores on Self-Assembly Behavior. Part 2: Domain Formation by Self-Assembly in Lipid Bilayer Membranes

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Stefan Werner

2017-09-01

Full Text Available Supramolecular self-assembly of membrane constituents within a phospholipid bilayer creates complex functional platforms in biological cells that operate in intracellular signaling, trafficking and membrane remodeling. Synthetic polyphilic compounds of macromolecular or small size can be incorporated into artificial phospholipid bilayers. Featuring three or four moieties of different philicities, they reach beyond ordinary amphiphilicity and open up avenues to new functions and interaction concepts. Here, we have incorporated a series of X-shaped bolapolyphiles into DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers of giant unilamellar vesicles. The bolapolyphiles consist of a rod-like oligo(phenylene ethynylene (OPE core, hydrophilic glycerol-based headgroups with or without oligo(ethylene oxide expansions at both ends and two lateral alkyl chains attached near the center of the OPE core. In the absence of DPPC and water, the compounds showed thermotropic liquid-crystalline behavior with a transition between polyphilic and amphiphilic assembly (see part 1 in this issue. In DPPC membranes, various trends in the domain morphologies were observed upon structure variations, which entailed branched alkyl chains of various sizes, alkyl chain semiperfluorination and size expansion of the headgroups. Observed effects on domain morphology are interpreted in the context of the bulk behavior (part 1 and of a model that was previously developed based on spectroscopic and physicochemical data.

Should the Endangered Status of the Giant Panda Really Be Reduced? The Case of Giant Panda Conservation in Sichuan, China.

Science.gov (United States)

Ma, Ben; Lei, Shuo; Qing, Qin; Wen, Yali

2018-05-03

The International Union for Conservation of Nature (IUCN) reduced the threat status of the giant panda from “endangered” to “vulnerable” in September 2016. In this study, we analyzed current practices for giant panda conservation at regional and local environmental scales, based on recent reports of giant panda protection efforts in Sichuan Province, China, combined with the survey results from 927 households within and adjacent to the giant panda reserves in this area. The results showed that household attitudes were very positive regarding giant panda protection efforts. Over the last 10 years, farmers’ dependence on the natural resources provided by giant panda reserves significantly decreased. However, socio-economic development increased resource consumption, and led to climate change, habitat fragmentation, environmental pollution, and other issues that placed increased pressure on giant panda populations. This difference between local and regional scales must be considered when evaluating the IUCN status of giant pandas. While the status of this species has improved in the short-term due to positive local attitudes, large-scale socio-economic development pressure could have long-term negative impacts. Consequently, the IUCN assessment leading to the classification of giant panda as “vulnerable” instead of “endangered”, should not affect its conservation intensity and effort, as such actions could negatively impact population recovery efforts, leading to the extinction of this charismatic species.

Should the Endangered Status of the Giant Panda Really Be Reduced? The Case of Giant Panda Conservation in Sichuan, China

Directory of Open Access Journals (Sweden)

Ben Ma

2018-05-01

Full Text Available The International Union for Conservation of Nature (IUCN reduced the threat status of the giant panda from “endangered” to “vulnerable” in September 2016. In this study, we analyzed current practices for giant panda conservation at regional and local environmental scales, based on recent reports of giant panda protection efforts in Sichuan Province, China, combined with the survey results from 927 households within and adjacent to the giant panda reserves in this area. The results showed that household attitudes were very positive regarding giant panda protection efforts. Over the last 10 years, farmers’ dependence on the natural resources provided by giant panda reserves significantly decreased. However, socio-economic development increased resource consumption, and led to climate change, habitat fragmentation, environmental pollution, and other issues that placed increased pressure on giant panda populations. This difference between local and regional scales must be considered when evaluating the IUCN status of giant pandas. While the status of this species has improved in the short-term due to positive local attitudes, large-scale socio-economic development pressure could have long-term negative impacts. Consequently, the IUCN assessment leading to the classification of giant panda as “vulnerable” instead of “endangered”, should not affect its conservation intensity and effort, as such actions could negatively impact population recovery efforts, leading to the extinction of this charismatic species.

A search for lithium-rich giant stars

International Nuclear Information System (INIS)

Brown, J.A.; Sneden, C.; Lambert, D.L.; Dutchover, E. Jr.

1989-01-01

Lithium abundances or upper limits have been determined for 644 bright G-K giant stars selected from the DDO photometric catalog. Two of these giants possess surface lithium abundances approaching the cosmic value of the interstellar medium and young main-sequence stars, and eight more giants have Li contents far in excess of standard predictions. At least some of these Li-rich giants are shown to be evolved to the stage of having convectively mixed envelopes, either from the direct evidence of low surface carbon isotope ratios, or from the indirect evidence of their H-R diagram positions. Suggestions are given for the unique conditions that might have allowed these stars to produce or accrete new lithium for their surface layers, or simply to preserve from destruction their initial lithium contents. The lithium abundance of the remaining stars demonstrates that giants only very rarely meet the expectations of standard first dredge-up theories; the average extra Li destruction required is about 1.5 dex. The evolutionary states of these giants and their average masses are discussed briefly, and the Li distribution of the giants is compared to predictions of Galactic chemical evolution. 110 refs

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

Antibody Binding Alters the Characteristics and Contents of Extracellular Vesicles Released by Histoplasma capsulatum

Energy Technology Data Exchange (ETDEWEB)

Baltazar, Ludmila M.; Nakayasu, Ernesto S.; Sobreira, Tiago; Choi, Hyungwon; Casadevall, Arturo; Nimrichter, Leonardo; Nosanchuk, Joshua D.

2016-03-30

Histoplasma capsulatumproduces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment ofH. capsulatumcells 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 bindH. capsulatumheat 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.

IMPORTANCEDiverse fungal species release extracellular vesicles, indicating that this is a

Macroscopic description of isoscalar giant multipole resonances

International Nuclear Information System (INIS)

Nix, J.R.; Sierk, A.J.

1980-01-01

On the basis of a simple macroscopic model, we calculate the isoscalar giant-resonance energy as a function of mass number and multipole degree. The restoring force is determined from the distortion of the Fermi surface, and the inertia is determined for the incompressible, irrotational flow of nucleons with unit effective mass. With no adjustable parameters, the resulting closed expression reproduces correctly the available experimental data, namely the magnitude and dependence upon mass number of the giant quadrupole energy and the magnitude of the giant octupole energy for 208 Pb. We also calculate the isoscalar giant-resonance width as a function of mass number and multipole degree for various macroscopic damping mechanisms, including two-body viscosity, one-body dissipation, and modified one-body dissipation. None of these damping mechanisms reproduces correctly all features of the available experimental data, namely the magnitude and dependence upon mass number of the giant quadrupole width and the magnitude of the giant octupole width for 208 Pb

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

Small-angle neutron scattering study of the n-decane effect on the bilayer thickness in extruded unilamellar dioleoylphosphatidylcholine liposomes.

