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Sample records for vesicles showed decreases

  1. PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance

    DEFF Research Database (Denmark)

    Holst, Birgitte; Madsen, Kenneth L; Jansen, Anna M

    2013-01-01

    by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate...

  2. PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance.

    Directory of Open Access Journals (Sweden)

    Birgitte Holst

    Full Text Available Secretory vesicles in endocrine cells store hormones such as growth hormone (GH and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs domain protein PICK1 (protein interacting with C kinase 1 as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of

  3. A multicenter study shows PTEN deletion is strongly associated with seminal vesicle involvement and extracapsular extension in localized prostate cancer.

    Science.gov (United States)

    Troyer, Dean A; Jamaspishvili, Tamara; Wei, Wei; Feng, Ziding; Good, Jennifer; Hawley, Sarah; Fazli, Ladan; McKenney, Jesse K; Simko, Jeff; Hurtado-Coll, Antonio; Carroll, Peter R; Gleave, Martin; Lance, Raymond; Lin, Daniel W; Nelson, Peter S; Thompson, Ian M; True, Lawrence D; Brooks, James D; Squire, Jeremy A

    2015-08-01

    Loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene is a promising marker of aggressive prostate cancer. Active surveillance and watchful waiting are increasingly recommended to patients with small tumors felt to be low risk, highlighting the difficulties of Gleason scoring in this setting. There is an urgent need for predictive biomarkers that can be rapidly deployed to aid in clinical decision-making. Our objectives were to assess the incidence and ability of PTEN alterations to predict aggressive disease in a multicenter study. We used recently developed probes optimized for sensitivity and specificity in a four-color FISH deletion assay to study the Canary Retrospective multicenter Prostate Cancer Tissue Microarray (TMA). This TMA was constructed specifically for biomarker validation from radical prostatectomy specimens, and is accompanied by detailed clinical information with long-term follow-up. In 612 prostate cancers, the overall rate of PTEN deletion was 112 (18.3%). Hemizygous PTEN losses were present in 55/612 (9.0%) of cancers, whereas homozygous PTEN deletion was observed in 57/612 (9.3%) of tumors. Significant associations were found between PTEN status and pathologic stage (P free survival (number of events = 189), pre-operative prostate specific antigen (PSA) (P free survival in multivariate models, as were seminal vesicle invasion, extracapsular extension, and Gleason score, and preoperative PSA. Furthermore, these data demonstrate that the assay can be readily introduced at first diagnosis in a cost effective manner analogous to the use of FISH for analysis of HER2/neu status in breast cancer. Combined with published research beginning 17 years ago, both the data and tools now exist to implement a PTEN assay in the clinic. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.

  4. Overexpression of synapsin Ia in the rat calyx of Held accelerates short-term plasticity and decreases synaptic vesicle volume and active zone area

    Directory of Open Access Journals (Sweden)

    Mariya eVasileva

    2013-12-01

    Full Text Available Synapsins are synaptic vesicle (SV proteins organizing a component of the reserve pool of vesicles at most central nervous system synapses. Alternative splicing of the three mammalian genes results in multiple isoforms that may differentially contribute to the organization and maintenance of the synaptic vesicle-pools. To address this, we first characterized the expression pattern of synapsin isoforms in the rat calyx of Held. At postnatal day 16, synapsins Ia, Ib, IIb and IIIa were present, while IIa – known to sustain repetitive transmission in glutamatergic terminals – was not detectable. To test if the synapsin I isoforms could mediate IIa-like effect, and if this depends on the presence of the E-domain, we overexpressed either synapsin Ia or synapsin Ib in the rat calyx of Held via recombinant adeno-associated virus-mediated gene transfer. Although the size and overall structure of the perturbed calyces remained unchanged, short-term depression and recovery from depression were accelerated upon overexpression of synapsin I isoforms. Thus, at the calyx of Held, synapsin Ia may not substitute for the synapsin IIa-function reported for hippocampal synapses. Using electron microscopic three-dimensional reconstructions we found a redistribution of SV clusters proximal to the active zones (AZ alongside with a decrease of both AZ area and SV volume. The number of SVs at individual AZs was strongly reduced. Hence, our data indicate that the amount of synapsin Ia expressed in the calyx regulates the rate and extent of short-term synaptic plasticity by affecting vesicle recruitment to the AZ. Finally, our study reveals a novel contribution of synapsin Ia to define the surface area of AZs.

  5. Rats with decreased brain cholecystokinin levels show increased responsiveness to peripheral electrical stimulation-induced analgesia.

    Science.gov (United States)

    Zhang, L X; Li, X L; Wang, L; Han, J S

    1997-01-16

    Using the P77PMC strain of rat, which is genetically prone to audiogenic seizures, and also has decreased levels of cholecystokinin (CCK), we examined the analgesic response to peripheral electrical stimulation, which is, in part, opiate-mediated. A number of studies have suggested that CCK may function as an antagonist to endogenous opiate effects. Therefore, we hypothesized that the P77PMC animals would show an enhanced analgesic response based on their decreased CCK levels producing a diminished endogenous opiate antagonism. We found that the analgesic effect on tail flick latency produced by 100 Hz peripheral electrical stimulation was more potent and longer lasting in P77PMC rats than in control rats. Moreover, the potency of the stimulation-produced analgesia correlated with the vulnerability to audiogenic seizures in these rats. We were able to block the peripheral electrical stimulation-induced analgesia (PSIA) using a cholecystokinin octapeptide (CCK-8) administered parenterally. Radioimmunoassay showed that the content of CCK-8 in cerebral cortex, hippocampus and periaqueductal gray was much lower in P77PMC rat than in controls. These results suggest that low CCK-8 content in the central nervous system of the P77PMC rats may be related to the high analgesic response to peripheral electrical stimulation, and further support the notion that CCK may be endogenous opiate antagonist.

  6. X-ray sensitive strains of CHO cells show decreased frequency of stable transfection

    International Nuclear Information System (INIS)

    Jeggo, P.; Smith, J.

    1987-01-01

    Six X-ray sensitive (xrs) strains of the Chinese hamster ovary cell line have previously been isolated and shown to have a defect in double strand break rejoining. In this study, these strains have been investigated for their ability to take up and integrate foreign DNA. All the xrs strains investigated so far have shown a decreased frequency of stable transfectants compared to their parent line, in experiments using the plasmid pSV2gpt, which contains the selectable bacterial gene, guanine phosphoribosyl transferase. This decreased frequency is observed over a wide range of DNA concentrations (0.1 to 20 μg DNA) but is more pronounced at higher DNA concentrations. In contrast, these xrs strains show the same level of transfection proficiency as the wild type parent using a transient transfection system with a plasmid containing the bacterial CAT (chloramphenicol acetyl transferase) gene. Since the level of CAT activity does not depend on integration of foreign DNA, this suggests that the xrs strains are able to take up the same amount of DNA as the parent strains, but have a defect in the integration of foreign DNA. Since this integration of foreign DNA probably occurs by non-homologous recombination, this may indicate a role of the xrs gene product in this process

  7. Ob/ob mouse livers show decreased oxidative phosphorylation efficiencies and anaerobic capacities after cold ischemia.

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    Michael J J Chu

    Full Text Available BACKGROUND: Hepatic steatosis is a major risk factor for graft failure in liver transplantation. Hepatic steatosis shows a greater negative influence on graft function following prolonged cold ischaemia. As the impact of steatosis on hepatocyte metabolism during extended cold ischaemia is not well-described, we compared markers of metabolic capacity and mitochondrial function in steatotic and lean livers following clinically relevant durations of cold preservation. METHODS: Livers from 10-week old leptin-deficient obese (ob/ob, n = 9 and lean C57 mice (n = 9 were preserved in ice-cold University of Wisconsin solution. Liver mitochondrial function was then assessed using high resolution respirometry after 1.5, 3, 5, 8, 12, 16 and 24 hours of storage. Metabolic marker enzymes for anaerobiosis and mitochondrial mass were also measured in conjunction with non-bicarbonate tissue pH buffering capacity. RESULTS: Ob/ob and lean mice livers showed severe (>60% macrovesicular and mild (<30% microvesicular steatosis on Oil Red O staining, respectively. Ob/ob livers had lower baseline enzymatic complex I activity but similar adenosine triphosphate (ATP levels compared to lean livers. During cold storage, the respiratory control ratio and complex I-fueled phosphorylation deteriorated approximately twice as fast in ob/ob livers compared to lean livers. Ob/ob livers also demonstrated decreased ATP production capacities at all time-points analyzed compared to lean livers. Ob/ob liver baseline lactate dehydrogenase activities and intrinsic non-bicarbonate buffering capacities were depressed by 60% and 40%, respectively compared to lean livers. CONCLUSIONS: Steatotic livers have impaired baseline aerobic and anaerobic capacities compared to lean livers, and mitochondrial function indices decrease particularly from after 5 hours of cold preservation. These data provide a mechanistic basis for the clinical recommendation of shorter cold storage durations in

  8. 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.)

  9. Fascicles and the interfascicular matrix show decreased fatigue life with ageing in energy storing tendons.

    Science.gov (United States)

    Thorpe, Chavaunne T; Riley, Graham P; Birch, Helen L; Clegg, Peter D; Screen, Hazel R C

    2017-07-01

    Tendon is composed of rope-like fascicles bound together by interfascicular matrix (IFM). The IFM is critical for the function of energy storing tendons, facilitating sliding between fascicles to allow these tendons to cyclically stretch and recoil. This capacity is required to a lesser degree in positional tendons. We have previously demonstrated that both fascicles and IFM in energy storing tendons have superior fatigue resistance compared with positional tendons, but the effect of ageing on the fatigue properties of these different tendon subunits has not been determined. Energy storing tendons become more injury-prone with ageing, indicating reduced fatigue resistance, hence we tested the hypothesis that the decline in fatigue life with ageing in energy storing tendons would be more pronounced in the IFM than in fascicles. We further hypothesised that tendon subunit fatigue resistance would not alter with ageing in positional tendons. Fascicles and IFM from young and old energy storing and positional tendons were subjected to cyclic fatigue testing until failure, and mechanical properties were calculated. The results show that both IFM and fascicles from the SDFT exhibit a similar magnitude of reduced fatigue life with ageing. By contrast, the fatigue life of positional tendon subunits was unaffected by ageing. The age-related decline in fatigue life of tendon subunits in energy storing tendons is likely to contribute to the increased risk of injury in aged tendons. Full understanding of the mechanisms resulting in this reduced fatigue life will aid in the development of treatments and interventions to prevent age-related tendinopathy. Understanding the effect of ageing on tendon-structure function relationships is crucial for the development of effective preventative measures and treatments for age-related tendon injury. In this study, we demonstrate for the first time that the fatigue resistance of the interfascicular matrix decreases with ageing in energy

  10. 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....

  11. 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.

  12. A single-nucleotide polymorphism of human neuropeptide s gene originated from Europe shows decreased bioactivity.

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    Cheng Deng

    Full Text Available Using accumulating SNP (Single-Nucleotide Polymorphism data, we performed a genome-wide search for polypeptide hormone ligands showing changes in the mature regions to elucidate genotype/phenotype diversity among various human populations. Neuropeptide S (NPS, a brain peptide hormone highly conserved in vertebrates, has diverse physiological effects on anxiety, fear, hyperactivity, food intake, and sleeping time through its cognate receptor-NPSR. Here, we report a SNP rs4751440 (L(6-NPS causing non-synonymous substitution on the 6(th position (V to L of the NPS mature peptide region. L(6-NPS has a higher allele frequency in Europeans than other populations and probably originated from European ancestors ~25,000 yrs ago based on haplotype analysis and Approximate Bayesian Computation. Functional analyses indicate that L(6-NPS exhibits a significant lower bioactivity than the wild type NPS, with ~20-fold higher EC50 values in the stimulation of NPSR. Additional evolutionary and mutagenesis studies further demonstrate the importance of the valine residue in the 6(th position for NPS functions. Given the known physiological roles of NPS receptor in inflammatory bowel diseases, asthma pathogenesis, macrophage immune responses, and brain functions, our study provides the basis to elucidate NPS evolution and signaling diversity among human populations.

  13. Ghrelin knockout mice show decreased voluntary alcohol consumption and reduced ethanol-induced conditioned place preference.

    Science.gov (United States)

    Bahi, Amine; Tolle, Virginie; Fehrentz, Jean-Alain; Brunel, Luc; Martinez, Jean; Tomasetto, Catherine-Laure; Karam, Sherif M

    2013-05-01

    Recent work suggests that stomach-derived hormone ghrelin receptor (GHS-R1A) antagonism may reduce motivational aspects of ethanol intake. In the current study we hypothesized that the endogenous GHS-R1A agonist ghrelin modulates alcohol reward mechanisms. For this purpose ethanol-induced conditioned place preference (CPP), ethanol-induced locomotor stimulation and voluntary ethanol consumption in a two-bottle choice drinking paradigm were examined under conditions where ghrelin and its receptor were blocked, either using ghrelin knockout (KO) mice or the specific ghrelin receptor (GHS-R1A) antagonist "JMV2959". We showed that ghrelin KO mice displayed lower ethanol-induced CPP than their wild-type (WT) littermates. Consistently, when injected during CPP-acquisition, JMV2959 reduced CPP-expression in C57BL/6 mice. In addition, ethanol-induced locomotor stimulation was lower in ghrelin KO mice. Moreover, GHS-R1A blockade, using JMV2959, reduced alcohol-stimulated locomotion only in WT but not in ghrelin KO mice. When alcohol consumption and preference were assessed using the two-bottle choice test, both genetic deletion of ghrelin and pharmacological antagonism of the GHS-R1A (JMV2959) reduced voluntary alcohol consumption and preference. Finally, JMV2959-induced reduction of alcohol intake was only observed in WT but not in ghrelin KO mice. Taken together, these results suggest that ghrelin neurotransmission is necessary for the stimulatory effect of ethanol to occur, whereas lack of ghrelin leads to changes that reduce the voluntary intake as well as conditioned reward by ethanol. Our findings reveal a major, novel role for ghrelin in mediating ethanol behavior, and add to growing evidence that ghrelin is a key mediator of the effects of multiple abused drugs. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. 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.

  15. 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...

  16. Dryland biological soil crust cyanobacteria show unexpected decreases in abundance under long-term elevated CO2

    Science.gov (United States)

    Steven, Blaire; Gallegos-Graves, La Verne; Yeager, Chris M.; Belnap, Jayne; Evans, R. David; Kuske, Cheryl R.

    2012-01-01

    Biological soil crusts (biocrusts) cover soil surfaces in many drylands globally. The impacts of 10 years of elevated atmospheric CO2 on the cyanobacteria in biocrusts of an arid shrubland were examined at a large manipulated experiment in Nevada, USA. Cyanobacteria-specific quantitative PCR surveys of cyanobacteria small-subunit (SSU) rRNA genes suggested a reduction in biocrust cyanobacterial biomass in the elevated CO2 treatment relative to the ambient controls. Additionally, SSU rRNA gene libraries and shotgun metagenomes showed reduced representation of cyanobacteria in the total microbial community. Taxonomic composition of the cyanobacteria was similar under ambient and elevated CO2 conditions, indicating the decline was manifest across multiple cyanobacterial lineages. Recruitment of cyanobacteria sequences from replicate shotgun metagenomes to cyanobacterial genomes representing major biocrust orders also suggested decreased abundance of cyanobacteria sequences across the majority of genomes tested. Functional assignment of cyanobacteria-related shotgun metagenome sequences indicated that four subsystem categories, three related to oxidative stress, were differentially abundant in relation to the elevated CO2 treatment. Taken together, these results suggest that elevated CO2 affected a generalized decrease in cyanobacteria in the biocrusts and may have favoured cyanobacteria with altered gene inventories for coping with oxidative stress.

  17. Transdermal delivery of flurbiprofen from surfactant-based vesicles: particle characterization and the effect of water on in vitro transport.

    Science.gov (United States)

    Uchino, Tomonobu; Matsumoto, Yuiko; Murata, Akiko; Oka, Toshihiko; Miyazaki, Yasunori; Kagawa, Yoshiyuki

    2014-04-10

    Flurbiprofen loaded rigid and elastic vesicles comprising the bilayer-forming surfactant sucrose-ester laurate were prepared by the film rehydration and extrusion method. The charge-inducing agent sodium dodecyl sulfate, and the micelle-forming surfactants, sorbitan monolaurate, polyethylene glycol monolaurate, and polysorbate 20, were used to enhance elasticity. Vesicle formulations were evaluated for size, zeta potential, (1)H and (19)F nuclear magnetic resonance (NMR) spectra, and in vitro skin permeation across Yucatan micropig (YMP) skin. Vesicle formulations were stable for 2 weeks and their mean sizes were 95-135 nm. NMR spectroscopy showed that flurbiprofen molecular mobility was restricted by interaction with vesicle components because of entrapment in vesicle bilayers. Moreover, sorbitan monolaurate-containing vesicles strongly retained flurbiprofen molecules. After non-occlusive application to YMP skin, flurbiprofen transport from all vesicle formulations was superior to that of flurbiprofen alone and remarkably decreased after water vaporization. Polarization microscopy and small-angle X-ray diffraction analysis showed that the vesicle formulation was transferred to liquid crystalline state. Suppression of vesicle transition to the liquid crystalline state was observed with applications of both large quantities and diluted samples. The presence of water in the formulations was associated with maintenance of the vesicle structure and greater flurbiprofen transport across YMP skin. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. 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.

  19. Simulations show decreasing carbon stocks and potential for carbon emissions in Rocky Mountain forests over the next century.

    Science.gov (United States)

    Boisvenue, Céline; Running, Steven W

    2010-07-01

    Climate change has altered the environment in which forests grow, and climate change models predict more severe alterations to come. Forests have already responded to these changes, and the future temperature and precipitation scenarios are of foremost concern, especially in the mountainous western United States, where forests occur in the dry environments that interface with grasslands. The objective of this study was to understand the trade-offs between temperature and water controls on these forested sites in the context of available climate projections. Three temperature and precipitation scenarios from IPCC AR4 AOGCMs ranging in precipitation levels were input to the process model Biome-BGC for key forested sites in the northern U.S. Rocky Mountains. Despite the omission of natural and human-caused disturbances in our simulations, our results show consequential effects from these conservative future temperature and precipitation scenarios. According to these projections, if future precipitation and temperatures are similar to or drier than the dry scenario depicted here, high-elevation forests on both the drier and wetter sites, which have in the absence of disturbance accumulated carbon, will reduce their carbon accumulation. Under the marginally drier climate projections, most forests became carbon sources by the end of the simulation horizon (2089). Under all three scenarios, growing season lengthened, the number of days with snow on the ground decreased, peak snow occurred earlier, and water stress increased through the projection horizon (1950-2089) for all sites, which represent the temperature and precipitation spectrum of forests in this region. The quantity, form, and timing of precipitation ultimately drive the carbon accumulation trajectory of forests in this region.

  20. 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

  1. Swedish and American studies show that initiatives to decrease maternal obesity could play a key role in reducing preterm birth.

    Science.gov (United States)

    Gould, Jeffrey B; Mayo, Jonathan; Shaw, Gary M; Stevenson, David K

    2014-06-01

    Maternal obesity is a major source of preventable perinatal morbidity, but studies of the relationship between obesity and preterm birth have been inconsistent. This review looks at two major studies covering just under 3.5 million births, from California, USA, and Sweden. Inconsistent findings in previous studies appear to stem from the complex relationship between obesity and preterm birth. Initiatives to decrease maternal obesity represent an important strategy in reducing preterm birth. ©2014 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

  2. Ataxia telangiectasia derived iPS cells show preserved x-ray sensitivity and decreased chromosomal instability

    OpenAIRE

    Fukawatase, Yoshihiro; Toyoda, Masashi; Okamura, Kohji; Nakamura, Ken-ichi; Nakabayashi, Kazuhiko; Takada, Shuji; Yamazaki-Inoue, Mayu; Masuda, Akira; Nasu, Michiyo; Hata, Kenichiro; Hanaoka, Kazunori; Higuchi, Akon; Takubo, Kaiyo; Umezawa, Akihiro

    2014-01-01

    Ataxia telangiectasia is a neurodegenerative inherited disease with chromosomal instability and hypersensitivity to ionizing radiation. iPS cells lacking ATM (AT-iPS cells) exhibited hypersensitivity to X-ray irradiation, one of the characteristics of the disease. While parental ataxia telangiectasia cells exhibited significant chromosomal abnormalities, AT-iPS cells did not show any chromosomal instability in vitro for at least 80 passages (560 days). Whole exome analysis also showed a compa...

  3. Hip Fractures: What Information Does the Evidence Show That Patients and Families Need to Decrease 30-Day Readmission?

    Science.gov (United States)

    Gardner, Kristin OʼMara

    2015-01-01

    The current bundled payment reimbursement from the Centers for Medicare & Medicaid Services will not cover the additional cost of hospital readmission for the same diagnosis, and patients with hip fractures have one of the highest cost-saving opportunities when compared with other admission reasons. Common reasons for readmission to the hospital after hip fracture include pneumonia, dehydration, and mobility issues. The learning modalities including visual, aural, read/write, and kinesthetic were used to make recommendations on how the education can be incorporated into the instruction of patients with hip fractures and their families. These learning techniques can be used to develop education to decrease possibility of 30-day readmission after hip fracture. Nurses must focus their education to meet the needs of each individual patient, adapting to different types of adult learners to increase the health literacy of patients with hip fractures and their families.

  4. Transcriptome of extracellular vesicles released by hepatocytes.

    Directory of Open Access Journals (Sweden)

    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.

  5. Non-expert listeners show decreased heart rate and increased blood pressure (fear bradycardia) in response to atonal music.

    Science.gov (United States)

    Proverbio, Alice M; Manfrin, Luigi; Arcari, Laura A; De Benedetto, Francesco; Gazzola, Martina; Guardamagna, Matteo; Lozano Nasi, Valentina; Zani, Alberto

    2015-01-01

    Previous studies suggested that listening to different types of music may modulate differently psychological mood and physiological responses associated with the induced emotions. In this study the effect of listening to instrumental classical vs. atonal contemporary music was examined in a group of 50 non-expert listeners. The subjects' heart rate and diastolic and systolic blood pressure values were measured while they listened to music of different style and emotional typologies. Pieces were selected by asking a group of composers and conservatory professors to suggest a list of the most emotional music pieces (from Renaissance to present time). A total of 214 suggestions from 20 respondents were received. Then it was asked them to identify which pieces best induced in the listener feelings of agitation, joy or pathos and the number of suggested pieces per style was computed. Atonal pieces were more frequently indicated as agitating, and tonal pieces as joyful. The presence/absence of tonality in a musical piece did not affect the affective dimension of pathos (being touching). Among the most frequently cited six pieces were selected that were comparable for structure and style, to represent each emotion and style. They were equally evaluated as unfamiliar by an independent group of 10 students of the same cohort) and were then used as stimuli for the experimental session in which autonomic parameters were recorded. Overall, listening to atonal music (independent of the pieces' emotional characteristics) was associated with a reduced heart rate (fear bradycardia) and increased blood pressure (both diastolic and systolic), possibly reflecting an increase in alertness and attention, psychological tension, and anxiety. This evidence fits with the results of the esthetical assessment showing how, overall, atonal music is perceived as more agitating and less joyful than tonal one.

  6. Connexin30-deficient mice show increased emotionality and decreased rearing activity in the open-field along with neurochemical changes.

    Science.gov (United States)

    Dere, E; De Souza-Silva, M A; Frisch, C; Teubner, B; Söhl, G; Willecke, K; Huston, J P

    2003-08-01

    Gap-junction channels in the brain, formed by connexin (Cx) proteins with a distinct regional/cell-type distribution, allow intercellular electrical and metabolic communication. In astrocytes, mainly the connexins 43, 26 and 30 are expressed. In addition, connexin30 is expressed in ependymal and leptomeningeal cells, as well as in skin and cochlea. The functional implications of the astrocytic gap-junctional network are not well understood and evidence regarding their behavioural relevance is lacking. Thus, we have tested groups of Cx30-/-, Cx30+/-, and Cx30+/+ mice in the open-field, an object exploration task, in the graded anxiety test and on the rotarod. The Cx30-/- mice showed reduced exploratory activity in terms of rearings but not locomotion in the open-field and object exploration task. Furthermore, Cx30-/- mice exhibited anxiogenic behaviour as shown by higher open-field centre avoidance and corner preference. Graded anxiety test and rotarod performance was similar across groups. The Cx30-/- mice had elevated choline levels in the ventral striatum, possibly related to their aberrant behavioural phenotypes. The Cx30+/- mice had lower dopamine and metabolite levels in the amygdala and ventral striatum and lower hippocampal 5-hydroxyindole acid (5-HIAA) concentrations relative to Cx30+/+ mice. Furthermore, the Cx30+/- mice had lower acetylcholine concentrations in the ventral striatum and higher choline levels in the neostriatum, relative to Cx30+/+ mice. Our data suggest that the elimination of connexin30 can alter the reactivity to novel environments, pointing to the importance of gap-junctional signalling in behavioural processes.

  7. Non-expert listeners show decreased heart rate and increased blood pressure (fear bradycardia in response to atonal music

    Directory of Open Access Journals (Sweden)

    Alice Mado eProverbio

    2015-10-01

    Full Text Available Previous studies suggested that listening to different types of music may modulate differently psychological mood and physiological responses associated with the induced emotions. In this study the effect of listening to instrumental classical vs. atonal contemporary music was examined in a group of 50 non-expert listeners. The subjects’ heart rate and diastolic and systolic blood pressure values were measured while they listened to music of different style and emotional typologies. Pieces were selected by asking a group of composers and conservatory professors to suggest a list of the most emotional music pieces (from Renaissance to present time. A total of 214 suggestions from 20 respondents was received. Then it was asked them to identify which pieces best induced in the listener feelings of agitation, joy or pathos and the number of suggested pieces per style was computed. Atonal pieces were more frequently indicated as agitating, and tonal pieces as joyful. The presence/absence of tonality in a musical piece did not affect the affective dimension of pathos (being touching. Among the most frequently cited six pieces were selected that were comparable for structure and style, to represent each emotion and style. They were equally evaluated as unfamiliar by an independent group of 10 students of the same cohort and were then used as stimuli for the experimental session in which autonomic parameters were recorded. Overall, listening to atonal music (independent of the pieces’ emotional characteristics was associated with a reduced heart rate (fear bradycardia and increased blood pressure (both diastolic and systolic, possibly reflecting an increase in alertness and attention, psychological tension, and anxiety. This evidence fits with the results of the aesthetical assessment showing how, overall, atonal music is perceived as more agitating and less joyful than tonal one.

  8. Non-expert listeners show decreased heart rate and increased blood pressure (fear bradycardia) in response to atonal music

    Science.gov (United States)

    Proverbio, Alice M.; Manfrin, Luigi; Arcari, Laura A.; De Benedetto, Francesco; Gazzola, Martina; Guardamagna, Matteo; Lozano Nasi, Valentina; Zani, Alberto

    2015-01-01

    Previous studies suggested that listening to different types of music may modulate differently psychological mood and physiological responses associated with the induced emotions. In this study the effect of listening to instrumental classical vs. atonal contemporary music was examined in a group of 50 non-expert listeners. The subjects’ heart rate and diastolic and systolic blood pressure values were measured while they listened to music of different style and emotional typologies. Pieces were selected by asking a group of composers and conservatory professors to suggest a list of the most emotional music pieces (from Renaissance to present time). A total of 214 suggestions from 20 respondents were received. Then it was asked them to identify which pieces best induced in the listener feelings of agitation, joy or pathos and the number of suggested pieces per style was computed. Atonal pieces were more frequently indicated as agitating, and tonal pieces as joyful. The presence/absence of tonality in a musical piece did not affect the affective dimension of pathos (being touching). Among the most frequently cited six pieces were selected that were comparable for structure and style, to represent each emotion and style. They were equally evaluated as unfamiliar by an independent group of 10 students of the same cohort) and were then used as stimuli for the experimental session in which autonomic parameters were recorded. Overall, listening to atonal music (independent of the pieces’ emotional characteristics) was associated with a reduced heart rate (fear bradycardia) and increased blood pressure (both diastolic and systolic), possibly reflecting an increase in alertness and attention, psychological tension, and anxiety. This evidence fits with the results of the esthetical assessment showing how, overall, atonal music is perceived as more agitating and less joyful than tonal one. PMID:26579029

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Hierarchical unilamellar vesicles of controlled compositional heterogeneity.

    Directory of Open Access Journals (Sweden)

    Maik Hadorn

    Full Text Available Eukaryotic life contains hierarchical vesicular architectures (i.e. organelles that are crucial for material production and trafficking, information storage and access, as well as energy production. In order to perform specific tasks, these compartments differ among each other in their membrane composition and their internal cargo and also differ from the cell membrane and the cytosol. Man-made structures that reproduce this nested architecture not only offer a deeper understanding of the functionalities and evolution of organelle-bearing eukaryotic life but also allow the engineering of novel biomimetic technologies. Here, we show the newly developed vesicle-in-water-in-oil emulsion transfer preparation technique to result in giant unilamellar vesicles internally compartmentalized by unilamellar vesicles of different membrane composition and internal cargo, i.e. hierarchical unilamellar vesicles of controlled compositional heterogeneity. The compartmentalized giant unilamellar vesicles were subsequently isolated by a separation step exploiting the heterogeneity of the membrane composition and the encapsulated cargo. Due to the controlled, efficient, and technically straightforward character of the new preparation technique, this study allows the hierarchical fabrication of compartmentalized giant unilamellar vesicles of controlled compositional heterogeneity and will ease the development of eukaryotic cell mimics that resemble their natural templates as well as the fabrication of novel multi-agent drug delivery systems for combination therapies and complex artificial microreactors.

  14. Seminal vesicle cycts

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  15. The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Mai [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan); Kitaguchi, Tetsuya [Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABOIS), Waseda University, 11 Biopolis Way, 05-01/02 Helios, Singapore 138667 (Singapore); Numano, Rika [The Electronics-Inspired Interdisciplinary Research Institute (EIIRIS), Toyohashi University of Technology, 1-1 Hibarigaoka, Tennpaku-cho, Toyohashi, Aichi 441-8580 (Japan); Ikematsu, Kazuya [Forensic Pathology and Science, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523 (Japan); Kakeyama, Masaki [Laboratory of Environmental Health Sciences, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Murata, Masayuki; Sato, Ken [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan); Tsuboi, Takashi, E-mail: takatsuboi@bio.c.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Regulation of exocytosis by Rho GTPase Cdc42. Black-Right-Pointing-Pointer Cdc42 increases the number of fusion events from newly recruited vesicles. Black-Right-Pointing-Pointer Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

  16. The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

    International Nuclear Information System (INIS)

    Sato, Mai; Kitaguchi, Tetsuya; Numano, Rika; Ikematsu, Kazuya; Kakeyama, Masaki; Murata, Masayuki; Sato, Ken; Tsuboi, Takashi

    2012-01-01

    Highlights: ► Regulation of exocytosis by Rho GTPase Cdc42. ► Cdc42 increases the number of fusion events from newly recruited vesicles. ► Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott–Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

  17. Direct Covalent Grafting of Phytate to Titanium Surfaces through Ti-O-P Bonding Shows Bone Stimulating Surface Properties and Decreased Bacterial Adhesion.

    Science.gov (United States)

    Córdoba, Alba; Hierro-Oliva, Margarita; Pacha-Olivenza, Miguel Ángel; Fernández-Calderón, María Coronada; Perelló, Joan; Isern, Bernat; González-Martín, María Luisa; Monjo, Marta; Ramis, Joana M

    2016-05-11

    Myo-inositol hexaphosphate, also called phytic acid or phytate (IP6), is a natural molecule abundant in vegetable seeds and legumes. Among other functions, IP6 inhibits bone resorption. It is adsorbed on the surface of hydroxyapatite, inhibiting its dissolution and decreasing the progressive loss of bone mass. We present here a method to directly functionalize Ti surfaces covalently with IP6, without using a cross-linker molecule, through the reaction of the phosphate groups of IP6 with the TiO2 layer of Ti substrates. The grafting reaction consisted of an immersion in an IP6 solution to allow the physisorption of the molecules onto the substrate, followed by a heating step to obtain its chemisorption, in an adaptation of the T-Bag method. The reaction was highly dependent on the IP6 solution pH, only achieving a covalent Ti-O-P bond at pH 0. We evaluated two acidic pretreatments of the Ti surface, to increase its hydroxylic content, HNO3 30% and HF 0.2%. The structure of the coated surfaces was characterized by X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, and ellipsometry. The stability of the IP6 coating after three months of storage and after sterilization with γ-irradiation was also determined. Then, we evaluated the biological effect of Ti-IP6 surfaces in vitro on MC3T3-E1 osteoblastic cells, showing an osteogenic effect. Finally, the effect of the surfaces on the adhesion and biofilm viability of oral microorganisms S. mutans and S. sanguinis was also studied, and we found that Ti-IP6 surfaces decreased the adhesion of S. sanguinis. A surface that actively improves osseointegration while decreasing the bacterial adhesion could be suitable for use in bone implants.

