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Sample records for vesicle acetylcholine release

  1. Amperometric detection of single vesicle acetylcholine release events from an artificial cell.

    Science.gov (United States)

    Keighron, Jacqueline D; Wigström, Joakim; Kurczy, Michael E; Bergman, Jenny; Wang, Yuanmo; Cans, Ann-Sofie

    2015-01-21

    Acetylcholine is a highly abundant nonelectroactive neurotransmitter in the mammalian central nervous system. Neurochemical release occurs on the millisecond time scale, requiring a fast, sensitive sensor such as an enzymatic amperometric electrode. Typically, the enzyme used for enzymatic electrochemical sensors is applied in excess to maximize signal. Here, in addition to sensitivity, we have also sought to maximize temporal resolution, by designing a sensor that is sensitive enough to work at near monolayer enzyme coverage. Reducing the enzyme layer thickness increases sensor temporal resolution by decreasing the distance and reducing the diffusion time for the enzyme product to travel to the sensor surface for detection. In this instance, the sensor consists of electrodeposited gold nanoparticle modified carbon fiber microelectrodes (CFMEs). Enzymes often are sensitive to curvature upon surface adsorption; thus, it was important to deposit discrete nanoparticles to maintain enzyme activity while depositing as much gold as possible to maximize enzyme coverage. To further enhance sensitivity, the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized onto the gold nanoparticles at the previously determined optimal ratio (1:10 AChE/ChO) for most efficient sequential enzymatic activity. This optimization approach has enabled the rapid detection to temporally resolve single vesicle acetylcholine release from an artificial cell. The sensor described is a significant advancement in that it allows for the recording of acetylcholine release on the order of the time scale for neurochemical release in secretory cells.

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

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    McCrea, P.D.

    1987-01-01

    Each of the five acetylcholine receptor (AChR) subunits, ..cap alpha../sub 2/..beta..-..gamma..delta, 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 delta 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 delta-delta desulfide, as evidenced by the transition of AChrR dimers to monomers, was quantitatively monitored on velocity sedimentation sucrose gradients. Alternatively, the reduction of delta/sub 2/ to delta 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 /sup 3/H-GSH efflux, demonstrated that intact reconstituted vesicles provide an adequate permeability barrier to GSH access of their intravesicular space.

  3. The readily releasable pool of synaptic vesicles.

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    Kaeser, Pascal S; Regehr, Wade G

    2017-04-01

    Each presynaptic bouton is densely packed with many vesicles, only a small fraction of which are available for immediate release. These vesicles constitute the readily releasable pool (RRP). The RRP size, and the probability of release of each vesicle within the RRP, together determine synaptic strength. Here, we discuss complications and recent advances in determining the size of the physiologically relevant RRP. We consider molecular mechanisms to generate and regulate the RRP, and discuss the relationship between vesicle docking and the RRP. We conclude that many RRP vesicles are docked, that some docked vesicles may not be part of the RRP, and that undocked vesicles can contribute to the RRP by rapid recruitment to unoccupied, molecularly activated ready-to-release sites. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. The effect of ketamine on intraspinal acetylcholine release

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    Abelson, Klas S P; Goldkuhl, Renée Röstlinger; Nylund, Anders

    2006-01-01

    The general anaesthetic ketamine affects the central cholinergic system in several manners, but its effect on spinal acetylcholine release, which may be an important transmitter in spinal antinociception, is unknown. This study aimed to investigate the effect of ketamine on spinal acetylcholine...... increased the acetylcholine release in high concentrations (100 microM to 10 mM). The results indicate that spinal nicotinic receptors are important for the ketamine-induced acetylcholine release, and that the effect is partly mediated at the spinal level....

  5. Decreased acetylcholine release delays the consolidation of object recognition memory.

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    De Jaeger, Xavier; Cammarota, Martín; Prado, Marco A M; Izquierdo, Iván; Prado, Vania F; Pereira, Grace S

    2013-02-01

    Acetylcholine (ACh) is important for different cognitive functions such as learning, memory and attention. The release of ACh depends on its vesicular loading by the vesicular acetylcholine transporter (VAChT). It has been demonstrated that VAChT expression can modulate object recognition memory. However, the role of VAChT expression on object recognition memory persistence still remains to be understood. To address this question we used distinct mouse lines with reduced expression of VAChT, as well as pharmacological manipulations of the cholinergic system. We showed that reduction of cholinergic tone impairs object recognition memory measured at 24h. Surprisingly, object recognition memory, measured at 4 days after training, was impaired by substantial, but not moderate, reduction in VAChT expression. Our results suggest that levels of acetylcholine release strongly modulate object recognition memory consolidation and appear to be of particular importance for memory persistence 4 days after training. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Coated vesicles as protein release mechanism in myeloma cells.

    Science.gov (United States)

    Trombetta, L D; Lazarus, S S

    An electron microscopic study was undertaken of the protein release mechanism within myeloma cells showing a very high degree of protein production. Smooth surfaced vesicles (50 millimicrons) were seen to originate from the outer margin of the perinuclear cistern. Similar vesicles were also associated with distended Golgi sacs. Possible function of these vesicles could not be determined. Coated vesicles (60 millimicrons) originated as evaginations from endoplasmic reticulum in the transitional region. They were present throughout the cytoplasm and were seen to fuse with the cell membrane discharging an electron dense material. These vesicles are, therefore, thought to transport protein from the rough endoplasmic reticulum and discharge it at the cell surface.

  7. Non-quantal acetylcholine release at mouse neuromuscular junction: effects of elevated quantal release and aconitine.

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    Yu, S P; Van der Kloot, W

    1990-09-04

    The rate of non-quantal acetylcholine (ACh) release was estimated at the mouse neuromuscular junction by observing the effect of (+)-tubocurarine on endplate membrane potential or current in preparations pretreated with an irreversible anti-acetylcholinesterase (anti-AChE). Voltage clamping was an effective method for measuring non-quantal release. Non-quantal release was markedly inhibited by 10 microM aconitine. Non-quantal release was not significantly increased by 10 microM dihyroouabain (DHO). (It has been reported that ouabain increases the leak). Non-quantal release was roughly doubled following exposure to hypertonic solution or to elevated K(+)-solution. This is in accord with the hypothesis that the leak is by way of ACh transporters incorporated into the terminal membrane following exocytosis, but other interpretations remain to be tested.

  8. TNF-? promotes extracellular vesicle release in mouse astrocytes through glutaminase

    OpenAIRE

    Wang, Kaizhe; Ye, Ling; Lu, Hongfang; Chen, Huili; Zhang, Yanyan; Huang, Yunlong; Zheng, Jialin C.

    2017-01-01

    Background Extracellular vesicles (EVs) are membrane-contained vesicles shed from cells. EVs contain proteins, lipids, and nucleotides, all of which play important roles in intercellular communication. The release of EVs is known to increase during neuroinflammation. Glutaminase, a mitochondrial enzyme that converts glutamine to glutamate, has been implicated in the biogenesis of EVs. We have previously demonstrated that TNF-? promotes glutaminase expression in neurons. However, the expressio...

  9. Intravenously administered lidocaine in therapeutic doses increases the intraspinal release of acetylcholine in rats

    DEFF Research Database (Denmark)

    Abelson, Klas S P; Höglund, A Urban

    2002-01-01

    The local anesthetic lidocaine suppresses different pain conditions when administered systemically. Part of the antinociceptive effect appears to be mediated via receptor mechanisms. We have previously shown that muscarinic and nicotinic agonists that produce antinociception increase the intraspi......The local anesthetic lidocaine suppresses different pain conditions when administered systemically. Part of the antinociceptive effect appears to be mediated via receptor mechanisms. We have previously shown that muscarinic and nicotinic agonists that produce antinociception increase...... the intraspinal release of acetylcholine. In the present study it was hypothesized that systemically administered lidocaine is acting through the same mechanisms as cholinergic agonists and affects the intraspinal release of acetylcholine. Microdialysis probes were placed in anesthetized rats for sampling...... of acetylcholine. Ten and 30 mg/kg lidocaine injected intravenously significantly increased the intraspinal release of acetylcholine. The effect of lidocaine could be reduced by pretreatment with intraspinally administered atropine or mecamylamine. Our results suggest that the antinociceptive effect produced...

  10. Calcium-dependent (/sup 3/H)acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

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    Marchi, M.; Caviglia, A.; Paudice, P.; Raiteri, M.

    1983-05-01

    A number of presynaptic cholinergic parameters (high affinity (/sup 3/H)choline uptake, (/sup 3/H)acetylcholine synthesis, (/sup 3/H)acetylcholine release, and autoinhibition of (/sup 3/H)acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of (/sup 3/H)acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca/sup 2 +/-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca/sup 2 +/-dependent release.

  11. GABA receptors and benzodiazepine binding sites modulate hippocampal acetylcholine release in vivo

    NARCIS (Netherlands)

    Moor, E; de Boer, P; Westerink, B.H.C.

    1998-01-01

    In the present study, the regulation of acetylcholine release from the ventral hippocampus by gamma-aminobutyric acid (GABA) was investigated in vivo. GABA receptor agonists and antagonists were administered locally in the medial septum and the adjacent vertical limb of the diagonal band of Broca,

  12. Concomitant release of ventral tegmental acetylcholine and accumbal dopamine by ghrelin in rats.

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    Elisabet Jerlhag

    Full Text Available Ghrelin, an orexigenic peptide, regulates energy balance specifically via hypothalamic circuits. Growing evidence suggest that ghrelin increases the incentive value of motivated behaviours via activation of the cholinergic-dopaminergic reward link. It encompasses the cholinergic afferent projection from the laterodorsal tegmental area (LDTg to the dopaminergic cells of the ventral tegmental area (VTA and the mesolimbic dopamine system projecting from the VTA to nucleus accumbens (N.Acc.. Ghrelin receptors (GHS-R1A are expressed in these reward nodes and ghrelin administration into the LDTg increases accumbal dopamine, an effect involving nicotinic acetylcholine receptors in the VTA. The present series of experiments were undertaken directly to test this hypothesis. Here we show that ghrelin, administered peripherally or locally into the LDTg concomitantly increases ventral tegmental acetylcholine as well as accumbal dopamine release. A GHS-R1A antagonist blocks this synchronous neurotransmitter release induced by peripheral ghrelin. In addition, local perfusion of the unselective nicotinic antagonist mecamylamine into the VTA blocks the ability of ghrelin (administered into the LDTg to increase N.Acc.-dopamine, but not VTA-acetylcholine. Collectively our data indicate that ghrelin activates the LDTg causing a release of acetylcholine in the VTA, which in turn activates local nicotinic acetylcholine receptors causing a release of accumbal dopamine. Given that a dysfunction in the cholinergic-dopaminergic reward system is involved in addictive behaviours, including compulsive overeating and alcohol use disorder, and that hyperghrelinemia is associated with such addictive behaviours, ghrelin-responsive circuits may serve as a novel pharmacological target for treatment of alcohol use disorder as well as binge eating.