Science.gov (United States)

Uhríková, D; Balgavý, P; Kucerka, N; Islamov, A; Gordeliy, V; Kuklin, A

2000-12-15

Dioleoylphosphatidylcholine (DOPC) and n-decane were mixed and hydrated afterwards in an excess of heavy water at 1 wt.% of DOPC. From this dispersion, unilamellar liposomes were prepared by extrusion through polycarbonate filter with 500-A pores. Small-angle neutron scattering (SANS) was conducted on these liposomes. From the Kratky-Porod plot ln[I(Q)Q2] vs. Q2 of SANS intensity I(Q) in the range of scattering vectors Q corresponding to the interval 0.001 A(-2) < or = Q2 < or = 0.006 A(-2), the liposome bilayer radius of gyration Rg and the bilayer thickness parameter d(g) = 12(0.5)Rg were obtained. The values of d(g) indicated that the bilayer thickness is within the experimental error constant up to n-decane/DOPC approximately 0.5 molar ratio, and then increases by 2.4 +/- 1.3 A up to n-decane/DOPC = 1.2 molar ratio.

Tests of the Giant Impact Hypothesis

Science.gov (United States)

Jones, J. H.

1998-01-01

The giant impact hypothesis has gained popularity as a means of explaining a volatile-depleted Moon that still has a chemical affinity to the Earth. As Taylor's Axiom decrees, the best models of lunar origin are testable, but this is difficult with the giant impact model. The energy associated with the impact would be sufficient to totally melt and partially vaporize the Earth. And this means that there should he no geological vestige of Barber times. Accordingly, it is important to devise tests that may be used to evaluate the giant impact hypothesis. Three such tests are discussed here. None of these is supportive of the giant impact model, but neither do they disprove it.

Magnetic resonance of seminal vesicles: a noninvasive study of seminal way

International Nuclear Information System (INIS)

Ocantos, J.A.; Rey Valzacchi, G.; Sinclair, M.E.; Loor Guadamud, G.

2010-01-01

The magnetic resonance without endorectal coil is an excellent diagnostic tool for studying the entire route of seminal non-invasive way in a single step diagnosis. We call magnetic resonance of seminal vesicles, but includes both the study of the seminal vesicles as the channels of the seminal way. [es

Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

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Yves Briers

Full Text Available Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

Science.gov (United States)

Briers, Yves; Staubli, Titu; Schmid, Markus C; Wagner, Michael; Schuppler, Markus; Loessner, Martin J

2012-01-01

Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

Residual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration dialysis enrichment.

Science.gov (United States)

Musante, Luca; Tataruch-Weinert, Dorota; Kerjaschki, Dontscho; Henry, Michael; Meleady, Paula; Holthofer, Harry

2017-01-01

Urinary extracellular vesicles (UEVs) appear an ideal source of biomarkers for kidney and urogenital diseases. The majority of protocols designed for their isolation are based on differential centrifugation steps. However, little is still known of the type and amount of vesicles left in the supernatant. Here we used an isolation protocol for UEVs which uses hydrostatic filtration dialysis as first pre-enrichment step, followed by differential centrifugation. Transmission electron microscopy (TEM), mass spectrometry (MS), western blot, ELISA assays and tuneable resistive pulse sensing (TRPS) were used to characterise and quantify UEVs in the ultracentrifugation supernatant. TEM showed the presence of a variety of small size vesicles in the supernatant while protein identification by MS matched accurately with the protein list available in Vesiclepedia. Screening and relative quantification for specific vesicle markers showed that the supernatant was preferentially positive for CD9 and TSG101. ELISA tests for quantification of exosome revealed that 14%, was left in the supernatant with a particle diameter of 110 nm and concentration of 1.54 × 10 10 /ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose.

A simplified method to recover urinary vesicles for clinical applications, and sample banking.

Science.gov (United States)

Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Benito-Martin, Alberto; Calzaferri, Giulio; Aherne, Sinead; Holthofer, Harry

2014-12-23

Urinary extracellular vesicles provide a novel source for valuable biomarkers for kidney and urogenital diseases: Current isolation protocols include laborious, sequential centrifugation steps which hampers their widespread research and clinical use. Furthermore, large individual urine sample volumes or sizable target cohorts are to be processed (e.g. for biobanking), the storage capacity is an additional problem. Thus, alternative methods are necessary to overcome such limitations. We have developed a practical vesicle isolation technique to yield easily manageable sample volumes in an exceptionally cost efficient way to facilitate their full utilization in less privileged environments and maximize the benefit of biobanking. Urinary vesicles were isolated by hydrostatic dialysis with minimal interference of soluble proteins or vesicle loss. Large volumes of urine were concentrated up to 1/100 of original volume and the dialysis step allowed equalization of urine physico-chemical characteristics. Vesicle fractions were found suitable to any applications, including RNA analysis. In the yield, our hydrostatic filtration dialysis system outperforms the conventional ultracentrifugation-based methods and the labour intensive and potentially hazardous step of ultracentrifugations are eliminated. Likewise, the need for trained laboratory personnel and heavy initial investment is avoided. Thus, our method qualifies as a method for laboratories working with urinary vesicles and biobanking.

The Function of Gas Vesicles in Halophilic Archaeaand Bacteria: Theories and Experimental Evidence

Science.gov (United States)

Oren, Aharon

2012-01-01

A few extremely halophilic Archaea (Halobacterium salinarum, Haloquadratum walsbyi, Haloferax mediterranei, Halorubrum vacuolatum, Halogeometricum borinquense, Haloplanus spp.) possess gas vesicles that bestow buoyancy on the cells. Gas vesicles are also produced by the anaerobic endospore-forming halophilic Bacteria Sporohalobacter lortetii and Orenia sivashensis. We have extensive information on the properties of gas vesicles in Hbt. salinarum and Hfx. mediterranei and the regulation of their formation. Different functions were suggested for gas vesicle synthesis: buoying cells towards oxygen-rich surface layers in hypersaline water bodies to prevent oxygen limitation, reaching higher light intensities for the light-driven proton pump bacteriorhodopsin, positioning the cells optimally for light absorption, light shielding, reducing the cytoplasmic volume leading to a higher surface-area-to-volume ratio (for the Archaea) and dispersal of endospores (for the anaerobic spore-forming Bacteria). Except for Hqr. walsbyi which abounds in saltern crystallizer brines, gas-vacuolate halophiles are not among the dominant life forms in hypersaline environments. There only has been little research on gas vesicles in natural communities of halophilic microorganisms, and the few existing studies failed to provide clear evidence for their possible function. This paper summarizes the current status of the different theories why gas vesicles may provide a selective advantage to some halophilic microorganisms. PMID:25371329

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Hadron excitation of giant resonances

International Nuclear Information System (INIS)

Morsch, H.-P.