  18. 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.

  19. Proteomic data show an increase in autoantibodies and alpha-fetoprotein and a decrease in apolipoprotein A-II with time in sera from senescence-accelerated mice

    Energy Technology Data Exchange (ETDEWEB)

    Guo, S.J. [Beijing Institute of Pharmacology and Toxicology, Beijing (China); Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Qi, C.H.; Zhou, W.X.; Zhang, Y.X. [Beijing Institute of Pharmacology and Toxicology, Beijing (China); Zhang, X.M.; Wang, J.; Wang, H.X. [National Center of Biomedical Analysis, Beijing (China)

    2013-04-12

    We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2-A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4{sup +} T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.

  20. Proteomic data show an increase in autoantibodies and alpha-fetoprotein and a decrease in apolipoprotein A-II with time in sera from senescence-accelerated mice

    International Nuclear Information System (INIS)

    Guo, S.J.; Qi, C.H.; Zhou, W.X.; Zhang, Y.X.; Zhang, X.M.; Wang, J.; Wang, H.X.

    2013-01-01

    We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2-A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4 + T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. Decreased anxiety- and depression-like behaviors and hyperactivity in a type 3 deiodinase-deficient mouse showing brain thyrotoxicosis and peripheral hypothyroidism.

    Science.gov (United States)

    Stohn, J Patrizia; Martinez, M Elena; Hernandez, Arturo

    2016-12-01

    Hypo- and hyperthyroid states, as well as functional abnormalities in the hypothalamic-pituitary-thyroid axis have been associated with psychiatric conditions like anxiety and depression. However, the nature of this relationship is poorly understood since it is difficult to ascertain the thyroid status of the brain in humans. Data from animal models indicate that the brain exhibits efficient homeostatic mechanisms that maintain local levels of the active thyroid hormone, triiodothyronine (T3) within a narrow range. To better understand the consequences of peripheral and central thyroid status for mood-related behaviors, we used a mouse model of type 3 deiodinase (DIO3) deficiency (Dio3 -/- mouse). This enzyme inactivates thyroid hormone and is highly expressed in the adult central nervous system. Adult Dio3 -/- mice exhibit elevated levels of T3-dependent gene expression in the brain, despite peripheral hypothyroidism as indicated by low circulating levels of thyroxine and T3. Dio3 -/- mice of both sexes exhibit hyperactivity and significantly decreased anxiety-like behavior, as measured by longer time spent in the open arms of the elevated plus maze and in the light area of the light/dark box. During the tail suspension, they stayed immobile for a significantly shorter time than their wild-type littermates, suggesting decreased depression-like behavior. These results indicate that increased thyroid hormone in the brain, not necessarily in peripheral tissues, correlates with hyperactivity and with decreases in anxiety and depression-like behaviors. Our results also underscore the importance of DIO3 as a determinant of behavior by locally regulating the brain levels of thyroid hormone. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. An asthma patient with steroid-resistant decrease in peak expiratory flow after the Great East Japan earthquake showing spontaneous recovery after 1 month.

    Science.gov (United States)

    Yanagimoto, Shintaro; Haida, Michiko; Suko, Matsunobu

    2012-01-01

    People living in Japan were affected in various ways after the Great East Japan earthquake of March 11, 2011. A 52-year-old female asthma patient not directly affected by the disaster experienced a decrease in peak expiratory flow (PEF) immediately after the earthquake. Despite increasing the inhaled and oral corticosteroid doses, her PEF did not recover. One month later, her PEF level abruptly returned to normal with minimal medications, which were previously ineffective, and the asthma-related symptoms vanished. The stabilization of her state of mind and actual social state seemed to be a part of the reason for the patient's recovery.

  9. 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.

  10. 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.

  11. 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...

  12. Spared pre-irradiated area in pustular lesions induced by icotinib showing decreased expressions of CD1a+ langerhans cells and FGFR2

    International Nuclear Information System (INIS)

    Zhao Qiong; Wang Yi Na; Wang Bo

    2013-01-01

    Icotinib hydrochloride, a novel inhibitor of epidermal growth factor receptor tyrosine kinase, has been approved by the State Food and Drug Administration for the treatment of advanced non-small-cell lung cancer. Up to date, cutaneous response to icotinib is largely unknown. Here we report an uncommon lesional phenomenon in a 56-year-old Chinese male with non-small-cell lung cancer, who received icotinib as a second-line treatment. Characteristic papulopustular rash on the chest and back was observed 4 days later. Interestingly, the rash completely spares a pre-irradiated area. The immunohistochemical study in the lesional skin area and spared skin area revealed a significant decrease in CD1a + Langerhans cells, Ki-67 as well as FGFR2 in the spared area than in the lesional area. Thus, the present case indicated that loss of the basal layer of proliferative cells and antigen-presenting cells (Langerhans cell), as well as the down-regulation of FGFR2 signaling in the pre-irradiated skin area, may join forces in inhibiting icotinib-associated cutaneous reactions. To our knowledge, this is the first report of both lesional area and lesion-spared area in a Chinese male receiving treatment with a new epidermal growth factor receptor-tyrosine kinase inhibitor (icotinib). The immunohistochemical reactions described here also provide new insight into the pathogenesis of epidermal growth factor receptor-tyrosine kinase inhibitor-related skin toxicities, and the role that other tyrosine kinase receptors (including FGFR) played in non-small-cell lung cancer. (author)

  13. Spared pre-irradiated area in pustular lesions induced by icotinib showing decreased expressions of CD1a+ langerhans cells and FGFR2.

    Science.gov (United States)

    Zhao, Qiong; Wang, Yi Na; Wang, Bo

    2013-02-01

    Icotinib hydrochloride, a novel inhibitor of epidermal growth factor receptor tyrosine kinase, has been approved by the State Food and Drug Administration for the treatment of advanced non-small-cell lung cancer. Up to date, cutaneous response to icotinib is largely unknown. Here we report an uncommon lesional phenomenon in a 56-year-old Chinese male with non-small-cell lung cancer, who received icotinib as a second-line treatment. Characteristic papulopustular rash on the chest and back was observed 4 days later. Interestingly, the rash completely spares a pre-irradiated area. The immunohistochemical study in the lesional skin area and spared skin area revealed a significant decrease in CD1a(+) Langerhans cells, Ki-67 as well as FGFR2 in the spared area than in the lesional area. Thus, the present case indicated that loss of the basal layer of proliferative cells and antigen-presenting cells (Langerhans cell), as well as the down-regulation of FGFR2 signaling in the pre-irradiated skin area, may join forces in inhibiting icotinib-associated cutaneous reactions. To our knowledge, this is the first report of both lesional area and lesion-spared area in a Chinese male receiving treatment with a new epidermal growth factor receptor-tyrosine kinase inhibitor (icotinib). The immunohistochemical reactions described here also provide new insight into the pathogenesis of epidermal growth factor receptor-tyrosine kinase inhibitor-related skin toxicities, and the role that other tyrosine kinase receptors (including FGFR) played in non-small-cell lung cancer.

  14. A snapshot of 3649 Web-based services published between 1994 and 2017 shows a decrease in availability after 2 years.

    Science.gov (United States)

    Osz, Ágnes; Pongor, Lorinc Sándor; Szirmai, Danuta; Gyorffy, Balázs

    2017-12-08

    The long-term availability of online Web services is of utmost importance to ensure reproducibility of analytical results. However, because of lack of maintenance following acceptance, many servers become unavailable after a short period of time. Our aim was to monitor the accessibility and the decay rate of published Web services as well as to determine the factors underlying trends changes. We searched PubMed to identify publications containing Web server-related terms published between 1994 and 2017. Automatic and manual screening was used to check the status of each Web service. Kruskall-Wallis, Mann-Whitney and Chi-square tests were used to evaluate various parameters, including availability, accessibility, platform, origin of authors, citation, journal impact factor and publication year. We identified 3649 publications in 375 journals of which 2522 (69%) were currently active. Over 95% of sites were running in the first 2 years, but this rate dropped to 84% in the third year and gradually sank afterwards (P Web services is 10.39 years. Working Web services were published in journals with higher impact factors (P = 4.8e-04). Services published before the year 2000 received minimal attention. The citation of offline services was less than for those online (P = 0.022). The majority of Web services provide analytical tools, and the proportion of databases is slowly decreasing. Conclusions. Almost one-third of Web services published to date went out of service. We recommend continued support of Web-based services to increase the reproducibility of published results. © The Author 2017. Published by Oxford University Press.

  15. Inertia-dependent dynamics of three-dimensional vesicles and red blood cells in shear flow.

    Science.gov (United States)

    Luo, Zheng Yuan; Wang, Shu Qi; He, Long; Xu, Feng; Bai, Bo Feng

    2013-10-28

    A three-dimensional (3D) simulation study of the effect of inertia on the dynamics of vesicles and red blood cells (RBCs) has not been reported. Here, we developed a 3D model based on the front tracking method to investigate how inertia affects the dynamics of spherical/non-spherical vesicles and biconcave-shaped RBCs with the Reynolds number ranging from 0.1 to 10. The results showed that inertia induced non-spherical vesicles transitioned from tumbling to swinging, which was not observed in previous 2D models. The critical viscosity ratio of inner/outer fluids for the tumbling–swinging transition remarkably increased with an increasing Reynolds number. The deformation of vesicles was greatly enhanced by inertia, and the frequency of tumbling and tank-treading was significantly decreased by inertia. We also found that RBCs can transit from tumbling to steady tank-treading through the swinging regime when the Reynolds number increased from 0.1 to 10. These results indicate that inertia needs to be considered at moderate Reynolds number (Re ~ 1) in the study of blood flow in the human body and the flow of deformable particle suspension in inertial microfluidic devices. The developed 3D model provided new insights into the dynamics of RBCs under shear flow, thus holding great potential to better understand blood flow behaviors under normal/disease conditions.

  16. High-anxious individuals show increased chronic stress burden, decreased protective immunity, and increased cancer progression in a mouse model of squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Firdaus S Dhabhar

    Full Text Available In spite of widespread anecdotal and scientific evidence much remains to be understood about the long-suspected connection between psychological factors and susceptibility to cancer. The skin is the most common site of cancer, accounting for nearly half of all cancers in the US, with approximately 2-3 million cases of non-melanoma cancers occurring each year worldwide. We hypothesized that a high-anxious, stress-prone behavioral phenotype would result in a higher chronic stress burden, lower protective-immunity, and increased progression of the immuno-responsive skin cancer, squamous cell carcinoma. SKH1 mice were phenotyped as high- or low-anxious at baseline, and subsequently exposed to ultraviolet-B light (1 minimal erythemal dose (MED, 3 times/week, 10-weeks. The significant strengths of this cancer model are that it uses a normal, immunocompetent, outbred strain, without surgery/injection of exogenous tumor cells/cell lines, and produces lesions that resemble human tumors. Tumors were counted weekly (primary outcome, and tissues collected during early and late phases of tumor development. Chemokine/cytokine gene-expression was quantified by PCR, tumor-infiltrating helper (Th, cytolytic (CTL, and regulatory (Treg T cells by immunohistochemistry, lymph node T and B cells by flow cytometry, adrenal and plasma corticosterone and tissue vascular-endothelial-growth-factor (VEGF by ELISA. High-anxious mice showed a higher tumor burden during all phases of tumor development. They also showed: higher corticosterone levels (indicating greater chronic stress burden, increased CCL22 expression and Treg infiltration (increased tumor-recruited immuno-suppression, lower CTACK/CCL27, IL-12, and IFN-γ gene-expression and lower numbers of tumor infiltrating Th and CTLs (suppressed protective immunity, and higher VEGF concentrations (increased tumor angiogenesis/invasion/metastasis. These results suggest that the deleterious effects of high trait anxiety

  17. 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

  18. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    Directory of Open Access Journals (Sweden)

    Zeng YX

    2016-04-01

    Full Text Available Yixuan Zeng,1,2,* Wenyuan Guo,1,* Guangqing Xu,3 Qinmei Wang,4 Luyang Feng,1,2 Simei Long,1 Fengyin Liang,1 Yi Huang,1 Xilin Lu,1 Shichang Li,5 Jiebin Zhou,5 Jean-Marc Burgunder,6 Jiyan Pang,5 Zhong Pei1,2 1Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Disease, The First Affiliated Hospital, Sun Yat-sen University, 2Guangzhou Center, Chinese Huntington’s Disease Network, 3Department of Rehabilitation, The First Affiliated Hospital, 4Key laboratory on Assisted Circulation, Ministry of Health, Department of Cardiovascular Medicine of the First Affiliated Hospital, 5School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; 6Swiss Huntington’s Disease Center, Department of Neurology, University of Bern, Bern, Switzerland *These authors contributed equally to this work Abstract: Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt. Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a

  19. Formation of Giant Protein Vesicles by a Lipid Cosolvent Method

    DEFF Research Database (Denmark)

    Hansen, Jesper S.; Vararattanavech, Ardcharaporn; Vissing, Thomas

    2011-01-01

    This paper describes a method to create giant protein vesicles (GPVs) of ≥10 μm by solvent‐driven fusion of large vesicles (0.1–0.2 μm) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein‐reconstituted large unilamellar vesicles (LUVs)...... of spinach SoPIP2;1 and E. coli AqpZ aquaporins. Our findings show that hydrophobic interactions within the bilayer of formed GPVs are influenced not only by the solvent partitioning propensity, but also by lipid composition and membrane protein isoform....

  20. 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)

  1. Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation.

    Science.gov (United States)

    Gamalier, Juliana P; Silva, Thiago P; Zarantonello, Victor; Dias, Felipe F; Melo, Rossana C N

    2017-01-01

    Secretion of membrane vesicles is an important biological process of both eukaryotic and prokaryotic cells. This process has been characterized in pathogenic bacteria, but is less clear in non-pathogenic bacteria from aquatic ecosystems. Here, we investigated, for the first time, the process of formation of outer membranes vesicles (OMVs), nanoscale vesicles extruded from the outer membrane (OM) of gram-negative bacteria, in cultures of freshwater bacteria after exposure or not to ultraviolet radiation (UVR) as an environmental stressor. Non-axenic cultures of freshwater bacteria isolated from a Brazilian aquatic ecosystem (Funil reservoir) were exposed or not to UVR (UVA+UVB) over a 3h period, during which cell density, viability and ultrastructure were analyzed. First, we showed that UVR induce bacterial death. UVR triggered significant negative effect on cell density after 3h of UVR treatment. This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe that enables the distinction of live/dead bacteria. Transmission electron microscopy (TEM) revealed changes indicative of cell death after 3h of UVR exposure, with significant increase of damaged cells compared to the control group. Second, we demonstrated that gram-negative bacteria release OMVs during normal growth and after UVR exposure. OMVs were clearly identified as round, membrane-bound vesicles budding off from the bacterial OM as isolated or clustered vesicles or free in the extracellular medium. Remarkably, quantitative TEM analyses showed that bacteria respond to UVR with increased formation of OMVs. Moreover, while OMVs numbers per intact or damaged cell did not differ in the untreated group, UVR led to a higher vesiculation by bacteria in process of death. This means that degenerating bacteria release OMVs before lysis and that this secretion might be an adaptive/protective response to rapid changes in environmental conditions such as UV radiation. Copyright

  2. 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.

  3. High energy irradiation of bacterial membrane vesicles

    International Nuclear Information System (INIS)

    De La Rosa, M.A.M.

    1977-01-01

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

  4. 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...

  5. 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...

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  7. SODIUM DI-N-DODECYL PHOSPHATE VESICLES IN AQUEOUS-SOLUTION - EFFECTS OF ETHANOL, PROPANOL, AND TETRAHYDROFURAN ON THE GEL TO LIQUID-PHASE TRANSITION

    NARCIS (Netherlands)

    BLANDAMER, MJ; BRIGGS, B; BUTT, MD; WATERS, M; CULLIS, PM; ENGBERTS, JBFN; HOEKSTRA, D; MOHANTY, RK

    1994-01-01

    For aqueous solutions containing vesicles formed by sodium di-n-dodecyl phosphate, the gel to liquid-crystal transition occurs near 35 degrees C, the temperature T-m. When ethanol is added, T-m decreases, but the scan shows evidence of several transitions as more alcohol is added. The effect of

  8. Glucose and lactate are equally effective in energizing activity-dependent synaptic vesicle turnover in purified cortical neurons.

    Science.gov (United States)

    Morgenthaler, F D; Kraftsik, R; Catsicas, S; Magistretti, P J; Chatton, J-Y

    2006-08-11

    This study examines the role of glucose and lactate as energy substrates to sustain synaptic vesicle cycling. Synaptic vesicle turnover was assessed in a quantitative manner by fluorescence microscopy in primary cultures of mouse cortical neurons. An electrode-equipped perfusion chamber was used to stimulate cells both by electrical field and potassium depolarization during image acquisition. An image analysis procedure was elaborated to select in an unbiased manner synaptic boutons loaded with the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43). Whereas a minority of the sites fully released their dye content following electrical stimulation, others needed subsequent K(+) depolarization to achieve full release. This functional heterogeneity was not significantly altered by the nature of metabolic substrates. Repetitive stimulation sequences of FM1-43 uptake and release were then performed in the absence of any metabolic substrate and showed that the number of active sites dramatically decreased after the first cycle of loading/unloading. The presence of 1 mM glucose or lactate was sufficient to sustain synaptic vesicle cycling under these conditions. Moreover, both substrates were equivalent for recovery of function after a phase of decreased metabolic substrate availability. Thus, lactate appears to be equivalent to glucose for sustaining synaptic vesicle turnover in cultured cortical neurons during activity.

  9. Uptake of 75Se-selenite by brush border membrane vesicles from chick duodenum stimulated by vitamin D

    International Nuclear Information System (INIS)

    Mykkanen, H.M.; Wasserman, R.H.

    1989-01-01

    Brush border membrane vesicles were isolated from mucosal homogenates of duodena from normal, rachitic and vitamin D-treated rachitic chicks using a discontinuous sucrose gradient, and further purified by glycerol gradient centrifugation. In vitro uptake of 75Se-selenite by purified brush border membrane vesicles was studied using a rapid filtration technique. The time course of 75Se uptake was non-linear; rapid initial binding was followed by a gradual decrease in the rate of uptake until an equilibrium value was reached at 60-120 min. The initial binding at 36 s was not affected by selenite concentration in the incubation buffer, while the fractional rate of uptake between the 36 s and 2 min time periods was clearly lower with 1 mM Se than with 4-100 microM Se. 75Se uptake did not show any dependency on the external Na-gradient, nor could it be inhibited by other anions (arsenate, phosphate). Treatment of rachitic chicks either with cholecalciferol (500 Iu, 72 h) or with 1,25(OH)2-cholecalciferol (0.5 microgram given 16 h prior to isolation of the vesicles) significantly enhanced 75Se uptake. A threefold excess of mannitol in the outside buffer reduced 75Se uptake by vesicles from vitamin D-deficient and D-treated chicks 60% and 35% respectively, but had no effect on vesicles from vitamin D-treated chicks preloaded with 75Se. Neither saponin treatment nor excess cold selenite could release the label from the vesicles preloaded with 75Se. These data are compatible with the hypothesis that selenite easily crosses the brush border membrane into the intravesicular space and, once inside, is tightly bound by the membrane

  10. Phospholipase D is involved in the formation of Golgi associated clathrin coated vesicles in human parotid duct cells.

    Directory of Open Access Journals (Sweden)

    Lorena Brito de Souza

    Full Text Available Phospholipase D (PLD has been implicated in many cellular functions, such as vesicle trafficking, exocytosis, differentiation, and proliferation. The aim of this study was to characterize the role of PLD in HSY cells, a human cell line originating from the intercalated duct of the parotid gland. As the function and intracellular localization of PLD varies according to cell type, initially, the intracellular localization of PLD1 and PLD2 was determined. By immunofluorescence, PLD1 and PLD2 both showed a punctate cytoplasmic distribution with extensive co-localization with TGN-46. PLD1 was also found in the nucleus, while PLD2 was associated with the plasma membrane. Treatment of cells with the primary alcohol 1-butanol inhibits the hydrolysis of phosphatidylcoline by PLD thereby suppressing phosphatidic acid (PA production. In untreated HSY cells, there was only a slight co-localization of PLD with the clathrin coated vesicles. When HSY cells were incubated with 1-butanol the total number of clathrin coated vesicles increased, especially in the juxtanuclear region and the co-localization of PLD with the clathrin coated vesicles was augmented. Transmission electron microscopy confirmed that the number of Golgi-associated coated vesicles was greater. Treatment with 1-butanol also affected the Golgi apparatus, increasing the volume of the Golgi saccules. The decrease in PA levels after treatment with 1-butanol likewise resulted in an accumulation of enlarged lysosomes in the perinuclear region. Therefore, in HSY cells PLD appears to be involved in the formation of Golgi associated clathrin coated vesicles as well as in the structural maintenance of the Golgi apparatus.

  11. Active elastohydrodynamics of vesicles in narrow blind constrictions

    Science.gov (United States)

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

    2017-11-01

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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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 ...

  18. 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.

  19. 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.

  20. Shear-induced formation of vesicles in membrane phases: Kinetics and size selection mechanisms, elasticity versus surface tension

    Science.gov (United States)

    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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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...

  6. Spontaneous Vesicle Recycling in the Synaptic Bouton

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. Preparation of wheat root plasma membrane vesicles and effect of water stress on 45Ca2+ transport activity

    International Nuclear Information System (INIS)

    Lu Jinyin; Gao Junfeng; Cao Cuiling

    1998-01-01

    The wheat roots plasma membrane (PM) vesicles were obtained by sucrose gradient centrifugation. The experiment results shows that the wheat roots of Zhengyin No.1 PM H + -ATPase latent activity was 24%, and PM inside-out vesicle (IOV) accounts for 76%. With -1.0 MPa stress of 24h, PM Ca 2+ -ATPase activity of both orientation wheat roots were increased. Under normal water condition and PEG stress, 62% and 53% of the enzyme activity was inhibited respectively by EGTA, radioactive calcium-45 transport amount was 22.09 nmol/mg pro and 4.17 nmol/mg pro. respectively with PM-IOV.PEG stress results in a decrease of 45 Ca 2+ transport amount of wheat roots PM-IOV by 81%

  14. 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

  15. 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

  16. Trafficking of astrocytic vesicles in hippocampal slices

    International Nuclear Information System (INIS)

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

    2009-01-01

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  17. Extracellular Vesicles in Hematological Disorders

    Directory of Open Access Journals (Sweden)

    Anat Aharon

    2014-10-01

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

  18. 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...

  19. 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.

  20. 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.

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

    Science.gov (United States)

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

    2015-03-03

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

  2. 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...

  3. 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.

  4. Overproduction of individual gas vesicle proteins perturbs flotation, antibiotic production and cell division in the enterobacterium Serratia sp. ATCC 39006.

    Science.gov (United States)

    Monson, Rita E; Tashiro, Yosuke; Salmond, George P C

    2016-09-01

    Gas vesicles are intracellular proteinaceous organelles that facilitate bacterial colonization of static water columns. In the enterobacterium Serratia sp. ATCC 39006, gas vesicle formation requires the proteins GvpA1, GvpF1, GvpG, GvpA2, GvpK, GvpA3, GvpF2 and GvpF3 and the three gas vesicle regulatory proteins GvrA, GvrB and GvrC. Deletion of gvpC alters gas vesicle robustness and deletion of gvpN or gvpV results in small bicone vesicles. In this work, we assessed the impacts on gas vesicle formation when each of these 14 essential proteins was overexpressed. Overproduction of GvpF1, GvpF2, GvrA, GvrB or GvrC all resulted in significantly reduced gas vesicle synthesis. Perturbations in gas vesicle formation were also observed when GvpV and GvpA3 were in excess. In addition to impacts on gas vesicle formation, overproduction of GvrA or GvrB led to elevated biosynthesis of the tripyrrole pigment, prodigiosin, a secondary metabolite of increasing medical interest due to its antimalarial and anticancer properties. Finally, when GvpG was overexpressed, gas vesicles were still produced, but the cells exhibited a growth defect. Further analysis showed that induction of GvpG arrested cell growth and caused a drop in viable count, suggesting a possible physiological role for this protein linking gas vesicle biogenesis and binary fission. These combined results demonstrate that the stoichiometry of individual gas vesicle proteins is crucially important for controlled organelle morphogenesis and flotation and provides evidence for the first link between gas vesicle assembly and cell division, to our knowledge.

  5. Responsive Polydiacetylene Vesicles for Biosensing Microorganisms

    Directory of Open Access Journals (Sweden)

    Estelle Lebègue

    2018-02-01

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

  6. 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

  7. 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.

  8. 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)

  9. 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.

  10. 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

  11. 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

  12. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    Directory of Open Access Journals (Sweden)

    Jifeng Zhang

    2015-01-01

    Full Text Available Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. Peripheral primitive neuroectodermal tumor of seminal vesicles: Is there a role for relatively aggressive treatment modalities?

    Directory of Open Access Journals (Sweden)

    Alessandro Crestani

    2014-12-01

    Full Text Available A 50 year old white man received an incidental ultrasound diagnosis of hypoechoic mass interesting the right seminal vesicle. A CT scan showed the presence of a 7.8 cm roundish cyst, originating from the right seminal vesicle. He had been followed by the removal of the right seminal vesicle and both the cystic lesion. The histological findings of the specimen documented the presence of small round cells compatible with Ewing’s sarcoma/PPNET. The patient received also adjuvant chemotherapy and radiation treatment. After 10 years, the follow-up is still negative.

  18. Apoptosis generates mechanical forces that close the lens vesicle in the chick embryo

    Science.gov (United States)

    Oltean, Alina; Taber, Larry A.

    2018-03-01

    During the initial stages of eye development, optic vesicles grow laterally outward from both sides of the forebrain and come into contact with the surrounding surface ectoderm (SE). Within the region of contact, these layers then thicken locally to create placodes and invaginate to form the optic cup (primitive retina) and lens vesicle (LV), respectively. This paper examines the biophysical mechanisms involved in LV formation, which consists of three phases: (1) lens placode formation; (2) invagination to create the lens pit (LP); and (3) closure to form a complete ellipsoidally shaped LV. Previous studies have suggested that extracellular matrix deposited between the SE and optic vesicle causes the lens placode to form by locally constraining expansion of the SE as it grows, while actomyosin contraction causes this structure to invaginate. Here, using computational modeling and experiments on chick embryos, we confirm that these mechanisms for Phases 1 and 2 are physically plausible. Our results also suggest, however, that they are not sufficient to close the LP during Phase 3. We postulate that apoptosis provides an additional mechanism by removing cells near the LP opening, thereby decreasing its circumference and generating tension that closes the LP. This hypothesis is supported by staining that shows a ring of cell death located around the LP opening during closure. Inhibiting apoptosis in cultured embryos using caspase inhibitors significantly reduced LP closure, and results from a finite-element model indicate that closure driven by cell death is plausible. Taken together, our results suggest an important mechanical role for apoptosis in lens development.

  19. 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)

  20. 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

    ABSTRACT

    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

  1. Squamous cell carcinoma of the seminal vesicle. Review of the related literature and case report

    Directory of Open Access Journals (Sweden)

    V. B. Matveev

    2015-01-01

    Full Text Available Seminal vesicle tumors are very rare malignancies which are not diagnosed in daily clinical oncology practice. Primary malignant tumors in seminal vesicle are difficult to define due to the lack of specific symptoms in the early stages of the disease. Another obstacle of proper diagnosis is the frequent invasion of tumors of the surrounding organs, especially the prostate, rectum and bladder which is difficult to differentiate. Very often seminal vesicle tumors are difficult to detect. Digital rectal examination as well as transrectal ultrasound scan (US could reveal a bulky mass in the retrovesical space. Computed tomography and magnetic resonance imaging (MRI are the main diagnostic methods which could help to reveal pathologic masses in the region of seminal vesicles. Levels of prostate-specific antigen, carcinoembryonic antigen and tumor markers specific for colorectal cancer are negative in seminal vesicle tumors.The world experience of treating seminal vesicle tumors is very limited. There is paucity of data regarding appropriate choice of surgical approach and further treatment strategy and most of the time the treatment is individualized and based on very scarce information. At the same time surgical approach may vary significantly from vesiculectomy to pelvic exenteration. Possibility of using any regimens of adjuvant radiation therapy, chemotherapy or hormone therapy is highly debatable. However, aggressive surgical approach with radical tumor removal followed by extended lymphodissection shows the most favorable results in survival of patients suffering from seminal vesicle cancer.Squamous cell carcinoma of the seminal vesicles is presumed to be an extremely rare disease as there are only 3 reports of it in the world literature. We report a case of patient B. suffering from squamous cell carcinoma of the right seminal vesicle whom we conducted an aggressive surgical approach – prostatovesiculectomy followed by resection of the

  2. 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

  3. 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...

  4. Synaptic Vesicle Endocytosis in Different Model Systems

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    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.

  5. A Phase of Liposomes with Entangled Tubular Vesicles

    Science.gov (United States)

    Chiruvolu, Shivkumar; Warriner, Heidi E.; Naranjo, Edward; Idziak, Stefan H. J.; Radler, Joachim O.; Plano, Robert J.; Zasadzinski, Joseph A.; Safinya, Cyrus R.

    1994-11-01

    An equilibrium phase belonging to the family of bilayer liposomes in ternary mixtures of dimyristoylphosphatidylcholine (DMPC), water, and geraniol (a biological alcohol derived from oil-soluble vitamins that acts as a cosurfactant) has been identified. Electron and optical microscopy reveal the phase, labeled Ltv, to be composed of highly entangled tubular vesicles. In situ x-ray diffraction confirms that the tubule walls are multilamellar with the lipids in the chain-melted state. Macroscopic observations show that the Ltv phase coexists with the well-known L_4 phase of spherical vesicles and a bulk L_α phase. However, the defining characteristic of the Ltv phase is the Weissenberg rod climbing effect under shear, which results from its polymer-like entangled microstructure.

  6. Primary leiomyosarcoma of the seminal vesicle: Case report and review of the literature

    International Nuclear Information System (INIS)

    Cauvin, Cécile; Moureau-Zabotto, Laurence; Chetaille, Bruno; Hilgers, Werner; Denoux, Yves; Jacquemier, Jocelyne; Guiramand, Jérôme; Sarran, Anthony; Bertucci, François

    2011-01-01

    Primary leiomyosarcoma of the seminal vesicle is exceedingly rare. We report a case of a 59-year-old man with tumour detected by rectal symptoms and ultrasonography. Computed tomography and magnetic resonance imaging suggested an origin in the right seminal vesicle. Transperineal biopsy of the tumour revealed leiomyosarcoma. A radical vesiculo-prostactectomy with bilateral pelvic lymphadenectomy was performed. Pathological examination showed a grade 2 leiomyosarcoma of the seminal vesicle. The patient received adjuvant radiotherapy. He developed distant metastases 29 months after diagnosis, and received chemotherapy. Metastatic disease was controlled by second-line gemcitabine-docetaxel combination. Fifty-one months after diagnosis of the primary tumour, and 22 months after the first metastases, the patient is alive with excellent performance status, and multiple asymptomatic stable lung and liver lesions. We report the eighth case of primary leiomyosarcoma of the seminal vesicle and the first one with a so long follow-up

  7. Primary leiomyosarcoma of the seminal vesicle: case report and review of the literature.

    Science.gov (United States)

    Cauvin, Cécile; Moureau-Zabotto, Laurence; Chetaille, Bruno; Hilgers, Werner; Denoux, Yves; Jacquemier, Jocelyne; Guiramand, Jérôme; Sarran, Anthony; Bertucci, François

    2011-07-29

    Primary leiomyosarcoma of the seminal vesicle is exceedingly rare. We report a case of a 59-year-old man with tumour detected by rectal symptoms and ultrasonography. Computed tomography and magnetic resonance imaging suggested an origin in the right seminal vesicle. Transperineal biopsy of the tumour revealed leiomyosarcoma. A radical vesiculo-prostactectomy with bilateral pelvic lymphadenectomy was performed. Pathological examination showed a grade 2 leiomyosarcoma of the seminal vesicle. The patient received adjuvant radiotherapy. He developed distant metastases 29 months after diagnosis, and received chemotherapy. Metastatic disease was controlled by second-line gemcitabine-docetaxel combination. Fifty-one months after diagnosis of the primary tumour, and 22 months after the first metastases, the patient is alive with excellent performance status, and multiple asymptomatic stable lung and liver lesions. We report the eighth case of primary leiomyosarcoma of the seminal vesicle and the first one with a so long follow-up.