  13. Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake.

    Science.gov (United States)

    Abels, Erik R; Breakefield, Xandra O

    2016-04-01

    Extracellular vesicles are a heterogeneous group of membrane-limited vesicles loaded with various proteins, lipids, and nucleic acids. Release of extracellular vesicles from its cell of origin occurs either through the outward budding of the plasma membrane or through the inward budding of the endosomal membrane, resulting in the formation of multivesicular bodies, which release vesicles upon fusion with the plasma membrane. The release of vesicles can facilitate intercellular communication by contact with or by internalization of contents, either by fusion with the plasma membrane or by endocytosis into "recipient" cells. Although the interest in extracellular vesicle research is increasing, there are still no real standards in place to separate or classify the different types of vesicles. This review provides an introduction into this expanding and complex field of research focusing on the biogenesis, nucleic acid cargo loading, content, release, and uptake of extracellular vesicles.

  14. Iontophoretic release of acetylcholine, noradrenaline, 5-hydroxytryptamine and D-lysergic acid diethylamide from micropipettes

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    Bradley, P. B.; Candy, J. M.

    1970-01-01

    1. The in vitro iontophoretic release of tritium-labelled acetylcholine and 5-hydroxytryptamine from large and small micropipettes and noradrenaline and D-lysergic acid diethylamide from small micropipettes was determined by liquid scintillation counting. 2. The release was directly proportional to the electrical charge passed in the range normally used in the iontophoretic study of these compounds. The transport numbers obtained for the large micropipettes were approximately double those with the small micropipettes. A very low transport number was found for D-lysergic acid diethylamide. 3. The spontaneous leakage was small and did not vary appreciably with time. 4. The iontophoretic release of acetylcholine in vitro agreed with the in vitro measurements. 5. The brain-stem tissue concentration of D-lysergic acid diethylamide after intravenous injection into intact and decerebrate cats was determined. PMID:5492892

  15. Microbubbles-Assisted Ultrasound Triggers the Release of Extracellular Vesicles

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    Yuana Yuana

    2017-07-01

    Full Text Available Microbubbles-assisted ultrasound (USMB has shown promise in improving local drug delivery. The formation of transient membrane pores and endocytosis are reported to be enhanced by USMB, and they contribute to cellular drug uptake. Exocytosis also seems to be linked to endocytosis upon USMB treatment. Based on this rationale, we investigated whether USMB triggers exocytosis resulting in the release of extracellular vesicles (EVs. USMB was performed on a monolayer of head-and-neck cancer cells (FaDu with clinically approved microbubbles and commonly used ultrasound parameters. At 2, 4, and 24 h, cells and EV-containing conditioned media from USMB and control conditions (untreated cells, cells treated with microbubbles and ultrasound only were harvested. EVs were measured using flow cytometric immuno-magnetic bead capture assay, immunogold electron microscopy, and western blotting. After USMB, levels of CD9 exposing-EVs significantly increased at 2 and 4 h, whereas levels of CD63 exposing-EVs increased at 2 h. At 24 h, EV levels were comparable to control levels. EVs released after USMB displayed a heterogeneous size distribution profile (30–1200 nm. Typical EV markers CD9, CD63, and alix were enriched in EVs released from USMB-treated FaDu cells. In conclusion, USMB treatment triggers exocytosis leading to the release of EVs from FaDu cells.

  16. Inhibition by halothane of potassium-stimulated acetylcholine release from rat cortical slices.

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    Griffiths, R.; Greiff, J. M.; Haycock, J.; Elton, C. D.; Rowbotham, D. J.; Norman, R. I.

    1995-01-01

    1. Cholinergic neurones in the basal forebrain are linked to cortical activation and arousal. 2. The present study was designed to examine the hypothesis that clinically relevant doses of halothane (0.1 to 5%) would significantly reduce depolarization-evoked acetylcholine (ACh) release from rat cortical slices. 3. ACh release was measured from rat cortical slices by a chemiluminescent technique. 4. Depolarization-evoked ACh release was inhibited significantly by halothane with an IC50 of 0.38%. This value equates to 0.3 MAC (the minimum alveolar concentration at which no movement occurs to a standard surgical stimulus in 50% of subjects) for the rat. 5. The potent effect of halothane on ACh release suggests that this mechanism may be a target for the action of volatile anaesthetic agents. This in vitro effect on ACh release is consistent with effects of halothane reported in vivo. PMID:8564265

  17. Dopamine modulates acetylcholine release via octopamine and CREB signaling in Caenorhabditis elegans.

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    Satoshi Suo

    Full Text Available Animals change their behavior and metabolism in response to external stimuli. cAMP response element binding protein (CREB is a signal-activated transcription factor that enables the coupling of extracellular signals and gene expression to induce adaptive changes. Biogenic amine neurotransmitters regulate CREB and such regulation is important for long-term changes in various nervous system functions, including learning and drug addiction. In Caenorhabditis elegans, the amine neurotransmitter octopamine activates a CREB homolog, CRH-1, in cholinergic SIA neurons, whereas dopamine suppresses CREB activation by inhibiting octopamine signaling in response to food stimuli. However, the physiological role of this activation is unknown. In this study, the effect of dopamine, octopamine, and CREB on acetylcholine signaling was analyzed using the acetylcholinesterase inhibitor aldicarb. Mutants with decreased dopamine signaling exhibited reduced acetylcholine signaling, and octopamine and CREB functioned downstream of dopamine in this regulation. This study demonstrates that the regulation of CREB by amine neurotransmitters modulates acetylcholine release from the neurons of C. elegans.

  18. Impaired acetylcholine release from the myenteric plexus of Trichinella-infected rats

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    Collins, S.M.; Blennerhassett, P.A.; Blennerhassett, M.G.; Vermillion, D.L. (McMaster Univ., Hamilton, Ontario (Canada))

    1989-12-01

    We examined the release of acetylcholine (ACh) from jejunal longitudinal muscle-myenteric plexus preparations in noninfected control rats and in rats infected 6, 23, or 40 days previously with Trichinella spiralis. ACh release was assessed by preincubating the tissue with ({sup 3}H)choline and measuring the evoked release of tritium. The uptake of {sup 3}H was significantly less in tissue from T. spiralis-infected rats compared with control. In tissues from either infected or control animals, electrical field stimulation (30 V, 0.5 ms, 10 Hz for 1 min), or veratridine (6-30 microM) induced {sup 3}H release that was tetrodotoxin sensitive. Depolarization by KCl (25-75 mM) also caused {sup 3}H release, but this was only partially reduced by tetrodotoxin. Radiochromatographic analysis indicated evoked release of {sup 3}H to be almost entirely ({sup 3}H)ACh. In rats infected 6 days previously with T. spiralis, ({sup 3}H)ACh release induced by KCl, veratridine, and field stimulation were decreased at least 80%. The suppression of ({sup 3}H)ACh release induced by veratridine or KCl was fully reversible after 40 days postinfection, but field-stimulated responses remained approximately 50% of control values. These results indicate that T. spiralis infection in the rat is accompanied by a reversible suppression of ACh release from the longitudinal muscle-myenteric plexus of the jejunum.

  19. Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells.

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    Castellano-Muñoz, Manuel; Schnee, Michael E; Ricci, Anthony J

    2016-01-01

    Hair cells from auditory and vestibular systems transmit continuous sound and balance information to the central nervous system through the release of synaptic vesicles at ribbon synapses. The high activity experienced by hair cells requires a unique mechanism to sustain recruitment and replenishment of synaptic vesicles for continuous release. Using pre- and postsynaptic electrophysiological recordings, we explored the potential contribution of calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents targeting endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs). Pharmacological treatments had no effect on hair cell resting potential or activation curves for calcium and potassium channels. However, these drugs exerted a reduction in vesicle release measured by dual-sine capacitance methods. In addition, calcium substitution by barium reduced release efficacy by delaying release onset and diminishing vesicle recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon synaptic transmission and suggest a novel contribution of CICR in hair cell vesicle recruitment. We hypothesize that calcium entry via calcium channels is tightly regulated to control timing of vesicle fusion at the synapse, whereas CICR is used to maintain a tonic calcium signal to modulate vesicle trafficking. Copyright © 2016 the American Physiological Society.

  20. Cholinergic mechanisms in canine narcolepsy--II. Acetylcholine release in the pontine reticular formation is enhanced during cataplexy.

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    Reid, M S; Siegel, J M; Dement, W C; Mignot, E

    1994-04-01

    Cataplexy in the narcoleptic canine has been shown to increase after local administration of carbachol into the pontine reticular formation. Rapid eye movement sleep has also been shown to increase after local administration of carbachol in the pontine reticular formation, and furthermore, acetylcholine release in the pontine tegmentum was found to increase during rapid eye movement sleep in rats. Therefore, in the present study we have investigated acetylcholine release in the pontine reticular formation during cataplexy in narcoleptic canines. Extracellular acetylcholine levels were measured in the pontine reticular formation of freely moving narcoleptic and control Doberman pinschers using in vivo microdialysis probes. Cataplexy was induced by the Food-Elicited Cataplexy Test and monitored using recordings of electroencephalogram, electrooculogram and electromyogram. Basal levels of acetylcholine in the microdialysis perfusates were approximately 0.5 pmol/10 min in both control and narcoleptic canines. Local perfusion with tetrodotoxin (10(-5) M) or artificial cerebrospinal fluid without Ca2+ produced a decrease, while intravenous injections of physostigmine (0.05 mg/kg) produced an increase in acetylcholine levels, indicating that the levels of acetylcholine levels measured are derived from neuronal release. During cataplexy induced by the Food-Elicited Cataplexy Test, acetylcholine levels increased by approximately 50% after four consecutive tests in narcoleptic canines, but did not change after four consecutive tests in control canines. Motor activity and feeding behavior, similar to that occurring during a Food-Elicited Cataplexy Test, had no effect on acetylcholine levels in the narcoleptic canines.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Enhanced nicotinic acetylcholine receptor-mediated [3H]norepinephrine release from neonatal rat hypothalamus.