1985-01-01

A review is given on giant resonance studies in heavy nuclei using scattering of different hadronic probes. Concerning isoscalar giant resonances compression modes are discussed with the possibility to obtain more detailed structure information. From detailed studies of α scattering the distribution of isoscalar strengths of multipolarity up to L=6 was obtained. Some recent aspects of heavy ion excitation of collective modes are mentioned. The possibility to study isovector giant resonances in hadron charge exchange reactions is discussed. Finally, a comparison is made between α and 200 MeV proton scattering from which isoscalar and spin-isospin continuum response are extracted. (orig.)

A study of the enhanced sensitizing capacity of a contact allergen in lipid vesicle formulations

International Nuclear Information System (INIS)

Simonsson, Carl; Madsen, Jakob Torp; Graneli, Annette; Andersen, Klaus E.; Karlberg, Ann-Therese; Jonsson, Charlotte A.; Ericson, Marica B.

2011-01-01

The growing focus on nanotechnology and the increased use of nano-sized structures, e.g. vesicles, in topical formulations has led to safety concerns. We have investigated the sensitizing capacity and penetration properties of a fluorescent model compound, rhodamine B isothiocyanate (RBITC), when administered in micro- and nano-scale vesicle formulations. The sensitizing capacity of RBITC was studied using the murine local lymph node assay (LLNA) and the skin penetration properties were compared using diffusion cells in combination with two-photon microscopy (TPM). The lymph node cell proliferation, an indicator of a compounds sensitizing capacity, increased when RBITC was applied in lipid vesicles as compared to an ethanol:water (Et:W) solution. Micro-scale vesicles showed a slightly higher cell proliferative response compared to nano-scale vesicles. TPM imaging revealed that the vesicle formulations improved the skin penetration of RBITC compared to the Et:W solution. A strong fluorescent region in the stratum corneum and upper epidermis implies elevated association of RBITC to these skin layers when formulated in lipid vesicles. In conclusion, the results indicate that there could be an elevated risk of sensitization when haptens are delivered in vehicles containing lipid vesicles. Although the size of the vesicles seems to be of minor importance, further studies are needed before a more generalized conclusion can be drawn. It is likely that the enhanced sensitizing capacity is a consequence of the improved penetration and increased formation of hapten-protein complexes in epidermis when RBITC is delivered in ethosomal formulations. - Graphical Abstract: Display Omitted

YOUNG SOLAR SYSTEM's FIFTH GIANT PLANET?

International Nuclear Information System (INIS)

Nesvorný, David

2011-01-01

Studies of solar system formation suggest that the solar system's giant planets formed and migrated in the protoplanetary disk to reach the resonant orbits with all planets inside ∼15 AU from the Sun. After the gas disk's dispersal, Uranus and Neptune were likely scattered by the gas giants, and approached their current orbits while dispersing the transplanetary disk of planetesimals, whose remains survived to this time in the region known as the Kuiper Belt. Here we performed N-body integrations of the scattering phase between giant planets in an attempt to determine which initial states are plausible. We found that the dynamical simulations starting with a resonant system of four giant planets have a low success rate in matching the present orbits of giant planets and various other constraints (e.g., survival of the terrestrial planets). The dynamical evolution is typically too violent, if Jupiter and Saturn start in the 3:2 resonance, and leads to final systems with fewer than four planets. Several initial states stand out in that they show a relatively large likelihood of success in matching the constraints. Some of the statistically best results were obtained when assuming that the solar system initially had five giant planets and one ice giant, with the mass comparable to that of Uranus and Neptune, and which was ejected to interstellar space by Jupiter. This possibility appears to be conceivable in view of the recent discovery of a large number of free-floating planets in interstellar space, which indicates that planet ejection should be common.

Young Solar System's Fifth Giant Planet?

Science.gov (United States)

Nesvorný, David

2011-12-01

Studies of solar system formation suggest that the solar system's giant planets formed and migrated in the protoplanetary disk to reach the resonant orbits with all planets inside ~15 AU from the Sun. After the gas disk's dispersal, Uranus and Neptune were likely scattered by the gas giants, and approached their current orbits while dispersing the transplanetary disk of planetesimals, whose remains survived to this time in the region known as the Kuiper Belt. Here we performed N-body integrations of the scattering phase between giant planets in an attempt to determine which initial states are plausible. We found that the dynamical simulations starting with a resonant system of four giant planets have a low success rate in matching the present orbits of giant planets and various other constraints (e.g., survival of the terrestrial planets). The dynamical evolution is typically too violent, if Jupiter and Saturn start in the 3:2 resonance, and leads to final systems with fewer than four planets. Several initial states stand out in that they show a relatively large likelihood of success in matching the constraints. Some of the statistically best results were obtained when assuming that the solar system initially had five giant planets and one ice giant, with the mass comparable to that of Uranus and Neptune, and which was ejected to interstellar space by Jupiter. This possibility appears to be conceivable in view of the recent discovery of a large number of free-floating planets in interstellar space, which indicates that planet ejection should be common.

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.

Giant multipole resonances: perspectives after ten years

International Nuclear Information System (INIS)

Bertrand, F.E.

1980-01-01

Nearly ten years ago evidence was published for the first of the so-called giant multipole resonances, the giant quadrupole resonance. During the ensuing years research in this field has spread to many nuclear physics laboratories throughout the world. The present status of electric giant multipole resonances is reviewed. 24 figures, 1 table

The Small-Angle Neutron Scattering Data Analysis of the Phospholipid Transport Nanosystem Structure

Science.gov (United States)

Zemlyanaya, E. V.; Kiselev, M. A.; Zhabitskaya, E. I.; Aksenov, V. L.; Ipatova, O. M.; Ivankov, O. I.

2018-05-01

The small-angle neutron scattering technique (SANS) is employed for investigation of structure of the phospholipid transport nanosystem (PTNS) elaborated in the V.N.Orekhovich Institute of Biomedical Chemistry (Moscow, Russia). The SANS spectra have been measured at the YuMO small-angle spectrometer of IBR-2 reactor (Joint Institute of Nuclear Research, Dubna, Russia). Basic characteristics of polydispersed population of PTNS unilamellar vesicles (average radius of vesicles, polydispersity, thickness of membrane, etc.) have been determined in three cases of the PTNS concentrations in D2O: 5%, 10%, and 25%. Numerical analysis is based on the separated form factors method (SFF). The results are discussed in comparison with the results of analysis of the small-angle X-ray scattering spectra collected at the Kurchatov Synchrotron Radiation Source of the National Research Center “Kurchatov Institute” (Moscow, Russia).