  8. Primary leiomyosarcoma of the seminal vesicle: Case report and review of the literature

    Directory of Open Access Journals (Sweden)

    Guiramand Jérôme

    2011-07-01

    Full Text Available Abstract Background Primary leiomyosarcoma of the seminal vesicle is exceedingly rare. Case Presentation We report a case of a 59-year-old man with tumour detected by rectal symptoms and ultrasonography. Computed tomography and magnetic resonance imaging suggested an origin in the right seminal vesicle. Transperineal biopsy of the tumour revealed leiomyosarcoma. A radical vesiculo-prostactectomy with bilateral pelvic lymphadenectomy was performed. Pathological examination showed a grade 2 leiomyosarcoma of the seminal vesicle. The patient received adjuvant radiotherapy. He developed distant metastases 29 months after diagnosis, and received chemotherapy. Metastatic disease was controlled by second-line gemcitabine-docetaxel combination. Fifty-one months after diagnosis of the primary tumour, and 22 months after the first metastases, the patient is alive with excellent performance status, and multiple asymptomatic stable lung and liver lesions. Conclusions We report the eighth case of primary leiomyosarcoma of the seminal vesicle and the first one with a so long follow-up.

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

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    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.

  10. 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.

  11. Towards traceable size determination of extracellular vesicles

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    Zoltán Varga

    2014-02-01

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

  12. 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.

  13. Dimensional characterization of extracellular vesicles using atomic force microscopy

    International Nuclear Information System (INIS)

    Sebaihi, N; De Boeck, B; Pétry, J; Yuana, Y; Nieuwland, R

    2017-01-01

    Extracellular vesicles (EV) are small biological entities released from cells into body fluids. EV are recognized as mediators in intercellular communication and influence important physiological processes. It has been shown that the concentration and composition of EV in body fluids may differ from healthy subjects to patients suffering from particular disease. So, EV have gained a strong scientific and clinical interest as potential biomarkers for diagnosis and prognosis of disease. Due to their small size, accurate detection and characterization of EV remain challenging. The aim of the presented work is to propose a characterization method of erythrocyte-derived EV using atomic force microscopy (AFM). The vesicles are immobilized on anti-CD235a-modified mica and analyzed by AFM under buffer liquid and dry conditions. EV detected under both conditions show very similar sizes namely ∼30 nm high and ∼90 nm wide. The size of these vesicles remains stable over drying time as long as 7 d at room temperature. Since the detected vesicles are not spherical, EV are characterized by their height and diameter, and not only by the height as is usually done for spherical nanoparticles. In order to obtain an accurate measurement of EV diameters, the geometry of the AFM tip was evaluated to account for the lateral broadening artifact inherent to AFM measurements. To do so, spherical polystyrene (PS) nanobeads and EV were concomitantly deposited on the same mica substrate and simultaneously measured by AFM under dry conditions. By applying this procedure, direct calibration of the AFM tip could be performed together with EV characterization under identical experimental conditions minimizing external sources of uncertainty on the shape and size of the tip, thus allowing standardization of EV measurement. (paper)

  14. Structural Disorder Provides Increased Adaptability for Vesicle Trafficking Pathways

    Science.gov (United States)

    Tompa, Peter

    2013-01-01

    Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (∼23%) than the other two, COPI (∼9%) and COPII (∼8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and

  15. Astrocytic Vesicle Mobility in Health and Disease

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    Robert Zorec

    2013-05-01

    Full Text Available Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide, (ii plasma membrane exchange of transporters and receptors (EAAT2, MHC-II, and (iii the involvement of vesicle mobility carrying aquaporins (AQP4 in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

  16. EXTRACELLULAR VESICLES: CLASSIFICATION, FUNCTIONS AND CLINICAL RELEVANCE

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    A. V. Oberemko

    2014-12-01

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

  17. 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...

  18. Pasteurization of breastmilk decreases the rate of postnatally acquired cytomegalovirus infections, but shows a nonsignificant trend to an increased rate of necrotizing enterocolitis in very preterm infants--a preliminary study.

    Science.gov (United States)

    Stock, Katharina; Griesmaier, Elke; Brunner, Barbara; Neubauer, Vera; Kiechl-Kohlendorfer, Ursula; Trawöger, Rudolf

    2015-03-01

    This study assessed whether feeding preterm infants unpasteurized breastmilk (1) decreases the rate of late-onset sepsis and necrotizing enterocolitis and (2) increases the rate of postnatally acquired cytomegalovirus infections. Between January 2008 and July 2013, preterm infants below 32 completed weeks of gestational age admitted to the neonatal intensive care unit of Innsbruck Medical University (Innsbruck, Austria) (n=344) were eligible for the study. Of those, 323 fed breastmilk were retrospectively enrolled in the study. Two groups were formed, with 164 infants being fed unpasteurized and 159 infants being fed pasteurized breastmilk. There was no significant difference in the rate of late-onset sepsis or necrotizing enterocolitis between the unpasteurized and pasteurized breastmilk groups (late-onset sepsis, 15.9% versus 15.1% [p=0.486]; necrotizing enterocolitis, 2.4% versus 4.4% [p=0.254]). The number of infants diagnosed with postnatally acquired cytomegalovirus infection was significantly higher in the unpasteurized group (39.3%) compared with the pasteurized group (4.2%) (p=0.008). Feeding preterm infants unpasteurized breastmilk increases the rate of postnatally acquired cytomegalovirus infections. However, we also demonstrate a nonsignificant trend to a decreased rate of necrotizing enterocolitis in the unpasteurized group, which needs to be confirmed in larger studies.

  19. 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...

  20. 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.

  1. Functionalization of Block Copolymer Vesicle Surfaces

    Directory of Open Access Journals (Sweden)

    Wolfgang Meier

    2011-01-01

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

  2. Irradiation-induced fusion between giant vesicles and photoresponsive large unilamellar vesicles containing malachite green derivative.

    Science.gov (United States)

    Uda, Ryoko M; Yoshikawa, Yuki; Kitaba, Moe; Nishimoto, Noriko

    2018-07-01

    Light-initiated fusion between vesicles has attracted much attention in the research community. In particular, fusion between photoresponsive and non-photoresponsive vesicles has been of much interest in the development of systems for the delivery of therapeutic agents to cells. We have performed fusion between giant vesicles (GVs) and photoresponsive smaller vesicles containing malachite green (MG) derivative, which undergoes ionization to afford a positive charge on the molecule by irradiation. The fusion proceeds as the concentration of GV lipid increases toward equimolarity with the lipid of the smaller vesicle. It is also dependent on the molar percentage of photoionized MG in the lipid of the smaller vesicle. On the other hand, the fusion is hardly affected by the anionic component of the GV. The photoinduced fusion was characterized by two methods, involving the mixing of lipid membranes and of aqueous contents. Fluorescence microscopy revealed that irradiation triggered the fusion of a single GV with the smaller vesicles containing MG. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Surface glycosylation profiles of urine extracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Jared Q Gerlach

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

  4. Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles

    International Nuclear Information System (INIS)

    Goldman, M.; Yaari, A.; Moran, A.; Doshnitzki, Z.; Cohen-Luria, R.

    2006-01-01

    Since the Gulf war exposure to depleted uranium, a known nephrotoxic agent, there is a renewed interest in the toxic effects of uranium in general and its mechanism of nephrotoxicity which is still largely unknown in particular. In order to investigate the mechanism responsible for uranium nephrotoxicity and the therapeutic effect of urine alkalization, we utilized rat renal brush border membrane vesicles (BBMV). Uranyl acetate (UA) caused a decrease in glucose transport in BBMV. The apparent K i of uranyl was 139±30 μg uranyl/mg protein of BBMV. Uranyl at 140 μg/mg protein of BBMV reduced the maximal capacity of the system to transport glucose [V max 2.2±0.2 and 0.96±0.16 nmol/mg protein for control and uranyl treated BBMV (P m (1.54±0.33 and 1.54±0.51 mM for control, and uranyl treated BBMV, respectively). This reduction in V max is at least partially due to a decrease in the number of sodium-coupled glucose transporters as apparent from the reduction in phlorizin binding to the uranyl treated membranes, V max was reduced from 247±13 pmol/mg protein in control BBMV to 119±3 pmol/mg protein in treated vesicles (P<0.001). The pH of the medium has a profound effect on the toxicity of UA on sodium-coupled glucose transport in BBMV: higher toxicity at neutral pH (around pH 7.0), and practically no toxicity at alkaline pH (7.6). This is the first report showing a direct inhibitory dose and pH dependent effect of uranyl on the glucose transport system in isolated apical membrane from kidney cortex. (orig.)

  5. Contraction and AICAR Stimulate IL-6 Vesicle Depletion From Skeletal Muscle Fibers In Vivo

    DEFF Research Database (Denmark)

    Lauritzen, Hans P M M; Brandauer, Josef; Schjerling, Peter

    2013-01-01

    muscle fibers and in live animals in vivo. Using confocal imaging to visualize endogenous IL-6 protein in fixed muscle fibers, we found IL-6 in small vesicle structures distributed throughout the fibers under basal (resting) conditions. To determine the kinetics of IL-6 secretion, intact quadriceps...... muscles were transfected with enhanced green fluorescent protein (EGFP)-tagged IL-6 (IL-6-EGFP), and 5 days later anesthetized mice were imaged before and after muscle contractions in situ. Contractions decreased IL-6-EGFP-containing vesicles and protein by 62% (P

  6. FIJI Macro 3D ART VeSElecT: 3D Automated Reconstruction Tool for Vesicle Structures of Electron Tomograms.

    Directory of Open Access Journals (Sweden)

    Kristin Verena Kaltdorf

    2017-01-01

    Full Text Available Automatic image reconstruction is critical to cope with steadily increasing data from advanced microscopy. We describe here the Fiji macro 3D ART VeSElecT which we developed to study synaptic vesicles in electron tomograms. We apply this tool to quantify vesicle properties (i in embryonic Danio rerio 4 and 8 days past fertilization (dpf and (ii to compare Caenorhabditis elegans N2 neuromuscular junctions (NMJ wild-type and its septin mutant (unc-59(e261. We demonstrate development-specific and mutant-specific changes in synaptic vesicle pools in both models. We confirm the functionality of our macro by applying our 3D ART VeSElecT on zebrafish NMJ showing smaller vesicles in 8 dpf embryos then 4 dpf, which was validated by manual reconstruction of the vesicle pool. Furthermore, we analyze the impact of C. elegans septin mutant unc-59(e261 on vesicle pool formation and vesicle size. Automated vesicle registration and characterization was implemented in Fiji as two macros (registration and measurement. This flexible arrangement allows in particular reducing false positives by an optional manual revision step. Preprocessing and contrast enhancement work on image-stacks of 1nm/pixel in x and y direction. Semi-automated cell selection was integrated. 3D ART VeSElecT removes interfering components, detects vesicles by 3D segmentation and calculates vesicle volume and diameter (spherical approximation, inner/outer diameter. Results are collected in color using the RoiManager plugin including the possibility of manual removal of non-matching confounder vesicles. Detailed evaluation considered performance (detected vesicles and specificity (true vesicles as well as precision and recall. We furthermore show gain in segmentation and morphological filtering compared to learning based methods and a large time gain compared to manual segmentation. 3D ART VeSElecT shows small error rates and its speed gain can be up to 68 times faster in comparison to manual

  7. Appetite - decreased

    Science.gov (United States)

    Loss of appetite; Decreased appetite; Anorexia ... Any illness can reduce appetite. If the illness is treatable, the appetite should return when the condition is cured. Loss of appetite can cause weight ...

  8. 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

  9. Extracellular vesicles: fundamentals and clinical relevance

    Directory of Open Access Journals (Sweden)

    Wael Nassar

    2015-01-01

    Full Text Available All types of cells of eukaryotic organisms produce and release small nanovesicles into their extracellular environment. Early studies have described these vesicles as ′garbage bags′ only to remove obsolete cellular molecules. Valadi and colleagues, in 2007, were the first to discover the capability of circulating extracellular vesicles (EVs to horizontally transfer functioning gene information between cells. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, chemoresistance, genetic exchange, and signaling pathway activation through growth factor/receptor transfer. EVs represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, signaling proteins, and RNAs. They contribute to physiology and pathology, and they have a myriad of potential clinical applications in health and disease. Moreover, vesicles can pass the blood-brain barrier and may perhaps even be considered as naturally occurring liposomes. These cell-derived EVs not only represent a central mediator of the disease microenvironment, but their presence in the peripheral circulation may serve as a surrogate for disease biopsies, enabling real-time diagnosis and disease monitoring. In this review, we′ll be addressing the characteristics of different types of extracellular EVs, as well as their clinical relevance and potential as diagnostic markers, and also define therapeutic options.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. Nanoplasmonic ruler to measure lipid vesicle deformation

    Czech Academy of Sciences Publication Activity Database

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

    2016-01-01

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

  15. Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis.

    Science.gov (United States)

    Burré, Jacqueline; Beckhaus, Tobias; Corvey, Carsten; Karas, Michael; Zimmermann, Herbert; Volknandt, Walter

    2006-09-01

    Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.

  16. Synaptic vesicle dynamic changes in a model of fragile X.

    Science.gov (United States)

    Broek, Jantine A C; Lin, Zhanmin; de Gruiter, H Martijn; van 't Spijker, Heleen; Haasdijk, Elize D; Cox, David; Ozcan, Sureyya; van Cappellen, Gert W A; Houtsmuller, Adriaan B; Willemsen, Rob; de Zeeuw, Chris I; Bahn, Sabine

    2016-01-01

    Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MS(E)) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS.

  17. 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.

  18. High-speed centrifugation induces aggregation of extracellular vesicles.

    Science.gov (United States)

    Linares, Romain; Tan, Sisareuth; Gounou, Céline; Arraud, Nicolas; Brisson, Alain R

    2015-01-01

    Plasma and other body fluids contain cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential biomedical applications. In order to isolate, concentrate and purify EVs, high-speed centrifugation is often used. We show here, using electron microscopy, receptor-specific gold labelling and flow cytometry, that high-speed centrifugation induces the formation of EV aggregates composed of a mixture of EVs of various phenotypes and morphologies. The presence of aggregates made of EVs of different phenotypes may lead to erroneous interpretation concerning the existence of EVs harbouring surface antigens from different cell origins.

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

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2008-01-01

    and eventually opens a pore to complete the fusion process. In pathway II, at higher tension, a stalk is formed during the fusion process that is then transformed by transmembrane pore formation into a fusion pore. Whereas the latter pathway II resembles stalk pathways as observed in other simulation studies......, fusion pathway I, which does not involve any stalk formation, has not been described previously to the best of our knowledge. A statistical analysis of the various processes shows that fusion is the dominant pathway for releasing the tension of the vesicles. The functional dependence of the observed...

  20. High-speed centrifugation induces aggregation of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Romain Linares

    2015-12-01

    Full Text Available Plasma and other body fluids contain cell-derived extracellular vesicles (EVs, which participate in physiopathological processes and have potential biomedical applications. In order to isolate, concentrate and purify EVs, high-speed centrifugation is often used. We show here, using electron microscopy, receptor-specific gold labelling and flow cytometry, that high-speed centrifugation induces the formation of EV aggregates composed of a mixture of EVs of various phenotypes and morphologies. The presence of aggregates made of EVs of different phenotypes may lead to erroneous interpretation concerning the existence of EVs harbouring surface antigens from different cell origins.

  1. Synthesis of Copper nanoparticles through vesicle template using gamma irradiation

    International Nuclear Information System (INIS)

    Noor Ezzah Rahimah Ahmad Samsuri

    2012-01-01

    Nano technology has gained attention for its application in life. This study was conducted to produce copper (Cu) nanoparticles using gamma ray irradiation through template vesicles. Cu nanoparticle has a variety of applications such as capacitor materials, catalyst, conductive coating, high thermal conductivity materials as well as lubricant additives. this study used gamma radiation compared to other methods because the use of gamma rays in producing nanoparticle is safer and environmental friendly. The purpose of this study was to see the effects of radiation on the formation of Cu nanoparticles. The radiation dose used was 80 kGy and 100 kGy. The vesicles were formed by mixing water, sodium n-lauroyl sarcosinat hydrated, 1-decanol and polyethylene glycol with certain ratio (85 %: 5 %: 7 %: 3 %). Analysis from the transmission electron microscopy (TEM) showed the production of multilammelar vesicles in size between 30 nm-80 nm. The formation of nanoparticles was analyzed using UV-Vis absorption spectroscopy, X-ray diffraction (XRD) and transmission electron microscopy (TEM). Analysis of UV-Vis absorption spectroscopy showed no resonance peak around 600 nm. XRD analysis confirmed the presence of Cu, Cu 2 O and CuO. Analysis and characterisation using transmission electron microscopy (TEM) also confirmed that nanoparticles were produced with different sizes according to the radiation dose. At the radiation dose of 80 kGy, nanoparticles size is found vary between 30 nm to 90 nm. While at the radiation dose of 100 kGy, nanoparticles size is found vary between 3 nm to 7 nm. From this study it can be concluded that higher radiation dse will produce smaller nanoparticles. (author)

  2. 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.

  3. 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....

  4. The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

    Directory of Open Access Journals (Sweden)

    Irini Topalidou

    2016-05-01

    Full Text Available The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.

  5. Synaptic vesicle exocytosis in hippocampal synaptosomes correlates directly with total mitochondrial volume

    Science.gov (United States)

    Ivannikov, Maxim V.; Sugimori, Mutsuyuki; Llinás, Rodolfo R.

    2012-01-01

    Synaptic plasticity in many regions of the central nervous system leads to the continuous adjustment of synaptic strength, which is essential for learning and memory. In this study, we show by visualizing synaptic vesicle release in mouse hippocampal synaptosomes that presynaptic mitochondria and specifically, their capacities for ATP production are essential determinants of synaptic vesicle exocytosis and its magnitude. Total internal reflection microscopy of FM1-43 loaded hippocampal synaptosomes showed that inhibition of mitochondrial oxidative phosphorylation reduces evoked synaptic release. This reduction was accompanied by a substantial drop in synaptosomal ATP levels. However, cytosolic calcium influx was not affected. Structural characterization of stimulated hippocampal synaptosomes revealed that higher total presynaptic mitochondrial volumes were consistently associated with higher levels of exocytosis. Thus, synaptic vesicle release is linked to the presynaptic ability to regenerate ATP, which itself is a utility of mitochondrial density and activity. PMID:22772899

  6. SNX9 - a prelude to vesicle release.

    Science.gov (United States)

    Lundmark, Richard; Carlsson, Sven R

    2009-01-01

    The sorting nexin SNX9 has, in the past few years, been singled out as an important protein that participates in fundamental cellular activities. SNX9 binds strongly to dynamin and is partly responsible for the recruitment of this GTPase to sites of endocytosis. SNX9 also has a high capacity for modulation of the membrane and might therefore participate in the formation of the narrow neck of endocytic vesicles before scission occurs. Once assembled on the membrane, SNX9 stimulates the GTPase activity of dynamin to facilitate the scission reaction. It has also become clear that SNX9 has the ability to activate the actin regulator N-WASP in a membrane-dependent manner to coordinate actin polymerization with vesicle release. In this Commentary, we summarize several aspects of SNX9 structure and function in the context of membrane remodeling, discuss its interplay with various interaction partners and present a model of how SNX9 might work in endocytosis.

  7. Stem cell extracellular vesicles and kidney injury

    OpenAIRE

    Grange, Cristina; Iampietro, Corinne; Bussolati, Benedetta

    2017-01-01

    Extracellular vesicles (EVs) appear as a new promising cell-free therapy for acute and chronic renal diseases. EVs retain characteristics of the cell of origin and those derived from stem cells may mimic their regenerative properties per se. In fact, EVs contain many active molecules such as proteins and RNA species that act on target cells through different mechanisms, stimulating proliferation and angiogenesis and reducing apoptosis and inflammation. There are several reports that demonstra...

  8. Endothelial microparticles: Sophisticated vesicles modulating vascular function

    Science.gov (United States)

    Curtis, Anne M; Edelberg, Jay; Jonas, Rebecca; Rogers, Wade T; Moore, Jonni S; Syed, Wajihuddin; Mohler, Emile R

    2015-01-01

    Endothelial microparticles (EMPs) belong to a family of extracellular vesicles that are dynamic, mobile, biological effectors capable of mediating vascular physiology and function. The release of EMPs can impart autocrine and paracrine effects on target cells through surface interaction, cellular fusion, and, possibly, the delivery of intra-vesicular cargo. A greater understanding of the formation, composition, and function of EMPs will broaden our understanding of endothelial communication and may expose new pathways amenable for therapeutic manipulation. PMID:23892447

  9. Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.

    Science.gov (United States)

    Zeelenberg, Ingrid S; Ostrowski, Matias; Krumeich, Sophie; Bobrie, Angélique; Jancic, Carolina; Boissonnas, Alexandre; Delcayre, Alain; Le Pecq, Jean-Bernard; Combadière, Béhazine; Amigorena, Sebastian; Théry, Clotilde

    2008-02-15

    Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

  10. 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.

  11. Light induced generation of a proton motive force and Ca++- transport in membrane vesicles of Streptococcus cremoris fused with bacteriorhodopsin proteoliposomes

    International Nuclear Information System (INIS)

    Driessen, A.J.M.; Hellingwerf, K.J.; Konings, W.N.

    1985-01-01

    This paper demonstrates that S. cremoris membrane vesicles efficiently fuse with Brh proteoliposomes at low pH which leads to a functional incorporation of Brh into S. cremoris membrane vesicle. The growth of the cells and preparation of the membrane vesicles are described. Fusion, binding, and calcium transport assays were examined. In order to test fusion between S. cremoris membrane vesicles and Brh proteoliposomes the authors applied the resonance energy transfer fusion assay which monitors changes in the spatial organization of two fluorescent lipid probes in the membrane. It is shown that mixing of equal quantities of S. cremoris membrane vesicles and Brh proteoliposomes at low pH resulted in a decrease of the fluorescence energy transfer efficiency, monitored as a nincrease in NBD fluorescence

  12. 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.

  13. Routes and mechanisms of extracellular vesicle uptake

    Directory of Open Access Journals (Sweden)

    Laura Ann Mulcahy

    2014-08-01

    Full Text Available Extracellular vesicles (EVs are small vesicles released by donor cells that can be taken up by recipient cells. Despite their discovery decades ago, it has only recently become apparent that EVs play an important role in cell-to-cell communication. EVs can carry a range of nucleic acids and proteins which can have a significant impact on the phenotype of the recipient. For this phenotypic effect to occur, EVs need to fuse with target cell membranes, either directly with the plasma membrane or with the endosomal membrane after endocytic uptake. EVs are of therapeutic interest because they are deregulated in diseases such as cancer and they could be harnessed to deliver drugs to target cells. It is therefore important to understand the molecular mechanisms by which EVs are taken up into cells. This comprehensive review summarizes current knowledge of EV uptake mechanisms. Cells appear to take up EVs by a variety of endocytic pathways, including clathrin-dependent endocytosis, and clathrin-independent pathways such as caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft–mediated internalization. Indeed, it seems likely that a heterogeneous population of EVs may gain entry into a cell via more than one route. The uptake mechanism used by a given EV may depend on proteins and glycoproteins found on the surface of both the vesicle and the target cell. Further research is needed to understand the precise rules that underpin EV entry into cells.

  14. Phospholipid transfer from vesicles to high density lipoproteins, catalyzed by human plasma phospholipid transfer protein

    International Nuclear Information System (INIS)

    Sweeny, S.A.

    1985-01-01

    Human plasma phospholipid transfer protein (PLTP) catalyzes the mass transfer of phosphatidylcholine (PC). Partial purification of PLTP yielded proteins with apparent M/sub r/ = 59,000 and 40,000 by SDS-PAGE. PLTP activity was measured by transfer of [ 14 C]L-α-dipalmitoyl PC from egg-PC vesicles to HDL. Activity was enhanced at low pH (4.5) upon addition of β-mercaptoethanol while Ca +2 and Na + had no effect. E/sub act/ for facilitated PC transfer was 18.2 +/- 2 kcal/mol. The donor specificity of PLTP was examined using vesicles containing egg-PC plus cholesterol or sphingomyelin. The fluidity of the donor membrane (measured by fluorescence polarization of diphenylhexatriene) correlated strongly with a decrease in PLTP activity. Phosphatidic acid did not affect activity. Increase in vesicle size reduced activity. The acceptor specificity of PLTP was examined using chemically modified HDL. PLTP activity increased up to 1.7-fold with an initial increase in negative charge and then decreased upon extensive modification. A mechanism is proposed where PLTP binds to vesicls and enhances the diffusion of PC into the medium where it is adsorbed by HDL

  15. Contraction and AICAR stimulate IL-6 vesicle depletion from skeletal muscle fibers in vivo.

    Science.gov (United States)

    Lauritzen, Hans P M M; Brandauer, Josef; Schjerling, Peter; Koh, Ho-Jin; Treebak, Jonas T; Hirshman, Michael F; Galbo, Henrik; Goodyear, Laurie J

    2013-09-01

    Recent studies suggest that interleukin 6 (IL-6) is released from contracting skeletal muscles; however, the cellular origin, secretion kinetics, and signaling mechanisms regulating IL-6 secretion are unknown. To address these questions, we developed imaging methodology to study IL-6 in fixed mouse muscle fibers and in live animals in vivo. Using confocal imaging to visualize endogenous IL-6 protein in fixed muscle fibers, we found IL-6 in small vesicle structures distributed throughout the fibers under basal (resting) conditions. To determine the kinetics of IL-6 secretion, intact quadriceps muscles were transfected with enhanced green fluorescent protein (EGFP)-tagged IL-6 (IL-6-EGFP), and 5 days later anesthetized mice were imaged before and after muscle contractions in situ. Contractions decreased IL-6-EGFP-containing vesicles and protein by 62% (P contraction. However, contraction-mediated IL-6-EGFP reduction was normal in muscle-specific AMP-activated protein kinase (AMPK) α2-inactive transgenic mice. In contrast, the AMPK activator AICAR decreased IL-6-EGFP vesicles, an effect that was inhibited in the transgenic mice. In conclusion, resting skeletal muscles contain IL-6-positive vesicles that are expressed throughout myofibers. Contractions stimulate the rapid reduction of IL-6 in myofibers, occurring through an AMPKα2-independent mechanism. This novel imaging methodology clearly establishes IL-6 as a contraction-stimulated myokine and can be used to characterize the secretion kinetics of other putative myokines.

  16. 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

  17. 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....

  18. Buffer-induced swelling and vesicle budding in binary lipid mixtures of dioleoylphosphatidylcholine:dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine:lysophosphatidylcholine using small-angle X-ray scattering and ³¹P static NMR.

    Science.gov (United States)

    Barriga, Hanna M G; Bazin, Richard; Templer, Richard H; Law, Robert V; Ces, Oscar

    2015-03-17

    A large variety of data exists on lipid phase behavior; however, it is mostly in nonbuffered systems over nonbiological temperature ranges. We present biophysical data on lipid mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and lysophosphatidylcholine (LysoPC) examining their behaviors in excess water and buffer systems over the temperature range 4-34 °C. These mixtures are commonly used to investigate the effects of spontaneous curvature on integral membrane proteins. Using small-angle X-ray scattering (SAXS) and (31)P NMR, we observed lamellar and vesicle phases, with the buffer causing an increase in the layer spacing. Increasing amounts of DOPE in a DOPC bilayer decreased the layer spacing of the mesophase, while the opposite trend was observed for increasing amounts of LysoPC. (31)P static NMR was used to analyze the DOPC:LysoPC samples to investigate the vesicle sizes present, with evidence of vesicle budding observed at LysoPC concentrations above 30 mol %. NMR line shapes were fitted using an adapted program accounting for the distortion of the lipids within the magnetic field. The distortion of the vesicle, because of magnetic susceptibility, varied with LysoPC content, and a discontinuity was found in both the water and buffer samples. Generally, the distortion increased with LysoPC content; however, at a ratio of DOPC:LysoPC 60:40, the sample showed a level of distortion of the vesicle similar to that of pure DOPC. This implies an increased flexibility in the membrane at this point. Commonly, the assumption is that for increasing LysoPC concentration there is a reduction in membrane tension, implying that estimations of membrane tension based on spontaneous curvature assumptions may not be accurate.

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

    Directory of Open Access Journals (Sweden)

    Francisco F De-Miguel

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

  20. 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.

  1. Intracellular vesicle acidification promotes maturation of infectious poliovirus particles.

    Directory of Open Access Journals (Sweden)

    Alexsia L Richards

    Full Text Available The autophagic pathway acts as part of the immune response against a variety of pathogens. However, several pathogens subvert autophagic signaling to promote their own replication. In many cases it has been demonstrated that these pathogens inhibit or delay the degradative aspect of autophagy. Here, using poliovirus as a model virus, we report for the first time bona fide autophagic degradation occurring during infection with a virus whose replication is promoted by autophagy. We found that this degradation is not required to promote poliovirus replication. However, vesicular acidification, which in the case of autophagy precedes delivery of cargo to lysosomes, is required for normal levels of virus production. We show that blocking autophagosome formation inhibits viral RNA synthesis and subsequent steps in the virus cycle, while inhibiting vesicle acidification only inhibits the final maturation cleavage of virus particles. We suggest that particle assembly, genome encapsidation, and virion maturation may occur in a cellular compartment, and we propose the acidic mature autophagosome as a candidate vesicle. We discuss the implications of our findings in understanding the late stages of poliovirus replication, including the formation and maturation of virions and egress of infectious virus from cells.

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

    Directory of Open Access Journals (Sweden)

    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

  3. 3D estimation of synaptic vesicle distributions in serial section transmission electron microscopy

    DEFF Research Database (Denmark)

    Khanmohammadi, Mahdieh; Darkner, Sune; Nava, Nicoletta

    inhomogeneity correction and image registration, we obtained the histogram of minimum distances of vesicles to the active zone with the traditional method and two proposed improvements. Comparing the traditional method with either of the two suggested methods using Wilcoxon signed rank test, shows a strong...

  4. 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...

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

    Science.gov (United States)

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

    2016-03-02

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

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

    OpenAIRE

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

    2016-01-01

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

  7. ARHGEF10 directs the localization of Rab8 to Rab6-positive executive vesicles.

    Science.gov (United States)

    Shibata, Satoshi; Kawanai, Tsubasa; Hara, Takayuki; Yamamoto, Asuka; Chaya, Taro; Tokuhara, Yasunori; Tsuji, Chinami; Sakai, Manabu; Tachibana, Taro; Inagaki, Shinobu

    2016-10-01

    The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)-Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si)RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner. © 2016. Published by The Company of Biologists Ltd.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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....

  13. Matrix vesicles in the fibrous cap of atherosclerotic plaque: possible contribution to plaque rupture.

    Science.gov (United States)

    Bobryshev, Y V; Killingsworth, M C; Lord, R S A; Grabs, A J

    2008-10-01

    Plaque rupture is the most common type of plaque complication and leads to acute ischaemic events such as myocardial infarction and stroke. Calcification has been suggested as a possible indicator of plaque instability. Although the role of matrix vesicles in the initial stages of arterial calcification has been recognized, no studies have yet been carried out to examine a possible role of matrix vesicles in plaque destabilization. Tissue specimens selected for the present study represented carotid specimens obtained from patients undergoing carotid endarterectomy. Serial frozen cross-sections of the tissue specimens were cut and mounted on glass slides. The thickness of the fibrous cap (FCT) in each advanced atherosclerotic lesion, containing a well developed lipid/necrotic core, was measured at its narrowest sites in sets of serial sections. According to established criteria, atherosclerotic plaque specimens were histologically subdivided into two groups: vulnerable plaques with thin fibrous caps (FCT <100 microm) and presumably stable plaques, in which fibrous caps were thicker than 100 microm. Twenty-four carotid plaques (12 vulnerable and 12 presumably stable plaques) were collected for the present analysis of matrix vesicles in fibrous caps. In order to provide a sufficient number of representative areas from each plaque, laser capture microdissection (LCM) was carried out. The quantification of matrix vesicles in ultrathin sections of vulnerable and stable plaques revealed that the numbers of matrix vesicles were significantly higher in fibrous caps of vulnerable plaques than those in stable plaques (8.908+0.544 versus 6.208+0.467 matrix vesicles per 1.92 microm2 standard area; P= 0.0002). Electron microscopy combined with X-ray elemental microanalysis showed that some matrix vesicles in atherosclerotic plaques were undergoing calcification and were characterized by a high content of calcium and phosphorus. The percentage of calcified matrix vesicles

  14. 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.

  15. Cholesterol affects the interaction between an ionic liquid and phospholipid vesicles. A study by differential scanning calorimetry and nanoplasmonic sensing.

    Science.gov (United States)

    Russo, Giacomo; Witos, Joanna; Rantamäki, Antti H; Wiedmer, Susanne K

    2017-12-01

    The present work aims at studying the interactions between cholesterol-rich phosphatidylcholine-based lipid vesicles and trioctylmethylphosphonium acetate ([P 8881 ][OAc]), a biomass dissolving ionic liquid (IL). The effect of cholesterol was assayed by using differential scanning calorimetry (DSC) and nanoplasmonic sensing (NPS) measurement techniques. Cholesterol-enriched dipalmitoyl-phosphatidylcholine vesicles were exposed to different concentrations of the IL, and the derived membrane perturbation was monitored by DSC. The calorimetric data could suggest that the binding and infiltration of the IL are delayed in the vesicles containing cholesterol. To clarify our findings, NPS was applied to quantitatively follow the resistance of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine incorporating 0, 10, and 50mol% of cholesterol toward the IL exposure over time. The membrane perturbation induced by different concentrations of IL was found to be a concentration dependent process on cholesterol-free lipid vesicles. Moreover, our results showed that lipid depletion in cholesterol-enriched lipid vesicles is inversely proportional to the increasing amount of cholesterol in the vesicles. These findings support that cholesterol-rich lipid bilayers are less susceptible toward membrane disrupting agents as compared to membranes that do not incorporate any sterols. This probably occurs because cholesterol tightens the phospholipid acyl chain packing of the plasma membranes, increasing their resistance and reducing their permeability. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Nonenzymatic glycation of phosphatidylethanolamine in erythrocyte vesicles

    International Nuclear Information System (INIS)

    Patkowska, M.J.; Horowitz, M.I.