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    O'Leary, K T; Leslie, F M

    2006-01-01

    Nicotinic acetylcholine receptor (nAChR)-evoked release of norepinephrine (NE) has been demonstrated in a number of brain regions that receive sole noradrenergic innervation from the locus coeruleus (LC). Many of these structures display enhanced nicotine-stimulated NE release in the neonate. We have examined the hypothalamus in order to determine if this region, which receives NE projections from both the LC and medullary catecholaminergic nuclei, also demonstrates maturational changes in nAChR-mediated NE release. Quantification of radiolabeled-NE release from rat hypothalamus slices by a maximally effective dose of nicotine revealed a peak response during the first postnatal week. This was followed by a decrease at postnatal day (P) 14, and a second peak at P21. Thereafter, release was equivalent to that observed at P14. Comparison of the pharmacological properties of nAChRs mediating NE release in neonatal (P7) and mature hypothalamus suggested involvement of different nAChR subtypes at the two ages. Using the selective toxin, DSP-4, nAChR-mediated NE release in the neonatal hypothalamus was shown to be from LC terminals. Our findings demonstrate an early sensitivity of hypothalamic LC terminals to nAChR regulation that may be associated with development of systems controlling critical homeostatic functions such as stress, feeding and cardiovascular regulation.

  2. Relationship between presynaptic membrane potential and acetylcholine release in synaptosomes from Torpedo electric organ.

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    Meunier, F M

    1984-01-01

    The membrane potential of purely cholinergic synaptosomes isolated from Torpedo electric organ was monitored with fluorescent carbocyanine dyes. An increased fluorescence was associated with depolarization and a quenching with hyperpolarization. Fluorescence data provided evidence that Torpedo synaptosomes have a membrane potential mainly driven by a K+ diffusion potential and a membrane potential of about -50 mV could be estimated after calibration of fluorescence signals with ionophore antibiotics. The release of acetylcholine (ACh) from Torpedo synaptosomes was monitored continuously by measuring the light emitted by a chemiluminescent method (Israël & Lesbats, 1981 a). Using fluorescence data, the release of ACh was expressed as a function of membrane potential. The relationship between presynaptic potential and transmitter release as determined by biochemical methods at cholinergic nerve endings showed striking similarities to that observed at the squid giant synapse. Several substances were also tested with regard to their depolarizing and releasing properties and it was found that the toxin isolated from the venom of the annelid Glycera convoluta, which induced a large increase in quantal release of transmitter (Manaranche, Thieffry, & Israël, 1980) promoted a depolarization of Torpedo synaptosomes in addition to ACh release. PMID:6207289

  3. Ryanodine-, IP3- and NAADP-dependent calcium stores control acetylcholine release.

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    Chameau, P; Van de Vrede, Y; Fossier, P; Baux, G

    2001-11-01

    Injections of inositol trisphosphate (IP3) or nicotinamide adenine dinucleotide phosphate (NAADP) into the presynaptic neurone of an identified cholinergic synapse in the buccal ganglion of Aplysia californica increased the amplitude of the inhibitory postsynaptic current evoked by a presynaptic action potential. This suggests that Ca2+ release from various Ca2+ stores can modulate acetylcholine (ACh) release. Specific blockade of the calcium-induced calcium release (CICR) mechanism with ryanodine, or of IP3-induced calcium release with heparin, abolished the effects of IP3, but not the effects of NAADP, suggesting the presence of an intracellular Ca2+ pool independent of those containing ryanodine receptors (RyR) or IP3 receptors. To reinforce electrophysiological observations, intracellular [Ca2+]i changes were measured using the fluorescent dye rhod-2. Injections of cyclic ADP-ribose (an activator of RyR), IP3 or NAADP into the presynaptic neurone induced transient increases in the free intracellular Ca2+ concentration. RyR- and IP3-induced increases were prevented by application of respective selective antagonists but not NAADP-induced increases. Our results show that RyR-dependent, IP3-dependent, and NAADP-dependent Ca2+ stores are present in the same presynaptic terminal but are differently involved in the regulation of the presynaptic Ca2+ concentration that triggers transmitter release.

  4. Acetylcholine-induced inhibition of presynaptic calcium signals and transmitter release in the frog neuromuscular junction

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    Eduard Khaziev

    2016-12-01

    Full Text Available Acetylcholine (ACh, released from axonal terminals of motor neurones in neuromuscular junctions regulates the efficacy of neurotransmission through activation of presynaptic nicotinic and muscarinic autoreceptors. Receptor-mediated presynaptic regulation could reflect either direct action on exocytotic machinery or modulation of Ca2+ entry and resulting intra-terminal Ca2+ dynamics. We have measured free intra-terminal cytosolic Ca2+ ([Ca2+]i using Oregon-Green 488 microfluorimetry, in parallel with voltage-clamp recordings of spontaneous (mEPC and evoked (EPC postsynaptic currents in post-junctional skeletal muscle fibre. Activation of presynaptic muscarinic and nicotinic receptors with exogenous acetylcholine and its non-hydrolized analogue carbachol reduced amplitude of the intra-terminal [Ca2+]i transients and decreased quantal content (calculated by dividing the area under EPC curve by the area under mEPC curve. Pharmacological analysis revealed the role of muscarinic receptors of M2 subtype as well as d-tubocurarine-sensitive nicotinic receptor in presynaptic modulation of [Ca2+]i transients. Modulation of synaptic transmission efficacy by ACh receptors was completely eliminated by pharmacological inhibition of N-type Ca2+ channels. We conclude that ACh receptor-mediated reduction of Ca2+ entry into the nerve terminal through N-type Ca2+ channels represents one of possible mechanism of presynaptic modulation in frog neuromuscular junction.

  5. A preliminary proteomic characterisation of extracellular vesicles released by the ovine parasitic nematode, Teladorsagia circumcincta.

    Science.gov (United States)

    Tzelos, Thomas; Matthews, Jacqueline B; Buck, Amy H; Simbari, Fabio; Frew, David; Inglis, Neil F; McLean, Kevin; Nisbet, Alasdair J; Whitelaw, C Bruce A; Knox, David P; McNeilly, Tom N

    2016-05-15

    Teladorsagia circumcincta is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The development of high levels of anthelmintic resistance in this nematode species challenges its future control. Recent research indicates that many parasite species release extracellular vesicles into their environment, many of which have been classified as endocytic in origin, termed exosomes. These vesicles are considered to play important roles in the intercellular communication between parasites and their hosts, and thus represent potentially useful targets for novel control strategies. Here, we demonstrate that exosome-like extracellular vesicles can be isolated from excretory-secretory (ES) products released by T. circumcincta fourth stage larvae (Tci-L4ES). Furthermore, we perform a comparative proteomic analysis of vesicle-enriched and vesicle-free Tci-L4ES. Approximately 73% of the proteins identified in the vesicle-enriched fraction were unique to this fraction, whilst the remaining 27% were present in both vesicle-enriched and vesicle-free fraction. These unique proteins included structural proteins, nuclear proteins, metabolic proteins, proteolytic enzymes and activation-associated secreted proteins. Finally, we demonstrate that molecules present within the vesicles-enriched material are targets of the IgA and IgG response in T. circumcincta infected sheep, and could potentially represent useful targets for future vaccine intervention studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Intravenously administered oxotremorine and atropine, in doses known to affect pain threshold, affect the intraspinal release of acetylcholine in rats

    DEFF Research Database (Denmark)

    Abelson, Klas S P; Höglund, A Urban

    2002-01-01

    /kg). Spinal microdialysis probes were placed intraspinally at approximately the C2-C5 spinal level for sampling of acetylcholine and dialysis delivery of atropine (0.1, 1, 10 nM). Additionally, the tail-flick behaviour was tested on conscious rats injected intraperitoneally with saline, atropine (10, 100...... muscarinic agonists and antagonists modify nociceptive threshold by affecting intraspinal release of acetylcholine (ACh). Catheters were inserted into the femoral vein in rats maintained on isoflurane anaesthesia for administration of oxotremorine (10-300 microg/kg) and atropine (0.1, 10, 5000 microg...... and 5000 microg/kg), or subcutaneously with oxotremorine (30, 100, 300 microg/kg). Subcutaneous administration of oxotremorine (30, 100, 300 microg/kg) significantly increased the tail-flick latency. These doses of oxotremorine dose-dependently increased the intraspinal release of acetylcholine...

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

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    Matos Baltazar, Ludmila; Nakayasu, Ernesto S; Sobreira, Tiago J P; Choi, Hyungwon; Casadevall, Arturo; Nimrichter, Leonardo; Nosanchuk, Joshua D

    2016-01-01

    Histoplasma capsulatum produces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment of H. capsulatum cells with monoclonal antibodies (MAbs) can change the outcome of infection in mice. We evaluated the sizes, enzymatic contents, and proteomic profiles of the vesicles released by fungal cells treated with either protective MAb 6B7 (IgG1) or nonprotective MAb 7B6 (IgG2b), both of which bind H. capsulatum heat shock protein 60 (Hsp60). Our results showed that treatment with either MAb was associated with changes in size and vesicle loading. MAb treatments reduced vesicle phosphatase and catalase activities compared to those of vesicles from untreated controls. We identified 1,125 proteins in vesicles, and 250 of these manifested differences in abundance relative to that of proteins in vesicles isolated from yeast cells exposed to Hsp60-binding MAbs, indicating that surface binding of fungal cells by MAbs modified protein loading in the vesicles. The abundance of upregulated proteins in vesicles upon MAb 7B6 treatment was 44.8% of the protein quantities in vesicles from fungal cells treated with MAb 6B7. Analysis of orthologous proteins previously identified in vesicles from other fungi showed that different ascomycete fungi have similar proteins in their extracellular milieu, many of which are associated with virulence. Our results demonstrate that antibody binding can modulate fungal cell responses, resulting in differential loading of vesicles, which could alter fungal cell susceptibility to host defenses. This finding provides additional evidence that antibody binding modulates microbial physiology and suggests a new function for specific immunoglobulins through alterations of fungal secretion. IMPORTANCE Diverse fungal species release extracellular vesicles, indicating that this is a common pathway for the delivery of molecules to the extracellular space. However, there has

  8. Calcium Assists Dopamine Release by Preventing Aggregation on the Inner Leaflet of Presynaptic Vesicles

    DEFF Research Database (Denmark)

    Mokkila, Sini; Postila, Pekka A.; Rissanen, Sami

    2017-01-01

    to a neutral (zwitterionic) phosphatidylcholine lipid bilayer model mimicking the inner leaflet of a presynaptic vesicle. We argue that the observed calcium-induced effect is likely in crucial role in the neurotransmitter release from the presynaptic vesicles docked in the active zone of nerve terminals....... The inner leaflets of presynaptic vesicles, which are responsible for releasing neurotransmitters into the synaptic cleft, are mainly composed of neutral lipids such as phosphatidylcholine and phosphatidylethanolamine. The neutrality of the lipid head group region, enhanced by a low pH level, should limit...