Porphyromonas gingivalis Outer Membrane Vesicles Mediate Coaggregation and Piggybacking of Treponema denticola and Lachnoanaerobaculum saburreum

Directory of Open Access Journals (Sweden)

Daniel Grenier

2013-01-01

Full Text Available Porphyromonas gingivalis sheds outer membrane vesicles that contain several virulence factors, including adhesins. In this study, we investigated the ability of P. gingivalis outer membrane vesicles to mediate the coaggregation and piggybacking of Treponema denticola and Lachnoanaerobaculum saburreum. Marked coaggregation between T. denticola and L. saburreum occurred in the presence of P. gingivalis outer membrane vesicles. Sucrose was an effective chemoattractant for the motile species T. denticola. The addition of outer membrane vesicles to a mixture of T. denticola and L. saburreum significantly increased the number of nonmotile bacteria that migrated into a sucrose-filled capillary tube immersed in the bacterial mixture. Under optimal conditions, the number of nonmotile L. saburreum in the capillary tube increased approximately 5-fold, whereas no increase occurred when boiled vesicles were used. This study showed that P. gingivalis outer membrane vesicles mediate coaggregation between T. denticola and L. saburreum and that nonmotile bacteria can be translocated by piggybacking on spirochetes.

Solitary ulcerated congenital giant juvenile xanthogranuloma

Directory of Open Access Journals (Sweden)

Su Yuen Ng

2015-01-01

Full Text Available A 3-month-old female patient with a giant ulcerated nodule over the back since birth was diagnosed as congenital giant juvenile xanthogranuloma (JXG based on clinical and histopathological examination. Congenital giant JXG with ulceration at birth is a rare presentation of JXG and commonly misdiagnosed. This case emphasizes the importance of being aware of the myriad presentations of JXG in order to make a correct diagnosis and avoid unnecessary investigations or treatment.

Giant resonances in heavy-ion reactions

International Nuclear Information System (INIS)

Hussein, M.S.

1982-11-01

The several roles of multipole giant resonances in heavy-ion reactions are discussed. In particular, the modifications in the effective ion-ion potencial due to the virtual excitation of giant resonances at low energies, are considered and estimated for several systems. Real excitation of giant resonances in heavy-ion reactions at intermediate energies are then discussed and their importance in the approach phase of deeply inelastic processes in emphasized. Several demonstrative examples are given. (Author) [pt

Onsager's variational principle for the dynamics of a vesicle in a Poiseuille flow

Science.gov (United States)

Oya, Yutaka; Kawakatsu, Toshihiro

2018-03-01

We propose a systematic formulation of the migration behaviors of a vesicle in a Poiseuille flow based on Onsager's variational principle, which can be used to determine the most stable steady state. Our model is described by a combination of the phase field theory for the vesicle and the hydrodynamics for the flow field. The dynamics is governed by the bending elastic energy and the dissipation functional, the latter being composed of viscous dissipation of the flow field, dissipation of the bending energy of the vesicle, and the friction between the vesicle and the flow field. We performed a series of simulations on 2-dimensional systems by changing the bending elasticity of the membrane and observed 3 types of steady states, i.e., those with slipper shape, bullet shape, and snaking motion, and a quasi-steady state with zig-zag motion. We show that the transitions among these steady states can be quantitatively explained by evaluating the dissipation functional, which is determined by the competition between the friction on the vesicle surface and the viscous dissipation in the bulk flow.

Swiss roll operation for giant fibroadenoma.

Science.gov (United States)

Soomro, Saleem A; Memon, Sohail A; Mohammad, Noor; Maher, Mumtaz

2009-01-01

Fibroadenoma 5 cm or more is called giant fibroadenoma. Giant fibroadenoma can distort the shape of breast and causes asymmetry, so it should be excised. There are several techniques for excision of giant fibroadenoma. In our technique we remove them through cosmetically acceptable circumareolar incision to maintain the shape and symmetry of breast. The objectives were to assess the cosmetic results of Swiss roll operation for giant fibroadenoma. The study was conducted for six years from January, 2002 to December, 2007. Seventy patients of giant fibroadenoma were included in this study. They were diagnosed on history and clinical examination supported by ultrasound and postoperative histopathological examination. Data were collected from outpatient department and operation theatre. Swiss roll operation was performed under general anaesthesia. Mean tumor size was 6.38 cm. Three cm and 4 cm incisions were used for tumour 6 cm in size respectively. Skin closed with Vicryl 3/0 subcuticular stitches. Sixteen out of 70 patients had no scar while others hadminimal scar. All patients had normal shape and symmetry of breast. On histopathology fibroadenoma was confirmed. Giant fibroadenoma should be removed through cosmetically acceptable cicumareolar incision especially in unmarried young females who have small breast. Swiss-roll operation is superior in maintaining the shape and symmetry of breast. No major complication was found in our series except seroma formation in 10 patients.

Giant serpentine intracranial aneurysm: a case report

International Nuclear Information System (INIS)

Park, Jae Seong; Lee, Myeong Sub; Kim, Myung Soon; Kim, Dong Jin; Park, Joong Wha; Whang, Kum

2001-01-01

The authors present a case of giant serpentine aneurysm (a partially thrombosed aneurysm containing tortuous vascular channels with a separate entrance and outflow pathway). Giant serpentine aneurysms form a subgroup of giant intracranial aneurysms, distinct from saccular and fusiform varieties, and in this case, too, the clinical presentation and radiographic features of CT, MR imaging and angiography were distinct

G protein betagamma-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties.