    1986-01-01

    Unsealed inside-out and right-side out vesicles were prepared from human red cells. The vesicles were incubated with D-glucose [ 14 C(U)] and sodium cyanoborohydride in phosphate buffer, pH 7.4. After incubation, lipids were extracted with 1-butanol and non-lipid contaminants removed by Sephadex G-25 chromatography. Phosphatidylethanolamine-sorbitol was purified by chromatography on columns of silicic acid and phenylboronate agarose gel. Phospholipase C (B. cereus) liberated phosphoethanolamine-sorbitol (I) which comigrated on TLC with synthetic I prepared by reductive condensation of phosphoethanolamine and D-glucose and also with the product of phospholipase C (B. cereus) hydrolysis of reference phosphatidylethanolamine-sorbitol. Exposure of I to alkaline phosphatase (E. coli) gave P/sub i/ and ethanolamine-sorbitol (II) which comigrated on TLC with synthetic II prepared by reductive condensation of ethanolamine and D-glucose or by phospholipase D hydrolysis of reference phosphatidylethanolamine-sorbitol. These studies demonstrate that vesicular phosphatidylethanolamine can be reductively glycated and illustrate the applicability of both phospholipase C and phospholipase D in characterizing glycated phosphoglycerides

  17. Mechanical collapse of confined fluid membrane vesicles.

    Science.gov (United States)

    Rim, Jee E; Purohit, Prashant K; Klug, William S

    2014-11-01

    Compact cylindrical and spherical invaginations are common structural motifs found in cellular and developmental biology. To understand the basic physical mechanisms that produce and maintain such structures, we present here a simple model of vesicles in confinement, in which mechanical equilibrium configurations are computed by energy minimization, balancing the effects of curvature elasticity, contact of the membrane with itself and the confining geometry, and adhesion. For cylindrical confinement, the shape equations are solved both analytically and numerically by finite element analysis. For spherical confinement, axisymmetric configurations are obtained numerically. We find that the geometry of invaginations is controlled by a dimensionless ratio of the adhesion strength to the bending energy of an equal area spherical vesicle. Larger adhesion produces more concentrated curvatures, which are mainly localized to the "neck" region where the invagination breaks away from its confining container. Under spherical confinement, axisymmetric invaginations are approximately spherical. For extreme confinement, multiple invaginations may form, bifurcating along multiple equilibrium branches. The results of the model are useful for understanding the physical mechanisms controlling the structure of lipid membranes of cells and their organelles, and developing tissue membranes.

  18. Yarrowia lipolytica vesicle-mediated protein transport pathways

    Directory of Open Access Journals (Sweden)

    Beckerich Jean-Marie

    2007-11-01

    Full Text Available Abstract Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii. These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular

  19. Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

    Science.gov (United States)

    Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

    2014-11-24

    Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

  20. Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane.

    Science.gov (United States)

    Roz, Netta; Rehavi, Moshe

    2003-06-13

    Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.

  1. Impact of titanium dioxide on androgen receptors, seminal vesicles and thyroid hormones of male rats: possible protective trial with aged garlic extract.

    Science.gov (United States)

    Abu Zeid, E H; Alam, R T M; Abd El-Hameed, N E

    2017-06-01

    The aim of this study was to evaluate the effect of titanium dioxide (TiO 2 ), a widely produced and consumed pigment in various food products, on the post-natal development of male albino rat seminal vesicle and thyroid hormones, as well as to evaluate the ameliorative effect of aged garlic extract (AGE) on TiO 2 -induced alterations. Forty male rat pups (3 weeks old) were divided into four equal groups. The 1st group received distilled water orally (control group), 2nd group was given 2 ml kg -1 AGE, 3rd group was administered TiO 2 (5 g kg -1 BW) day after day for 65 days, and the 4th group administered AGE 6 hr prior to TiO 2 gavage. TiO 2 -exposed rats showed nonsignificant changes in the serum testosterone, TSH, T 3 and T 4 , while serum glucose showed a significant decrease. Androgen receptor (AR) mRNA expression was significantly down-regulated and weak signal of AR immune labelling. Histopathologically, the epithelium cell lining of seminal vesicles showed focal areas of necrosis and fibrous tissue with the prominent fibrous stroma of the atrophied glands. Meanwhile, AGE supplementation ameliorated the deleterious effects of TiO 2 intoxication through protecting the tissues from oxidative stress caused by TiO 2 . In summary, oral administration of TiO 2 resulted in abnormal developmental events in male rat seminal vesicle and AGE able to reduce TiO 2 toxicity. © 2016 Blackwell Verlag GmbH.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. Model of separated form factors for unilamellar vesicles

    International Nuclear Information System (INIS)

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

    2001-01-01

    A new model of separated form factors is proposed for the evaluation of small-angle neutron scattering curves from large unilamellar vesicles. The validity of the model was checked via comparison with the model of a hollow sphere. The model of separated form factors and the hollow sphere model give a reasonable agreement in the evaluation of vesicle parameters

  8. 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

  9. 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

  10. Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system.

    Science.gov (United States)

    Lerner, Natalie; Avissar, Sofia; Beit-Yannai, Elie

    2017-01-01

    Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood. Exosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot. Exosomes derived from NPCE cells were purified and detected as small rounded 50-140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118-1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of

  11. Membrane Protrusion Coarsening and Nanotubulation within Giant Unilamellar Vesicles

    KAUST Repository

    Węgrzyn, Ilona

    2011-11-16

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

  12. 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.

  13. 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

  14. Chloroquine causes similar electroretinogram modifications, neuronal phospholipidosis and marked impairment of synaptic vesicle transport in Albino and Pigmented Rats

    International Nuclear Information System (INIS)

    Lezmi, Stéphane; Rokh, Najla; Saint-Macary, Gérard; Pino, Michael; Sallez, Valérie; Thevenard, Françoise; Roome, Nigel; Rosolen, Serge

    2013-01-01

    Retinal toxicity of chloroquine has been known for several years, but the mechanism(s) of toxicity remain controversial; some author support the idea that the binding of chloroquine to melanin pigments in the retinal pigmented epithelium (RPE) play a major toxic role by concentrating the drug in the eye. In our study, 12 albinos Sprague-Dawley (SD) and 12 pigmented Brown Norway (BN) rats were treated orally for 3 months with chloroquine to compare functional and pathological findings. On Flash electroretinograms (ERG) performed in scotopic conditions, similar and progressive (time-dependent) delayed onset and decreased amplitudes of oscillatory potentials (from Day 71) and b-waves (on Day 92) were identified in both BN and SD rats. In both strains, identical morphological changes consisted of neuronal phospholipidosis associated with UV auto-fluorescence without evidence of retinal degeneration and gliosis; the RPE did not show any morphological lesions or autofluorescence. IHC analyses demonstrated a decrease in GABA expression in the inner nuclear layer. In addition, a marked accumulation of synaptic vesicles coupled with a marked disruption of neurofilaments in the optic nerve fibers was identified. In conclusion, ERG observations were very similar to those described in humans. Comparable ERG modifications, histopathology and immunohistochemistry findings were observed in the retina of both rat strains suggesting that melanin pigment is unlikely involved. chloroquine-induced impairment of synaptic vesicle transport, likely related to disruption of neurofilaments was identified and non-previously reported. This new mechanism of toxicity may also be responsible for the burry vision described in humans chronically treated with chloroquine

  15. Chloroquine causes similar electroretinogram modifications, neuronal phospholipidosis and marked impairment of synaptic vesicle transport in albino and pigmented rats.

    Science.gov (United States)

    Lezmi, Stéphane; Rokh, Najla; Saint-Macary, Gérard; Pino, Michael; Sallez, Valérie; Thevenard, Françoise; Roome, Nigel; Rosolen, Serge

    2013-06-07

    Retinal toxicity of chloroquine has been known for several years, but the mechanism(s) of toxicity remain controversial; some author support the idea that the binding of chloroquine to melanin pigments in the retinal pigmented epithelium (RPE) play a major toxic role by concentrating the drug in the eye. In our study, 12 albinos Sprague-Dawley (SD) and 12 pigmented Brown Norway (BN) rats were treated orally for 3 months with chloroquine to compare functional and pathological findings. On Flash electroretinograms (ERG) performed in scotopic conditions, similar and progressive (time-dependent) delayed onset and decreased amplitudes of oscillatory potentials (from Day 71) and b-waves (on Day 92) were identified in both BN and SD rats. In both strains, identical morphological changes consisted of neuronal phospholipidosis associated with UV auto-fluorescence without evidence of retinal degeneration and gliosis; the RPE did not show any morphological lesions or autofluorescence. IHC analyses demonstrated a decrease in GABA expression in the inner nuclear layer. In addition, a marked accumulation of synaptic vesicles coupled with a marked disruption of neurofilaments in the optic nerve fibers was identified. In conclusion, ERG observations were very similar to those described in humans. Comparable ERG modifications, histopathology and immunohistochemistry findings were observed in the retina of both rat strains suggesting that melanin pigment is unlikely involved. chloroquine-induced impairment of synaptic vesicle transport, likely related to disruption of neurofilaments was identified and non-previously reported. This new mechanism of toxicity may also be responsible for the burry vision described in humans chronically treated with chloroquine. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  16. Presence of Cx43 in extracellular vesicles reduces the cardiotoxicity of the anti-tumour therapeutic approach with doxorubicin

    Directory of Open Access Journals (Sweden)

    Tania Martins-Marques

    2016-09-01

    Full Text Available Extracellular vesicles (EVs are major conveyors of biological information, mediating local and systemic cell-to-cell communication under physiological and pathological conditions. These endogenous vesicles have been recognized as prominent drug delivery vehicles of several therapeutic cargoes, including doxorubicin (dox, presenting major advantages over the classical approaches. Although dox is one of the most effective anti-tumour agents in the clinical practice, its use is very often hindered by its consequent dramatic cardiotoxicity. Despite significant advances witnessed in the past few years, more comprehensive studies, supporting the therapeutic efficacy of EVs, with decreased side effects, are still scarce. The main objective of this study was to evaluate the role of the gap junction protein connexin43 (Cx43 in mediating the release of EV content into tumour cells. Moreover, we investigated whether Cx43 improves the efficiency of dox-based anti-tumour treatment, with a concomitant decrease of cardiotoxicity. In the present report, we demonstrate that the presence of Cx43 in EVs increases the release of luciferin from EVs into tumour cells in vitro and in vivo. In addition, using cell-based approaches and a subcutaneous mouse tumour model, we show that the anti-tumour effect of dox incorporated into EVs is similar to the administration of the free drug, regardless the presence of Cx43. Strikingly, we demonstrate that the presence of Cx43 in dox-loaded EVs reduces the cardiotoxicity of the drug. Altogether, these results bring new insights into the concrete potential of EVs as therapeutic vehicles and open new avenues toward the development of strategies that help to reduce unwanted side effects.

  17. Nanodesign of olein vesicles for the topical delivery of the antioxidant resveratrol.

    Science.gov (United States)

    Pando, Daniel; Caddeo, Carla; Manconi, Maria; Fadda, Anna Maria; Pazos, Carmen

    2013-08-01

    The ex-vivo percutaneous absorption of the natural antioxidant resveratrol in liposomes and niosomes was investigated. The influence of vesicle composition on their physicochemical properties and stability was evaluated. Liposomes containing resveratrol were formulated using soy phosphatidylcholine (Phospholipon90G). Innovative niosomes were formulated using mono- or diglycerides: glycerol monooleate (Peceol) and polyglyceryl-3 dioleate (Plurol OleiqueCC), respectively, two suitable skin-compatible oleins used in pharmaceutical formulations as penetration enhancers. Small, negatively charged vesicles with a mean size of approximately 200 nm were prepared. The accelerated stability of vesicles was evaluated using Turbiscan Lab Expert, and the bilayer deformability was also assessed. Ex-vivo transdermal experiments were carried out in Franz diffusion cells, on newborn pig skin, to study the influence of the different vesicle formulations on resveratrol skin delivery. Results indicated a high cutaneous accumulation and a low transdermal delivery of resveratrol, especially when Peceol niosomes were used. Overall, niosomes formulated with Plurol oleique or Peceol showed a better behaviour than liposomes in the cutaneous delivery of resveratrol. © 2013 Royal Pharmaceutical Society.

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

    Directory of Open Access Journals (Sweden)

    Bartosz Różycki

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

  19. 6K2-induced vesicles can move cell to cell during turnip mosaic virus infection

    Directory of Open Access Journals (Sweden)

    Romain eGrangeon

    2013-12-01

    Full Text Available To successfully infect plants, viruses replicate in an initially infected cell and then move to neighboring cells through plasmodesmata (PDs. However, the nature of the viral entity that crosses over the cell barrier into non-infected ones is not clear. The membrane-associated 6K2 protein of turnip mosaic virus (TuMV induces the formation of vesicles involved in the replication and intracellular movement of viral RNA. This study shows that 6K2-induced vesicles trafficked towards the plasma membrane and were associated with plasmodesmata (PD. We demonstrated also that 6K2 moved cell-to-cell into adjoining cells when plants were infected with TuMV. 6K2 was then fused to photo-activable GFP (6K2:PAGFP to visualize how 6K2 move intercellularly during TuMV infection. After activation, 6K2:PAGFP-tagged vesicles moved to the cell periphery and across the cell wall into adjacent cells. These vesicles were shown to contain the viral RNA-dependent RNA polymerase and viral RNA. Symplasmic movement of TuMV may thus be achieved in the form of a membrane-associated viral RNA complex induced by 6K2.

  20. Joint small-angle X-ray and neutron scattering data analysis of asymmetric lipid vesicles

    International Nuclear Information System (INIS)

    Eicher, Barbara; Heberle, Frederick A.; Marquardt, Drew T.

    2017-01-01

    Low- and high-resolution models describing the internal transbilayer structure of asymmetric lipid vesicles have been developed. These models can be used for the joint analysis of small-angle neutron and X-ray scattering data. The models describe the underlying scattering length density/electron density profiles either in terms of slabs or through the so-called scattering density profile, previously applied to symmetric lipid vesicles. Both models yield structural details of asymmetric membranes, such as the individual area per lipid, and the hydrocarbon thickness of the inner and outer bilayer leaflets. The scattering density profile model, however, comes at a cost of increased computational effort but results in greater structural resolution, showing a slightly lower packing of lipids in the outer bilayer leaflet of ~120 nm diameter palmitoyloleoyl phosphatidylcholine (POPC) vesicles, compared to the inner leaflet. Here, analysis of asymmetric dipalmitoyl phosphatidylcholine/POPC vesicles did not reveal evidence of transbilayer coupling between the inner and outer leaflets at 323 K,i.e.above the melting transition temperature of the two lipids.

  1. 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.

  2. A perspective on extracellular vesicles proteomics

    Science.gov (United States)

    Rosa-Fernandes, Livia; Rocha, Victória Bombarda; Carregari, Victor Corasolla; Urbani, Andrea; Palmisano, Giuseppe

    2017-11-01

    Increasing attention has been given to secreted extracellular vesicles (EVs) in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieve from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.

  3. Gas Vesicle Nanoparticles for Antigen Display

    Directory of Open Access Journals (Sweden)

    Shiladitya DasSarma

    2015-09-01

    Full Text Available Microorganisms like the halophilic archaeon Halobacterium sp. NRC-1 produce gas-filled buoyant organelles, which are easily purified as protein nanoparticles (called gas vesicles or GVNPs. GVNPs are non-toxic, exceptionally stable, bioengineerable, and self-adjuvanting. A large gene cluster encoding more than a dozen proteins has been implicated in their biogenesis. One protein, GvpC, found on the exterior surface of the nanoparticles, can accommodate insertions near the C-terminal region and results in GVNPs displaying the inserted sequences on the surface of the nanoparticles. Here, we review the current state of knowledge on GVNP structure and biogenesis as well as available studies on immunogenicity of pathogenic viral, bacterial, and eukaryotic proteins and peptides displayed on the nanoparticles. Recent improvements in genetic tools for bioengineering of GVNPs are discussed, along with future opportunities and challenges for development of vaccines and other applications.

  4. Periodic-cylinder vesicle with minimal energy

    International Nuclear Information System (INIS)

    Xiao-Hua, Zhou

    2010-01-01

    We give some details about the periodic cylindrical solution found by Zhang and Ou-Yang in [1996 Phys. Rev. E 53 4206] for the general shape equation of vesicle. Three different kinds of periodic cylindrical surfaces and a special closed cylindrical surface are obtained. Using the elliptic functions contained in mathematic, we find that this periodic shape has the minimal total energy for one period when the period–amplitude ratio β ≈ 1.477, and point out that it is a discontinuous deformation between plane and this periodic shape. Our results also are suitable for DNA and multi-walled carbon nanotubes (MWNTs). (cross-disciplinary physics and related areas of science and technology)

  5. Methods to isolate extracellular vesicles for diagnosis

    Science.gov (United States)

    Kang, Hyejin; Kim, Jiyoon; Park, Jaesung

    2017-12-01

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

  6. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    Directory of Open Access Journals (Sweden)

    Thomas Kieselbach

    Full Text Available Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT and leukotoxin (LtxA into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs. To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e using liquid chromatography-tandem mass spectrometry (LC-MS/MS. This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.

  7. Vesicle Origami and the Influence of Cholesterol on Lipid Packing.

    Science.gov (United States)

    Tanasescu, Radu; Lanz, Martin A; Mueller, Dennis; Tassler, Stephanie; Ishikawa, Takashi; Reiter, Renate; Brezesinski, Gerald; Zumbuehl, Andreas

    2016-05-17

    The artificial phospholipid Pad-PC-Pad was analyzed in 2D (monolayers at the air/water interface) and 3D (aqueous lipid dispersions) systems. In the gel phase, the two leaflets of a Pad-PC-Pad bilayer interdigitate completely, and the hydrophobic bilayer region has a thickness comparable to the length of a single phospholipid acyl chain. This leads to a stiff membrane with no spontaneous curvature. Forced into a vesicular structure, Pad-PC-Pad has faceted geometry, and in its extreme form, tetrahedral vesicles were found as predicted a decade ago. Above the main transition temperature, a noninterdigitated Lα phase with fluid chains has been observed. The addition of cholesterol leads to a slight decrease of the main transition temperature and a gradual decrease in the transition enthalpy until the transition vanishes at 40 mol % cholesterol in the mixture. Additionally, cholesterol pulls the chains apart, and a noninterdigitated gel phase is observed. In monolayers, cholesterol has an ordering effect on liquid-expanded phases and disorders condensed phases. The wavenumbers of the methylene stretching vibration indicate the formation of a liquid-ordered phase in mixtures with 40 mol % cholesterol.

  8. Development and dosimetric evaluation of radiochromic PCDA vesicle gel dosimeters

    International Nuclear Information System (INIS)

    Sun, P.; Fu, Y.C.; Hu, J.; Hao, N.; Huang, W.; Jiang, B.

    2016-01-01

    The gel dosimeter has the unique capacity in recording radiation dose distribution in three dimensions (3D), which has the specific advantages in dosimetry measurements where steep dose gradients exist, such as in intensity-modulated radiation therapy (IMRT), brachytherapy and so on. Some 3D dosimeters, such as Fricke gel dosimeters, polymer gel dosimeters, the PRESAGE plastic dosimeters and micelle gel dosimeters have appeared recently. However, there are several disadvantages of these 3D dosimeters limit their application in radiotherapy dose verification. In this study, a novel radiochromic gel dosimeter for 3D dose verification of radiotherapy was developed by dispersing nanovesicles self-assembled by 10,12-pentacosadiynoic acid (PCDA) into the tissue equivalence gel matrix. The characteristics of radiochromic PCDA vesicle gel dosimeters were evaluated. The results indicate that these radiochromic gel dosimeters have good linear dose response to X-ray irradiation in the dose range of 2–100 Gy. In addition, the radiochromic gel dosimeters breakthrough the limitations of the existing gel dosimeters such as diffusion effect, post-radiation effect, and poor forming ability. The response of the gel dosimeter does not show any dose rate dependence, energy dependence and temperature effect, and there was no obvious difference in the gel response between single and cumulative dose of fractional irradiation. Hence, the radiochromic PCDA vesicle gel dosimeters developed in this study could be generally applied to 3D dose verification in radiotherapy. - Highlights: • A novel radiochromic gel dosimeter was developed by dispersing PCDA nanovesicles into the tissue equivalence gel matrix. • This nanovesicle overcomes the dose image blurring caused by the diffusion of monomer molecules. • This nanovesicle limits the polymer chain growth, so as to reduce the post-radiation effect. • The gel matrixes possess excellent tissue equivalence and elastic strength, which

  9. In Candida albicans hyphae, Sec2p is physically associated with SEC2 mRNA on secretory vesicles.

    Science.gov (United States)

    Caballero-Lima, David; Hautbergue, Guillaume M; Wilson, Stuart A; Sudbery, Peter E

    2014-11-01

    Candida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans-Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  10. An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles.

    Science.gov (United States)

    Heyward, Catherine A; Pettitt, Trevor R; Leney, Sophie E; Welsh, Gavin I; Tavaré, Jeremy M; Wakelam, Michael J O

    2008-05-20

    Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

  11. An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles

    Directory of Open Access Journals (Sweden)

    Welsh Gavin I

    2008-05-01

    Full Text Available Abstract Background Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Results Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. Conclusion The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

  12. 3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

    Science.gov (United States)

    Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

    2012-05-01

    3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2014-10-01

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

  14. 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

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

    Directory of Open Access Journals (Sweden)

    Azusa Oshima

    2017-09-01

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

  16. Taurocholate transport by brush-border membrane vesicles from the developing rabbit ileum: Structure/function relationships

    International Nuclear Information System (INIS)

    Schwarz, S.M.; Watkins, J.B.; Ling, S.C.

    1990-01-01

    To examine the ontogenesis of bile acid transport in the rabbit ileum, brush-border membrane vesicles (12- to 20-fold purified) were prepared from 14- to 49-day-old animals. Taurocholate uptake was characterized by the emergence of secondary active, Na(+)-dependent transport at the start of weaning (21 days). Transient intravesicular accumulation (overshoot) of taurocholate occurred at 5-10 s of incubation, and the overshoot maximum increased significantly from 21 days (349.2 +/- 22.4 nmol/mg protein) to 35 days (569.0 +/- 84.3 nmol/mg protein; p less than 0.001), without further increase at maturity (49 days, not equal to 607.6 +/- 136.7 nmol/mg protein). No significant taurocholate active uptake component was noted at 14 days; however, ileal vesicles from sucklings showed carrier-mediated, Na+ D-glucose cotransport. In greater than or equal to 35-day-old rabbits, osmolarity studies at 20 s of incubation showed that only approximately 12% of [14C]taurocholate uptake was secondary to bile acid-to-membrane binding. Conversely, at 20 min, greater than 95% of radiolabel incorporation represented solute bound to the external and/or internal membrane surface. Arrhenius plots establish brush-border membrane taurocholate uptake as an intrinsic, lipid-dependent process, with a slope discontinuity between 24 and 28 degrees C, similar to the membrane lipid thermotropic transition region. Steady-state fluorescence polarization studies (1,6-diphenyl-1,3,5-hexatriene) demonstrate a temporal association between the maturation of taurocholate uptake and age-related decreases in ileal brush-border membrane fluidity. These data indicate that maturation of bile acid secondary active transport in the rabbit ileum may be regulated, at least in part, by changes in brush-border membrane lipid dynamics

  17. Circulating extracellular vesicles with specific proteome and liver microRNAs are potential biomarkers for liver injury in experimental fatty liver disease.

    Directory of Open Access Journals (Sweden)

    Davide Povero

    Full Text Available Nonalcoholic fatty liver disease (NAFLD is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy.Choline Deficient L-Amino Acid (CDAA and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens.We observed statistically significant differences in the level of extracellular vesicles (EVs in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05, fibrosis (r2 = 0.66, p<0.05 and pathological angiogenesis (r2 = 0.71, p<0.05. Extensive characterization of blood EVs identified both microparticles (MPs and exosomes (EXO present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192--two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver.These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.

  18. Glioblastoma extracellular vesicles: reservoirs of potential biomarkers

    Directory of Open Access Journals (Sweden)

    Redzic JS

    2014-02-01

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

  19. Ovalbumin with Glycated Carboxyl Groups Shows Membrane-Damaging Activity

    Directory of Open Access Journals (Sweden)

    Ching-Chia Tang

    2017-02-01

    Full Text Available The aim of the present study was to investigate whether glycated ovalbumin (OVA showed novel activity at the lipid-water interface. Mannosylated OVA (Man-OVA was prepared by modification of the carboxyl groups with p-aminophenyl α-dextro (d-mannopyranoside. An increase in the number of modified carboxyl groups increased the membrane-damaging activity of Man-OVA on cell membrane-mimicking vesicles, whereas OVA did not induce membrane permeability in the tested phospholipid vesicles. The glycation of carboxyl groups caused a notable change in the gross conformation of OVA. Moreover, owing to their spatial positions, the Trp residues in Man-OVA were more exposed, unlike those in OVA. Fluorescence quenching studies suggested that the Trp residues in Man-OVA were located on the interface binds with the lipid vesicles, and their microenvironment was abundant in positively charged residues. Although OVA and Man-OVA showed a similar binding affinity for lipid vesicles, the lipid-interacting feature of Man-OVA was distinct from that of OVA. Chemical modification studies revealed that Lys and Arg residues, but not Trp residues, played a crucial role in the membrane-damaging activity of Man-OVA. Taken together, our data suggest that glycation of carboxyl groups causes changes in the structural properties and membrane-interacting features of OVA, generating OVA with membrane-perturbing activities at the lipid-water interface.

  20. Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

    DEFF Research Database (Denmark)

    Blans, Kristine Ingrid Marie; Hansen, Maria Stenum; Sørensen, Laila V.

    2017-01-01

    -marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides...... accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles...... from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV...

  1. The SNARE protein vti1a functions in dense-core vesicle biogenesis

    DEFF Research Database (Denmark)

    Walter, Alexander M; Kurps, Julia; de Wit, Heidi

    2014-01-01

    overlapping with syntaxin-6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca(2+)-channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin-2 content. In contrast, release kinetics and Ca(2+)-sensitivity remain unchanged, indicating that the final fusion......The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans-Golgi network, partially...... reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long-term re-expression of vti1a (days...

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

    Science.gov (United States)

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  5. Biological properties of extracellular vesicles and their physiological functions

    NARCIS (Netherlands)

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

    2015-01-01

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

  6. 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...

  7. 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

  8. 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.

  9. Extracellular vesicles provide a means for tissue crosstalk during exercise

    DEFF Research Database (Denmark)

    Whitham, Martin; Parker, Benjamin L; Friedrichsen, Martin

    2018-01-01

    Exercise stimulates the release of molecules into the circulation, supporting the concept that inter-tissue signaling proteins are important mediators of adaptations to exercise. Recognizing that many circulating proteins are packaged in extracellular vesicles (EVs), we employed quantitative prot...

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  11. 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).

  12. 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.

  13. 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.

  14. 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)

  15. The longitudinal effect of ejaculation on seminal vesicle fluid volume and whole-prostate ADC as measured on prostate MRI

    Energy Technology Data Exchange (ETDEWEB)

    Barrett, Tristan; Gallagher, Ferdia A. [Addenbrooke' s Hospital and University of Cambridge, Department of Radiology, Cambridge (United Kingdom); Addenbrooke' s Hospital and University of Cambridge, CamPARI Clinic, Cambridge (United Kingdom); Tanner, James; Gill, Andrew B.; Slough, Rhys A. [Addenbrooke' s Hospital and University of Cambridge, Department of Radiology, Cambridge (United Kingdom); Wason, James [University of Cambridge, MRC Biostatistics Unit, Cambridge (United Kingdom)

    2017-12-15

    To prospectively investigate the longitudinal effect of ejaculatory abstinence on MRI-measured seminal vesicle (SV) volume and whole-prostate ADC over consecutive days. 15 healthy male volunteers (mean 35.9 years, range 27-53) underwent 3-T MRI at baseline and 1, 2 and 3 days post-ejaculation. Prostate and SV volumes were derived by volume segmentation and whole-gland apparent diffusion coefficient (ADC) values calculated. A mixed-effects linear regression compared ADC values and prostate/seminal vesicle volumes in each volunteer between studies in a pairwise manner. All subjects completed the four MRIs. Mean prostate volume was 22.45 cm{sup 3} (range 13.04-31.21 cm{sup 3}), with no change between the four studies (p = 0.89-0.99). 13/15 subjects showed SV volume reduction from baseline to day 1, with group-mean decreasing from 6.45 to 4.80 cm{sup 3} (-25.6%, p < 0.001), and a significant reduction from baseline to day 2 (-18.1%, p = 0.002). There was a significant volume increase from both day 1 (+21.3%, p = 0.006) and day 2 (+10.2%, p = 0.022) to day 3 post-ejaculation. There was a significant reduction in ADC from 1.105 at baseline to 1.056 x 10{sup -3} mm{sup 2}/s at day 1 (mean -4.3%, p = 0.009). The longitudinal effect of ejaculation on SV volume was demonstrated. Significant reductions in SV volume and whole-gland ADC were observed post-ejaculation, supporting a 3-day period of abstinence before prostate MRI. (orig.)

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  17. Effect of ethanol amine plasmalogens on Fe-induced peroxidation of arachidonic acid in dipalmitoylphosphatidylcholine vesicles.

    Science.gov (United States)

    Omodeo Salè, M F; Rizzo, A M; Masserini, M

    2000-12-01

    We have investigated the influence of ethanolamine plasmalogens on iron-induced oxidation of arachidonic acid in dipalmitoylphosphatidylcholine (DPPC) vesicles. Lipoperoxidation was induced by the addition of 50 microM FeSO4 and studied above (50 degrees C) and below (15 degrees C) the gel-to liquid transition temperature of the vesicles, at two different pH values (7.4 or 6.4). The extent of peroxidation was measured as thiobarbituric reactive product formed and the influence exerted by ethanolamine plasmalogens (PEPL) in this process was compared to that of dipalmitoylphosphatidylethanolamine (DPPE) and diacylphosphatidylethanolamines (DAPE). The extent of peroxidation of arachidonic acid embedded in DPPC vesicles was similar at the two temperatures and greater at 50 degrees C under acidic conditions. However, the peroxidative process was significantly decreased at 50 degrees C in the presence of PEPL, but not of DPPE or DAPE and the inhibitory effect was enhanced at pH 6.4. The possibility that a different phase distribution of the phospholipids, namely a transition from a lamellar to a hexagonal phase, may play a role in the scavenger effect of ethanolamine plasmalogens is discussed.

  18. Effect of nitrate supply and mycorrhizal inoculation on characteristics of tobacco root plasma membrane vesicles.

    Science.gov (United States)

    Moche, Martin; Stremlau, Stefanie; Hecht, Lars; Göbel, Cornelia; Feussner, Ivo; Stöhr, Christine

    2010-01-01

    Plant plasma membrane (pm) vesicles from mycorrhizal tobacco (Nicotiana tabacum cv. Samsun) roots were isolated with negligible fungal contamination by the aqueous two-phase partitioning technique as proven by fatty acid analysis. Palmitvaccenic acid became apparent as an appropriate indicator for fungal membranes in root pm preparations. The pm vesicles had a low specific activity of the vanadate-sensitive ATPase and probably originated from non-infected root cells. In a phosphate-limited tobacco culture system, root colonisation by the vesicular arbuscular mycorrhizal fungus, Glomus mosseae, is inhibited by external nitrate in a dose-dependent way. However, detrimental high concentrations of 25 mM nitrate lead to the highest colonisation rate observed, indicating that the defence system of the plant is impaired. Nitric oxide formation by the pm-bound nitrite:NO reductase increased in parallel with external nitrate supply in mycorrhizal roots in comparison to the control plants, but decreased under excess nitrate. Mycorrhizal pm vesicles had roughly a twofold higher specific activity as the non-infected control plants when supplied with 10-15 mM nitrate.

  19. 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....

  20. 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.

  1. Pardaxin permeabilizes vesicles more efficiently by pore formation than by disruption

    DEFF Research Database (Denmark)

    Vad, Brian S; Bertelsen, Kresten; Johansen, Charlotte Hau

    2010-01-01

    Pardaxin is a 33-amino-acid neurotoxin from the Red Sea Moses sole Pardachirus marmoratus, whose mode of action shows remarkable sensitivity to lipid chain length and charge, although the effect of pH is unclear. Here we combine optical spectroscopy and dye release experiments with laser scanning...... is more efficient than lysis of anionic vesicles, suggesting that electrostatic interactions may trap pardaxin in several suboptimal interconverting conformations on the membrane surface....