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

    Science.gov (United States)

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

    2015-08-01

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

  10. Distinctive Modulation of Dopamine Release in the Nucleus Accumbens Shell Mediated by Dopamine and Acetylcholine Receptors.

    Science.gov (United States)

    Shin, Jung Hoon; Adrover, Martin F; Alvarez, Veronica A

    2017-11-15

    Nucleus accumbens (NAc) shell shows unique dopamine (DA) signals in vivo and plays a unique role in DA-dependent behaviors such as reward-motivated learning and the response to drugs of abuse. A disynaptic mechanism for DA release was reported and shown to require synchronized firing of cholinergic interneurons (CINs) and activation of nicotinic acetylcholine (ACh) receptors (nAChRs) in DA neuron (DAN) axons. The properties of this disynaptic mechanism of DA transmission are not well understood in the NAc shell. In this study, in vitro fast-scan cyclic voltammetry was used to examine the modulation of DA transmission evoked by CINs firing in the shell of mice and compared with other striatal regions. We found that DA signals in the shell displayed significant degree of summation in response to train stimulation of CINs, contrary to core and dorsal striatum. The summation was amplified by a D2-like receptor antagonist and experiments with mice with targeted deletion of D2 receptors to DANs or CINs revealed that D2 receptors in CINs mediate a fast inhibition observed within 100 ms of the first pulse, whereas D2 autoreceptors in DAN terminals are engaged in a slower inhibition that peaks at ∼500 ms. ACh also contributes to the use-dependent inhibition of DA release through muscarinic receptors only in the shell, where higher activity of acetylcholinesterase minimizes nAChR desensitization and promotes summation. These findings show that DA signals are modulated differentially by endogenous DA and ACh in the shell, which may underlie the unique features of shell DA signals in vivoSIGNIFICANCE STATEMENT The present study reports that dopamine (DA) release evoked by activation of cholinergic interneurons displays a high degree of summation in the shell and shows unique modulation by endogenous DA and acetylcholine. Desensitization of nicotinic receptors, which is a prevailing mechanism for use-dependent inhibition in the nucleus accumbens core and dorsal striatum, is

  11. Proteomic profiling of extracellular vesicles released from vascular smooth muscle cells during initiation of phosphate-induced mineralization.

    Science.gov (United States)

    Chaudhary, Sandeep C; Khalid, Sana; Smethurst, Victoria; Monier, Daisy; Mobley, James; Huet, Alexis; Conway, James F; Napierala, Dobrawa

    2018-02-22

    Elevated serum phosphate is one of the major factors contributing to vascular calcification. Studies suggested that extracellular vesicles released from vascular smooth muscle cells significantly contribute to the initiation and progression of this pathology. Recently, we have demonstrated that elevated phosphate stimulates release of extracellular vesicles from osteogenic cells at the initiation of the mineralization process. Here, we used MOVAS cell line as an in vitro model of vascular calcification to examine whether vascular smooth muscle cells respond to high phosphate levels in a similar way and increase formation of extracellular vesicles. Vesicles residing in extracellular matrix as well as vesicles released to culture medium were evaluated by nanoparticle tracking analyses. In addition, using mass spectrometry and protein profiling, protein composition of extracellular vesicles released by MOVAS cells under standard growth conditions and upon exposure to high phosphate was compared. Significant increase of the number of extracellular vesicles was detected after 72 hours of exposure of cells to high phosphate. Elevated phosphate levels also affected protein composition of extracellular vesicles released from MOVAS cells. Finally, the comparative analyses of proteins in extracellular vesicles isolated from extracellular matrix and from conditioned medium identified significant differences in protein composition in these two groups of extracellular vesicles. In conclusion, results of this study demonstrate that exposure of MOVAS cells to high phosphate levels stimulates the release of extracellular vesicles and changes their protein composition.

  12. Quantitative and qualitative analysis of nano-sized vesicles released by dendritic cells and T cells. Towards deciphering the role of extracellular vesicles in immune cell communication

    NARCIS (Netherlands)

    van der Vlist, E.J.|info:eu-repo/dai/nl/314640908

    2013-01-01

    Many cell types release nano-sized vesicles, which can be found in body fluids as well as in cell culture-conditioned medium. These extracellular vesicles (EV) have been identified as vehicles for intercellular communication and are thought to be involved in many (patho)physiological processes. They

  13. Differential Acetylcholine Release in the Prefrontal Cortex and Hippocampus During Pavlovian Trace and Delay Conditioning

    Science.gov (United States)

    Flesher, M. Melissa; Butt, Allen E.; Kinney-Hurd, Brandee L.

    2011-01-01

    Pavlovian trace conditioning critically depends on the medial prefrontal cortex (mPFC) and hippocampus (HPC), whereas delay conditioning does not depend on these brain structures. Given that the cholinergic basal forebrain system modulates activity in both the mPFC and HPC, it was reasoned that the level of acetylcholine (ACh) release in these regions would show distinct profiles during testing in trace and delay conditioning paradigms. To test this assumption, microdialysis probes were implanted unilaterally into the mPFC and HPC of rats that were pre-trained in appetitive trace and delay conditioning paradigms using different conditional stimuli in the two tasks. On the day of microdialysis testing, dialysate samples were collected during a quiet baseline interval before trials were initiated, and again during performance in separate blocks of trace and delay conditioning trials in each animal. ACh levels were quantified using high performance liquid chromatography and electrochemical detection techniques. Consistent with our hypothesis, results showed that ACh release in the mPFC was greater during trace conditioning than during delay conditioning. The level of ACh released during trace conditioning in the HPC was also greater than the levels observed during delay conditioning. While ACh efflux in both the mPFC and HPC selectively increased during trace conditioning, ACh levels in the mPFC during trace conditioning testing showed the greatest increases observed. These results demonstrate a dissociation in cholinergic activation of the mPFC and HPC during performance in trace but not delay appetitive conditioning, where this cholinergic activity may contribute to attentional mechanisms, adaptive response timing, or memory consolidation necessary for successful trace conditioning. PMID:21514394

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

    Energy Technology Data Exchange (ETDEWEB)

    Rengasamy, A.; Feinberg, H.

    1988-02-15

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

  16. Neurotransmitter Release: The Last Millisecond in the Life of a Synaptic Vesicle

    Science.gov (United States)

    Südhof, Thomas C.

    2013-01-01

    During an action potential, Ca2+ entering a presynaptic terminal triggers synaptic vesicle exocytosis and neurotransmitter release in less than a millisecond. How does Ca2+ stimulate release so rapidly and precisely? Work over the last decades revealed that Ca2+-binding to synaptotagmin triggers release by stimulating synaptotagmin-binding to a core machinery composed of SNARE and SM proteins that mediates membrane fusion during exocytosis. Complexin adaptor proteins assist synaptotagmin by activating and clamping this core fusion machinery. Synaptic vesicles containing synaptotagmin are positioned at the active zone, the site of vesicle fusion, by a protein complex containing RIM proteins. RIM proteins simultaneously activate docking and priming of synaptic vesicles and recruit Ca2+-channels to active zones, thereby connecting in a single complex primed synaptic vesicles to Ca2+-channels. This architecture allows direct flow of Ca2+-ions from Ca2+-channels to synaptotagmin, which then triggers fusion, thus mediating tight millisecond coupling of an action potential to neurotransmitter release. PMID:24183019

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

    Science.gov (United States)

    Deatherage, Brooke L; Cookson, Brad T

    2012-06-01

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

  18. Non-quantal release of acetylcholine in rat atrial myocardium is inhibited by noradrenaline.

    Science.gov (United States)

    Borodinova, Anastasia A; Abramochkin, Denis V; Sukhova, Galina S

    2013-12-01

    In the mammalian myocardium, ACh, which is the main neurotransmitter of cardiac parasympathetic postganglionic fibres, can be released via both quantal (vesicular) and non-quantal (non-vesicular) mechanisms of secretion. Non-quantal release is continuous and independent of vagus activity and exocytosis of ACh-containing vesicles. During the incubation of myocardium in the presence of acetylcholinesterase (AChE) inhibitors, non-quantal ACh release leads to accumulation of ACh in the myocardium and cholinergic effects, which are proportional to the intensity of non-quantal secretion. The aim of the present study was to reveal whether non-quantal release of ACh can be modulated by another major cardioregulator, noradrenaline, or whether it represents uncontrolled leakage of ACh from cholinergic fibres. Cholinergic changes of electrical activity induced by the AChE inhibitor paraoxon (5 × 10(-6) M) in isolated rat right atrial preparations were determined by means of a standard microlectrode technique and used as a measure of the intensity of non-quantal release. Noradrenaline (10(-7) and 10(-6) M) substantially suppressed, but did not abolish, effects of paraoxon via stimulation of α-adrenoceptors, because all experiments were conducted in the presence of the β-blocker propranolol (5 × 10(-6) M). A blocker of ganglionic transmission, hexamethonium bromide (10(-4) M), failed to alter the inhibitory effect of noradrenaline, indicating that only non-quantal ACh release is suppressed by this neurotransmitter. The effects of noradrenaline could be reduced by the α2-antagonist yohimbine (10(-6) M). However, both the α1-agonist phenylephrine (10(-6) M) and the α2-agonist clonidine (10(-6) M) significantly inhibited the cholinergic effects of paraoxon, indicating the possible involvement of both α-adrenoceptor subtypes in mediation of the adrenergic inhibition of non-quantal ACh release. Thus, cardiac non-quantal ACh release can be negatively regulated by

  19. Synaptopathy under conditions of altered gravity: changes in synaptic vesicle fusion and glutamate release.

    Science.gov (United States)

    Krisanova, N V; Trikash, I O; Borisova, T A

    2009-12-01

    Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO ( approximately 10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[(14)C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca(2+) or Mg(2+)/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.