Science.gov (United States)

Photowala, Huzefa; Blackmer, Trillium; Schwartz, Eric; Hamm, Heidi E; Alford, Simon

2006-03-14

Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

miR-200–containing extracellular vesicles promote breast cancer cell metastasis

Science.gov (United States)

Le, Minh T.N.; Hamar, Peter; Guo, Changying; Basar, Emre; Perdigão-Henriques, Ricardo; Balaj, Leonora; Lieberman, Judy

2014-01-01

Metastasis is associated with poor prognosis in breast cancer patients. Not all cancer cells within a tumor are capable of metastasizing. The microRNA-200 (miR-200) family, which regulates the mesenchymal-to-epithelial transition, is enriched in the serum of patients with metastatic cancers. Ectopic expression of miR-200 can confer metastatic ability to poorly metastatic tumor cells in some settings. Here, we investigated whether metastatic capability could be transferred between metastatic and nonmetastatic cancer cells via extracellular vesicles. miR-200 was secreted in extracellular vesicles from metastatic murine and human breast cancer cell lines, and miR-200 levels were increased in sera of mice bearing metastatic tumors. In culture, murine and human metastatic breast cancer cell extracellular vesicles transferred miR-200 microRNAs to nonmetastatic cells, altering gene expression and promoting mesenchymal-to-epithelial transition. In murine cancer and human xenograft models, miR-200–expressing tumors and extracellular vesicles from these tumors promoted metastasis of otherwise weakly metastatic cells either nearby or at distant sites and conferred to these cells the ability to colonize distant tissues in a miR-200–dependent manner. Together, our results demonstrate that metastatic capability can be transferred by the uptake of extracellular vesicles. PMID:25401471

The BAR Domain Protein PICK1 Controls Vesicle Number and Size in Adrenal Chromaffin Cells

DEFF Research Database (Denmark)

da Silva Pinheiro, Paulo César; Jansen, Anna M; de Wit, Heidi

2014-01-01

, a marker for immature granules. In chromaffin cells isolated from a PICK1 knockout (KO) mouse the amount of exocytosis was reduced, while release kinetics and Ca(2+) sensitivity were unaffected. Vesicle-fusion events had a reduced frequency and released lower amounts of transmitter per vesicle (i...... in vesicle number and size, whereas the fusion competence of generated vesicles was unaffected by the absence of PICK1. Viral rescue experiments demonstrated that long-term re-expression of PICK1 is necessary to restore normal vesicular content and secretion, while short-term overexpression is ineffective...

Stellar oscillations in planet-hosting giant stars

Energy Technology Data Exchange (ETDEWEB)

Hatzes, Artie P; Zechmeister, Mathias [Thueringer Landessternwarte, Sternwarte 5, D-07778 (Germany)], E-mail: artie@tls-tautenburg.de

2008-10-15

Recently a number of giant extrasolar planets have been discovered around giant stars. These discoveries are important because many of these giant stars have intermediate masses in the range 1.2-3 Msun. Early-type main sequence stars of this mass range have been avoided by radial velocity planet search surveys due the difficulty of getting the requisite radial velocity precision needed for planet discoveries. Thus, giant stars can tell us about planet formation for stars more massive than the sun. However, the determination of stellar masses for giant stars is difficult due to the fact that evolutionary tracks for stars covering a wide range of masses converge to the same region of the H-R diagram. We report here on stellar oscillations in three planet-hosting giant stars: HD 13189, {beta} Gem, and {iota} Dra. Precise stellar radial velocity measurements for these stars show variations whose periods and amplitudes are consistent with solar-like p-mode oscillations. The implied stellar masses for these objects based on the characteristics of the stellar oscillations are consistent with the predictions of stellar isochrones. An investigation of stellar oscillations in planet hosting giant stars offers us the possibility of getting an independent determination of the stellar mass for these objects which is of crucial importance for extrasolar planet studies.

Giant nuclear resonances

International Nuclear Information System (INIS)

Snover, K.A.

1989-01-01

Giant nuclear resonances are elementary mods of oscillation of the whole nucleus, closely related to the normal modes of oscillation of coupled mechanical systems. They occur systematically in most if not all nuclei, with oscillation energies typically in the range 10-30 MeV. One of the best - known examples is the giant electric dipole (El) resonance, in which all the protons and all the neutrons oscillate with opposite phase, producing a large time - varying electric dipole moment which acts as an effective antenna for radiating gamma ray. This paper discusses this mode as well as quadrupole and monopole modes

Polymerization of styrene in DODAB vesicles : a small-angle neutron scattering study

NARCIS (Netherlands)

Jung, M.; Robinson, B.H.; Steytler, D.C.; German, A.L.; Heenan, R.K.

2002-01-01

The polymerization of styrene, located in the bilayer of dioctadecyldimethylammonium bromide (DODAB) vesicles, gives rise to phase separation between the growing polymer and the bilayer. The result is a small (20-30 nm) bead of polymer located in the bilayer of each vesicle giving them a

Measuring Precise Radii of Giants Orbiting Giants to Distinguish Between Planet Evolution Models

Science.gov (United States)

Grunblatt, Samuel; Huber, Daniel; Lopez, Eric; Gaidos, Eric; Livingston, John

2017-10-01

Despite more than twenty years since the initial discovery of highly irradiated gas giant planets, the mechanism for planet inflation remains unknown. However, proposed planet inflation mechanisms can now be separated into two general classes: those which allow for post-main sequence planet inflation by direct irradiation from the host star, and those which only allow for slowed cooling of the planet over its lifetime. The recent discovery of two inflated warm Jupiters orbiting red giant stars with the NASA K2 Mission allows distinction between these two classes, but uncertainty in the planet radius blurs this distinction. Observing transits of these planets with the Spitzer Space Telescope would reduce stellar variability and thus planet radius uncertainties by approximately 50% relative to K2, allowing distinction between the two planet inflation model classes at a 3-sigma level. We propose to observe one transit of both known warm Jupiters orbiting red giant stars, K2-97b and EPIC228754001.01, to distinguish between planet model inflation classes and measure the planetary heating efficiency to 3-sigma precision. These systems are benchmarks for the upcoming NASA TESS Mission, which is predicted to discover an order of magnitude more red giant planet systems after launching next year.

Manganese and Gd-DTPA stearyl liposomes as reticuloendothelial-system-specific MR imaging contrast agents

International Nuclear Information System (INIS)

Wuthrich, R.; Schwendener, R.; Duewell, S.; VonSchulthess, G.K.; Fuchs, W.A.

1988-01-01

Liposomes can be used to target metal ions as MR contrast agents to liver and spleen. It was the aim of this work to examine unilamellar liposomes containing manganese and gadolinium ions with respect to their targetting ability, contrast enhancement, and in vivo kinetics in rats and dogs. Unilamellar liposomes containing DTPA stearate were complexed with Mn/sup 2+/ and Gd/sup 3+/ resulting in vesicles of 30-40 nm. Injected into rats, approximately 35% of manganese liposomes were present in the liver after 30-60 minutes, and after 24 hours more than 80% had been eliminated. The pharmacokinetics of gadolinium were more protracted. In MR imaging, a reduction in the T1 of the liver parenchyma from 450 to 170 and 280 msec was observed for manganese and gadolinium liposomes (0.03 mmol/kg body weight), respectively, with the liver appearing as bright as fat. Manganese (and Gd-DTPA) stearyl liposomes are potential organ-selective contrast agents for liver and spleen and are eliminated through a hepatobiliary route

Determination of giant resonance strengths

International Nuclear Information System (INIS)

Serr, F.E.