  2. 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.

  3. 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.

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

    Science.gov (United States)

    Pors, Susanne E; Pedersen, Ida J; Skjerning, Ragnhild Bager; Thøfner, Ida C N; Persson, Gry; Bojesen, Anders M

    2016-11-15

    Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have previously shown promising results in protection against infections and we hypothesized that OMVs could serve as an immunogen to protect egg-laying hens against G. anatis. To investigate the immunogenic potential of G. anatis OMVs, two in vivo studies in egg-laying hens were made. The trials assessedthe degree of protection provided by immunization with G. anatis OMV against challenge and the IgY responses in serum after immunization and challenge, respectively. A total of 64 egg-laying hens were included in the trials. OMVs for immunization were produced and purified from a high-producing G. anatis ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re-isolation remained unchanged. Furthermore, a high OMV-specific IgY response was induced by immunization and subsequent challenge of the hens. The results strongly indicate that immunization with G. anatis OMVs provides significant protection against G. anatis challenge and induces specific antibody responses with high titers of OMV-specific IgY in serum. The results therefore show great promise for OMV based vaccines aiming at providing protecting against G. anatis in egg-laying hens. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Suppression of autophagy by extracellular vesicles promotes myofibroblast differentiation in COPD pathogenesis

    Directory of Open Access Journals (Sweden)

    Yu Fujita

    2015-11-01

    Full Text Available Extracellular vesicles (EVs, such as exosomes and microvesicles, encapsulate proteins and microRNAs (miRNAs as new modulators of both intercellular crosstalk and disease pathogenesis. The composition of EVs is modified by various triggers to maintain physiological homeostasis. In response to cigarette smoke exposure, the lungs develop emphysema, myofibroblast accumulation and airway remodelling, which contribute to chronic obstructive pulmonary disease (COPD. However, the lung disease pathogenesis through modified EVs in stress physiology is not understood. Here, we investigated an EV-mediated intercellular communication mechanism between primary human bronchial epithelial cells (HBECs and lung fibroblasts (LFs and discovered that cigarette smoke extract (CSE-induced HBEC-derived EVs promote myofibroblast differentiation in LFs. Thorough evaluations of the modified EVs and COPD lung samples showed that cigarette smoke induced relative upregulation of cellular and EV miR-210 expression of bronchial epithelial cells. Using co-culture assays, we showed that HBEC-derived EV miR-210 promotes myofibroblast differentiation in LFs. Surprisingly, we found that miR-210 directly regulates autophagy processes via targeting ATG7, and expression levels of miR-210 are inversely correlated with ATG7 expression in LFs. Importantly, autophagy induction was significantly decreased in LFs from COPD patients, and silencing ATG7 in LFs led to myofibroblast differentiation. These findings demonstrate that CSE triggers the modification of EV components and identify bronchial epithelial cell-derived miR-210 as a paracrine autophagy mediator of myofibroblast differentiation that has potential as a therapeutic target for COPD. Our findings show that stressor exposure changes EV compositions as emerging factors, potentially controlling pathological disorders such as airway remodelling in COPD.

  6. Decreasing Relative Risk Premium

    DEFF Research Database (Denmark)

    Hansen, Frank

    relative risk premium in the small implies decreasing relative risk premium in the large, and decreasing relative risk premium everywhere implies risk aversion. We finally show that preferences with decreasing relative risk premium may be equivalently expressed in terms of certain preferences on risky......We consider the risk premium demanded by a decision maker with wealth x in order to be indifferent between obtaining a new level of wealth y1 with certainty, or to participate in a lottery which either results in unchanged present wealth or a level of wealth y2 > y1. We define the relative risk...... premium as the quotient between the risk premium and the increase in wealth y1–x which the decision maker puts on the line by choosing the lottery in place of receiving y1 with certainty. We study preferences such that the relative risk premium is a decreasing function of present wealth, and we determine...

  7. Regional differences in actomyosin contraction shape the primary vesicles in the embryonic chicken brain

    International Nuclear Information System (INIS)

    Filas, Benjamen A; Oltean, Alina; Majidi, Shabnam; Bayly, Philip V; Taber, Larry A; Beebe, David C

    2012-01-01

    In the early embryo, the brain initially forms as a relatively straight, cylindrical epithelial tube composed of neural stem cells. The brain tube then divides into three primary vesicles (forebrain, midbrain, hindbrain), as well as a series of bulges (rhombomeres) in the hindbrain. The boundaries between these subdivisions have been well studied as regions of differential gene expression, but the morphogenetic mechanisms that generate these constrictions are not well understood. Here, we show that regional variations in actomyosin-based contractility play a major role in vesicle formation in the embryonic chicken brain. In particular, boundaries did not form in brains exposed to the nonmuscle myosin II inhibitor blebbistatin, whereas increasing contractile force using calyculin or ATP deepened boundaries considerably. Tissue staining showed that contraction likely occurs at the inner part of the wall, as F-actin and phosphorylated myosin are concentrated at the apical side. However, relatively little actin and myosin was found in rhombomere boundaries. To determine the specific physical mechanisms that drive vesicle formation, we developed a finite-element model for the brain tube. Regional apical contraction was simulated in the model, with contractile anisotropy and strength estimated from contractile protein distributions and measurements of cell shapes. The model shows that a combination of circumferential contraction in the boundary regions and relatively isotropic contraction between boundaries can generate realistic morphologies for the primary vesicles. In contrast, rhombomere formation likely involves longitudinal contraction between boundaries. Further simulations suggest that these different mechanisms are dictated by regional differences in initial morphology and the need to withstand cerebrospinal fluid pressure. This study provides a new understanding of early brain morphogenesis. (paper)

  8. Characterization of rat primary trigeminal satellite glial cells and associated extracellular vesicles under normal and inflammatory conditions

    DEFF Research Database (Denmark)

    Vinterhøj, Hye Sook Han; Stensballe, Allan; Duroux, Meg

    2018-01-01

    Satellite glial cells (SGCs) in sensory ganglia contribute to the pathogenesis of chronic pain, potentially through mediating extracellular or paracrine signaling. Recently, extracellular vesicles (EVs) in the form of exosomes have been found to play an important role in cell-cell communication....... Results demonstrated that SGCs shed vesicles in the size range of exosomes (>150 nm) but with altered protein expression upon LPS-activation. Proteomic profiling of SGCs-shed EVs showed that a number of proteins were differentially regulated upon LPS stimulation such as junction plakoglobin and myosin 9...

  9. Comparative Analysis of Membrane Vesicles from Three Piscirickettsia salmonis Isolates Reveals Differences in Vesicle Characteristics.

    Directory of Open Access Journals (Sweden)

    Julia I Tandberg

    Full Text Available Membrane vesicles (MVs are spherical particles naturally released from the membrane of Gram-negative bacteria. Bacterial MV production is associated with a range of phenotypes including biofilm formation, horizontal gene transfer, toxin delivery, modulation of host immune responses and virulence. This study reports comparative profiling of MVs from bacterial strains isolated from three widely disperse geographical areas. Mass spectrometry identified 119, 159 and 142 proteins in MVs from three different strains of Piscirickettsia salmonis isolated from salmonids in Chile (LF-89, Norway (NVI 5692 and Canada (NVI 5892, respectively. MV comparison revealed several strain-specific differences related to higher virulence capability for LF-89 MVs, both in vivo and in vitro, and stronger similarities between the NVI 5692 and NVI 5892 MV proteome. The MVs were similar in size and appearance as analyzed by electron microscopy and dynamic light scattering. The MVs from all three strains were internalized by both commercial and primary immune cell cultures, which suggest a potential role of the MVs in the bacterium's utilization of leukocytes. When MVs were injected into an adult zebrafish infection model, an upregulation of several pro-inflammatory genes were observed in spleen and kidney, indicating a modulating effect on the immune system. The present study is the first comparative analysis of P. salmonis derived MVs, highlighting strain-specific vesicle characteristics. The results further illustrate that the MV proteome from one bacterial strain is not representative of all bacterial strains within one species.

  10. 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.

  11. 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

  12. Magnetically Triggered Release From Giant Unilamellar Vesicles: Visualization By Means Of Confocal Microscopy

    KAUST Repository

    Nappini, Silvia

    2011-04-07

    Magnetically triggered release from magnetic giant unilamellar vesicles (GUVs) loaded with Alexa fluorescent dye was studied by means of confocal laser scanning microscopy (CLSM) under a low-frequency alternating magnetic field (LF-AMF). Core/shell cobalt ferrite nanoparticles coated with rhodamine B isothiocyanate (MP@SiO 2(RITC)) were prepared and adsorbed on the GUV membrane. The MP@SiO 2(RITC) location and distribution on giant lipid vesicles were determined by 3D-CLSM projections, and their effect on the release properties and GUV permeability under a LF-AMF was investigated by CLSM time-resolved experiments. We show that the mechanism of release of the fluorescent dye during the LF-AMF exposure is induced by magnetic nanoparticle energy and mechanical vibration, which promote the perturbation of the GUV membrane without its collapse. © 2011 American Chemical Society.

  13. Instability of a Lamellar Phase under Shear Flow: Formation of Multilamellar Vesicles

    Science.gov (United States)

    Courbin, L.; Delville, J. P.; Rouch, J.; Panizza, P.

    2002-09-01

    The formation of closed-compact multilamellar vesicles (referred to in the literature as the ``onion texture'') obtained upon shearing lamellar phases is studied using small-angle light scattering and cross-polarized microscopy. By varying the shear rate γ ˙, the gap cell D, and the smectic distance d, we show that: (i)the formation of this structure occurs homogeneously in the cell at a well-defined wave vector qi, via a strain-controlled process, and (ii)the value of qi varies as (dγ ˙/D)1/3. These results strongly suggest that formation of multilamellar vesicles may be monitored by an undulation (buckling) instability of the membranes, as expected from theory.

  14. Readily releasable pool of synaptic vesicles measured at single synaptic contacts.

    Science.gov (United States)

    Trigo, Federico F; Sakaba, Takeshi; Ogden, David; Marty, Alain

    2012-10-30

    To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7.

  15. Benefits of a vesicle re-education program on elderly hospitalized patients

    Directory of Open Access Journals (Sweden)

    Silvia del Rey Contreras

    2007-05-01

    Full Text Available Urinary incontinence is a geriatric syndrome of high prevalence in elderly people, which very frequently is not diagnosed and many times appears when suffering a sharp incapacitate Pathology.The aim of the research is to discover the response to the treatment of vesicle re-education in patients with various pathologies and to observe if the benefit is permanent.A group of 372 patients of the “Middle-Stay Unit” of the Red Cross Hospital were evaluated from October 1, 2001 to December 31, 2002 using objective instruments of physical and mental evaluation. The results showed that the prevalence of the incontinence at patients’ admission was very high and the vesicle re-education was proved to be very effective since the continence was recovered in a significant number of patients permanently.

  16. Seminal vesicle involvement at salvage radical prostatectomy.

    Science.gov (United States)

    Meeks, Joshua J; Walker, Marc; Bernstein, Melanie; Eastham, James A

    2013-06-01

    To describe the incidence and clinical outcomes of seminal vesicle invasion (SVI) at salvage radical prostatectomy (SRP) and to describe the accuracy of SV biopsy. As SRP is used after biochemical recurrence (BCR) of prostate cancer after radiotherapy (RT) to gain local oncological control. The SVs receive lower doses of radiation from external-beam RT (EBRT) to avoid rectal exposure and are not targeted with brachytherapy (BT) with low-risk prostate cancer. SRP was performed on 206 men with BCR after RT at a tertiary care institution between 1998 and 2011. Post-RT biopsy and SRP specimens were reviewed by a genitourinary pathologist. SVI was detected in 65 (32%) of 206 patients. No difference was found between EBRT alone (65% vs 63%) and BT (29% vs 31%) with or without EBRT in patients with SVI. Men with SVI had higher rates of cT3 disease (20% vs 8%) and Gleason score ≥ 8 at SRP (52% vs 21%). BCR-free survival at 5 years was 18% and 56% in patients with and without SVI (hazard ratio 2.85, 95% confidence interval 1.87-4.36, P < 0.001), yet the rate of local recurrence was low (11%). Prostate cancer was identified in nine of 18 patients who underwent SV biopsy and was the only location of prostate cancer in two patients. SVI is a prognostic indicator for BCR after SRP, but local recurrence in patients with SVI after SRP remains low. We recommend SV biopsy to improve staging and cancer detection in men with BCR after radiotherapy. © 2013 BJU International.

  17. Plant plasma membrane aquaporins in natural vesicles as potential stabilizers and carriers of glucosinolates.

    Science.gov (United States)

    Martínez-Ballesta, Maria Del Carmen; Pérez-Sánchez, Horacio; Moreno, Diego A; Carvajal, Micaela

    2016-07-01

    Their biodegradable nature and ability to target cells make biological vesicles potential nanocarriers for bioactives delivery. In this work, the interaction between proteoliposomes enriched in aquaporins derived from broccoli plants and the glucosinolates was evaluated. The vesicles were stored at different temperatures and their integrity was studied. Determination of glucosinolates, showed that indolic glucosinolates were more sensitive to degradation in aqueous solution than aliphatic glucosinolates. Glucoraphanin was stabilized by leaf and root proteoliposomes at 25°C through their interaction with aquaporins. An extensive hydrogen bond network, including different aquaporin residues, and hydrophobic interactions, as a consequence of the interaction between the linear alkane chain of glucoraphanin and Glu31 and Leu34 protein residues, were established as the main stabilizing elements. Combined our results showed that plasma membrane vesicles from leaf and root tissues of broccoli plants may be considered as suitable carriers for glucosinolate which stabilization can be potentially attributed to aquaporins. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

    Science.gov (United States)

    Blans, Kristine; Hansen, Maria S; Sørensen, Laila V; Hvam, Michael L; Howard, Kenneth A; Möller, Arne; Wiking, Lars; Larsen, Lotte B; Rasmussen, Jan T

    2017-01-01

    Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

  19. 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

  20. 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

  1. 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

  2. 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

  3. Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles.

    Directory of Open Access Journals (Sweden)

    Stefanie Fischer

    Full Text Available The biological relevance of extracellular vesicles (EV in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.

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

    Science.gov (United States)

    Ascenso, Andreia; Raposo, Sara; Batista, Cátia; Cardoso, Pedro; Mendes, Tiago; Praça, Fabíola Garcia; Bentley, Maria Vitória Lopes Badra; Simões, Sandra

    2015-01-01

    Ultradeformable vesicles (UDV) have recently become a promising tool for the development of improved and innovative dermal and transdermal therapies. The aim of this work was to study three related UDV: transfersomes, ethosomes, and transethosomes for the incorporation of actives of distinct polarities, namely, vitamin E and caffeine, and to evaluate the effect of the carrier on skin permeation and penetration. These actives were incorporated in UDV formulations further characterized for vesicles imaging by transmission electron microscopy; mean vesicle size and polydispersity index by photon correlation spectroscopy; zeta potential by laser-Doppler anemometry; deformability by pressure-driven transport; and incorporation efficiency (IE) after actives quantification by high-performance liquid chromatography. Topical delivery studies were performed in order to compare UDV formulations regarding the release, skin permeation, and penetration profiles. All UDV formulations showed size values within the expected range, except transethosomes prepared by "transfersomal method", for which size was smaller than 100 nm in contrast to that obtained for vesicles prepared by "ethosomal method". Zeta potential was negative and higher for formulations containing sodium cholate. The IE was much higher for vitamin E- than caffeine-loaded UDV as expected. For flux measurements, the following order was obtained: transethosomes (TE) > ethosomes (E) ≥ transfersomes (T). This result was consistent with the release and skin penetration profiles for Vitamin E-loaded UDV. However, the releasing results were totally the opposite for caffeine-loaded UDV, which might be explained by the solubility and thermodynamic activity of this active in each formulation instead of the UDV deformability attending to the higher non-incorporated fraction of caffeine. Anyway, a high skin penetration and permeation for all caffeine-loaded UDV were obtained. Transethosomes were more deformable than ethosomes

  5. A novel multiplex bead-based platform highlights the diversity of extracellular vesicles.

    Science.gov (United States)

    Koliha, Nina; Wiencek, Yvonne; Heider, Ute; Jüngst, Christian; Kladt, Nikolay; Krauthäuser, Susanne; Johnston, Ian C D; Bosio, Andreas; Schauss, Astrid; Wild, Stefan

    2016-01-01

    The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell-derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.

  6. 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.

  7. 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.

  8. 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.

  9. LRRK2 affects vesicle trafficking, neurotransmitter extracellular level and membrane receptor localization.

    Directory of Open Access Journals (Sweden)

    Rossana Migheli

    Full Text Available The leucine-rich repeat kinase 2 (LRRK2 gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson's disease (PD. LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2(G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2(G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.

  10. Comparative miRNA Analysis of Urine Extracellular Vesicles Isolated through Five Different Methods

    Directory of Open Access Journals (Sweden)

    Felix Royo

    2016-12-01

    Full Text Available Urine extracellular vesicles are a valuable low-invasive source of information, especially for the cells of the genitourinary tract. In the search for biomarkers, different techniques have been developed to isolate and characterize the cargo of these vesicles. In the present work, we compare five of these different isolation methods (three commercial isolation kits, ultracentrifugation, and lectin-based purification and perform miRNA profiling using a multiplex miRNA assay. The results showed high correlation through all isolation techniques, and 48 out of 68 miRNAs were detected above the detection limit at least 10 times. The results obtained by multiplex assay were validated through Taqman qPCR. In addition, using this technique combined with a clinically friendly extracellular vesicle (uEV-enrichment method, we performed the analysis of selected miRNAs in urine from patients affected with bladder cancer, benign prostate hyperplasia, or prostate cancer. Importantly, we found that those miRNAs could be detected in almost 100% of the samples, and no significant differences were observed between groups. Our results support the feasibility of analyzing exosomes-associated miRNAs using a methodology that requires a small volume of urine and is compatible with a clinical environment and high-throughput analysis.

  11. 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

  12. Presynaptic mechanisms of lead neurotoxicity: effects on vesicular release, vesicle clustering and mitochondria number.

    Science.gov (United States)

    Zhang, Xiao-Lei; Guariglia, Sara R; McGlothan, Jennifer L; Stansfield, Kirstie H; Stanton, Patric K; Guilarte, Tomás R

    2015-01-01

    Childhood lead (Pb2+) intoxication is a global public health problem and accounts for 0.6% of the global burden of disease associated with intellectual disabilities. Despite the recognition that childhood Pb2+ intoxication contributes significantly to intellectual disabilities, there is a fundamental lack of knowledge on presynaptic mechanisms by which Pb2+ disrupts synaptic function. In this study, using a well-characterized rodent model of developmental Pb2+ neurotoxicity, we show that Pb2+ exposure markedly inhibits presynaptic vesicular release in hippocampal Schaffer collateral-CA1 synapses in young adult rats. This effect was associated with ultrastructural changes which revealed a reduction in vesicle number in the readily releasable/docked vesicle pool, disperse vesicle clusters in the resting pool, and a reduced number of presynaptic terminals with multiple mitochondria with no change in presynaptic calcium influx. These studies provide fundamental knowledge on mechanisms by which Pb2+ produces profound inhibition of presynaptic vesicular release that contribute to deficits in synaptic plasticity and intellectual development.

  13. Development of a magnetic bead-based method for the collection of circulating extracellular vesicles.

    Science.gov (United States)

    Shih, Chun-Liang; Chong, Kowit-Yu; Hsu, Shih-Che; Chien, Hsin-Jung; Ma, Ching-Ting; Chang, John Wen-Cheng; Yu, Chia-Jung; Chiou, Chiuan-Chian

    2016-01-25

    Cells release different types of extracellular vesicles (EVs). These EVs contain biomolecules, including proteins and nucleic acids, from their parent cells, which can be useful for diagnostic applications. The aim of this study was to develop a convenient procedure to collect circulating EVs with detectable mRNA or other biomolecules. Magnetic beads coated with annexin A5 (ANX-beads), which bound to phosphatidylserine moieties on the surfaces of most EVs, were tested for their ability to capture induced apoptotic bodies in vitro and other phosphatidylserine-presenting vesicles in body fluids. Our results show that up to 60% of induced apoptotic bodies could be captured by the ANX-beads. The vesicles captured from cultured media or plasma contained amplifiable RNA. Suitable blood samples for EV collection included EDTA-plasma and serum but not heparin-plasma. In addition, EVs in plasma were labile to freeze-and-thaw cycles. In rodents xenografted with human cancer cells, tumor-derived mRNA could be detected in EVs captured from serum samples. Active proteins could be detected in EVs captured from ascites but not from plasma. In conclusion, we have developed a magnetic bead-based procedure for the collection of EVs from body fluids and proved that captured EVs contain biomolecules from their parent cells, and therefore have great potential for disease diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Acute isoproterenol induces anxiety-like behavior in rats and increases plasma content of extracellular vesicles.

    Science.gov (United States)

    Leo, Giuseppina; Guescini, Michele; Genedani, Susanna; Stocchi, Vilberto; Carone, Chiara; Filaferro, Monica; Sisti, Davide; Marcoli, Manuela; Maura, Guido; Cortelli, Pietro; Guidolin, Diego; Fuxe, Kjell; Agnati, Luigi Francesco

    2015-04-01

    Several clinical observations have demonstrated a link between heart rate and anxiety or panic disorders. In these patients, β-adrenergic receptor function was altered. This prompted us to investigate whether the β-adrenergic receptor agonist isoproterenol, at a dose that stimulates peripheral β-adrenergic system but has no effects at the central nervous system, can induce anxiety-like behavior in rats. Moreover, some possible messengers involved in the peripheral to brain communication were investigated. Our results showed that isoproterenol (5 mg kg(-1) i.p.) increased heart rate, evoked anxiety-like behavior, did not result in motor impairments and increased extracellular vesicle content in the blood. Plasma corticosterone level was unmodified as well as vesicular Hsp70 content. Vesicular miR-208 was also unmodified indicating a source of increased extracellular vesicles different from cardiomyocytes. We can hypothesize that peripheral extracellular vesicles might contribute to the β-adrenergic receptor-evoked anxiety-like behavior, acting as peripheral signals in modulating the mental state. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Nucleation in mesoscopic systems under transient conditions: Peptide-induced pore formation in vesicles

    Science.gov (United States)

    Zhdanov, Vladimir P.; Höök, Fredrik

    2013-04-01

    Attachment of lytic peptides to the lipid membrane of virions or bacteria is often accompanied by their aggregation and pore formation, resulting eventually in membrane rupture and pathogen neutralization. The membrane rupture may occur gradually via formation of many pores or abruptly after the formation of the first pore. In academic studies, this process is observed during interaction of peptides with lipid vesicles. We present an analytical model and the corresponding Monte Carlo simulations focused on the pore formation in such situations. Specifically, we calculate the time of the first nucleation-limited pore-formation event and show the distribution of this time in the regime when the fluctuations of the number of peptides attached to a vesicle are appreciable. The results obtained are used to clarify the mechanism of the pore formation and membrane destabilization observed recently during interaction of highly active α-helical peptide with sub-100-nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature. The model proposed and the analysis presented are generic and may be applicable to other meso- and nanosystems.

  16. 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

  17. Variability in prostate and seminal vesicle delineations defined on magnetic resonance images, a multi-observer, -center and -sequence study

    DEFF Research Database (Denmark)

    Nyholm, Tufve; Jonsson, Joakim; Söderström, Karin

    2013-01-01

    and approximately equal for the prostate and seminal vesicles. Large differences in variability were observed for individual patients, and also for individual imaging sequences used at the different centers. There was however no indication of decreased variability with higher field strength. CONCLUSION: The overall......BACKGROUND: The use of magnetic resonance (MR) imaging as a part of preparation for radiotherapy is increasing. For delineation of the prostate several publications have shown decreased delineation variability using MR compared to computed tomography (CT). The purpose of the present work....... Two physicians from each center delineated the prostate and the seminal vesicles on each of the 25 image sets. The variability between the delineations was analyzed with respect to overall, intra- and inter-physician variability, and dependence between variability and origin of the MR images, i...

  18. 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.

  19. 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.

  20. A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death.

    Science.gov (United States)

    Vdovikova, Svitlana; Luhr, Morten; Szalai, Paula; Nygård Skalman, Lars; Francis, Monika K; Lundmark, Richard; Engedal, Nikolai; Johansson, Jörgen; Wai, Sun N

    2017-01-01

    Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes , a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo . We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae , VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.

  1. L-alanine uptake in membrane vesicles from Mytilus edulis gills

    International Nuclear Information System (INIS)

    Pajor, A.M.; Wright, S.H.

    1986-01-01

    Previous studies have shown that gills from M. edulis can accumulate L-alanine from seawater by a saturable process specific for α-neutral amino acids. This uptake occurs against chemical gradients in excess of 10 6 to 1. To further characterize this uptake, membrane vesicles were prepared from M. edulis gill tissue by differential centrifugation. Enrichments of putative enzyme markers (relative to that in combined initial fractions) were as follows: γ-Glutamyltranspeptidase, 25-30x; Alkaline Phosphatase, 5-6x; K + -dependent para-Nitrophenyl Phosphatase, 3-5x; Succinate Dehydrogenase 0.1-0.2x. These results suggest that the preparation is enriched in plasma membranes, although histochemical studies will be needed to verify this. The time course of 14 C-L-alanine uptake in the presence of inwardly-directed Na + gradient showed a transient overshoot (3-5 fold) at 10 minutes which decreased to equilibrium after six hours. The size of the overshoot and early uptake rates depended on the size of the inwardly-directed Na + gradient. No overshoot was seen in the presence of inwardly-directed gradients of LiCl or choline-Cl, or with equilibrium concentrations NaCl or mannitol. A reduced overshoot was seen with a gradient of NaSCN. A small overshoot was seen with an inwardly-directed gradient of KCl. Transport of L-alanine included saturable and diffusive components. Uptake of 6 μM L-alanine was inhibited more than 80% by 100 μM α-zwitterionic amino acids (alanine, leucine, glycine); by 30 to 75% by proline, aspartate and lysine; and less than 20% by a β-amino acid, taurine. The results of these experiments agree with those from intact gill studies and support the hypothesis that L-alanine is transported into gill epithelial cells by a secondary active transport process involving Na +

  2. A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death

    Directory of Open Access Journals (Sweden)

    Svitlana Vdovikova

    2017-05-01

    Full Text Available Bacterial membrane vesicle (MV production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO, is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts. Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.

  3. 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

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

    Science.gov (United States)

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

    2011-05-01

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

  5. Kinetic partitioning between aggregation and vesicle permeabilization by modified ADan

    DEFF Research Database (Denmark)

    Nesgaard, Lise W.; Vad, Brian; Christiansen, Gunna

    2009-01-01

    The neurodegenerative illness Familial Danish Dementia (FDD) is linked to formation and aggregation of the 34-residue ADan peptide, whose cytotoxicity may be mediated by membrane interactions. Here we characterize the derived peptide SerADan, in which the two cysteines found in ADan have been....... Aggregation is prevented at neutral/acidic pH and low ionic strength by anionic lipid vesicles. These vesicles are permeabilized by monomeric SerADan assembling on the membrane to form stable beta-sheet structures which are different from the solution aggregates. In contrast, solution ageing of SerADan first...

  6. 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)

  7. 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.)

  8. Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

    International Nuclear Information System (INIS)

    Kasai, M.; Muto, S.

    1990-01-01

    Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment. 45Ca2+ transport was assayed by membrane filtration technique. Results showed that Ca2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca2+ transport system had a high affinity for Ca2+(Km(Ca2+) = 0.4 microM) and ATP(Km(ATP) = 3.9 microM), and showed pH optimum at 7.5. ATP-dependent Ca2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl- or NO3-. Quenching of quinacrine fluorescence showed that these anions also induced H+ transport into the vesicles. The Ca2+ uptake stimulated by Cl- was dependent on the activity of H+ transport into the vesicles. However, carbonylcyanide m-chlorophenylhydrazone (CCCP) and VO4(3-) which is known to inhibit the H+ pump associated with the plasma membrane, canceled almost all of the Cl(-)-stimulated Ca2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca2+ uptake into the vesicles. These results suggest that the Cl(-)-stimulated Ca2+ uptake is caused by the efflux of H+ from the vesicles by the operation of Ca2+/H+ antiport system in the plasma membrane. In Cl(-)-free medium, H+ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca2+ uptake into the vesicles. These results suggest that two Ca2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca2+ transport system (Ca2+ pump) and the other is a Ca2+/H+ antiport system. Little difference in characteristics of Ca2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves

  9. Endothelial Plasmalemma Vesicle Associated Protein regulates the homeostasis of splenic immature B cell and B1 B cells

    Science.gov (United States)

    Elgueta, Raul; Tse, Dan; Deharvengt, Sophie J.; Luciano, Marcus R.; Carriere, Catherine; Noelle, Randolph J.; Stan, Radu V.

    2016-01-01

    Plasmalemma vesicle associated protein (Plvap) is an endothelial protein with roles in endothelial diaphragm formation and maintenance of basal vascular permeability. At the same time Plvap has roles in immunity by facilitating leukocyte diapedesis at inflammatory sites and controlling peripheral lymph node morphogenesis and the entry of soluble antigens into lymph node conduits. Based on its postulated role in diapedesis, we have investigated the role of Plvap in hematopoiesis and show that deletion of Plvap results in a dramatic decrease of IgM+IgDlo B cells in both the spleen and peritoneal cavity. Tissue specific deletion of Plvap demonstrates that the defect is B cell extrinsic, as B cell and pan hematopoietic Plvap deletion has no effect on IgM+IgDlo B cell numbers. Endothelial specific deletion of Plvap in the embryo or at adult stage recapitulates the full Plvap knockout phenotype whereas endothelial specific reconstitution of Plvap under the Chd5 promoter rescues the IgM+IgDlo B cell phenotype. Taken together, these results show that Plvap expression in endothelial cells is important in the maintenance of IgM+ B cells in the spleen and peritoneal cavity. PMID:27742829

  10. Endothelial Plasmalemma Vesicle-Associated Protein Regulates the Homeostasis of Splenic Immature B Cells and B-1 B Cells.

    Science.gov (United States)

    Elgueta, Raul; Tse, Dan; Deharvengt, Sophie J; Luciano, Marcus R; Carriere, Catherine; Noelle, Randolph J; Stan, Radu V

    2016-11-15

    Plasmalemma vesicle-associated protein (Plvap) is an endothelial protein with roles in endothelial diaphragm formation and maintenance of basal vascular permeability. At the same time, Plvap has roles in immunity by facilitating leukocyte diapedesis at inflammatory sites and controlling peripheral lymph node morphogenesis and the entry of soluble Ags into lymph node conduits. Based on its postulated role in diapedesis, we have investigated the role of Plvap in hematopoiesis and show that deletion of Plvap results in a dramatic decrease of IgM + IgD lo B cells in both the spleen and the peritoneal cavity. Tissue-specific deletion of Plvap demonstrates that the defect is B cell extrinsic, because B cell and pan-hematopoietic Plvap deletion has no effect on IgM + IgD lo B cell numbers. Endothelial-specific deletion of Plvap in the embryo or at adult stage recapitulates the full Plvap knockout phenotype, whereas endothelial-specific reconstitution of Plvap under the Chd5 promoter rescues the IgM + IgD lo B cell phenotype. Taken together, these results show that Plvap expression in endothelial cells is important in the maintenance of IgM + B cells in the spleen and peritoneal cavity. Copyright © 2016 by The American Association of Immunologists, Inc.

  11. Roles of head group architecture and side chain length on colorimetric response of polydiacetylene vesicles to temperature, ethanol and pH.

    Science.gov (United States)

    Charoenthai, Nipaphat; Pattanatornchai, Thanutpon; Wacharasindhu, Sumrit; Sukwattanasinitt, Mongkol; Traiphol, Rakchart

    2011-08-15

    In this contribution, we report the relationship between molecular structures of polydiacetylene (PDA) vesicles, fabricated by using three monomers, 10,12-tricosadiynoic acid (TCDA), 10,12-pentacosadiynoic acid (PCDA) and N-(2-aminoethyl)pentacosa-10,12-diynamide (AEPCDA), and their color-transition behaviors. The modification of side chain length and head group of the PDA vesicles strongly affects the colorimetric response to temperature, ethanol and pH. A shorter side chain of poly(TCDA) yields weaker inter- and intra-chain dispersion interactions in the bilayers compared to the system of poly(PCDA), which in turn results in a faster color transition upon exposure to all stimuli. A change of head group in poly(AEPCDA) slightly reduces the transition temperature. Interestingly, the colorimetric response of poly(AEPCDA) vesicles to the addition of ethanol is found to occur in a two-step fashion while the response of poly(PCDA) vesicles takes place in a one-step process. The amount of ethanol required for inducing complete color-transition of poly(AEPCDA) vesicles is also much higher, about 87% v/v. The increase of pH to ~9 and ~10 causes a color-transition of poly(TCDA) and poly(PCDA) vesicles, respectively. The poly(AEPCDA) vesicles, on the other hand, change color upon decreasing pH to ~0. The colorimetric response also occurs in a multi-step fashion. These discrepancies are attributed to the architecture of surface layers of poly(AEPCDA), constituting amine and amide groups separated by ethyl linkers. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ascenso A

    2015-09-01

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

  13. Ricinus communis agglutinin-mediated agglutination and fusion of glycolipid-containing phospholipid vesicles: effect of carbohydrate head group size, calcium ions, and spermine.