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

    Science.gov (United States)

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

    2017-10-14

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

  1. 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.

  2. Anterior Thalamic Lesions Alter Both Hippocampal-Dependent Behavior and Hippocampal Acetylcholine Release in the Rat

    Science.gov (United States)

    Savage, Lisa M.; Hall, Joseph M.; Vetreno, Ryan P.

    2011-01-01

    The anterior thalamic nuclei (ATN) are important for learning and memory as damage to this region produces a persistent amnestic syndrome. Dense connections between the ATN and the hippocampus exist, and importantly, damage to the ATN can impair hippocampal functioning. Acetylcholine (ACh) is a key neurotransmitter in the hippocampus, and in vivo…

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

    Directory of Open Access Journals (Sweden)

    Oscarsson Jan

    2008-01-01

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

  4. Synaptic vesicle release regulates myelin sheath number of individual oligodendrocytes in vivo.

    Science.gov (United States)

    Mensch, Sigrid; Baraban, Marion; Almeida, Rafael; Czopka, Tim; Ausborn, Jessica; El Manira, Abdeljabbar; Lyons, David A

    2015-05-01

    The myelination of axons by oligodendrocytes markedly affects CNS function, but how this is regulated by neuronal activity in vivo is not known. We found that blocking synaptic vesicle release impaired CNS myelination by reducing the number of myelin sheaths made by individual oligodendrocytes during their short period of formation. We also found that stimulating neuronal activity increased myelin sheath formation by individual oligodendrocytes. These data indicate that neuronal activity regulates the myelinating capacity of single oligodendrocytes.

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

    OpenAIRE

    Deatherage, Brooke L.; Cookson, Brad T.

    2012-01-01

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

  6. TNF-α promotes extracellular vesicle release in mouse astrocytes through glutaminase.

    Science.gov (United States)

    Wang, Kaizhe; Ye, Ling; Lu, Hongfang; Chen, Huili; Zhang, Yanyan; Huang, Yunlong; Zheng, Jialin C

    2017-04-20

    Extracellular vesicles (EVs) are membrane-contained vesicles shed from cells. EVs contain proteins, lipids, and nucleotides, all of which play important roles in intercellular communication. The release of EVs is known to increase during neuroinflammation. Glutaminase, a mitochondrial enzyme that converts glutamine to glutamate, has been implicated in the biogenesis of EVs. We have previously demonstrated that TNF-α promotes glutaminase expression in neurons. However, the expression and the functionality of glutaminase in astrocytes during neuroinflammation remain unknown. We posit that TNF-α can promote the release of EVs in astrocytes through upregulation of glutaminase expression. Release of EVs, which was demonstrated by electron microscopy, nanoparticle tracking analysis (NTA), and Western Blot, increased in mouse astrocytes when treated with TNF-α. Furthermore, TNF-α treatment significantly upregulated protein levels of glutaminase and increased the production of glutamate, suggesting that glutaminase activity is increased after TNF-α treatment. Interestingly, pretreatment with a glutaminase inhibitor blocked TNF-α-mediated generation of reactive oxygen species in astrocytes, which indicates that glutaminase activity contributes to stress in astrocytes during neuroinflammation. TNF-α-mediated increased release of EVs can be blocked by either the glutaminase inhibitor, antioxidant N-acetyl-L-cysteine, or genetic knockout of glutaminase, suggesting that glutaminase plays an important role in astrocyte EV release during neuroinflammation. These findings suggest that glutaminase is an important metabolic factor controlling EV release from astrocytes during neuroinflammation.

  7. Selective release of circRNAs in platelet-derived extracellular vesicles.

    Science.gov (United States)

    Preußer, Christian; Hung, Lee-Hsueh; Schneider, Tim; Schreiner, Silke; Hardt, Martin; Moebus, Anna; Santoso, Sentot; Bindereif, Albrecht

    2018-01-01

    Circular RNAs (circRNAs) are a novel class of noncoding RNAs present in all eukaryotic cells investigated so far and generated by a special mode of alternative splicing of pre-mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form. CircRNAs are cell-type specifically expressed, are unusually stable, and can be found in various body fluids such as blood and saliva. Here we analysed circRNAs and the corresponding linear splice isoforms from human platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles from purified and in vitro activated human platelets, using density-gradient centrifugation, followed by RNA-seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and may be involved in signalling pathways. Since platelets are essential for central physiological processes such as haemostasis, wound healing, inflammation and cancer metastasis, these findings should greatly extend the potential of circRNAs as prognostic and diagnostic biomarkers.

  8. Aspects of dopamine and acetylcholine release induced by glutamate receptors; Aspectos das liberacoes de dopamina e acetilcolina mediadas por receptores de glutamato

    Energy Technology Data Exchange (ETDEWEB)

    Paes, Paulo Cesar de Arruda

    2002-07-01

    The basal ganglia play an important role in the motor control of rats and humans. This control involves different neurotransmitters and the mutual control of these key elements has been subject to several studies. In this work we determined the role of glutamate on the release of radioactively labelled dopamine and acetylcholine from chopped striatal tissue in vitro. The values of Effective Concentration 50% for glutamate, NMDA, kainic, quisqualic acids and AMPA on the release of dopamine and acetylcholine were obtained. The inhibitory effects of magnesium, tetrodotoxin, MK-801, AP5 and MCPG, as well as the effects of glycin were evaluated. The results suggested that dopamine is influenced by the NMDA type glutamate receptor while acetylcholine seems to be influenced by NMDA, kainate and AMPA receptors. Tetrodotoxin experiments suggested that kainate receptors are both present in cholinergic terminals and cell bodies while AMPA and NMDA receptors are preferentially distributed in cell bodies. Magnesium effectively blocked the NMDA stimulation and unexpectedly also AMPA- and quisqualate-induced acetylcholine release. The latter could not be blocked by MCPG ruling out the participation of methabotropic receptors. MK-801 also blocked NMDA-receptors. Results point out the importance of the glutamic acid control of dopamine and acetylcholine release in striatal tissue. (author)

  9. 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.

  10. 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

  11. Presynaptic α4β2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus.

    Science.gov (United States)

    Garduño, Julieta; Galindo-Charles, Luis; Jiménez-Rodríguez, Javier; Galarraga, Elvira; Tapia, Dagoberto; Mihailescu, Stefan; Hernandez-Lopez, Salvador

    2012-10-24

    Several behavioral effects of nicotine are mediated by changes in serotonin (5-HT) release in brain areas that receive serotonergic afferents from the dorsal raphe nucleus (DRN). In vitro experiments have demonstrated that nicotine increases the firing activity in the majority of DRN 5-HT neurons and that DRN contains nicotinic acetylcholine receptors (nAChRs) located at both somata and presynaptic elements. One of the most common presynaptic effects of nicotine is to increase glutamate release. Although DRN receives profuse glutamatergic afferents, the effect of nicotine on glutamate release in the DRN has not been studied in detail. Using whole-cell recording techniques, we investigated the effects of nicotine on the glutamatergic input to 5-HT DRN neurons in rat midbrain slices. Low nicotine concentrations, in the presence of bicuculline and tetrodotoxin (TTX), increased the frequency but did not change the amplitude of glutamate-induced EPSCs, recorded from identified 5-HT neurons. Nicotine-induced increase of glutamatergic EPSC frequency persisted 10-20 min after drug withdrawal. This nicotinic effect was mimicked by exogenous administration of acetylcholine (ACh) or inhibition of ACh metabolism. In addition, the nicotine-induced increase in EPSC frequency was abolished by blockade of α4β2 nAChRs, voltage-gated calcium channels, or intracellular calcium signaling but not by α7 nAChR antagonists. These data suggest that both nicotine and endogenous ACh can increase glutamate release through activation of presynaptic α4β2 but not α7 nAChRs in the DRN. The effect involves long-term changes in synaptic function, and it is dependent on voltage-gated calcium channels and presynaptic calcium stores.

  12. Alterations in acetylcholine, PGE2 and IL6 release from urothelial cells following treatment with pyocyanin and lipopolysaccharide.

    Science.gov (United States)

    McDermott, C; Chess-Williams, R; Mills, K A; Kang, S H; Farr, S E; Grant, G D; Perkins, A V; Davey, A K; Anoopkumar-Dukie, S

    2013-09-01

    The effects of pseudomonal virulence factor pyocyanin, and LPS from Pseudomonas aeruginosa and Escherichia coli on urothelial mediator release and cytokine production were examined. RT4 urothelial cells were treated with pyocyanin (1-100 μM) or LPS (1-100 ng/mL) for 24-h. Effects were measured in terms of changes in cell viability, basal and stretch-induced acetylcholine (Ach) and PGE2 release, and inflammatory cytokines (IL-6 and IL-12) production. Twenty-four hour pyocyanin (100 μM) treatment significantly decreased urothelial cell viability, while stretch-induced Ach release response was inhibited. E. coli LPS (100 ng/mL) produced a similar response with an additional significant increase in basal Ach release. All three virulence factors significantly increased urothelial PGE2 release; under basal release for pyocyanin (100 μM), stretch-induced release for pseudomonal LPS (≥ 10 ng/mL) and both basal and stimulated release for E. coli LPS (≥ 10 ng/mL). IL-6 and IL-12 were not detected in control samples, however 24h treatment with pyocyanin (100 μM) or LPS (100 ng/mL) resulted in IL-6 release from urothelial cells. The changes in urothelial Ach and PGE2, and release of inflammatory cytokine IL-6 induced by exposure to the bacterial virulence factors may play a role in the symptoms of pain and urinary urgency experienced with urinary tract infections. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Time-Resolved SAXS Studies of the Kinetics of Thermally Triggered Release of Encapsulated Silica Nanoparticles from Block Copolymer Vesicles.