1983-01-01

Using theoretical strength functions to describe the different giant resonances expected at excitation energies of the order of (60-85)/Asup(1/3) MeV, we calculate the double differential cross sections d 2 sigma/dΩ dE associated with the reactions 208 Pb(α, α') and 90 Zr(α, α') (Esub(α) = 152 MeV). The angular distributions for the giant quadrupole and giant monopole resonances obtained from fits to these spectra, making simple, commonly used assumptions for the peak shapes and background, are compared to the original angular distributions. The differences between them are an indication of some of the uncertainties affecting the giant resonance strengths extracted from hadron inelastic scattering data. Fits to limited angular regions lead to errors of up to 50% in the value of the energy-weighted sum rule, depending on the angles examined. While it seems possible to extract the correct EWSR for the GMR by carrying out the analyses at 0 0 , no single privileged angle seems to exist in the case of the GQR. (orig.)

Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

Science.gov (United States)

Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

2014-01-01

How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

Extracellular Vesicles as Biomarkers and Therapeutics in Dermatology: A Focus on Exosomes.

Science.gov (United States)

McBride, Jeffrey D; Rodriguez-Menocal, Luis; Badiavas, Evangelos V

2017-08-01

Extracellular vesicles (exosomes, microvesicles, and apoptotic bodies) are ubiquitous in human tissues, circulation, and body fluids. Of these vesicles, exosomes are of growing interest among investigators across multiple fields, including dermatology. The characteristics of exosomes, their associated cargo (nucleic acids, proteins, and lipids), and downstream functions are vastly different, depending on the cell origin. Here, we review concepts in extracellular vesicle biology, with a focus on exosomes, highlighting recent studies in the field of dermatology. Furthermore, we highlight emerging technical issues associated with isolating and measuring exosomes. Extracellular vesicles, including exosomes, have immediate potential for serving as biomarkers and therapeutics in dermatology over the next decade. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

CMB lensing and giant rings

Energy Technology Data Exchange (ETDEWEB)

Rathaus, Ben; Itzhaki, Nissan, E-mail: nitzhaki@post.tau.ac.il, E-mail: ben.rathaus@gmail.com [Raymond and Beverly Sackler Faculty of Exact Sciences, School of Physics and Astronomy, Tel-Aviv University, Ramat-Aviv, 69978 (Israel)

2012-05-01

We study the CMB lensing signature of a pre-inationary particle (PIP), assuming it is responsible for the giant rings anomaly that was found recently in the WMAP data. Simulating Planck-like data we find that generically the CMB lensing signal to noise ratio associated with such a PIP is quite small and it would be difficult to cross correlate the temperature giant rings with the CMB lensing signal. However, if the pre-inationary particle is also responsible for the bulk flow measured from the local large scale structure, which happens to point roughly at the same direction as the giant rings, then the CMB lensing signal to noise ratio is fairly significant.

Morphological changes in vesicles and release of an encapsulated compound triggered by a photoresponsive Malachite Green leuconitrile derivative.

Science.gov (United States)

Uda, Ryoko M; Hiraishi, Eri; Ohnishi, Ryo; Nakahara, Yoshio; Kimura, Keiichi

2010-04-20

Photoinduced morphological changes in phosphatidylcholine vesicles are triggered by a Malachite Green leuconitrile derivative dissolved in the lipidic membrane, and are observed at Malachite Green derivative/lipid ratios Malachite Green derivative is a photoresponsive compound that undergoes ionization to afford a positive charge on the molecule by UV irradiation. The Malachite Green derivative exhibits amphiphilicity when ionized photochemically, whereas it behaves as a lipophilic compound under dark conditions. Cryo-transmission electron microscopy was used to determine vesicle morphology. The effects of the Malachite Green derivative on vesicles were studied by dynamic light scattering and fluorescence resonance energy transfer. Irradiation of vesicles containing the Malachite Green derivative induces nonspherical vesicle morphology, fusion of vesicles, and membrane solubilization, depending on conditions. Furthermore, irradiation of the Malachite Green derivative induces the release of a vesicle-encapsulated compound.

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

Science.gov (United States)

Noguchi, Hiroshi

2013-02-01

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

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation*

OpenAIRE

Nojiri, Mari; Loyet, Kelly M.; Klenchin, Vadim A.; Kabachinski, Gregory; Martin, Thomas F. J.

2009-01-01

CAPS (Ca2+-dependent activator protein for secretion) functions in priming Ca2+-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca2+-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in...

α-Synuclein may cross-bridge v-SNARE and acidic phospholipids to facilitate SNARE-dependent vesicle docking.

Science.gov (United States)

Lou, Xiaochu; Kim, Jaewook; Hawk, Brenden J; Shin, Yeon-Kyun

2017-06-06

Misfolded α-synuclein (A-syn) is widely recognized as the primal cause of neurodegenerative diseases including Parkinson's disease and dementia with Lewy bodies. The normal cellular function of A-syn has, however, been elusive. There is evidence that A-syn plays multiple roles in the exocytotic pathway in the neuron, but the underlying molecular mechanisms are unclear. A-syn has been known to interact with negatively charged phospholipids and with vesicle SNARE protein VAMP2. Using single-vesicle docking/fusion assays, we find that A-syn promotes SNARE-dependent vesicles docking significantly at 2.5 µM. When phosphatidylserine (PS) is removed from t-SNARE-bearing vesicles, the docking enhancement by A-syn disappears and A-syn instead acts as an inhibitor for docking. In contrast, subtraction of PS from the v-SNARE-carrying vesicles enhances vesicle docking even further. Moreover, when we truncate the C-terminal 45 residues of A-syn that participates in interacting with VAMP2, the promotion of vesicle docking is abrogated. Thus, the results suggest that the A-syn's interaction with v-SNARE through its C-terminal tail and its concurrent interaction with PS in trans through its amphipathic N-terminal domain facilitate SNARE complex formation, whereby A-syn aids SNARE-dependent vesicle docking. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Proton-stimulated Cl-HCO3 antiport by basolateral membrane vesicles of lobster hepatopancreas

International Nuclear Information System (INIS)

Ahearn, G.A.; Grover, M.L.; Tsuji, R.T.; Clay, L.P.