    Science.gov (United States)

    Hoekstra, D; Düzgüneş, N

    1986-03-25

    The glycolipids galactosylcerebroside (GalCer), lactosylceramide (LacCer), and trihexosylceramide (Gb3) were inserted into phospholipid vesicles, consisting of phosphatidylethanolamine and phosphatidic acid. The extent to which their carbohydrate head groups protruded beyond the vesicle surface and their interference with membrane approach were examined by determining vesicle susceptibility toward type I Ricinus communis agglutinin (RCA1) induced agglutination and Ca2+- and spermine-induced aggregation and fusion either in the presence or in the absence of the lectin. The initial agglutination rates increased in the order GalCer much less than LacCer less than Gb3, while a reversed order was obtained for Ca2+- and spermine-induced aggregation and fusion, indicating an enhanced steric interference on close approach of bilayers with increasing head group size. The lectin-mediated agglutination rates for LacCer- and Gb3-containing vesicles increased by an order of magnitude when Ca2+ was also included in the medium, at a concentration that did not induce aggregation per se. Charge neutralization could not account for this observation as the polyvalent cation spermine did not display this synergistic effect with RCA1. Addition of Ca2+ to preagglutinated vesicles substantially reduced the threshold cation concentration for fusion (micromolar vs. millimolar). Quantitatively, this concentration decreased with decreasing carbohydrate head group size, indicating that the head group protrusion determined the interbilayer distance within the vesicle aggregate. The distinct behavior of Ca2+ vs. spermine on RCA1-induced agglutination on the one hand and fusion on the other indicated that Ca2+ regulates the steric orientation of the carbohydrate head group, which appears to be related to its ability to dehydrate the bilayer. As a result, lectin agglutinability becomes enhanced while fusion will be interrupted as the interbilayer distance increases, the threshold head group size

  14. 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.

  15. Show-Bix &

    DEFF Research Database (Denmark)

    2014-01-01

    The anti-reenactment 'Show-Bix &' consists of 5 dias projectors, a dial phone, quintophonic sound, and interactive elements. A responsive interface will enable the Dias projectors to show copies of original dias slides from the Show-Bix piece ”March på Stedet”, 265 images in total. The copies are...

  16. Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons.

    Science.gov (United States)

    Lapatsina, Liudmila; Jira, Julia A; Smith, Ewan St J; Poole, Kate; Kozlenkov, Alexey; Bilbao, Daniel; Lewin, Gary R; Heppenstall, Paul A

    2012-06-01

    A complex of stomatin-family proteins and acid-sensing (proton-gated) ion channel (ASIC) family members participate in sensory transduction in invertebrates and vertebrates. Here, we have examined the role of the stomatin-family protein stomatin-like protein-3 (STOML3) in this process. We demonstrate that STOML3 interacts with stomatin and ASIC subunits and that this occurs in a highly mobile vesicle pool in dorsal root ganglia (DRG) neurons and Chinese hamster ovary cells. We identify a hydrophobic region in the N-terminus of STOML3 that is required for vesicular localization of STOML3 and regulates physical and functional interaction with ASICs. We further characterize STOML3-containing vesicles in DRG neurons and show that they are Rab11-positive, but not part of the early-endosomal, lysosomal or Rab14-dependent biosynthetic compartment. Moreover, uncoupling of vesicles from microtubules leads to incorporation of STOML3 into the plasma membrane and increased acid-gated currents. Thus, STOML3 defines a vesicle pool in which it associates with molecules that have critical roles in sensory transduction. We suggest that the molecular features of this vesicular pool may be characteristic of a 'transducosome' in sensory neurons.

  17. Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.

    Directory of Open Access Journals (Sweden)

    Robert D Lagow

    2007-04-01

    Full Text Available Both constitutive secretion and Ca(2+-regulated exocytosis require the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.

  18. The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy.

    Science.gov (United States)

    Drabik, Dominik; Przybyło, Magda; Sikorski, Aleksander; Langner, Marek

    2016-03-01

    Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or βBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.

  19. 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.

  20. 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

  1. MRI, CT and TRUS imaging of seminal vesicle metastasis

    International Nuclear Information System (INIS)

    Larsson, P.; Blomqvist, L.; Norming, U.

    1997-01-01

    We present a case of a testicular germ-cell metastasis in the seminal vesicle. Diagnostic imaging with transrectal ultrasonography (TRUS), CT, and MRI was performed. This case emphasizes the role of MRI in the evaluation of patients with pathology in the pelvic region. (orig.)

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

    NARCIS (Netherlands)

    Williams, Charles; Royo, Felix; Aizpurua-Olaizola, Oier; Pazos, Raquel; Boons, Geert-Jan; Reichardt, Niels-Christian; Falcon-Perez, Juan M

    2018-01-01

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

  3. Continuous fabrication of polymeric vesicles and nanotubes with fluidic channe

    NARCIS (Netherlands)

    Peng, F.; Deng, N.-N.; Tu, Y.; van Hest, J.C.M.; Wilson, D.A.

    2017-01-01

    Fluidic channels were employed to induce the self-assembly of poly(ethylene glycol)-b-polystyrene into polymeric vesicles and nanotubes. The laminar flow in the device enables controlled diffusion of two miscible liquids at the phase boundary, leading to the formation of homogeneous polymeric

  4. Primary malignancy of seminal vesicle: A rare entity

    Directory of Open Access Journals (Sweden)

    Rajaraman Ramamurthy

    2011-01-01

    Full Text Available We report a rare case of seminal vesicle malignancy (primitive neuro ectodermal tumor in a 40-year-old male patient. He was treated with enbloc resection of the tumor and ureteric reimplantation. In view of the rarity of this entity, management of these tumors should be individualized.

  5. 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...

  6. Seminal vesicle abscess causing unilateral hydroureteronephrosis: A case report

    Directory of Open Access Journals (Sweden)

    Vittorio Imperatore

    2017-12-01

    Full Text Available Seminal vesicle abscess (SVA is a rare urologic entity. It mainly occurs in subjects with predisposing factors and may be associated with other urogenital infections. We describe the case of a diabetic subject with SVA associated with funiculitis, epididymitis and obstructive pyelonephritis. Treatment consisted of laparotomic surgical drainage of the abscess and ureteral stent placement.

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

    Directory of Open Access Journals (Sweden)

    Jong Suk Jin

    2011-12-01

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

  8. Removal of Vesicle Structures from Transmission Electron Microscope Images

    DEFF Research Database (Denmark)

    Jensen, Katrine Hommelhoff; Sigworth, Fred; Brandt, Sami Sebastian

    2015-01-01

    In this paper, we address the problem of imaging membrane proteins for single-particle cryo-electron microscopy reconstruction of the isolated protein structure. More precisely, we propose a method for learning and removing the interfering vesicle signals from the micrograph, prior to reconstruct...

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

    DEFF Research Database (Denmark)

    Goettsch, Claudia; Hutscheson, JD; Aikawa, M

    2016-01-01

    obscure. Here, we have demonstrated that sortilin is a key regulator of smooth muscle cell (SMC) calcification via its recruitment to extracellular vesicles. Sortilin localized to calcifying vessels in human and mouse atheromata and participated in formation of microcalcifications in SMC culture. Sortilin...

  10. Ultrasound-guided seminal vesicle biopsies in prostate cancer

    NARCIS (Netherlands)

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

    2000-01-01

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

  11. 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

  12. Inflammation leads to distinct populations of extracellular vesicles from microglia

    DEFF Research Database (Denmark)

    Yang, Yiyi; Boza-Serrano, Antonio; Dunning, Christopher J.R.

    2018-01-01

    Background: Activated microglia play an essential role in inflammatory responses elicited in the central nervous system (CNS). Microglia-derived extracellular vesicles (EVs) are suggested to be involved in propagation of inflammatory signals and in the modulation of cell-to-cell communication...

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

    Directory of Open Access Journals (Sweden)

    P.F. Wong

    2014-09-01

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

  14. 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.

  15. 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.

  16. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

  18. Insight into the template effect of vesicles on the laccase-catalyzed oligomerization of N-phenyl-1,4-phenylenediamine from Raman spectroscopy and cyclic voltammetry measurements

    Science.gov (United States)

    Ležaić, Aleksandra Janoševic; Luginbühl, Sandra; Bajuk-Bogdanović, Danica; Pašti, Igor; Kissner, Reinhard; Rakvin, Boris; Walde, Peter; Ćirić-Marjanović, Gordana

    2016-08-01

    We report about the first Raman spectroscopy study of a vesicle-assisted enzyme-catalyzed oligomerization reaction. The aniline dimer N-phenyl-1,4-phenylenediamine (= p-aminodiphenylamine, PADPA) was oxidized and oligomerized with Trametes versicolor laccase and dissolved O2 in the presence of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) vesicles (80-100 nm diameter) as templates. The conversion of PADPA into oligomeric products, poly(PADPA), was monitored during the reaction by in situ Raman spectroscopy. The results obtained are compared with UV/vis/NIR and EPR measurements. All three complementary methods indicate that at least some of the poly(PADPA) products, formed in the presence of AOT vesicles, resemble the conductive emeraldine salt form of polyaniline (PANI-ES). The Raman measurements also show that structural units different from those of “ordinary” PANI-ES are present too. Without vesicles PANI-ES-like products are not obtained. For the first time, the as-prepared stable poly(PADPA)-AOT vesicle suspension was used directly to coat electrodes (without product isolation) for investigating redox activities of poly(PADPA) by cyclic voltammetry (CV). CV showed that poly(PADPA) produced with vesicles is redox active not only at pH 1.1-as expected for PANI-ES-but also at pH 6.0, unlike PANI-ES and poly(PADPA) synthesized without vesicles. This extended pH range of the redox activity of poly(PADPA) is important for applications.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. Hydrogen Bonded Supramolecular Polymers in Both Apolar and Aqueous Media: Self-Assembly and Reversible Conversion of Vesicles and Gels%Hydrogen Bonded Supramolecular Polymers in Both Apolar and Aqueous Media: Self-Assembly and Reversible Conversion of Vesicles and Gels

    Institute of Scientific and Technical Information of China (English)

    杜平; 孔军; 王贵涛; 赵新; 李光玉; 蒋锡夔; 黎占亭

    2011-01-01

    In a preliminary letter (Tetrahedron Lett. 2010, 51, 188), we reported two new hydrazide-based quadruple hydrogen-bonding motifs, this is, two monopodal (la and lb) and five dipodal (2a, 2b and 3a--3c) aromatic hydrazide derivatives, and the formation of supramolecular polymers and vesicles from the dipodal motifs in hydrocarbons. In this paper, we present a full picture on the properties of these hydrogen-bonding motifs with an emphasis on their self-assembling behaviors in aqueous media. SEM, AFM, TEM and fluorescent micrographs indicate that all the dipodal compounds also form vesicles in polar methanol and water-methanol (up to 50% of water) mixtures. Control experiments show that lb does not form vesicles in same media. Addition of lb to the solution of the dipodal compounds inhibits the latter's capacity of forming vesicles. At high concentrations, 3b and 3c also gelate discrete solvents, including hydrocarbons, esters, methanol, and methanol-water mixture. Concentration-dependent SEM investigations reveal that the vesicles of 3b and 3c fuse to form gels and the gel of 3c can de-aggregate to form the vesicles reversibly.

  4. Improved stability of highly fluorinated phospholipid-based vesicles in the presence of bile salts.

    Science.gov (United States)

    Gadras, C; Santaella, C; Vierling, P

    1999-01-04

    The stability of fluorinated phospholipid-based vesicles in terms of detergent-induced release of encapsulated carboxyfluorescein has been evaluated. The fluorinated liposomes are substantially more resistant towards the lytic action of sodium taurocholate than conventional DSPC or even DSPC/CH 1/1 liposomes. Concerning structure/permeability relationships, the larger the fluorination degree of the membrane, the higher the resistance of the fluorinated liposomes to their destruction by the detergent. These results show that fluorinated liposomes have a promising potential as drug carrier and delivery systems for oral administration.

  5. Activity Induces Fmr1-Sensitive Synaptic Capture of Anterograde Circulating Neuropeptide Vesicles.

    Science.gov (United States)

    Cavolo, Samantha L; Bulgari, Dinara; Deitcher, David L; Levitan, Edwin S

    2016-11-16

    Synaptic neuropeptide and neurotrophin stores are maintained by constitutive bidirectional capture of dense-core vesicles (DCVs) as they circulate in and out of the nerve terminal. Activity increases DCV capture to rapidly replenish synaptic neuropeptide stores following release. However, it is not known whether this is due to enhanced bidirectional capture. Here experiments at the Drosophila neuromuscular junction, where DCVs contain neuropeptides and a bone morphogenic protein, show that activity-dependent replenishment of synaptic neuropeptides following release is evident after inhibiting the retrograde transport with the dynactin disruptor mycalolide B or photobleaching DCVs entering a synaptic bouton by retrograde transport. In contrast, photobleaching anterograde transport vesicles entering a bouton inhibits neuropeptide replenishment after activity. Furthermore, tracking of individual DCVs moving through boutons shows that activity selectively increases capture of DCVs undergoing anterograde transport. Finally, upregulating fragile X mental retardation 1 protein (Fmr1, also called FMRP) acts independently of futsch/MAP-1B to abolish activity-dependent, but not constitutive, capture. Fmr1 also reduces presynaptic neuropeptide stores without affecting activity-independent delivery and evoked release. Therefore, presynaptic motoneuron neuropeptide storage is increased by a vesicle capture mechanism that is distinguished from constitutive bidirectional capture by activity dependence, anterograde selectivity, and Fmr1 sensitivity. These results show that activity recruits a separate mechanism than used at rest to stimulate additional synaptic capture of DCVs for future release of neuropeptides and neurotrophins. Synaptic release of neuropeptides and neurotrophins depends on presynaptic accumulation of dense-core vesicles (DCVs). At rest, DCVs are captured bidirectionally as they circulate through Drosophila motoneuron terminals by anterograde and retrograde

  6. Characterization of Extracellular Vesicles by Size-Exclusion High-Performance Liquid Chromatography (HPLC).

    Science.gov (United States)

    Huang, Tao; He, Jiang

    2017-01-01

    Extracellular vesicles (EVs) have recently attracted substantial attention due to the potential diagnostic and therapeutic relevance. Although a variety of techniques have been used to isolate and analyze EVs, it is still far away from satisfaction. Size-exclusion chromatography (SEC), which separates subjects by size, has been widely applied in protein purification and analysis. The purpose of this chapter is to show the applications of size-exclusion high-performance liquid chromatography (HPLC) as methods for EV characterization of impurities or contaminants of small size, and thus for quality assay for the purity of the samples of EVs.

  7. The role of hair cells, cilia and ciliary motility in otolith formation in the zebrafish otic vesicle.

    Science.gov (United States)

    Stooke-Vaughan, Georgina A; Huang, Peng; Hammond, Katherine L; Schier, Alexander F; Whitfield, Tanya T

    2012-05-01

    Otoliths are biomineralised structures required for the sensation of gravity, linear acceleration and sound in the zebrafish ear. Otolith precursor particles, initially distributed throughout the otic vesicle lumen, become tethered to the tips of hair cell kinocilia (tether cilia) at the otic vesicle poles, forming two otoliths. We have used high-speed video microscopy to investigate the role of cilia and ciliary motility in otolith formation. In wild-type ears, groups of motile cilia are present at the otic vesicle poles, surrounding the immotile tether cilia. A few motile cilia are also found on the medial wall, but most cilia (92-98%) in the otic vesicle are immotile. In mutants with defective cilia (iguana) or ciliary motility (lrrc50), otoliths are frequently ectopic, untethered or fused. Nevertheless, neither cilia nor ciliary motility are absolutely required for otolith tethering: a mutant that lacks cilia completely (MZovl) is still capable of tethering otoliths at the otic vesicle poles. In embryos with attenuated Notch signalling [mindbomb mutant or Su(H) morphant], supernumerary hair cells develop and otolith precursor particles bind to the tips of all kinocilia, or bind directly to the hair cells' apical surface if cilia are absent [MZovl injected with a Su(H)1+2 morpholino]. However, if the first hair cells are missing (atoh1b morphant), otolith formation is severely disrupted and delayed. Our data support a model in which hair cells produce an otolith precursor-binding factor, normally localised to tether cell kinocilia. We also show that embryonic movement plays a minor role in the formation of normal otoliths.

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

    Science.gov (United States)

    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.

  9. Focus on Extracellular Vesicles: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Bin Zhang

    2016-02-01

    Full Text Available The intense research focus on stem and progenitor cells could be attributed to their differentiation potential to generate new cells to replace diseased or lost cells in many highly intractable degenerative diseases, such as Alzheimer disease, multiple sclerosis, and heart diseases. However, experimental and clinical studies have increasingly attributed the therapeutic efficacy of these cells to their secretion. While stem and progenitor cells secreted many therapeutic molecules, none of these molecules singly or in combination could recapitulate the functional effects of stem cell transplantations. Recently, it was reported that extracellular vesicles (EVs could recapitulate the therapeutic effects of stem cell transplantation. Based on the observations reported thus far, the prevailing hypothesis is that stem cell EVs exert their therapeutic effects by transferring biologically active molecules such as proteins, lipids, mRNA, and microRNA from the stem cells to injured or diseased cells. In this respect, stem cell EVs are similar to EVs from other cell types. They are both primarily vehicles for intercellular communication. Therefore, the differentiating factor is likely due to the composition of their cargo. The cargo of EVs from different cell types are known to include a common set of proteins and also proteins that reflect the cell source of the EVs and the physiological or pathological state of the cell source. Hence, elucidation of the stem cell EV cargo would provide an insight into the multiple physiological or biochemical changes necessary to affect the many reported stem cell-based therapeutic outcomes in a variety of experimental models and clinical trials.

  10. 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...

  11. Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion

    Energy Technology Data Exchange (ETDEWEB)

    Petrov, Alexey M., E-mail: fysio@rambler.ru; Zakyrjanova, Guzalija F., E-mail: guzik121192@mail.ru; Yakovleva, Anastasia A., E-mail: nastya1234qwer@mail.ru; Zefirov, Andrei L., E-mail: zefiroval@rambler.ru

    2015-01-02

    Highlights: • We examine the involvement of PKC in MCD induced synaptic vesicle exocytosis. • PKC inhibitor does not decrease the effect MCD on MEPP frequency. • PKC inhibitor prevents MCD induced FM1-43 unloading. • PKC activation may switch MCD induced exocytosis from kiss-and-run to a full mode. • Inhibition of phospholipase C does not lead to similar change in exocytosis. - Abstract: Previous studies demonstrated that depletion of membrane cholesterol by 10 mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis.

  12. Shear thinning and shear thickening of a confined suspension of vesicles

    Science.gov (United States)

    Nait Ouhra, A.; Farutin, A.; Aouane, O.; Ez-Zahraouy, H.; Benyoussef, A.; Misbah, C.

    2018-01-01

    Widely regarded as an interesting model system for studying flow properties of blood, vesicles are closed membranes of phospholipids that mimic the cytoplasmic membranes of red blood cells. In this study we analyze the rheology of a suspension of vesicles in a confined geometry: the suspension, bound by two planar rigid walls on each side, is subject to a shear flow. Flow properties are then analyzed as a function of shear rate γ ˙, the concentration of the suspension ϕ , and the viscosity contrast λ =ηin/ηout , where ηin and ηout are the fluid viscosities of the inner and outer fluids, respectively. We find that the apparent (or effective viscosity) of the suspension exhibits both shear thinning (decreasing viscosity with shear rate) or shear thickening (increasing viscosity with shear rate) in the same concentration range. The shear thinning or thickening behaviors appear as subtle phenomena, dependant on viscosity contrast λ . We provide physical arguments on the origins of these behaviors.

  13. Talking with TV shows

    DEFF Research Database (Denmark)

    Sandvik, Kjetil; Laursen, Ditte

    2014-01-01

    User interaction with radio and television programmes is not a new thing. However, with new cross-media production concepts such as X Factor and Voice, this is changing dramatically. The second-screen logic of these productions encourages viewers, along with TV’s traditional one-way communication...... mode, to communicate on interactive (dialogue-enabling) devices such as laptops, smartphones and tablets. Using the TV show Voice as our example, this article shows how the technological and situational set-up of the production invites viewers to engage in new ways of interaction and communication...

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have...... ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re...

  15. Talk Show Science.

    Science.gov (United States)

    Moore, Mitzi Ruth

    1992-01-01

    Proposes having students perform skits in which they play the roles of the science concepts they are trying to understand. Provides the dialog for a skit in which hot and cold gas molecules are interviewed on a talk show to study how these properties affect wind, rain, and other weather phenomena. (MDH)

  16. Obesity in show cats.

    Science.gov (United States)

    Corbee, R J

    2014-12-01

    Obesity is an important disease with a high prevalence in cats. Because obesity is related to several other diseases, it is important to identify the population at risk. Several risk factors for obesity have been described in the literature. A higher incidence of obesity in certain cat breeds has been suggested. The aim of this study was to determine whether obesity occurs more often in certain breeds. The second aim was to relate the increased prevalence of obesity in certain breeds to the official standards of that breed. To this end, 268 cats of 22 different breeds investigated by determining their body condition score (BCS) on a nine-point scale by inspection and palpation, at two different cat shows. Overall, 45.5% of the show cats had a BCS > 5, and 4.5% of the show cats had a BCS > 7. There were significant differences between breeds, which could be related to the breed standards. Most overweight and obese cats were in the neutered group. It warrants firm discussions with breeders and cat show judges to come to different interpretations of the standards in order to prevent overweight conditions in certain breeds from being the standard of beauty. Neutering predisposes for obesity and requires early nutritional intervention to prevent obese conditions. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

  17. Honored Teacher Shows Commitment.

    Science.gov (United States)

    Ratte, Kathy

    1987-01-01

    Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

  18. 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.

  19. 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)

  20. 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.

  1. 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.

  2. 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.

  3. Location of the carbohydrates present in the HK-ATPase vesicles isolated from hog gastric mucosa

    International Nuclear Information System (INIS)

    Hall, K.; Perez, G.; Anderson, D.; Gutierrez, C.; Munson, K.; Hersey, S.J.; Kaplan, J.H.; Sachs, G.

    1990-01-01

    The glycosylation of H+K(+)-ATPase vesicles isolated from hog gastric mucosa was investigated by various methods. Following protein separation on sodium dodecyl sulfate reducing gels and transfer to poly(vinyl difluoride) membranes, binding of concanavalin A was confined to the 94-kDa band which corresponds to the catalytic subunit. In contrast, wheat germ agglutinin binding occurred in a region below the 94-kDa subunit, corresponding to the 60-85-kDa region, and also to protein just above the catalytic subunit. Treatment with glycopeptidase F removed most of the concanavalin A staining and also the wheat germ agglutinin staining found below the 94-kDa region, but spared the higher molecular weight wheat germ agglutinin reactive material. During the deglycosylation experiments a protein of 35-kDa was produced. Sequencing analysis of V8 protease generated peptide fragments of the 35-kDa protein show at least 30% homology with the Na+K(+)-ATPase beta-subunits. Labeling of the carbohydrates by galactosyltransferase and [3H]uridine diphosphate-galactose showed that the sites of labeling were extracellular and were confined to the wheat germ agglutinin staining regions. Two molecular weight regions, below the 94-kDa region, of 60 and 85 kDa were identified. Electron microscopy using postembedding staining techniques showed that both concanavalin A and wheat germ agglutinin staining occurred on the extracellular face of the gastric vesicles

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

    Science.gov (United States)

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

    2015-09-01

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

  5. The energy show

    International Nuclear Information System (INIS)

    1988-01-01

    The Energy Show is a new look at the problems of world energy, where our supplies come from, now and in the future. The programme looks at how we need energy to maintain our standards of living. Energy supply is shown as the complicated set of problems it is - that Fossil Fuels are both raw materials and energy sources, that some 'alternatives' so readily suggested as practical options are in reality a long way from being effective. (author)

  6. Three-dimensional computed tomography-guided monotherapeutic pararectal brachytherapy of prostate cancer with seminal vesicle invasion

    International Nuclear Information System (INIS)

    Koutrouvelis, Panos; Lailas, Niko; Hendricks, Fred; Gil-Montero, Guillermo; Sehn, James; Katz, Stuart

    2001-01-01

    Purpose: To treat patients with prostate cancer and seminal vesicle invasion with monotherapeutic three dimensional computed tomography (3-DCT)-guided posterior pararectal brachytherapy. Methods and materials: Three hundred and sixty two patients with clinical stage T1 a,b or T2 a,b of prostate cancer were referred for 3-DCT-guided brachytherapy. Each underwent further staging with 3-D CT-guided pararectal biopsy of the seminal vesicles under local anesthesia during the pre-treatment CT-planning. Forty-three patients (12%) were upstaged to T3 cNoMo disease. In the set of 43 patients, Eight had Gleason's score≤6, 24 Gleason's score=7, and 11 patients ≥8. Initial PSA was 20 in 18 patients. Of the 43 patients, 37 patients were treated monotherapeutically with 3-D CT-guided brachytherapy. No patients received hormone therapy after the implant. The prescribed dosage to the seminal vesicles and prostate is 120 Gy with Pd-103 seeds and 144 Gy with 1-125 seeds. Results: The prescribed dosage was achieved in all 37 patient's throughout the seminal vesicles whose range of target radiation extended 5-10 mm outside the target in the adjacent fat as calculated with post-implant CT-dosimetry with Varian Brachy Vision or MMS software. Prostate Specific Antigen (PSA) outcome data were available in 34 patients treated with monotherapy and follow up ranged from 12-56 months (median, 24 months). Decreased PSA levels were stratified into six groups based on the presenting Gleason's score and initial PSA. In the first group (with Gleason's score≤6 and initial PSA 20 ng/ml), PSA decreased to less than 0.5 ng/ml in four out of eight patients (50%). All of the patients in the fourth group (with Gleason's score≥8 and initial PSA 20 ng/ml). There were no patients with Gleason's score of 1-6 and greater than 20 ng/ml initial PSA. Patients, irrespective of the Gleason's score and PSA, had an overall response of decreased PSA (less than 1 ng/ml) of 79%. Conclusion: 3-D CT

  7. Radiotherapy effect on the release of tumor micro-vesicles by glioblastoma cells

    International Nuclear Information System (INIS)

    Ding, Haixia

    2014-01-01

    Radiation therapy is a major therapeutic tool for glioblastoma (GBM). However, the post-radiation recurrence is almost inevitable, due to the emergence of a subpopulation of radioresistant cancer cells with greater proliferative, invasive, and pro-angiogenic capacities. The objective of this study was to investigate in vitro how irradiated cancer cells affect the function of untreated neighboring tumor cells and endothelial cells, focusing on signals exchange initiated by irradiation, such as soluble factors and tumor micro-vesicles (TMVs). Radiotherapy has slowed down the proliferation of GBM cells (T98G, U87) and induced mitotic death of 50-60%, without significant apoptosis. Through long-term monitoring of cell growth (xCELLigence) and wound-healing assay, we have confirmed that surviving GBM cells after irradiation release signals that can change the functions of endothelial cells HUVEC and non-irradiated tumor cells. In addition to the secretion of known soluble factors (VEGF, uPA), we were able to show using scanning electron microscopy and the Nanoparticle Tracking Analysis (NTA), the release of tumor micro-vesicles (TMVS), whose size was generally less than 500 nm. By NTA and flow cytometry, we have shown that the release of TMVs (exosome + 'shedding vesicles') can be significantly stimulated by irradiation in two lines, in a time-dependent manner. According to the proteomics analysis, soluble factors such as VEGF or IL-8, well known as pro-angiogenic factors, rather contribute to promote the survival or proliferation of HUVEC, while the released TMVs after irradiation, significantly altered the migration abilities of non-irradiated HUVEC and tumor cells. The pro-migratory properties of TMVs could thus contribute to glioblastoma recurrence after irradiation. (author) [fr

  8. Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells

    International Nuclear Information System (INIS)

    Mulvey, Hillary E.; Chang, Audrey; Adler, Jason; Del Tatto, Michael; Perez, Kimberly; Quesenberry, Peter J.; Chatterjee, Devasis

    2015-01-01

    Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype. EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB. This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs. Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer

  9. The interactions between ionic surfactants and phosphatidylcholine vesicles: Conductometry

    Science.gov (United States)

    Tsao, Heng-Kwong; Tseng, Wen Liang

    2001-11-01

    The interaction between ionic surfactants and phosphatidylcholine vesicles, which are prepared without addition of buffer and salt, is investigated by conductivity measurements. On the basis of the vesicle acting as a trap of charge carriers, the bilayer/aqueous phase partition coefficient K and the surfactant/lipid molar ratio Re of nine surfactants are determined. The thermodynamic consistency is satisfied by the measured parameters. The effects of the alkyl chain length (C10-C16) and ionic head group are then studied. The inverse partition coefficient K-1 is linearly related to the critical micelle concentration. The solubilizing ability Reb is a consequence of the competition between the surfactant incorporation into the bilayer and the formation of micelles. Consequently, the K parameter rises whereas the Reb parameter declines as the chain length is increased. The influence due to addition of salt is also discussed.

  10. Thermal and active fluctuations of a compressible bilayer vesicle

    Science.gov (United States)

    Sachin Krishnan, T. V.; Yasuda, Kento; Okamoto, Ryuichi; Komura, Shigeyuki

    2018-05-01

    We discuss thermal and active fluctuations of a compressible bilayer vesicle by using the results of hydrodynamic theory for vesicles. Coupled Langevin equations for the membrane deformation and the density fields are employed to calculate the power spectral density matrix of membrane fluctuations. Thermal contribution is obtained by means of the fluctuation dissipation theorem, whereas active contribution is calculated from exponentially decaying time correlation functions of active random forces. We obtain the total power spectral density as a sum of thermal and active contributions. An apparent response function is further calculated in order to compare with the recent microrheology experiment on red blood cells. An enhanced response is predicted in the low-frequency regime for non-thermal active fluctuations.

  11. Imaging and Quantification of Extracellular Vesicles by Transmission Electron Microscopy.

    Science.gov (United States)

    Linares, Romain; Tan, Sisareuth; Gounou, Céline; Brisson, Alain R

    2017-01-01

    Extracellular vesicles (EVs) are cell-derived vesicles that are present in blood and other body fluids. EVs raise major interest for their diverse physiopathological roles and their potential biomedical applications. However, the characterization and quantification of EVs constitute major challenges, mainly due to their small size and the lack of methods adapted for their study. Electron microscopy has made significant contributions to the EV field since their initial discovery. Here, we describe the use of two transmission electron microscopy (TEM) techniques for imaging and quantifying EVs. Cryo-TEM combined with receptor-specific gold labeling is applied to reveal the morphology, size, and phenotype of EVs, while their enumeration is achieved after high-speed sedimentation on EM grids.

  12. Importance of vesicle release stochasticity in neuro-spike communication.

    Science.gov (United States)

    Ramezani, Hamideh; Akan, Ozgur B

    2017-07-01

    Aim of this paper is proposing a stochastic model for vesicle release process, a part of neuro-spike communication. Hence, we study biological events occurring in this process and use microphysiological simulations to observe functionality of these events. Since the most important source of variability in vesicle release probability is opening of voltage dependent calcium channels (VDCCs) followed by influx of calcium ions through these channels, we propose a stochastic model for this event, while using a deterministic model for other variability sources. To capture the stochasticity of calcium influx to pre-synaptic neuron in our model, we study its statistics and find that it can be modeled by a distribution defined based on Normal and Logistic distributions.

  13. The Role of Extracellular Vesicles in Bone Metastasis

    Directory of Open Access Journals (Sweden)

    Michela Rossi

    2018-04-01

    Full Text Available Multiple types of cancer have the specific ability to home to the bone microenvironment and cause metastatic lesions. Despite being the focus of intense investigation, the molecular and cellular mechanisms that regulate the metastasis of disseminated tumor cells still remain largely unknown. Bone metastases severely impact quality of life since they are associated with pain, fractures, and bone marrow aplasia. In this review, we will summarize the recent discoveries on the role of extracellular vesicles (EV in the regulation of bone remodeling activity and bone metastasis occurrence. Indeed, it was shown that extracellular vesicles, including exosomes and microvesicles, released from tumor cells can modify the bone microenvironment, allowing the formation of osteolytic, osteosclerotic, and mixed mestastases. In turn, bone-derived EV can stimulate the proliferation of tumor cells. The inhibition of EV-mediated crosstalk between cancer and bone cells could represent a new therapeutic target for bone metastasis.