    Science.gov (United States)

    Mable, Charlotte J; Derry, Matthew J; Thompson, Kate L; Fielding, Lee A; Mykhaylyk, Oleksandr O; Armes, Steven P

    2017-06-13

    Silica-loaded poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) diblock copolymer vesicles are prepared in the form of concentrated aqueous dispersions via polymerization-induced self-assembly (PISA). As the concentration of silica nanoparticles present during the PISA synthesis is increased up to 35% w/w, higher degrees of encapsulation of this component within the vesicles can be achieved. After centrifugal purification to remove excess non-encapsulated silica nanoparticles, SAXS, DCP, and TGA analysis indicates encapsulation of up to hundreds of silica nanoparticles per vesicle. In the present study, the thermally triggered release of these encapsulated silica nanoparticles is examined by cooling to 0 °C for 30 min, which causes in situ vesicle dissociation. Transmission electron microscopy studies confirm the change in diblock copolymer morphology and also enable direct visualization of the released silica nanoparticles. Time-resolved small-angle X-ray scattering is used to quantify the extent of silica release over time. For an initial silica concentration of 5% w/w, cooling induces a vesicle-to-sphere transition with subsequent nanoparticle release. For higher silica concentrations (20 or 30% w/w) cooling only leads to perforation of the vesicle membranes, but silica nanoparticles are nevertheless released through the pores. For vesicles prepared in the presence of 30% w/w silica, the purified silica-loaded vesicles were cooled to 0 °C for 30 min, and SAXS patterns were collected every 15 s. A new SAXS model has been developed to determine both the mean volume fraction of encapsulated silica within the vesicles and the scattering length density. Satisfactory data fits to the experimental SAXS patterns were obtained using this model.

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

    Directory of Open Access Journals (Sweden)

    Zhuo Li

    2016-07-01

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

  15. Mechanisms involved in nicotinic acetylcholine receptor-induced neurotransmitter release from sympathetic nerve terminals in the mouse vas deferens.

    Directory of Open Access Journals (Sweden)

    Damian J Williams

    Full Text Available Prejunctional nicotinic acetylcholine receptors (nAChRs amplify postganglionic sympathetic neurotransmission, and there are indications that intraterminal Ca(2+ stores might be involved. However, the mechanisms by which nAChR activation stimulates neurotransmitter release at such junctions is unknown. Rapid local delivery (picospritzing of the nAChR agonist epibatidine was combined with intracellular sharp microelectrode recording to monitor spontaneous and field-stimulation-evoked neurotransmitter release from sympathetic nerve terminals in the mouse isolated vas deferens. Locally applied epibatidine (1 µM produced 'epibatidine-induced depolarisations' (EIDs that were similar in shape to spontaneous excitatory junction potentials (SEJPs and were abolished by nonselective nAChR antagonists and the purinergic desensitizing agonist α,β-methylene ATP. The amplitude distribution of EIDs was only slightly shifted towards lower amplitudes by the selective α7 nAChR antagonists α-bungarotoxin and methyllcaconitine, the voltage-gated Na(+ channel blocker tetrodotoxin or by blocking voltage-gated Ca(2+ channels with Cd(2+. Lowering the extracellular Ca(2+ concentration reduced the frequency of EIDs by 69%, but more surprisingly, the Ca(2+-induced Ca(2+ release blocker ryanodine greatly decreased the amplitude (by 41% and the frequency of EIDs by 36%. Ryanodine had no effect on electrically-evoked neurotransmitter release, paired-pulse facilitation, SEJP frequency, SEJP amplitude or SEJP amplitude distribution. These results show that activation of non-α7 nAChRs on sympathetic postganglionic nerve terminals induces high-amplitude junctional potentials that are argued to represent multipacketed neurotransmitter release synchronized by intraterminal Ca(2+-induced Ca(2+ release, triggered by Ca(2+ influx directly through the nAChR. This nAChR-induced neurotransmitter release can be targeted pharmacologically without affecting spontaneous or electrically

  16. Titration of Syntaxin1 in mammalian synapses reveals multiple roles in vesicle docking, priming, and release probability.

    Science.gov (United States)

    Arancillo, Marife; Min, Sang-Won; Gerber, Stefan; Münster-Wandowski, Agnieszka; Wu, Yuan-Ju; Herman, Melissa; Trimbuch, Thorsten; Rah, Jong-Cheol; Ahnert-Hilger, Gudrun; Riedel, Dietmar; Südhof, Thomas C; Rosenmund, Christian

    2013-10-16

    Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms.

  17. 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.

  18. Nephrin is expressed on the surface of insulin vesicles and facilitates glucose-stimulated insulin release.

    Science.gov (United States)

    Fornoni, Alessia; Jeon, Jongmin; Varona Santos, Javier; Cobianchi, Lorenzo; Jauregui, Alexandra; Inverardi, Luca; Mandic, Slavena A; Bark, Christina; Johnson, Kevin; McNamara, George; Pileggi, Antonello; Molano, R Damaris; Reiser, Jochen; Tryggvason, Karl; Kerjaschki, Dontscho; Berggren, Per-Olof; Mundel, Peter; Ricordi, Camillo

    2010-01-01

    Nephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic beta-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in beta-cell function. Nephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling. Islets from diabetic (n = 5) and nondiabetic (n = 7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were used to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of green fluorescent protein (GFP)-nephrin-transfected cells was used to study nephrin endocytosis. Nephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared with nondiabetic control subjects. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin-transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin-positive effect on insulin release. Our data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes.

  19. D2‐dopamine receptor‐mediated inhibition of intracellular Ca2+ mobilization and release of acetylcholine from guinea‐pig neostriatal slices

    National Research Council Canada - National Science Library

    Fujiwara, Hiroshi; Kato, Noriko; Shuntoh, Hisato; Tanaka, Chikako

    1987-01-01

    ... (a laevorotatory enantiomer of LY‐141865: N‐propyl tricyclic pyrazole) at 10 −6 m inhibited electrical stimulation‐and high K + ‐evoked release of [ 3 H]‐acetylcholine ([ 3 H]‐ACh) to 47.7 ± 6.0% and 54.1 ± 5.0...

  20. Modes and nodes explain the mechanism of action of vortioxetine, a multimodal agent (MMA): blocking 5HT3 receptors enhances release of serotonin, norepinephrine, and acetylcholine.

    Science.gov (United States)

    Stahl, Stephen M

    2015-10-01

    Vortioxetine is an antidepressant with multiple pharmacologic modes of action at targets where serotonin neurons connect with other neurons. 5HT3 receptor antagonism is one of these actions, and this leads to increased release of norepinephrine (NE), acetylcholine (ACh), and serotonin (5HT) within various brain circuits.

  1. Nicotine increases stress-induced serotonin release by stimulating nicotinic acetylcholine receptor in rat striatum.

    Science.gov (United States)

    Takahashi, H; Takada, Y; Nagai, N; Urano, T; Takada, A

    1998-03-01

    We used a microdialysis technique to analyze the effects of footshock stress on the release of serotonin (5-hydroxytryptamine: 5-HT) in the striatum or prefrontal cortex (PFC) in rats that were pretreated with nicotine. Neither nicotine administration alone nor stress application alone changed 5-HT release. During stress application, however, both chronic nicotine administration and local infusion of nicotine to the striatum significantly increased 5-HT release in the striatum, though not in the PFC. These increases in 5-HT release were eradicated by a local infusion of mecamylamine. Release of 5-HT increased in the striatum during stress application when nicotine was injected to the striatum, while nicotinic injection to the dorsal raphe nucleus did not increase 5-HT release in the striatum. The present study demonstrates that nicotine induced a release of 5-HT upon stress application by stimulating presynaptic nicotinic receptors in the striatum.

  2. Using Dynamic Covalent Chemistry To Drive Morphological Transitions: Controlled Release of Encapsulated Nanoparticles from Block Copolymer Vesicles.

    Science.gov (United States)

    Deng, Renhua; Derry, Matthew J; Mable, Charlotte J; Ning, Yin; Armes, Steven P

    2017-06-07

    Dynamic covalent chemistry is exploited to drive morphological order-order transitions to achieve the controlled release of a model payload (e.g., silica nanoparticles) encapsulated within block copolymer vesicles. More specifically, poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) (PGMA-PHPMA) diblock copolymer vesicles were prepared via aqueous polymerization-induced self-assembly in either the presence or absence of silica nanoparticles. Addition of 3-aminophenylboronic acid (APBA) to such vesicles results in specific binding of this reagent to some of the pendent cis-diol groups on the hydrophilic PGMA chains to form phenylboronate ester bonds in mildly alkaline aqueous solution (pH ∼ 10). This leads to a subtle increase in the effective volume fraction of this stabilizer block, which in turn causes a reduction in the packing parameter and hence induces a vesicle-to-worm (or vesicle-to-sphere) morphological transition. The evolution in copolymer morphology (and the associated sol-gel transitions) was monitored using dynamic light scattering, transmission electron microscopy, oscillatory rheology, and small-angle X-ray scattering. In contrast to the literature, in situ release of encapsulated silica nanoparticles is achieved via vesicle dissociation at room temperature; moreover, the rate of release can be fine-tuned by varying the solution pH and/or the APBA concentration. Furthermore, this strategy also works (i) for relatively thick-walled vesicles that do not normally exhibit stimulus-responsive behavior and (ii) in the presence of added salt. This novel molecular recognition strategy to trigger morphological transitions via dynamic covalent chemistry offers considerable scope for the design of new stimulus-responsive copolymer vesicles (and hydrogels) for targeted delivery and controlled release of cargoes. In particular, the conditions used in this new approach are relevant to liquid laundry formulations, whereby enzymes require

  3. A comparison of chemiluminescent and radioenzymatic methods for the measurement of acetylcholine released from a rat phrenic nerve-hemidiaphragm preparation.

    Science.gov (United States)

    Ehler, K W; Hoops, E A; Storella, R J; Bierkamper, G G

    1986-11-01

    A chemiluminescent assay coupled to a periodide extraction method is described for the measurement of acetylcholine release from the vascular perfused rat phrenic nerve-hemidiaphragm preparation. A direct comparison of the chemiluminescent assay with an established radioenzymatic assay for acetylcholine demonstrates that the two assays are quantitatively equivalent and yield similar limits of sensitivity of approximately 2 pmol, and that the periodide extraction/chemiluminescent assay method is more consistent than the tetraphenylboron extraction/radioenzymatic assay method. Additionally, cholinergic drug interference with the chemiluminescent assay is minimal. The absence of radioactivity and the reduced cost of the chemiluminescent assay make it an attractive alternative to the radioenzymatic assay.

  4. Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles.

    Directory of Open Access Journals (Sweden)

    Taulant Bacaj

    2015-10-01

    Full Text Available In forebrain neurons, Ca(2+ triggers exocytosis of readily releasable vesicles by binding to synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both synaptotagmin-1 and -7. Although maintenance of RRP size was Ca(2+-independent, mutations in Ca(2+-binding sequences of synaptotagmin-1 or synaptotagmin-7--which are contained in flexible top-loop sequences of their C2 domains--blocked the ability of these synaptotagmins to maintain the RRP size. Both synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca(2+ triggering of different phases of release.