1987-01-01

Purified epithelial basolateral membrane vesicles were prepared from lobster hepatopancreas by sorbitol gradient centrifugation. Na+-K+-adenosinetriphosphatase, alkaline phosphatase, and cytochrome-c oxidase enzyme activities in the final membrane preparation were enriched 9.6-, 1.4-, and 0.4-fold, respectively, compared with their activities in the original tissue homogenate. Vesicle osmotic reactivity was demonstrated using 60-min equilibrium 36 Cl uptake experiments at a variety of transmembrane osmotic gradients. 36 Cl uptake into vesicles preloaded with HCO 3 was significantly greater than into vesicles lacking HCO 3 . This exchange process was stimulated by a transmembrane proton gradient (internal pH greater than external pH). Proton-gradient-dependent Cl-HCO 3 exchange was potential sensitive and stimulated by an electrically negative vesicle interior. 36 Cl influx (4-s exposures) into HCO 3 -loaded vesicles occurred by the combination of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid sensitive, carrier-mediated transfer and apparent diffusion. 36 Cl influx was a hyperbolic function of both internal [HCO 3 ] and internal [Cl]. The two internal anions displayed a 100-fold difference in apparent affinity constants with HCO 3 being strongly preferred. 36 Cl influx was stimulated more by preloaded monovalent than by divalent anions. Na was an inhibitor of proton-dependent anion antiport, whereas K had no effect. A model for HCl-HCO 3 antiport is suggested that employs combined transmembrane concentration gradients of Cl and HCO 3 to power anion exchange and transfer protons against a concentration gradient

Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery.

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Yang, Yoosoo; Hong, Yeonsun; Cho, Eunji; Kim, Gi Beom; Kim, In-San

2018-01-01

Membrane proteins are of great research interest, particularly because they are rich in targets for therapeutic application. The suitability of various membrane proteins as targets for therapeutic formulations, such as drugs or antibodies, has been studied in preclinical and clinical studies. For therapeutic application, however, a protein must be expressed and purified in as close to its native conformation as possible. This has proven difficult for membrane proteins, as their native conformation requires the association with an appropriate cellular membrane. One solution to this problem is to use extracellular vesicles as a display platform. Exosomes and microvesicles are membranous extracellular vesicles that are released from most cells. Their membranes may provide a favourable microenvironment for membrane proteins to take on their proper conformation, activity, and membrane distribution; moreover, membrane proteins can cluster into microdomains on the surface of extracellular vesicles following their biogenesis. In this review, we survey the state-of-the-art of extracellular vesicle (exosome and small-sized microvesicle)-based therapeutics, evaluate the current biological understanding of these formulations, and forecast the technical advances that will be needed to continue driving the development of membrane protein therapeutics.

Role of nature reserves in giant panda protection.

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Kang, Dongwei; Li, Junqing

2018-02-01

Giant panda (Ailuropoda melanoleuca) is a flagship species in nature conservation of the world; to protect this species, 67 nature reserves have been established in China. To evaluate the protection effect of giant panda nature reserves, we analyzed the variation of giant panda number and habitat area of 23 giant panda nature reserves of Sichuan province based on the national survey data released by State Forestry Administration and Sichuan Forestry Department. Results showed that from the third national survey to the fourth, giant panda number and habitat area of 23 giant panda nature reserves of Sichuan province failed to realize the significant increase. Furthermore, we found that the total population growth rate of 23 nature reserves in the last 12 years was lower than those of the province total of Sichuan and the national total of China, and the total habitat area of the 23 nature reserves was decreasing in the last 12 years, but the province total and national total were all increasing. We propose that giant panda protection should pay more attention to how to improve the protective effects of nature reserves.

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)

Shear-induced formation of vesicles in membrane phases: Kinetics and size selection mechanisms, elasticity versus surface tension

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Courbin, L.; Panizza, P.

2004-02-01

Multilamellar vesicles can be formed upon shearing lamellar phases (Lα) and phase-separated lamellar-sponge (Lα/L3) mixtures. In the first case, the vesicle volume fraction is always 100% and the vesicle size is monitored by elasticity (“onion textures”). In the second system the vesicle volume fraction can be tuned from 0 to 100% and the mean size results from a balance between capillary and viscous forces (“Taylor droplets”). However, despite these differences, in both systems we show that the formation of vesicles is a strain-controlled process monitored by a universal primary buckling instability of the lamellae.

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

DEFF Research Database (Denmark)

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

2014-01-01

as exosomes and microvesicles. These vesicles contain various types of RNAs and proteins, suggested to transfer health-promoting messages from mother to offspring. However, the variety of the vesicles in milk is less understood and, additionally, complicated by the complexity of more pronounced milk...... components. Here we present a novel strategy for a short, gentle and non-denaturing isolation of skim milk-derived membrane vesicles. Methods: Untreated fresh bovine milk was defatted to remove milk fat globules. The resulting skim milk was subjected to ultracentrifugation. The resulting ochre...

Modeling Impacts of Climate Change on Giant Panda Habitat

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Melissa Songer

2012-01-01

Full Text Available Giant pandas (Ailuropoda melanoleuca are one of the most widely recognized endangered species globally. Habitat loss and fragmentation are the main threats, and climate change could significantly impact giant panda survival. We integrated giant panda habitat information with general climate models (GCMs to predict future geographic distribution and fragmentation of giant panda habitat. Results support a major general prediction of climate change—a shift of habitats towards higher elevation and higher latitudes. Our models predict climate change could reduce giant panda habitat by nearly 60% over 70 years. New areas may become suitable outside the current geographic range but much of these areas is far from the current giant panda range and only 15% fall within the current protected area system. Long-term survival of giant pandas will require the creation of new protected areas that are likely to support suitable habitat even if the climate changes.

Routes and mechanisms of extracellular vesicle uptake

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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.

Introducing a prognostic score for pretherapeutic assessment of seminal vesicle invasion in patients with clinically localized prostate cancer

International Nuclear Information System (INIS)

Salomon, Laurent; Porcher, Raphaeel; Anastasiadis, Aristotelis G.; Levrel, Olivier; Saint, Fabian; Taille, Alexandre de la; Vordos, Dimitrios; Cicco, Antony; Hoznek, Andras; Chopin, Dominique; Abbou, Clement-Claude; Lagrange, Jean-Leon