  14. Lubrication synergy: Mixture of hyaluronan and dipalmitoylphosphatidylcholine (DPPC) vesicles

    DEFF Research Database (Denmark)

    Raj, Akanksha; Wang, Min; Zander, Thomas

    2017-01-01

    consisting of non-homogeneous phospholipid bilayer with hyaluronan/DPPC aggregates on top. The presence of these aggregates generates a long-range repulsive surface force as two such surfaces are brought together. However, the aggregates are easily deformed, partly rearranged into multilayer structures......Phospholipids and hyaluronan have been implied to fulfil important roles in synovial joint lubrication. Since both components are present in synovial fluid, self-assembly structures formed by them should also be present. We demonstrate by small angle X-ray scattering that hyaluronan associates...... with the outer shell of dipalmitoylphophatidylcholine (DPPC) vesicles in bulk solution. Further, we follow adsorption to silica from mixed hyaluronan/DPPC vesicle solution by Quartz Crystal Microbalance with Dissipation measurements. Atomic Force Microscope imaging visualises the adsorbed layer structure...

  15. 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)

  16. Optimized microviscosimeter for detection and characterization of biological vesicles

    International Nuclear Information System (INIS)

    Gaiffe, O; Cretin, B; Boireau, W; Baudouy, J C; Vairac, P

    2008-01-01

    In this paper, we report on studies aimed at sensing the stiffness of biological membranes, in particular in the case of lipidic vesicles. To obtain pertinent results, we have developed and checked a specific sensor based on a vibrating sphere. The near-field acoustic wave generated by this vibrating sphere enables us to characterize biological particles which change the apparent viscosity and density of the surrounding fluid. The microsphere is well suited for very small volumes of liquid (typically about a few microlitres). The experimental results demonstrate the high sensitivity of the sensor to small variations of the composition of the aqueous media, particularly in the case of various populations of lipidic nanoparticles. Finally, this microviscosimeter demonstrates its ability to discriminate the population of vesicles on the basis of their global viscous properties

  17. The dense core vesicle protein IA-2, but not IA-2β, is required for active avoidance learning.

    Science.gov (United States)

    Carmona, G N; Nishimura, T; Schindler, C W; Panlilio, L V; Notkins, A L

    2014-06-06

    The islet-antigens IA-2 and IA-2β are major autoantigens in type-1 diabetes and transmembrane proteins in dense core vesicles (DCV). Recently we showed that deletion of both IA-2 and IA-2β alters the secretion of hormones and neurotransmitters and impairs behavior and learning. The present study was designed to evaluate the contribution to learning of each of these genes by using single knockout (SKO) and double knockout (DKO) mice in an active avoidance test. After 5 days of training, wild-type (WT) mice showed 60-70% active avoidance responses, whereas the DKO mice showed only 10-15% active avoidance responses. The degree of active avoidance responses in the IA-2 SKO mice was similar to that of the DKO mice, but in contrast, the IA-2β SKO mice behaved like WT mice showing 60-70% active avoidance responses. Molecular studies revealed a marked decrease in the phosphorylation of the cAMP response element-binding protein (CREB) and Ca(2+)/calmodulin-dependent protein kinase II (CAMKII) in the striatum and hippocampus of the IA-2 SKO and DKO mice, but not in the IA-2β SKO mice. To evaluate the role of CREB and CAMKII in the SKO and DKO mice, GBR-12909, which selectively blocks the dopamine uptake transporter and increases CREB and CAMKII phosphorylation, was administered. GBR-12909 restored the phosphorylation of CREB and CAMKII and increased active avoidance learning in the DKO and IA-2 SKO to near the normal levels found in the WT and IA-2β SKO mice. We conclude that in the absence of the DCV protein IA-2, active avoidance learning is impaired. Published by Elsevier Ltd.

  18. Phosphoproteins in extracellular vesicles as candidate markers for breast cancer

    OpenAIRE

    Chen, I-Hsuan; Xue, Liang; Hsu, Chuan-Chih; Paez, Juan Sebastian Paez; Pan, Li; Andaluz, Hillary; Wendt, Michael K.; Iliuk, Anton B.; Zhu, Jian-Kang; Tao, W. Andy

    2017-01-01

    Protein phosphorylation is a major regulatory mechanism for many cellular functions, but no phosphoprotein in biofluids has been developed for disease diagnosis because of the presence of active phosphatases. This study presents a general strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs from small volumes of plasma sam...

  19. The role of extracellular vesicles in phenotypic cancer transformation:

    OpenAIRE

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

    2013-01-01

    Background. Cancer has traditionally been considered as a disease resulting from gene mutations. New findings in biology are challenging gene-centered explanations of cancer progression and redirecting them to the non-genetic origins of tumorigenicity. It has become clear that intercellular communication plays a crucial role in cancer progression. Among the most intriguing ways of intercellular communication is that via extracellular vesicles (EVs). EVs are membrane structures released from v...

  20. 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)

  1. Assets of the non-pathogenic microorganism Dictyostelium discoideum as a model for the study of eukaryotic extracellular vesicles [v1; ref status: indexed, http://f1000r.es/pa

    Directory of Open Access Journals (Sweden)

    Irène Tatischeff

    2013-03-01

    Full Text Available Dictyostelium discoideum microvesicles have recently been presented as a valuable model for eukaryotic extracellular vesicles. Here, the advantages of D. discoideum for unraveling important biological functions of extracellular vesicles in general are detailed. D. discoideum, a non-pathogenic eukaryotic microorganism, belongs to a billion-year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split. During growth and early starvation-induced development, it presents analogies with lymphocytes and macrophages with regard to motility and phagocytosis capability, respectively. Its 6-chromosome genome codes for about 12,500 genes, some showing analogies with human genes. The presence of extracellular vesicles during cell growth has been evidenced as a detoxification mechanism of various structurally unrelated drugs. Controls led to the discovery of constitutive extracellular vesicle secretion in this microorganism, which was an important point. It means that the secretion of extracellular vesicles occurs, in the absence of any drug, during both cell growth and early development. This constitutive secretion of D. discoideum cells is very likely to play a role in intercellular communication. The detoxifying secreted vesicles, which can transport drugs outside the cells, can also act as "Trojan horses", capable of transferring these drugs not only into naïve D. discoideum cells, but into human cells as well. Therefore, these extracellular vesicles were proposed as a new biological drug delivery tool. Moreover, Dictyostelium, chosen by the NIH (USA as a new model organism for biomedical research, has already been used for studying some human diseases. These cells, which are much easier to manipulate than human cells, can be easily designed in simple conditioned medium experiments. Owing to the increasing consensus that extracellular vesicles are probably important mediators of intercellular communication, D

  2. The Role of Extracellular Vesicles in Metastasis

    Science.gov (United States)

    2017-10-01

    transferred via ESVs to osteoblasts. These bone cells represent the most common tissue target for breast cancer metastasis, and we will mimic ESV...separation of beads will result in good separation of bead-complexed exosomes and microvesicles. Furthermore, we show that we can use the same device to...proteins for 3 common exosome tetraspanin markers (CD9, CD63, and CD81) [Andreu & Yanez-Mo 2014] in tandem (pLLNL-exo-GFP) in order to both increase

  3. Royal Society Scientific Meeting: Extracellular vesicles in the tumour microenvironment.

    Science.gov (United States)

    Pink, Ryan Charles; Elmusrati, Areeg A; Lambert, Daniel; Carter, David Raul Francisco

    2018-01-05

    Cancer cells do not grow as an isolated homogeneous mass; tumours are, in fact, complex and heterogeneous collections of cancer and surrounding stromal cells, collectively termed the tumour microenvironment. The interaction between cancer cells and stromal cells in the tumour microenvironment has emerged as a key concept in the regulation of cancer progression. Understanding the intercellular dialogue in the tumour microenvironment is therefore an important goal. One aspect of this dialogue that has not been appreciated until recently is the role of extracellular vesicles (EVs). EVs are small vesicles released by cells under both normal and pathological conditions; they can transfer biological molecules between cells leading to changes in phenotype. EVs have emerged as important regulators of biological processes and can be dysregulated in diseases such as cancer; rapidly growing interest in their biology and therapeutic potential led to the Royal Society hosting a Scientific Meeting to explore the roles of EVs in the tumour microenvironment. This cross-disciplinary meeting explored examples of how aberrant crosstalk between tumour and stromal cells can promote cancer progression, and how such signalling can be targeted for diagnostic, prognostic and therapeutic benefit. In this review, and the special edition of Philosophical Transactions of the Royal Society B that follows, we will provide an overview of the content and outcomes of this exciting meeting.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'. © 2017 The Author(s).

  4. Myeloid extracellular vesicles: messengers from the demented brain

    Directory of Open Access Journals (Sweden)

    Annamaria eNigro

    2016-01-01

    Full Text Available Blood-borne monocyte derived cells play a pivotal, initially unrecognized, role in most central nervous system disorders, including diseases initially classified as purely neurodegenerative (i.e. AD, PD, and ALS. Their trafficking to the brain and spinal cord has been extensively studied in classical neuroinflammatory disorders such as multiple sclerosis. Central nervous system resident myeloid cells, namely microglia and perivascular macrophages, also are in the spotlight of investigations on neurological disorders. Myeloid cells, such as infiltrating macrophages and microglia, have been described as having both protective and destructive features in neurological disorders, thus identification of their functional phenotype during disease evolution would be of paramount importance. Extracellular vesicles, namely exosomes and shed vesicles, are released by virtually any cell type and can be detected and identified in terms of cell origin in biological fluids. They therefore constitute an ideal tool to access information on cells residing in an inaccessible site such as the brain. We will review here available information on extracellular vesicles detection in neurological disorders with special emphasis on neurodegenerative diseases.

  5. 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.

  6. A Hierarchical Convolutional Neural Network for vesicle fusion event classification.

    Science.gov (United States)

    Li, Haohan; Mao, Yunxiang; Yin, Zhaozheng; Xu, Yingke

    2017-09-01

    Quantitative analysis of vesicle exocytosis and classification of different modes of vesicle fusion from the fluorescence microscopy are of primary importance for biomedical researches. In this paper, we propose a novel Hierarchical Convolutional Neural Network (HCNN) method to automatically identify vesicle fusion events in time-lapse Total Internal Reflection Fluorescence Microscopy (TIRFM) image sequences. Firstly, a detection and tracking method is developed to extract image patch sequences containing potential fusion events. Then, a Gaussian Mixture Model (GMM) is applied on each image patch of the patch sequence with outliers rejected for robust Gaussian fitting. By utilizing the high-level time-series intensity change features introduced by GMM and the visual appearance features embedded in some key moments of the fusion process, the proposed HCNN architecture is able to classify each candidate patch sequence into three classes: full fusion event, partial fusion event and non-fusion event. Finally, we validate the performance of our method on 9 challenging datasets that have been annotated by cell biologists, and our method achieves better performances when comparing with three previous methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Supramolecular "Big Bang" in a Single-Ionic Surfactant/Water System Driven by Electrostatic Repulsion: From Vesicles to Micelles.

    Science.gov (United States)

    Leclercq, Loïc; Bauduin, Pierre; Nardello-Rataj, Véronique

    2017-04-11

    In aqueous solution, dimethyldi-n-octylammonium chloride, [DiC 8 ][Cl], spontaneously forms dimers at low concentrations (1-10 mM) to decrease the strength of the hydrophobic-water contact. Dimers represent ideal building blocks for the abrupt edification of vesicles at 10 mM. These vesicles are fully characterized by dynamic and static light scattering, self-diffusion nuclear magnetic resonance, and freeze-fracture transmission electron microscopy. An increase in concentration leads to electrostatic repulsion between vesicles that explode into small micelles at 30 mM. These transitions are detected by means of surface tension, conductivity, and solubility of hydrophobic solutes as well as by isothermal titration microcalorimetry. These unusual supramolecular transitions emerge from the surfactant chemical structure that combines two contradictory features: (i) the double-chain structure tending to form low planar aggregates with low water solubility and (ii) the relatively short chains giving high hydrophilicity. The well-balanced hydrophilic-hydrophobic character of [DiC 8 ][Cl] is then believed to be at the origin of the unusual supramolecular sequence offering new opportunities for drug delivery systems.

  8. Comparative study on the 3H-thymidine index of dorsal epidermis, buccal mucosa, and seminal vesicles in senile male rats

    International Nuclear Information System (INIS)

    Hornstein, O.P.; Schell, H.

    1975-01-01

    To supplement previous investigations on endogenous fluctuations of DNA synthesis in male rat dorsal epidermis, buccal mucosa and seminal vesicle epithelium from birth to sexual maturity, the labelling indices (L.I.) of these tissues in senile male rats from the same breed, studied under analogous experimental conditions, were evaluated as well as compared to the data obtained from rats in puberal and early mature age. In the dorsal epidermis and buccal mucosa of the old animals the medium L.I. were found to be at about the same level as those measured after puberty. In the aged seminal vesicle epithelium the medium L.I. was found to be decreased. The maintenance of epidermal and buccal DNA synthesis in senile rats as well as the results of previous studies with male rats subjected to castration or long-term administration of cyproteron acetate furnish evidence that a deficiency of testosteron does not diminish the rate of DNA synthesis in epidermis and buccal mucosa. However, from the decreased L.I. in seminal vesicle epithelium a reduced blood level or stimulating capacity, respectively, of testosteron in senile rats can be concluded. Furhtermore, withdrawal of testosteron by orchidectomy or administration of cyproteron acetate is appropriate to suppress significantly the proliferative activity of seminal vesicles epithelium. (orig.) [de

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

    Directory of Open Access Journals (Sweden)

    Rossella Grande

    2017-06-01

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

  10. Long-term culture of astrocytes attenuates the readily releasable pool of synaptic vesicles.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kawano

    Full Text Available The astrocyte is a major glial cell type of the brain, and plays key roles in the formation, maturation, stabilization and elimination of synapses. Thus, changes in astrocyte condition and age can influence information processing at synapses. However, whether and how aging astrocytes affect synaptic function and maturation have not yet been thoroughly investigated. Here, we show the effects of prolonged culture on the ability of astrocytes to induce synapse formation and to modify synaptic transmission, using cultured autaptic neurons. By 9 weeks in culture, astrocytes derived from the mouse cerebral cortex demonstrated increases in β-galactosidase activity and glial fibrillary acidic protein (GFAP expression, both of which are characteristic of aging and glial activation in vitro. Autaptic hippocampal neurons plated on these aging astrocytes showed a smaller amount of evoked release of the excitatory neurotransmitter glutamate, and a lower frequency of miniature release of glutamate, both of which were attributable to a reduction in the pool of readily releasable synaptic vesicles. Other features of synaptogenesis and synaptic transmission were retained, for example the ability to induce structural synapses, the presynaptic release probability, the fraction of functional presynaptic nerve terminals, and the ability to recruit functional AMPA and NMDA glutamate receptors to synapses. Thus the presence of aging astrocytes affects the efficiency of synaptic transmission. Given that the pool of readily releasable vesicles is also small at immature synapses, our results are consistent with astrocytic aging leading to retarded synapse maturation.

  11. Transmission of HBV DNA Mediated by Ceramide-Triggered Extracellular VesiclesSummary

    Directory of Open Access Journals (Sweden)

    Takahiro Sanada

    2017-03-01

    Full Text Available Background & Aims: An extracellular vesicle (EV is a nanovesicle that shuttles proteins, nucleic acids, and lipids, thereby influencing cell behavior. A recent crop of reports have shown that EVs are involved in infectious biology, influencing host immunity and playing a role in the viral life cycle. In the present work, we investigated the EV-mediated transmission of hepatitis B virus (HBV infection. Methods: We investigated the EV-mediated transmission of HBV infection by using a HBV infectious culture system that uses primary human hepatocytes derived from humanized chimeric mice (PXB-cells. Purified EVs were isolated by ultracentrifugation. To analyze the EVs and virions, we used stimulated emission depletion microscopy. Results: Purified EVs from HBV-infected PXB-cells were shown to contain HBV DNA and to be capable of transmitting HBV DNA to naive PXB-cells. These HBV-DNA–transmitting EVs were shown to be generated through a ceramide-triggered EV production pathway. Furthermore, we showed that these HBV-DNA–transmitting EVs were resistant to antibody neutralization; stimulated emission depletion microscopy showed that EVs lacked hepatitis B surface antigen, the target of neutralizing antibodies. Conclusions: These findings suggest that EVs harbor a DNA cargo capable of transmitting viral DNA into hepatocytes during HBV infection, representing an additional antibody-neutralization–resistant route of HBV infection. Keywords: HBV, Extracellular Vesicles, Transmission Pathway

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

    International Nuclear Information System (INIS)

    Pszon-Bartosz, Kamila; Hansen, Jesper S.; Stibius, Karin B.; Groth, Jesper S.; Emneus, Jenny; Geschke, Oliver; Helix-Nielsen, Claus

    2011-01-01

    Research highlights: → We have established a vesicle fusion efficacy assay based on the major non-specific porin of Fusobacterium nucleatum (FomA). → Maximal fusion obtained was almost 150,000 porin insertions during 20 min. → Incorporation can be either first order or exponential kinetics which has implications for establishing protein delivery to biomimetic membranes. -- Abstract: Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10 5 FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm 2 within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.

  13. Investigation of nano lipid vesicles of methotrexate for anti-rheumatoid activity

    Directory of Open Access Journals (Sweden)

    Prabhu P

    2012-01-01

    was a significant reduction in edema volume in the rat group administered with the test stealth liposomal formulations and chitosan-coated conventional liposomes (PEGylated and chitosan-coated conventional compared to that of the control and standard (administered with free MTX group of rats. PEGylated liposomes showed almost equal efficacy as that of the chitosan-coated conventional liposomes.Conclusion: Lipid nano vesicles of MTX can be administered by intravenous route, whereby the drug selectively reaches the target site with reduced toxicity to other organs.Keywords: methotrexate, stealth liposomes, conventional liposomes, chitosan coating, targeted delivery, anti-rheumatoid efficacy

  14. Showing Value (Editorial

    Directory of Open Access Journals (Sweden)

    Denise Koufogiannakis

    2009-06-01

    Full Text Available When Su Cleyle and I first decided to start Evidence Based Library and Information Practice, one of the things we agreed upon immediately was that the journal be open access. We knew that a major obstacle to librarians using the research literature was that they did not have access to the research literature. Although Su and I are both academic librarians who can access a wide variety of library and information literature from our institutions, we belong to a profession where not everyone has equal access to the research in our field. Without such access to our own body of literature, how can we ever hope for practitioners to use research evidence in their decision making? It would have been contradictory to the principles of evidence based library and information practice to do otherwise.One of the specific groups we thought could use such an open access venue for discovering research literature was school librarians. School librarians are often isolated and lacking access to the research literature that may help them prove to stakeholders the importance of their libraries and their role within schools. Certainly, school libraries have been in decline and the use of evidence to show value is needed. As Ken Haycock noted in his 2003 report, The Crisis in Canada’s School Libraries: The Case for Reform and Reinvestment, “Across the country, teacher-librarians are losing their jobs or being reassigned. Collections are becoming depleted owing to budget cuts. Some principals believe that in the age of the Internet and the classroom workstation, the school library is an artifact” (9. Within this context, school librarians are looking to our research literature for evidence of the impact that school library programs have on learning outcomes and student success. They are integrating that evidence into their practice, and reflecting upon what can be improved locally. They are focusing on students and showing the impact of school libraries and

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  16. 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...

  17. Reversible Self-Assembly of Supramolecular Vesicles and Nanofibers Driven by Chalcogen-Bonding Interactions.

    Science.gov (United States)

    Chen, Liang; Xiang, Jun; Zhao, Yue; Yan, Qiang

    2018-05-29

    Chalcogen-bonding interactions have been viewed as new noncovalent forces in supramolecular chemistry. However, harnessing chalcogen bonds to drive molecular self-assembly processes is still unexplored. Here we report for the first time a novel class of supra-amphiphiles formed by Te···O or Se···O chalcogen-bonding interactions, and their self-assembly into supramolecular vesicles and nanofibers. A quasi-calix[4]chalcogenadiazole (C4Ch) as macrocyclic donor and a tailed pyridine N-oxide surfactant as molecular acceptor are designed to construct the donor-acceptor complex via chalcogen-chalcogen connection between the chalcogenadiazole moieties and oxide anion. The affinity of such chalcogen-bonding can dictate the geometry of supra-amphiphiles, driving diverse self-assembled morphologies. Furthermore, the reversible disassembly of these nanostructures can be promoted by introducing competing anions, such as halide ions, or by decreasing the systemic pH value.

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

    Science.gov (United States)

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

    2018-01-01

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

  19. Influence of testosterone on the distribution of 65Zn-binding proteins in the prostate and seminal vesicles of rats

    International Nuclear Information System (INIS)

    Arlt, R.; Foerster, R.; Scherr, F.; Guenther, T.

    1977-01-01

    65 Zn (7.4 MBq; 200 μCi) was injected intravenously into normal, castrated and castrated, testosteronesubstituted rats. After 1,24 and 48 hours, the distribution of 65 Zn-binding proteins in the 100,000 g supernatant of the prostate and seminal vesicles was investigated by separation on Sephadex G 100. The prostate and seminal vesicles from any one rat showed the same distribution pattern of 65 Zn-proteins. In castrated rats, the incorporation of 65 Zn was, however, 5-6 times lower than in the normal or castrated, testosterone-substituted rats. One hour after the injection, the highest activity of 65 Zn was found in proteins in the molecular weight range above 100,000. After 48 hours the greatest proportion of 65 Zn was present in the protein peak corresponding to 28,000 Daltons. (orig.) 891 AJ [de

  20. Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation.

    Science.gov (United States)

    Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Lasserre, Rémi; Agüera-Gonzalez, Sonia; Cuche, Céline; Danckaert, Anne; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

    2016-06-01

    The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation. © 2016 The Authors.

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

    Science.gov (United States)

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

    2010-01-01

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

  2. 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)).

  3. Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

    Science.gov (United States)

    Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H G; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

    2016-06-02

    Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

  4. Loss of Huntingtin stimulates capture of retrograde dense-core vesicles to increase synaptic neuropeptide stores.

    Science.gov (United States)

    Bulgari, Dinara; Deitcher, David L; Levitan, Edwin S

    2017-08-01

    The Huntington's disease protein Huntingtin (Htt) regulates axonal transport of dense-core vesicles (DCVs) containing neurotrophins and neuropeptides. DCVs travel down axons to reach nerve terminals where they are either captured in synaptic boutons to support later release or reverse direction to reenter the axon as part of vesicle circulation. Currently, the impact of Htt on DCV dynamics in the terminal is unknown. Here we report that knockout of Drosophila Htt selectively reduces retrograde DCV flux at proximal boutons of motoneuron terminals. However, initiation of retrograde transport at the most distal bouton and transport velocity are unaffected suggesting that synaptic capture rate of these retrograde DCVs could be altered. In fact, tracking DCVs shows that retrograde synaptic capture efficiency is significantly elevated by Htt knockout or knockdown. Furthermore, synaptic boutons contain more neuropeptide in Htt knockout larvae even though bouton size, single DCV fluorescence intensity, neuropeptide release in response to electrical stimulation and subsequent activity-dependent capture are unaffected. Thus, loss of Htt increases synaptic capture as DCVs travel by retrograde transport through boutons resulting in reduced transport toward the axon and increased neuropeptide in the terminal. These results therefore identify native Htt as a regulator of synaptic capture and neuropeptide storage. Copyright © 2017 Elsevier GmbH. All rights reserved.

  5. Getting into the flow: Red cells go on a roll, two-component vesicles swing

    Science.gov (United States)

    Viallat, Annie; Dupire, Jules; Khelloufi, Kamel; Al Halifa, Al Hair; Adhesion and Inflammation Team

    2013-11-01

    Red blood cells are soft capsules. Under shear flow, their two known motions were ``tumbling'' and ``swinging-tank treading,'' depending on cell mechanics and flow conditions. We reveal new wobbling regimes, among which the ``rolling'' regime, where red cells move as wheels on a road. We show, by coupling two video-microscopy approaches providing multi-directional cell pictures that the orientation of cells flipping into the flow is determined by the shear rate. Rolling permits to avoid energetically costly cellular deformations and is a true signature of the cytoskeleton elasticity. We highlight two transient dynamics: an intermittent regime during the ``tank-treading-to-flipping'' transition and a Frisbee-like ``spinning'' regime during the ``rolling-to-tank-treading'' transition. We find that the biconcave red cell shape is very stable under moderate shear stresses, and we interpret this result in terms of shape memory and elastic buckling. Finally, we generate lipid vesicles with a shape memory by using two lipids with different bending rigidities. These vesicles swing in shear flow similarly to red blood cells but their non-axisymmetric stress-free shape changes the periodicity of the motion and induces specific features.

  6. ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes.

    Science.gov (United States)

    Liu, Jun; Liu, Wenjing; Yang, Jun

    2016-02-11

    We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1-3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca(2+)-dependent lysosomal exocytosis.

  7. Skin penetration and retention of L-ascorbic acid 2-phosphate using multilamellar vesicles.

    Science.gov (United States)

    Yoo, Juno; Shanmugam, Srinivasan; Song, Chung-Kil; Kim, Dae-Duk; Choi, Han-Gon; Yong, Chul-Soon; Woo, Jong-Soo; Yoo, Bong Kyu

    2008-12-01

    Transdermal formulation of L-ascorbic acid 2-phosphate magnesium salt (A2P) was prepared using multilamellar vesicles (MLV). A2P was either physically mixed with or entrapped into three different MLVs of neutral, cationic, and anionic liposome vesicles. For the preparation of neutral MLVs, phosphatidylcholine (PC) and cholesterol (CH) were used. For cationic and anionic MLVs, dioleoyl-trimethylammonium-propane and dimyristoyl glycerophosphate were added as surface charge inducers, respectively, in addition to PC and CH. Particle size of the three A2P-loaded MLVs was submicron, and polydispersity index revealed homogenous distribution of the prepared MLVs except neutral ones. Skin penetration study with hairless mouse skin showed that both physical mixtures of A2P with empty MLVs and A2P-loaded MLVs increased penetration of the drug compared to aqueous A2P solution. During the penetration, however, significant amount of the drug was metabolized into L-ascorbic acid, which has no beneficial effect on stimulation of hair growth. Out of the physical mixtures and A2P-loaded MLVs tested, physical mixture of A2P with empty cationic MLV resulted in the greatest skin penetration and retention in hairless mouse skin.

  8. Staphylococcus aureus produces membrane-derived vesicles that induce host cell death.

    Directory of Open Access Journals (Sweden)

    Mamata Gurung

    Full Text Available Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

  9. Outer Membrane Vesicles From Probiotic and Commensal Escherichia coli Activate NOD1-Mediated Immune Responses in Intestinal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    María-Alexandra Cañas

    2018-03-01

    Full Text Available Gut microbiota plays a critical role in maintaining human intestinal homeostasis and host health. Bacterial extracellular vesicles are key players in bacteria–host communication, as they allow delivery of effector molecules into the host cells. Outer membrane vesicles (OMVs released by Gram-negative bacteria carry many ligands of pattern recognition receptors that are key components of innate immunity. NOD1 and NOD2 cytosolic receptors specifically recognize peptidoglycans present within the bacterial cell wall. These intracellular immune receptors are essential in host defense against bacterial infections and in the regulation of inflammatory responses. Recent contributions show that NODs are also fundamental to maintain intestinal homeostasis and microbiota balance. Peptidoglycan from non-invasive pathogens is delivered to cytosolic NODs through OMVs, which are internalized via endocytosis. Whether this pathway could be used by microbiota to activate NOD receptors remains unexplored. Here, we report that OMVs isolated from the probiotic Escherichia coli Nissle 1917 and the commensal ECOR12 activate NOD1 signaling pathways in intestinal epithelial cells. NOD1 silencing and RIP2 inhibition significantly abolished OMV-mediated activation of NF-κB and subsequent IL-6 and IL-8 expression. Confocal fluorescence microscopy analysis confirmed that endocytosed OMVs colocalize with NOD1, trigger the formation of NOD1 aggregates, and promote NOD1 association with early endosomes. This study shows for the first time the activation of NOD1-signaling pathways by extracellular vesicles released by gut microbiota.

  10. Platelet activating factor enhances synaptic vesicle exocytosis via PKC, elevated intracellular calcium, and modulation of synapsin 1 dynamics and phosphorylation

    Directory of Open Access Journals (Sweden)

    Jennetta W Hammond

    2016-01-01

    Full Text Available Platelet activating factor (PAF is an inflammatory phospholipid signaling molecule implicated in synaptic plasticity, learning and memory and neurotoxicity during neuroinflammation. However, little is known about the intracellular mechanisms mediating PAF’s physiological or pathological effects on synaptic facilitation. We show here that PAF receptors are localized at the synapse. Using fluorescent reporters of presynaptic activity we show that a non-hydrolysable analogue of PAF (cPAF enhances synaptic vesicle release from individual presynaptic boutons by increasing the size or release of the readily releasable pool and the exocytosis rate of the total recycling pool. cPAF also activates previously silent boutons resulting in vesicle release from a larger number of terminals. The underlying mechanism involves elevated calcium within presynaptic boutons and protein kinase C (PKC activation. Furthermore, cPAF increases synapsin I phosphorylation at sites 1 and 3, and increases dispersion of synapsin I from the presynaptic compartment during stimulation, freeing synaptic vesicles for subsequent release. These findings provide a conceptual framework for how PAF, regardless of its cellular origin, can modulate synapses during normal and pathologic synaptic activity.

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

    Directory of Open Access Journals (Sweden)

    Daniela eBoassa

    2015-02-01

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

  12. The Influence of Cholesterol on Fast Dynamics Inside of Vesicle and Planar Phospholipid Bilayers Measured with 2D IR Spectroscopy.

    Science.gov (United States)

    Kel, Oksana; Tamimi, Amr; Fayer, Michael D

    2015-07-23

    Phospholipid bilayers are frequently used as models for cell membranes. Here the influence of cholesterol on the structural dynamics in the interior of 1,2-dilauroyl-sn-glycero-3-phosphocholine (dilauroylphosphatidylcholine, DLPC) vesicles and DLPC planar bilayers are investigated as a function of cholesterol concentration. 2D IR vibrational echo spectroscopy was performed on the antisymmetric CO stretch of the vibrational probe molecule tungsten hexacarbonyl, which is located in the interior alkyl regions of the bilayers. The 2D IR experiments measure spectral diffusion, which is caused by the structural fluctuations of the bilayers. The 2D IR measurements show that the bilayer interior alkyl region dynamics occur on time scales ranging from a few picoseconds to many tens of picoseconds. These are the time scales of the bilayers' structural dynamics, which act as the dynamic solvent bath for chemical processes of membrane biomolecules. The results suggest that at least a significant fraction of the dynamics arise from density fluctuations. Samples are studied in which the cholesterol concentration is varied from 0% to 40% in both the vesicles (72 nm diameter) and fully hydrated planar bilayers in the form of aligned multibilayers. At all cholesterol concentrations, the structural dynamics are faster in the curved vesicle bilayers than in the planar bilayers. As the cholesterol concentration is increased, at a certain concentration there is a sudden change in the dynamics, that is, the dynamics abruptly slow down. However, this change occurs at a lower concentration in the vesicles (between 10% and 15% cholesterol) than in the planar bilayers (between 25% and 30% cholesterol). The sudden change in the dynamics, in addition to other IR observables, indicates a structural transition. However, the results show that the cholesterol concentration at which the transition occurs is influenced by the curvature of the bilayers.

  13. 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.

  14. 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...

  15. 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.

  16. 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

  17. 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...

  18. Cellular therapy without cells: extracellular vesicles promote activation of stem cells after irradiation

    International Nuclear Information System (INIS)

    Lange, C.

    2016-01-01

    Mesenchymal stromal cells from the bone marrow (MSC) have been shown to be effective in several cell therapeutic treatments. However, MSC accumulate in lungs after i.v. injection. How do MSC transfer their potential to organs with therapeutic need? We show that released extracellular vesicles (EV) might be playing an active role in this transfer. EV were isolated from MSC supernatant and characterized with flow cytometry, proteomics and next generation sequencing. Our data showed the transfer of RNAs, clustering into several protective gene groups. Besides, we repeatedly detected genomic DNA on vesicles. Using a plant - derived detector gene we showed horizontal DNA transfer via EV. Furthermore, we showed that EV were able to salvage stem/progenitor cells in vitro from radiation suppression. Three selected proteins from proteomics data were examined for stem cell protection after irradiation. EV derived from down-regulated producer MSC showed a substantial loss of protection in irradiated stem cells supporting their relevance for stem cell protection. Finally, we showed that EV after i.v. injection into lethally irradiated animals colocalize within 2-4 hours with hematopoietic stem cells in the bone marrow giving hint to direct protection of stem cells by EV. In conclusion, EV derived from bone marrow MSC were able to transfer several cargo compounds leading potentially to change of the genetic properties. Importantly, EV protect irradiated hematopoietic stem cells, stimulate their recovery and proliferation and rescue lethally irradiated animals long-term. Thus, EV might be an alternative for future cell therapeutic treatment particularly in radiation-based events. (author)

  19. FAT/CD36 is localized in sarcolemma and in vesicle-like structures in subsarcolemma regions but not in mitochondria

    DEFF Research Database (Denmark)

    Jeppesen, Jacob; Mogensen, Martin; Prats, Clara

    2010-01-01

    was performed on single muscle fibers dissected from soleus muscle of lean and obese Zucker rats and from the vastus lateralis muscle from humans. Co-staining against FAT/CD36 and MitoNEET clearly show that FAT/CD36 is highly present in sarcolemma and it also associates with some vesicle-like intracellular...