  5. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation.

    Science.gov (United States)

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G; Enghild, Jan J; Praetorius, Jeppe; Borregaard, Niels; Petersen, Steen V

    2016-08-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication.

    Science.gov (United States)

    Wang, Juan; Silva, Malan; Haas, Leonard A; Morsci, Natalia S; Nguyen, Ken C Q; Hall, David H; Barr, Maureen M

    2014-03-03

    Cells release extracellular vesicles (ECVs) that play important roles in intercellular communication and may mediate a broad range of physiological and pathological processes. Many fundamental aspects of ECV biogenesis and signaling have yet to be determined, with ECV detection being a challenge and obstacle due to the small size (100 nm) of the ECVs. We developed an in vivo system to visualize the dynamic release of GFP-labeled ECVs. We show here that specific Caenorhabdidits elegans ciliated sensory neurons shed and release ECVs containing GFP-tagged polycystins LOV-1 and PKD-2. These ECVs are also abundant in the lumen surrounding the cilium. Electron tomography and genetic analysis indicate that ECV biogenesis occurs via budding from the plasma membrane at the ciliary base and not via fusion of multivesicular bodies. Intraflagellar transport and kinesin-3 KLP-6 are required for environmental release of PKD-2::GFP-containing ECVs. ECVs isolated from wild-type animals induce male tail-chasing behavior, while ECVs isolated from klp-6 animals and lacking PKD-2::GFP do not. We conclude that environmentally released ECVs play a role in animal communication and mating-related behaviors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Sulfanilamide in solution and liposome vesicles; in vitro release and UV-stability studies

    Directory of Open Access Journals (Sweden)

    Sanja Petrović

    2017-12-01

    Full Text Available The main goal of this study was to develop a liposome formulation with sulfanilamide and to investigate the liposomes impact on its release and stability to the UV-A/UV-B and UV-C irradiation. Liposome dispersions with incorporated sulfanilamide were prepared by thin-film hydration method and liposomes role to the sulfanilamide release was investigated by using a dialysis method. Comparatively, sulfanilamide in phosphate buffer solution was subject to release study as well to the UV irradiation providing for the possibilities of kinetics analysis. In vitro drug release study demonstrated that 20% of sulfanilamide was released from liposomes within 1 h that is approximately twice as slower as in the case of dissolved sulfanilamide in phosphate buffer solution. The kinetic release process can be described by Korsmeyer–Peppas model and according to the value of diffusion release exponent it can be concluded that drug release mechanism is based on the phenomenon of diffusion. The sulfanilamide degradation in phosphate buffer solution and liposomes is related to the formation of UV-induced degradation products that are identified by UHPLC/MS analysis as: sulfanilic acid, aniline and benzidine. The UV-induced sulfanilamide degradation in the phosphate buffer solution and liposome vesicles fits the first- order kinetic model. The degradation rate constants are dependent on the involved UV photons energy input as well as sulfanilamide microenvironment. Liposome microenvironment provides better irradiation sulfanilamide stability. The obtained results suggest that liposomes might be promising carriers for delayed sulfanilamide delivery and may serve as a basis for further research.

  8. Fluoxetine Alleviates Behavioral Depression while Decreasing Acetylcholine Release in the Nucleus Accumbens Shell

    Science.gov (United States)

    Chau, David T; Rada, Pedro V; Kim, Kay; Kosloff, Rebecca A; Hoebel, Bartley G

    2011-01-01

    Selective serotonin reuptake inhibitors, such as fluoxetine, have demonstrated the ability to alleviate behavioral depression in the forced swim test; however, the sites and mechanisms of their actions remain to be further elucidated. Previous studies have suggested that behavioral depression in the swim test is mediated in part by acetylcholine (ACh) stimulating the cholinergic M1 receptors in the nucleus accumbens (NAc) shell. The current study tested whether acute, local, and chronic, subcutaneous fluoxetine treatments increase escape motivation during the swim test while simultaneously lowering extracellular ACh in the NAc shell. Experiment 1: Fluoxetine (1.0 mM) infused unilaterally in the NAc shell for 40 min reduced extracellular ACh while simultaneously increasing swimming time. Experiment 2: Fluoxetine (0.2, 0.5, and 0.75 mM) infused bilaterally in the NAc shell on day 3 dose-dependently decreased immobility and increased the total escape attempts (swimming and climbing) compared with Ringer given on day 2. Experiment 3: Fluoxetine (0.5 mM) infused bilaterally in the NAc for 40 min did not affect activities in an open field. Experiment 4: Chronic systemic fluoxetine treatment decreased immobility scores and increased total escape attempt scores compared with control saline treatment. In all, 14 days after the initial swim test, basal extracellular ACh in the shell was still elevated in the saline-treated group, but not in the fluoxetine-treated group. In summary, these data suggest that one of the potential mechanisms by which fluoxetine alleviates behavioral depression in the forced swim test may be to suppress cholinergic activities in the NAc shell. PMID:21525864

  9. PML-RARa modulates the vascular signature of extracellular vesicles released by acute promyelocytic leukemia cells.

    Science.gov (United States)

    Fang, Yi; Garnier, Delphine; Lee, Tae Hoon; D'Asti, Esterina; Montermini, Laura; Meehan, Brian; Rak, Janusz

    2016-01-01

    Oncogenic transformation is believed to impact the vascular phenotype and microenvironment in cancer, at least in part, through mechanisms involving extracellular vesicles (EVs). We explored these questions in the context of acute promyelocytic leukemia cells (NB4) expressing oncogenic fusion protein, PML-RARa and exquisitely sensitive to its clinically used antagonist, the all-trans retinoic acid (ATRA). We report that NB4 cells produce considerable numbers of EVs, which are readily taken up by cultured endothelial cells triggering their increased survival. NB4 EVs contain PML-RARa transcript, but no detectable protein, which is also absent in endothelial cells upon the vesicle uptake, thereby precluding an active intercellular trafficking of this oncogene in this setting. ATRA treatment changes the emission profile of NB4-related EVs resulting in preponderance of smaller vesicles, an effect that occurs in parallel with the onset of cellular differentiation. ATRA also increases IL-8 mRNA and protein content in NB4 cells and their EVs, while decreasing the levels of VEGF and tissue factor (TF). Endothelial cell uptake of NB4-derived EVs renders these cells more TF-positive and procoagulant, and this effect is diminished by pre-treatment of EV donor cells with ATRA. Profiling angiogenesis-related transcripts in intact and ATRA-treated APL cells and their EVs reveals multiple differences attributable to cellular responses and EV molecular packaging. These observations point to the potential significance of changes in the angiogenic signature and activity associated with EVs released from tumor cells subjected to targeted therapy.

  10. Respiratory infections cause the release of extracellular vesicles: implications in exacerbation of asthma/COPD.

    Directory of Open Access Journals (Sweden)

    Suffwan Eltom

    Full Text Available Infection-related exacerbations of respiratory diseases are a major health concern; thus understanding the mechanisms driving them is of paramount importance. Despite distinct inflammatory profiles and pathological differences, asthma and COPD share a common clinical facet: raised airway ATP levels. Furthermore, evidence is growing to suggest that infective agents can cause the release of extracellular vesicle (EVs in vitro and in bodily fluids. ATP can evoke the P2X7/caspase 1 dependent release of IL-1β/IL-18 from EVs; these cytokines are associated with neutrophilia and are increased during exacerbations. Thus we hypothesized that respiratory infections causes the release of EVs in the airway and that the raised ATP levels, present in respiratory disease, triggers the release of IL-1β/IL-18, neutrophilia and subsequent disease exacerbations.To begin to test this hypothesis we utilised human cell-based assays, ex vivo murine BALF, in vivo pre-clinical models and human samples to test this hypothesis.Data showed that in a murine model of COPD, known to have increased airway ATP levels, infective challenge causes exacerbated inflammation. Using cell-based systems, murine models and samples collected from challenged healthy subjects, we showed that infection can trigger the release of EVs. When exposed to ATP the EVs release IL-1β/IL-18 via a P2X7/caspase-dependent mechanism. Furthermore ATP challenge can cause a P2X7 dependent increase in LPS-driven neutrophilia.This preliminary data suggests a possible mechanism for how infections could exacerbate respiratory diseases and may highlight a possible signalling pathway for drug discovery efforts in this area.

  11. Intrahippocampal Infusions of Anisomycin Produce Amnesia: Contribution of Increased Release of Norepinephrine, Dopamine, and Acetylcholine

    Science.gov (United States)

    Qi, Zhenghan; Gold, Paul E.

    2009-01-01

    Intra-amygdala injections of anisomycin produce large increases in the release of norepinephrine (NE), dopamine (DA), and serotonin in the amygdala. Pretreatment with intra-amygdala injections of the beta-adrenergic receptor antagonist propranolol attenuates anisomycin-induced amnesia without reversing the inhibition of protein synthesis, and…

  12. ELKS controls the pool of readily releasable vesicles at excitatory synapses through its N-terminal coiled-coil domains.

    Science.gov (United States)

    Held, Richard G; Liu, Changliang; Kaeser, Pascal S

    2016-06-02

    In a presynaptic nerve terminal, synaptic strength is determined by the pool of readily releasable vesicles (RRP) and the probability of release (P) of each RRP vesicle. These parameters are controlled at the active zone and vary across synapses, but how such synapse specific control is achieved is not understood. ELKS proteins are enriched at vertebrate active zones and enhance P at inhibitory hippocampal synapses, but ELKS functions at excitatory synapses are not known. Studying conditional knockout mice for ELKS, we find that ELKS enhances the RRP at excitatory synapses without affecting P. Surprisingly, ELKS C-terminal sequences, which interact with RIM, are dispensable for RRP enhancement. Instead, the N-terminal ELKS coiled-coil domains that bind to Liprin-α and Bassoon are necessary to control RRP. Thus, ELKS removal has differential, synapse-specific effects on RRP and P, and our findings establish important roles for ELKS N-terminal domains in synaptic vesicle priming.