2003-01-01

Purpose: To identify prostate cancer patients who will have the most likely benefit from sparing the seminal vesicles during 3D conformal radiation therapy. Methods and materials: From 1988 to 2001, 532 patients underwent radical prostatectomy for clinically localized prostate cancer. Primary endpoint was the pathological evidence of seminal vesicle invasion. Variables for univariate and multivariate analyses were age, prostate weight, clinical stage, PSA level, Gleason score, number and site of positive prostate sextant biopsies. Multivariate logistic regression with backward stepwise variable selection was used to identify a set of independent predictors of seminal vesicle invasion, and the variable selection procedure was validated by non-parametric bootstrap. Results: Seminal vesicle invasion was reported in 14% of the cases. In univariate analysis, all variables except age and prostate weight were predictors of seminal vesicle invasion. In multivariate analysis, only the number of positive biopsies (P<0.0001), Gleason score (P<0.007) and PSA (P<0.0001) were predictors for seminal vesicles invasion. Based on the multivariate model, we were able to develop a prognostic score for seminal vesicle invasion, which allowed us to discriminate two patient groups: A group with low risk of seminal vesicles invasion (5.7%), and the second with a higher risk of seminal vesicles invasion (32.7%). Conclusions: Using the number of positive biopsies, Gleason score and PSA, it is possible to identify patients with low risk of seminal vesicles invasion. In this population, seminal vesicles might be excluded as a target volume in radiation therapy of prostate cancer

3D imaging of vesicles in hyaloclastic fragments - clues to syn-eruptive shear conditions

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Helo, C.; Flaws, A.; Hess, K.; Franz, A.; Clague, D. A.; Dingwell, D. B.

2011-12-01

3D imaging of stretched vesicles in hyaloclastic fragments has been used to investigate the shear environment of mild pyroclastic eruptions at mid-ocean ridges. X-ray computed tomography offers an attractive non-invasive method to investigate geomaterials at a high resolution for the geometry of the different phases. In this study, we have imaged vesicles within two types of basaltic glass fragments. Stretched, ellipsoid-shaped vesicles in thin limu o Pele and tubular vesicles in a pumiceous fragment. Both types originate from pyroclastic activity on Axial Seamount, Juan de Fuca ridge. Rapid quenching of the glass has prevented extensive bubble relaxation and information about syn-eruptive shear and differential stress conditions is stored, as the dimensions of a stretched bubble directly relates to the extent and mode of shearing. The X-ray tomography data was processed using a set of codes based on edge detection and ellipsoid fitting to acquire quantitative information on the shape of the stretched vesicles. Preliminary results demonstrate, that the geometry of the stretched vesicles, e.g., the elongation of the vesicle with respect to the calculated undeformed radius, is in accordance with simple shear scenarios. Stored differential stress ranges from 5 kPa to 90 kPa with shear rates between 3.2x102 s-1 and 5.7x3 s-1 within a single limu o Pele fragment. This range may be explained by either variable time available for relaxation as the cooling front proceeds through the fragment, complex interplay in space and time between fragmentation and quenching, bubble clusters mutually inhibiting each others extend of deformation, or any combination of these. Bubble relaxation time scales are less then 0.005 s providing constraints on the timeframe for cooling to the glass transition. Qualitative analyses of the tube pumice indicates that the tubular structures grow in length by coalescence of vertically aligned ellipsoid-shaped vesicles, and in width by coalescence of

Studies on liposomes with Chlorophyll for monitoring the electromagnetic influence at molecular level

International Nuclear Information System (INIS)

Tugulea, Laura; Miclaus, Simona; Iacovache, Ioan

2001-01-01

The liposomes with Chlorophyll are excellent model membranes and could be successfully used to study the electromagnetic influence at molecular level. The strong visible absorption and fluorescence of Chlorophyll allow its use as sensor for the interactions at molecular level and as a fluorescence marker; it reflects certain aspects of the supramolecular structure of the lipid phase: fluidity, lipid and liposomes aggregation. The objective of our work was to evidence athermal effect of low level, pulsed microwave (MW) fields on liposomes and to evidence the possible mechanism of interaction at molecular level. Unilamellar liposomes were obtained from multilamellar vesicles by the hand-shaken method and sonication for 30 minutes. The multilamellar vesicles were prepared using Chla /lipid films with specific molar ratio (lipid/Chla 1/10 and 1/100) and different lipids (Dipalmitoyl phosphatidylcholine, Dimirystoyl Phosphatidylcholine and Dioleoyl Phosphatidylcholine-Sigma). The films were dispersed in buffer solutions of different pH (6.2 - 7.6). The Chlorophyll was freshly extracted from spinach leaves and separated by the chromatographic method. Portions of liposome suspension (0.6 ml) were inserted into Teflon cuvettes. The samples were irradiated in series, for periods of 5-30 minutes. The exposure system was: MW generator + adapted load (shortened rectangular waveguide) + Teflon cuvette filled with sample liquid. The effect of MW irradiation is not observable on multilamellar vesicles, but only on small unilamellar vesicles. The MW effect is athermal, verified by conventional heating in the same range of temperatures and results in enlarging the size of vesicles. The enlarging effect of MW is opposed to the effect of ultrasounds exposure. It is not clear if effects due to MW are proportional with exposure duration; it seems that this mostly depends on the type of lipid in vesicles. The UV and VIS spectra were recorded to observe the oxidation state of the

Giant lobelias exemplify convergent evolution

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Givnish Thomas J

2010-01-01

Full Text Available Abstract Giant lobeliads on tropical mountains in East Africa and Hawaii have highly unusual, giant-rosette growth forms that appear to be convergent on each other and on those of several independently evolved groups of Asteraceae and other families. A recent phylogenetic analysis by Antonelli, based on sequencing the widest selection of lobeliads to date, raises doubts about this paradigmatic example of convergent evolution. Here I address the kinds of evidence needed to test for convergent evolution and argue that the analysis by Antonelli fails on four points. Antonelli's analysis makes several important contributions to our understanding of lobeliad evolution and geographic spread, but his claim regarding convergence appears to be invalid. Giant lobeliads in Hawaii and Africa represent paradigmatic examples of convergent evolution.

Surgical treatment for giant incisional hernia

DEFF Research Database (Denmark)

Eriksson, A; Rosenberg, J; Bisgaard, T

2014-01-01

INTRODUCTION: Repair for giant incisional hernias is a challenge due to unacceptable high morbidity and recurrence rates. Several surgical techniques are available, but all are poorly documented. This systematic review was undertaken to evaluate the existing literature on repair for giant...... % with a wide range between studies of 4-100 %. The mortality ranged from 0 to 5 % (median 0 %) and recurrence rate ranged from 0 to 53 % (median 5 %). Study follow-up ranged from 15 to 97 months (median 36 months). Mesh repair should always be used for patients undergoing repair for a giant hernia......, and the sublay position may have advantages over onlay positioning. To avoid tension, it may be advisable to use a mesh in combination with a component separation technique. Inlay positioning of the mesh and repair without a mesh should be avoided. CONCLUSIONS: Evidence to optimise repair for giant hernias...

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

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