  20. 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.

  1. 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.

  2. Ultrastructral studies of epidermis in acute radiation dermatitis. Basal lamina thickening and coated vesicles. [X-ray

    Energy Technology Data Exchange (ETDEWEB)

    Oguchi, M.; Komura, J.; Ofuji, S.

    1978-06-29

    Fine structural changes of keratinocytes by x-ray were examined in normal skin area around a lesion of Bowen's disease. The area was exposed to about 600 r over 28 days. The findings were: a decreased number of desmosomes and microvilli, formation of cytoplasmic vaculoes with or without membrane, perinuclear aggregation of tonofibrils, intracytoplasmic desmosomes and gap junction, cytoplasmic occurrence of dense bodies, lipid droplet and glycogen particles, changes in mitochondria, endoplasmic reticulum and Golgi complex, and deep invagination of the nuclear membrane. Possible exocytotic nature of coated vesicles in x-ray irradiated keratinocytes is discussed.

  3. 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.

  4. 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...

  5. 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.

  6. 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.

  7. 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.

  8. Extracellular Vesicles in Heart Disease: Excitement for the Future?

    Directory of Open Access Journals (Sweden)

    Kirsty M. Danielson

    2014-01-01

    Full Text Available Extracellular vesicles (EV, including exosomes, microvesicles and apoptotic bodies, are released from numerous cell types and are involved in intercellular communication, physiological functions and the pathology of disease. They have been shown to carry and transfer a wide range of cargo including proteins, lipids and nucleic acids. The role of EVs in cardiac physiology and heart disease is an emerging field that has produced intriguing findings in recent years. This review will outline what is currently known about EVs in the cardiovascular system, including cellular origins, functional roles and utility as biomarkers and potential therapeutics.

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

    Directory of Open Access Journals (Sweden)

    Soni Priya Valeru

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jian Wu

    2015-01-01

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

  11. HIV internalization into oral and genital epithelial cells by endocytosis and macropinocytosis leads to viral sequestration in the vesicles

    Science.gov (United States)

    Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina; Lien, Kathy; Tugizov, Sharof M.

    2018-01-01

    Recently, we showed that HIV-1 is sequestered, i.e., trapped, in the intracellular vesicles of oral and genital epithelial cells. Here, we investigated the mechanisms of HIV-1 sequestration in vesicles of polarized tonsil, foreskin and cervical epithelial cells. HIV-1 internalization into epithelial cells is initiated by multiple entry pathways, including clathrin-, caveolin/lipid raft-associated endocytosis and macropinocytosis. Inhibition of HIV-1 attachment to galactosylceramide and heparan sulfate proteoglycans, and virus endocytosis and macropinocytosis reduced HIV-1 sequestration by 30–40%. T-cell immunoglobulin and mucin domain 1 (TIM-1) were expressed on the apical surface of polarized tonsil, cervical and foreskin epithelial cells. However, TIM-1-associated HIV-1 macropinocytosis and sequestration were detected mostly in tonsil epithelial cells. Sequestered HIV-1 was resistant to trypsin, pronase, and soluble CD4, indicating that the sequestered virus was intracellular. Inhibition of HIV-1 intraepithelial sequestration and elimination of vesicles containing virus in the mucosal epithelium may help in the prevention of HIV-1 mucosal transmission. PMID:29277006

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We...... establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein...... reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR)=50 more than 105 FomA proteins could be incorporated...

  13. Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs

    Science.gov (United States)

    Phinney, Donald G.; Di Giuseppe, Michelangelo; Njah, Joel; Sala, Ernest; Shiva, Sruti; St Croix, Claudette M.; Stolz, Donna B.; Watkins, Simon C.; Di, Y. Peter; Leikauf, George D.; Kolls, Jay; Riches, David W. H.; Deiuliis, Giuseppe; Kaminski, Naftali; Boregowda, Siddaraju V.; McKenna, David H.; Ortiz, Luis A.

    2015-01-01

    Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs. PMID:26442449

  14. Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs.

    Science.gov (United States)

    Phinney, Donald G; Di Giuseppe, Michelangelo; Njah, Joel; Sala, Ernest; Shiva, Sruti; St Croix, Claudette M; Stolz, Donna B; Watkins, Simon C; Di, Y Peter; Leikauf, George D; Kolls, Jay; Riches, David W H; Deiuliis, Giuseppe; Kaminski, Naftali; Boregowda, Siddaraju V; McKenna, David H; Ortiz, Luis A

    2015-10-07

    Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs.

  15. ATP Modifies the Proteome of Extracellular Vesicles Released by Microglia and Influences Their Action on Astrocytes

    Directory of Open Access Journals (Sweden)

    Francesco Drago

    2017-12-01

    Full Text Available Extracellular ATP is among molecules promoting microglia activation and inducing the release of extracellular vesicles (EVs, which are potent mediators of intercellular communication between microglia and the microenvironment. We previously showed that EVs produced under ATP stimulation (ATP-EVs propagate a robust inflammatory reaction among astrocytes and microglia in vitro and in mice with subclinical neuroinflammation (Verderio et al., 2012. However, the proteome of EVs released upon ATP stimulation has not yet been elucidated. In this study we applied a label free proteomic approach to characterize the proteome of EVs released constitutively and during microglia activation with ATP. We show that ATP drives sorting in EVs of a set of proteins implicated in cell adhesion/extracellular matrix organization, autophagy-lysosomal pathway and cellular metabolism, that may influence the response of recipient astrocytes to EVs. These data provide new clues to molecular mechanisms involved in microglia response to ATP and in microglia signaling to the environment via EVs.

  16. Complexin II plays a positive role in Ca2+-triggered exocytosis by facilitating vesicle priming

    DEFF Research Database (Denmark)

    Cai, Haijiang; Reim, Kerstin; Varoqueaux, Frederique

    2008-01-01

    SNARE-mediated exocytosis is a multistage process central to synaptic transmission and hormone release. Complexins (CPXs) are small proteins that bind very rapidly and with a high affinity to the SNARE core complex, where they have been proposed recently to inhibit exocytosis by clamping the comp......SNARE-mediated exocytosis is a multistage process central to synaptic transmission and hormone release. Complexins (CPXs) are small proteins that bind very rapidly and with a high affinity to the SNARE core complex, where they have been proposed recently to inhibit exocytosis by clamping......), while showing no change in the kinetics of fusion pore dilation or morphological vesicle docking. Overexpression of WT CPX II-but not of SNARE-binding-deficient mutants-restores the size of the the releasable pools in knockout cells, and in WT cells it markedly enlarges them. Our results show that CPXs...

  17. 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.

  18. Decreasing Serial Cost Sharing

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Østerdal, Lars Peter

    The increasing serial cost sharing rule of Moulin and Shenker [Econometrica 60 (1992) 1009] and the decreasing serial rule of de Frutos [Journal of Economic Theory 79 (1998) 245] have attracted attention due to their intuitive appeal and striking incentive properties. An axiomatic characterization...... of the increasing serial rule was provided by Moulin and Shenker [Journal of Economic Theory 64 (1994) 178]. This paper gives an axiomatic characterization of the decreasing serial rule...

  19. Decreasing serial cost sharing

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Østerdal, Lars Peter Raahave

    2009-01-01

    The increasing serial cost sharing rule of Moulin and Shenker (Econometrica 60:1009-1037, 1992) and the decreasing serial rule of de Frutos (J Econ Theory 79:245-275, 1998) are known by their intuitive appeal and striking incentive properties. An axiomatic characterization of the increasing serial...... rule was provided by Moulin and Shenker (J Econ Theory 64:178-201, 1994). This paper gives an axiomatic characterization of the decreasing serial rule....

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

    International Nuclear Information System (INIS)

    Goldsmith, M.H.M.

    1986-01-01

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  2. Sulfate uptake by crustacean hepatopancreatic brush border membrane vesicles

    International Nuclear Information System (INIS)

    Gerencser, G.A.; Cattey, M.A; Ahearn, G.A.

    1990-01-01

    Purified brush border membrane vesicles (BBMV) were prepared from Atlantic lobster (Homarus americanus) hepatopancreas using differential centrifugation and Mg +2 precipitation techniques. Uptake of 0.1 mM 35 SO 4 -2 was stimulated by pre-loading vesicles with Cl - leading to a transient accumulation of isotope more than twice that at equilibrium. Pre-loading with HCO 3 - or gluconate had no effect on sulfate uptake. No stimulation of 35 SO 4 -2 was observed in the presence of inwardly directed Na + or tetramethylammonium + gradients. Uptake of the divalent anion was strongly stimulated by inwardly directed proton gradients (pH o i ) and markedly inhibited by outwardly directed proton gradients (pH o > pH i ). 35 SO 4 -2 /Cl - exchange was enhanced by imposing a transmembrane inside positive K + diffusion potential and inhibited by a membrane potential of the opposite polarity (K + /valinomycin). Results suggest the presence of a proton-dependent, electrogenic anion antiport mechanism in BBMV isolated from the crustacean hepatopancreas

  3. The computational route from bilayer membranes to vesicle fusion

    International Nuclear Information System (INIS)

    Shillcock, Julian C; Lipowsky, Reinhard

    2006-01-01

    Biological membranes are examples of 'smart' materials whose properties and behaviour emerge from the propagation across many scales of the molecular characteristics of their constituents. Artificial smart materials, such as drug delivery vehicles and biosensors, often rely on modifying naturally occurring soft matter, such as polymers and lipid vesicles, so that they possess useful behaviour. However, the complexity of natural membranes, both in their static properties, exemplified in their phase behaviour, and in their dynamic properties, as in the kinetics of their formation and interactions, hinders their rational modification. Mesoscopic simulations, such as dissipative particle dynamics (DPD), allow in silico experiments to be easily and cheaply performed on complex, soft materials requiring as input only the molecular structure of the constituents at a coarse-grained level. They can therefore act as a guide to experimenters prior to performing costly assays. Additionally, mesoscopic simulations provide the only currently feasible window on the length- and timescales relevant to important biophysical processes such as vesicle fusion. We review here the development of computational models of bilayer membranes, and in particular the use of mesoscopic simulations to follow the molecular rearrangements that occur during membrane fusion

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

    International Nuclear Information System (INIS)

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-15

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

  6. Intraluminal proteome and peptidome of human urinary extracellular vesicles.

    Science.gov (United States)

    Liu, Xinyu; Chinello, Clizia; Musante, Luca; Cazzaniga, Marta; Tataruch, Dorota; Calzaferri, Giulio; James Smith, Andrew; De Sio, Gabriele; Magni, Fulvio; Zou, Hequn; Holthofer, Harry

    2015-06-01

    Urinary extracellular vesicles (UEVs) are a novel source for disease biomarker discovery. However, Tamm-Horsfall protein (THP) is still a challenge for proteomic analysis since it can inhibit detection of low-abundance proteins. Here, we introduce a new approach that does not involve an ultracentrifugation step to enrich vesicles and that reduces the amount of THP to manageable levels. UEVs were dialyzed and ultrafiltered after reduction and alkylation. The retained fraction was digested with trypsin to reduce the remaining THP and incubated with deoxycholate (DOC). The internal peptidome and internal proteome were analyzed by LC-ESI-MS. A total of 942 different proteins and 3115 unique endogenous peptide fragments deriving from 973 different protein isoforms were identified. Around 82% of the key endosomal sorting complex required for transport components of UEVs generation could be detected from the intraluminal content. Our UEVs preparation protocol provides a simplified way to investigate the intraluminal proteome and peptidome, in particular the subpopulation of UEVs of the trypsin-resistant class of exosomes (positive for tumor susceptibility gene101) and eliminates the majority of interfering proteins such as THP. This method allows the possibility to study endoproteome and endopeptidome of UEVs, thus greatly facilitating biomarker discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Vesicle computers: Approximating a Voronoi diagram using Voronoi automata

    International Nuclear Information System (INIS)

    Adamatzky, Andrew; De Lacy Costello, Ben; Holley, Julian; Gorecki, Jerzy; Bull, Larry

    2011-01-01

    Highlights: → We model irregular arrangements of vesicles filled with chemical systems. → We examine influence of precipitation threshold on the system's computational potential. → We demonstrate computation of Voronoi diagram and skeleton. - Abstract: Irregular arrangements of vesicles filled with excitable and precipitating chemical systems are imitated by Voronoi automata - finite-state machines defined on a planar Voronoi diagram. Every Voronoi cell takes four states: resting, excited, refractory and precipitate. A resting cell excites if it has at least one neighbour in an excited state. The cell precipitates if the ratio of excited cells in its neighbourhood versus the number of neighbours exceeds a certain threshold. To approximate a Voronoi diagram on Voronoi automata we project a planar set onto the automaton lattice, thus cells corresponding to data-points are excited. Excitation waves propagate across the Voronoi automaton, interact with each other and form precipitate at the points of interaction. The configuration of the precipitate represents the edges of an approximated Voronoi diagram. We discover the relationship between the quality of the Voronoi diagram approximation and the precipitation threshold, and demonstrate the feasibility of our model in approximating Voronoi diagrams of arbitrary-shaped objects and in constructing a skeleton of a planar shape.

  8. Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.

    Directory of Open Access Journals (Sweden)

    Hina Kalra

    Full Text Available Extracellular vesicles (EVs are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

  9. 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.

  10. Exocytosis from chromaffin cells: hydrostatic pressure slows vesicle fusion

    Science.gov (United States)

    Stühmer, Walter

    2015-01-01

    Pressure affects reaction kinetics because chemical transitions involve changes in volume, and therefore pressure is a standard thermodynamic parameter to measure these volume changes. Many organisms live in environments at external pressures other than one atmosphere (0.1 MPa). Marine animals have adapted to live at depths of over 7000 m (at pressures over 70 MPa), and microorganisms living in trenches at over 110 MPa have been retrieved. Here, kinetic changes in secretion from chromaffin cells, measured as capacitance changes using the patch-clamp technique at pressures of up to 20 MPa are presented. It is known that these high pressures drastically slow down physiological functions. High hydrostatic pressure also affects the kinetics of ion channel gating and the amount of current carried by them, and it drastically slows down synaptic transmission. The results presented here indicate a similar change in volume (activation volume) of 390 ± 57 Å3 for large dense-core vesicles undergoing fusion in chromaffin cells and for degranulation of mast cells. It is significantly larger than activation volumes of voltage-gated ion channels in chromaffin cells. This information will be useful in finding possible protein conformational changes during the reactions involved in vesicle fusion and in testing possible molecular dynamic models of secretory processes. PMID:26009771

  11. 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.

  12. 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.

  13. 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.

  14. Partition thermodynamics of ionic surfactants between phosphatidylcholine vesicle and water phases

    Science.gov (United States)

    Chu, Shin-Chi; Hung, Chia-Hui; Wang, Shun-Cheng; Tsao, Heng-Kwong

    2003-08-01

    The partition of ionic surfactants (sodium alkyl sulfate and alkyl trimethyl ammonium bromide) between phosphatidylcholine vesicles and aqueous phase is investigated by simple conductometry under different temperatures. The experimental results can be well represented by the proposed regular solution theory and the thermodynamic parameters satisfy the thermodynamic consistency. The deviation from ideal partition is manifested through the effective interaction energy between lipid and surfactant wb, which is O(kT) large. It is found that wb rises as the alkyl chain is decreased for a specified head group. This is attributed to significant mismatch of chain lengths between surfactant and lipid molecules. The partition coefficient K declines with increasing temperature. The energy barrier from bilayer to aqueous phase, Δμ/kT∝ln K, is in the range of 16-26 kJ/mol. As the alkyl chain length is decreased for a given head group, Δμ is lowered by 1.3-1.5 kJ/mol per methylene group. Two independent analyses are employed to confirm this result. Using the thermodynamic parameters determined from experiments, the internal energy, entropy, and free energy of the partition process can be derived. Partition is essentially driven by the internal energy gain. The solubilizing ability, which is represented by the maximum surfactant-lipid ratio in the bilayer, Reb also decreases in accord with the K parameter. It is because the change in temperature influences the surfactant incorporation into the bilayer more than the formation of micelles.

  15. Significant changes of T2 value in the peripheral zone and seminal vesicles after ejaculation

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Takeshi [Dokkyo Medical University, Department of Radiology, Tochigi (Japan); Dokkyo Medical University Koshigaya Hospital, Department of Urology, Saitama (Japan); Kaji, Yasushi [Dokkyo Medical University, Department of Radiology, Tochigi (Japan); Shukuya, Toshiro; Nozaki, Miwako [Dokkyo Medical University Koshigaya Hospital, Department of Radiology, Saitama (Japan); Soh, Shigehiro; Okada, Hiroshi [Dokkyo Medical University Koshigaya Hospital, Department of Urology, Saitama (Japan)

    2018-03-15

    To analyse the quantitative changes of the prostate and seminal vesicles (SV) on magnetic resonance imaging (MRI) after ejaculation. Ten healthy young males were enrolled for T2-weighted and T2 mapping MRI before and after two consecutive ejaculations. T2 values of the peripheral zone (PZ) and the central gland (CG) at the midgland of the prostate were compared before and after ejaculation, respectively. T2 values of the PZ at the apex and base were also compared before and after, respectively. Pre- and post-ejaculation SV volumes were compared. The Wilcoxon's signed rank test with Bonferroni adjustment was used for comparison. After ejaculation, T2 values of the PZ significantly decreased (mean, 119±20 vs. 105±21, p=0.002) while those of the CG did not significantly change at the midgland. At the apex, T2 values of the PZ also decreased significantly (mean, 114±9 vs. 94±7, p=0.002). On the other hand, T2 values of the PZ did not change at the base. SV volumes were significantly reduced after ejaculation (mean, 11.1±7.7mL vs. 7.2±6.7mL, p=0.002). Ejaculation decreases T2 values of the PZ at the midgland and apex, and reduces SV volumes. Abstinence periods should be considered in evaluating the prostate and SV on MRI. (orig.)

  16. 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.

  17. Free amino acids in synaptic vesicles isolated from the cerebellum and cerebral hemispheres of control and neonatally X-irradiated rats

    International Nuclear Information System (INIS)

    Valcana, T.; Hudson, D.B.; Timiras, P.S.

    1984-01-01

    X-irradiation of the rat brain (1000R, at two days of age), suppresses the normal age-related increase in the weight of the cerebellum and cerebral hemispheres and influences amino acid levels. The decrease in glutamic acid concentration, particularly in the cerebellum, supports the previously advanced proposition that this amino acid may be associated with or may be the transmitter of, the rat cerebellar granule cells. Subfractionation of the cerebellar tissue reveals that the decrease in the glutamic acid level consequent to the loss of granule cells, is reflected in the cytoplasmic fraction but not in the synaptic vesicle subfraction, where glutamic acid was increased. The reduced weight gain in the cerebral hemispheres after irradiation, is accompanied by a significant decrease of aspartate in the cytoplasmic fraction, changes which suggest that a specific cell type, with aspartic acid as its neurotransmitter (possibly in the hippocampus), may also be radiosensitive in the early postnatal period. In contrast, in the synaptic vesicle fraction from cerebral hemispheres, all free amino acids, with the exception of glutamine, increased significantly. Overall, the changes in free amino acid concentration induced by X-irradiation in the cytoplasmic fraction in both brain regions studied are opposite to those found in the synaptic vesicle fraction and although they may indicate changes in specific cell populations, as proposed above, they could also reflect changes in cellular compartmentalization and metabolism or changes in the relative axonal arborization of the affected regions

  18. Inflammaging and Frailty Status Do Not Result in an Increased Extracellular Vesicle Concentration in Circulation

    Directory of Open Access Journals (Sweden)

    Ainhoa Alberro

    2016-07-01

    Full Text Available In the last decades extracellular vesicles (EVs have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed “inflammaging” has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process.

  19. Inflammaging and Frailty Status Do Not Result in an Increased Extracellular Vesicle Concentration in Circulation.

    Science.gov (United States)

    Alberro, Ainhoa; Sáenz-Cuesta, Matías; Muñoz-Culla, Maider; Mateo-Abad, Maider; Gonzalez, Esperanza; Carrasco-Garcia, Estefania; Araúzo-Bravo, Marcos J; Matheu, Ander; Vergara, Itziar; Otaegui, David

    2016-07-20

    In the last decades extracellular vesicles (EVs) have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed "inflammaging" has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process.

  20. Biological interaction of living cells with COSAN-based synthetic vesicles.

    Science.gov (United States)

    Tarrés, Màrius; Canetta, Elisabetta; Paul, Eleanor; Forbes, Jordan; Azzouni, Karima; Viñas, Clara; Teixidor, Francesc; Harwood, Adrian J

    2015-01-15

    Cobaltabisdicarbollide (COSAN) [3,3'-Co(1,2-C2B9H11)2](-), is a complex boron-based anion that has the unusual property of self-assembly into membranes and vesicles. These membranes have similar dimensions to biological membranes found in cells, and previously COSAN has been shown to pass through synthetic lipid membranes and those of living cells without causing breakdown of membrane barrier properties. Here, we investigate the interaction of this inorganic membrane system with living cells. We show that COSAN has no immediate effect on cell viability, and cells fully recover when COSAN is removed following exposure for hours to days. COSAN elicits a range of cell biological effects, including altered cell morphology, inhibition of cell growth and, in some cases, apoptosis. These observations reveal a new biology at the interface between inorganic, synthetic COSAN membranes and naturally occurring biological membranes.

  1. Environmental Decontamination of a Chemical Warfare Simulant Utilizing a Membrane Vesicle-Encapsulated Phosphotriesterase.

    Science.gov (United States)

    Alves, Nathan J; Moore, Martin; Johnson, Brandy J; Dean, Scott N; Turner, Kendrick B; Medintz, Igor L; Walper, Scott A

    2018-05-09

    While technologies for the remediation of chemical contaminants continue to emerge, growing interest in green technologies has led researchers to explore natural catalytic mechanisms derived from microbial species. One such method, enzymatic degradation, offers an alternative to harsh chemical catalysts and resins. Recombinant enzymes, however, are often too labile or show limited activity when challenged with nonideal environmental conditions that may vary in salinity, pH, or other physical properties. Here, we demonstrate how phosphotriesterase encapsulated in a bacterial outer membrane vesicle can be used to degrade the organophosphate chemical warfare agent (CWA) simulant paraoxon in environmental water samples. We also carried out remediation assays on solid surfaces, including glass, painted metal, and fabric, that were selected as representative materials, which could potentially be contaminated with a CWA.

  2. 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

  3. 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.

  4. The early postnatal pattern of vesicle formation in different regions of the porcien small intestine

    DEFF Research Database (Denmark)

    Elbrønd, Vibeke Sødring; Weström, B.R.

    2007-01-01

    applied to visualize cytoplasmatic subcellular components such as fat (Oil red O) and carbohydrates (PAS). Appearance and morphology of the epithelial vesicles were compared. In the proximal region several small supranuclear and a single large subnuclear electron dense, eosinophilic and PAS+ vesicle were...

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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).

  10. Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

    DEFF Research Database (Denmark)

    Shupliakov, Oleg; Bloom, Ona; Gustafsson, Jenny S

    2002-01-01

    Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin ...

  11. 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...

  12. 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.

  13. 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.

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

    Science.gov (United States)

    Leung, Cheuk Yui Curtis

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

  15. Decreasing relative risk premium

    DEFF Research Database (Denmark)

    Hansen, Frank

    2007-01-01

    such that the corresponding relative risk premium is a decreasing function of present wealth, and we determine the set of associated utility functions. We find a new characterization of risk vulnerability and determine a large set of utility functions, closed under summation and composition, which are both risk vulnerable...

  16. Decreasing asthma morbidity

    African Journals Online (AJOL)

    1994-12-12

    Dec 12, 1994 ... Apart from the optimal use of drugs, various supplementary methods have been tested to decrease asthma morbidity, usually in patients from reiatively affluent socio-economic backgrounds. A study of additional measures taken in a group of moderate to severe adult asthmatics from very poor socio- ...

  17. 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.

  18. 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

  19. 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

  20. A model for growth of a single fungal hypha based on well-mixed tanks in series: simulation of nutrient and vesicle transport in aerial reproductive hyphae.

    Science.gov (United States)

    Balmant, Wellington; Sugai-Guérios, Maura Harumi; Coradin, Juliana Hey; Krieger, Nadia; Furigo Junior, Agenor; Mitchell, David Alexander

    2015-01-01

    Current models that describe the extension of fungal hyphae and development of a mycelium either do not describe the role of vesicles in hyphal extension or do not correctly describe the experimentally observed profile for distribution of vesicles along the hypha. The present work uses the n-tanks-in-series approach to develop a model for hyphal extension that describes the intracellular transport of nutrient to a sub-apical zone where vesicles are formed and then transported to the tip, where tip extension occurs. The model was calibrated using experimental data from the literature for the extension of reproductive aerial hyphae of three different fungi, and was able to describe different profiles involving acceleration and deceleration of the extension rate. A sensitivity analysis showed that the supply of nutrient to the sub-apical vesicle-producing zone is a key factor influencing the rate of extension of the hypha. Although this model was used to describe the extension of a single reproductive aerial hypha, the use of the n-tanks-in-series approach to representing the hypha means that the model has the flexibility to be extended to describe the growth of other types of hyphae and the branching of hyphae to form a complete mycelium.

  1. 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

  2. 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.

  3. 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.

  4. Maturation arrest of human oocytes at germinal vesicle stage

    Directory of Open Access Journals (Sweden)

    Zhi Qin Chen

    2010-01-01

    Full Text Available Maturation arrest of human oocytes may occur at various stages of the cell cycle. A total failure of human oocytes to complete meiosis is rarely observed during assisted conception cycles. We describe here a case of infertile couples for whom all oocytes repeatedly failed to mature at germinal vesicle (GV stage during in vitro fertilization/Intra cytoplasmic sperm injection (IVF/ICSI. The patient underwent controlled ovarian stimulation followed by oocyte retrieval and IVF/ICSI. The oocytes were stripped off cumulus cells prior to the ICSI procedure and their maturity status was defined. The oocyte maturation was repeatedly arrested at the GV. Oocyte maturation arrest may be the cause of infertility in this couple. The recognition of oocyte maturation arrest as a specific medical condition may contribute to the characterization of the currently known as "oocyte factor." The cellular and genetic mechanisms causing oocyte maturation arrest should be the subject for further investigation.

  5. Phosphoproteins in extracellular vesicles as candidate markers for breast cancer.

    Science.gov (United States)

    Chen, I-Hsuan; Xue, Liang; Hsu, Chuan-Chih; Paez, Juan Sebastian Paez; Pan, Li; Andaluz, Hillary; Wendt, Michael K; Iliuk, Anton B; Zhu, Jian-Kang; Tao, W Andy

    2017-03-21

    The state of protein phosphorylation can be a key determinant of cellular physiology such as early-stage cancer, but the development of phosphoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate a strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs isolated from small volumes of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer compared with healthy controls. Several biomarkers were validated in individual patients using paralleled reaction monitoring for targeted quantitation. This study demonstrates that the development of phosphoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring.

  6. Electrogenic sulfate uptake by crustacean hepatopancreatic basolateral membrane vesicles

    International Nuclear Information System (INIS)

    Cattey, M.A.; Gerencser, G.A.; Aheam, G.A.

    1990-01-01

    Basolateral membrane vesicles (BLMV) were isolated from Atlantic lobster (Homarus americanus) hepatopancreas and purified by discontinuous sucrose gradient centrifugation. BLMV prepared in this fashion were osmotically reactive exhibiting linear dependence of vesicular 35 SO 4 -2 uptake to increasing external osmotic pressure with negligible non-specific isotope binding. Under short circuited conditions (valinomycin/K + ) BLMV responded to either a HCO 3 - gradient directed out or equilibrated HCO 3 - (10 mM) by displaying short term accumulation of sulfate above that of equilibrium. Uptake of divalent anion was unaffected by an inwardly directed transmembrane Na + or tetramethylammonium + gradient. 35 SO 4 -2 /HCO 3 - exchange in the presence of valinomycin was stimulated by transient inside positive K + diffusion potentials and inhibited by transient inside negative K + diffusion potentials. The role of electrogenic anion exchange by hepatopancreas BLMV in transcellular sulfate transport is discussed

  7. Stem Cell Extracellular Vesicles: Extended Messages of Regeneration

    Science.gov (United States)

    Riazifar, Milad; Pone, Egest J.; Lötvall, Jan; Zhao, Weian

    2017-01-01

    Stem cells are critical to maintaining steady-state organ homeostasis and regenerating injured tissues. Recent intriguing reports implicate extracellular vesicles (EVs) as carriers for the distribution of morphogens and growth and differentiation factors from tissue parenchymal cells to stem cells, and conversely, stem cell–derived EVs carrying certain proteins and nucleic acids can support healing of injured tissues. We describe approaches to make use of engineered EVs as technology platforms in therapeutics and diagnostics in the context of stem cells. For some regenerative therapies, natural and engineered EVs from stem cells may be superior to single-molecule drugs, biologics, whole cells, and synthetic liposome or nanoparticle formulations because of the ease of bioengineering with multiple factors while retaining superior biocompatibility and biostability and posing fewer risks for abnormal differentiation or neoplastic transformation. Finally, we provide an overview of current challenges and future directions of EVs as potential therapeutic alternatives to cells for clinical applications. PMID:27814025

  8. 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.

  9. Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds

    Science.gov (United States)

    Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H. G.; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

    2016-06-01

    Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of

  10. Spontaneous vesicle phase formation by pseudogemini surfactants in aqueous solutions.

    Science.gov (United States)

    Sun, Nan; Shi, Lijuan; Lu, Fei; Xie, Shuting; Zheng, Liqiang

    2014-08-14

    The phase behavior of a kind of pseudogemini surfactant in aqueous solutions, formed by the mixture of sodium dodecyl benzene sulfonate (SDBS) and butane-1,4-bis (methylimidazolium bromide) ([mim-C4-mim]Br2) or butane-1,4-bis(methylpyrrolidinium bromide) ([mpy-C4-mpy]Br2) in a molar ratio of 2 : 1, is reported in the present work. When [mim-C4-mim]Br2 or [mpy-C4-mpy]Br2 is mixed with SDBS in aqueous solutions, one cationic [mim-C4-mim]Br2 or [mpy-C4-mpy]Br2 molecule "bridges" two SDBS molecules by noncovalent interactions (e.g. electrostatic, π-π stacking, and σ-π interactions), behaving like a pseudogemini surfactant. Vesicles can be formed by this kind of pseudogemini surfactant, determined by freeze-fracture transmission electron microscopy (FF-TEM) or cryogenic-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). The mixed system of sodium dodecyl sulfate (SDS) with [mim-C4-mim]Br2 or [mpy-C4-mpy]Br2 was also constructed, and only micelles were observed. We infer that a pseudogemini surfactant is formed under the synergic effect of electrostatic, π-π stacking, and σ-π interactions in the SDBS/[mim-C4-mim]Br2/H2O system, while electrostatic attraction and hydrophobic interactions may provide the directional force for vesicle formation in the SDBS/[mpy-C4-mpy]Br2/H2O system.

  11. A VESICLE TRAFFICKING PROTEIN αSNAP REGULATES PANETH CELL DIFFERENTIATION IN VIVO

    Science.gov (United States)

    Lechuga, Susana; Naydenov, Nayden G.; Feygin, Alex; Jimenez, Antonio J.; Ivanov, Andrei I.

    2017-01-01

    A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. PMID:28359759

  12. A vesicle trafficking protein αSNAP regulates Paneth cell differentiation in vivo.

    Science.gov (United States)

    Lechuga, Susana; Naydenov, Nayden G; Feygin, Alex; Jimenez, Antonio J; Ivanov, Andrei I

    2017-05-13

    A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. On-chip immunoelectrophoresis of extracellular vesicles released from human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Takanori Akagi

    Full Text Available Extracellular vesicles (EVs including exosomes and microvesicles have attracted considerable attention in the fields of cell biology and medicine. For a better understanding of EVs and further exploration of their applications, the development of analytical methods for biological nanovesicles has been required. In particular, considering the heterogeneity of EVs, methods capable of measuring individual vesicles are desired. Here, we report that on-chip immunoelectrophoresis can provide a useful method for the differential protein expression profiling of individual EVs. Electrophoresis experiments were performed on EVs collected from the culture supernatant of MDA-MB-231 human breast cancer cells using a measurement platform comprising a microcapillary electrophoresis chip and a laser dark-field microimaging system. The zeta potential distribution of EVs that reacted with an anti-human CD63 (exosome and microvesicle marker antibody show