  13. Molecular machines regulating the release probability of synaptic vesicles at the active zone.

    Directory of Open Access Journals (Sweden)

    Christoph eKoerber

    2016-03-01

    Full Text Available The fusion of synaptic vesicles (SVs with the plasma membrane of the active zone (AZ upon arrival of an action potential (AP at the presynaptic compartment is a tightly regulated probabil-istic process crucial for information transfer. The probability of a SV to release its transmitter content in response to an AP, termed release probability (Pr, is highly diverse both at the level of entire synapses and individual SVs at a given synapse. Differences in Pr exist between different types of synapses, between synapses of the same type, synapses originating from the same axon and even between different SV subpopulations within the same presynaptic terminal. The Pr of SVs at the AZ is set by a complex interplay of different presynaptic properties including the availability of release-ready SVs, the location of the SVs relative to the voltage-gated calcium channels (VGCCs at the AZ, the magnitude of calcium influx upon arrival of the AP, the buffer-ing of calcium ions as well as the identity and sensitivity of the calcium sensor. These properties are not only interconnected, but can also be regulated dynamically to match the requirements of activity patterns mediated by the synapse. Here, we review recent advances in identifying mole-cules and molecular machines taking part in the determination of vesicular Pr at the AZ.

  14. CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

    NARCIS (Netherlands)

    S. Basu (Saonli); S. Sladecek (Stefan); I.M. De La Peña Y Valenzuela (Isabel Martinez); M. Akaaboune (Mohammed); I. Smal (Ihor); K. Martin (Katrin); N.J. Galjart (Niels); H.R. Brenner (Hans Rudolf)

    2015-01-01

    textabstractA hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to

  15. Caffeine Modulates Vesicle Release and Recovery at Cerebellar Parallel Fibre Terminals, Independently of Calcium and Cyclic AMP Signalling.

    Science.gov (United States)

    Dobson, Katharine L; Jackson, Claire; Balakrishnan, Saju; Bellamy, Tomas C

    2015-01-01

    Cerebellar parallel fibres release glutamate at both the synaptic active zone and at extrasynaptic sites-a process known as ectopic release. These sites exhibit different short-term and long-term plasticity, the basis of which is incompletely understood but depends on the efficiency of vesicle release and recycling. To investigate whether release of calcium from internal stores contributes to these differences in plasticity, we tested the effects of the ryanodine receptor agonist caffeine on both synaptic and ectopic transmission. Whole cell patch clamp recordings from Purkinje neurons and Bergmann glia were carried out in transverse cerebellar slices from juvenile (P16-20) Wistar rats. Caffeine caused complex changes in transmission at both synaptic and ectopic sites. The amplitude of postsynaptic currents in Purkinje neurons and extrasynaptic currents in Bergmann glia were increased 2-fold and 4-fold respectively, but paired pulse ratio was substantially reduced, reversing the short-term facilitation observed under control conditions. Caffeine treatment also caused synaptic sites to depress during 1 Hz stimulation, consistent with inhibition of the usual mechanisms for replenishing vesicles at the active zone. Unexpectedly, pharmacological intervention at known targets for caffeine--intracellular calcium release, and cAMP signalling--had no impact on these effects. We conclude that caffeine increases release probability and inhibits vesicle recovery at parallel fibre synapses, independently of known pharmacological targets. This complex effect would lead to potentiation of transmission at fibres firing at low frequencies, but depression of transmission at high frequency connections.

  16. Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream.

    Directory of Open Access Journals (Sweden)

    Michele Guescini

    Full Text Available In the past few years, skeletal muscle has emerged as an important secretory organ producing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine effects. Moreover, recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly characterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs, which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and alpha-sarcoglycan (SGCA, and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs account for 1-5% of the total and that 60-65% of these EVs were also positive for the exosomal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and -181a-5p were significantly up-regulated after acute exercise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis.

  17. Extracellular vesicles released following heat stress induce bystander effect in unstressed populations.

    Science.gov (United States)

    Bewicke-Copley, Findlay; Mulcahy, Laura Ann; Jacobs, Laura Ann; Samuel, Priya; Akbar, Naveed; Pink, Ryan Charles; Carter, David Raul Francisco

    2017-01-01

    Cells naïve to stress can display the effects of stress, such as DNA damage and apoptosis, when they are exposed to signals from stressed cells; this phenomenon is known as the bystander effect. We previously showed that bystander effect induced by ionising radiation are mediated by extracellular vesicles (EVs). Bystander effect can also be induced by other types of stress, including heat shock, but it is unclear whether EVs are involved. Here we show that EVs released from heat shocked cells are also able to induce bystander damage in unstressed populations. Naïve cells treated with media conditioned by heat shocked cells showed higher levels of DNA damage and apoptosis than cells treated with media from control cells. Treating naïve cells with EVs derived from media conditioned by heat shocked cells also induced a bystander effect when compared to control, with DNA damage and apoptosis increasing whilst the level of cell viability was reduced. We demonstrate that treatment of naïve cells with heat shocked cell-derived EVs leads to greater invasiveness in a trans-well Matrigel assay. Finally, we show that naïve cells treated with EVs from heat-shocked cells are more likely to survive a subsequent heat shock, suggesting that these EVs mediate an adaptive response. We propose that EVs released following stress mediate an intercellular response that leads to apparent stress in neighbouring cells but also greater robustness in the face of a subsequent insult.

  18. Lipid-Induced Signaling Causes Release of Inflammatory Extracellular Vesicles From Hepatocytes.

    Science.gov (United States)

    Hirsova, Petra; Ibrahim, Samar H; Krishnan, Anuradha; Verma, Vikas K; Bronk, Steven F; Werneburg, Nathan W; Charlton, Michael R; Shah, Vijay H; Malhi, Harmeet; Gores, Gregory J

    2016-04-01

    Hepatocyte cellular dysfunction and death induced by lipids and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in the release of extracellular vesicles (EVs) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype. Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice also were given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative polymerase chain reaction. Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EVs, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) inhibition. Hepatocyte-derived EVs contained tumor necrosis factor-related apoptosis-inducing ligand and induced expression of interleukin 1β and interleukin 6 messenger RNAs in mouse bone marrow-derived macrophages. Activation of macrophages required DR5 and receptor-interacting protein kinase 1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EVs; this reduction was associated with decreased liver injury, inflammation, and fibrosis. Lipids, which stimulate DR5, induce release of hepatocyte EVs, which

  19. D2-dopamine receptor-mediated inhibition of intracellular Ca2+ mobilization and release of acetylcholine from guinea-pig neostriatal slices.

    OpenAIRE

    Fujiwara, H.; Kato, N; Shuntoh, H; Tanaka, C

    1987-01-01

    The effect of dopamine receptor activation on electrically- or high K+ (30 mM)-evoked neurotransmitter release and rise in intracellular Ca2+ concentration was investigated using slices of guinea-pig neostriatum. A specific D2-dopamine receptor agonist, LY-171555 (a laevorotatory enantiomer of LY-141865: N-propyl tricyclic pyrazole) at 10(-6) M inhibited electrical stimulation- and high K+-evoked release of [3H]-acetylcholine ([3H]-ACh) to 47.7 +/- 6.0% and 54.1 +/- 5.0% of control, respectiv...

  20. Proteomic and Bioinformatic Characterization of Extracellular Vesicles Released from Human Macrophages upon Influenza A Virus Infection.

    Science.gov (United States)

    Cypryk, Wojciech; Lorey, Martina; Puustinen, Anne; Nyman, Tuula A; Matikainen, Sampsa

    2017-01-06

    Influenza A viruses (IAVs) are aggressive pathogens that cause acute respiratory diseases and annual epidemics in humans. Host defense against IAV infection is initiated by macrophages, which are the principal effector cells of the innate immune system. We have previously shown that IAV infection of human macrophages is associated with robust secretion of proteins via conventional and unconventional protein release pathways. Here we have characterized unconventional, extracellular vesicle (EV)-mediated protein secretion in human macrophages during IAV infection using proteomics, bioinformatics, and functional studies. We demonstrate that at 9 h postinfection a robust EV-mediated protein secretion takes place. We identified 2359 human proteins from EVs of IAV-infected macrophages compared with 1448 proteins identified from EVs of control cells. Bioinformatic analysis shows that many proteins involved in translation, like components of spliceosome machinery and the ribosome, are secreted in EVs in response to IAV infection. Our data also shows that EVs derived from IAV-infected macrophages contain fatty acid-binding proteins, antiviral cytokines, copper metabolism Murr-1 domain proteins, and autophagy-related proteins. In addition, our data suggest that secretory autophagy plays a role in activating EV-mediated protein secretion during IAV infection.

  1. Mechanisms and functions of extracellular vesicle release in vivo-What we can learn from flies and worms.

    Science.gov (United States)

    Beer, Katharina B; Wehman, Ann Marie

    2017-03-04

    Cells from bacteria to man release extracellular vesicles (EVs) that contain signaling molecules like proteins, lipids, and nucleic acids. The content, formation, and signaling roles of these conserved vesicles are diverse, but the physiological relevance of EV signaling in vivo is still debated. Studies in classical genetic model organisms like C. elegans and Drosophila have begun to reveal the developmental and behavioral roles for EVs. In this review, we discuss the emerging evidence for the in vivo signaling roles of EVs. Significant effort has also been made to understand the mechanisms behind the formation and release of EVs, specifically of exosomes derived from exocytosis of multivesicular bodies and of microvesicles derived from plasma membrane budding called ectocytosis. In this review, we detail the impact of flies and worms on understanding the proteins and lipids involved in EV biogenesis and highlight the open questions in the field.

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

    Directory of Open Access Journals (Sweden)

    Bas van de Waterbeemd

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

  3. Caffeine Modulates Vesicle Release and Recovery at Cerebellar Parallel Fibre Terminals, Independently of Calcium and Cyclic AMP Signalling

    Science.gov (United States)

    Dobson, Katharine L.; Jackson, Claire; Balakrishnan, Saju; Bellamy, Tomas C.

    2015-01-01

    Background Cerebellar parallel fibres release glutamate at both the synaptic active zone and at extrasynaptic sites—a process known as ectopic release. These sites exhibit different short-term and long-term plasticity, the basis of which is incompletely understood but depends on the efficiency of vesicle release and recycling. To investigate whether release of calcium from internal stores contributes to these differences in plasticity, we tested the effects of the ryanodine receptor agonist caffeine on both synaptic and ectopic transmission. Methods Whole cell patch clamp recordings from Purkinje neurons and Bergmann glia were carried out in transverse cerebellar slices from juvenile (P16-20) Wistar rats. Key Results Caffeine caused complex changes in transmission at both synaptic and ectopic sites. The amplitude of postsynaptic currents in Purkinje neurons and extrasynaptic currents in Bergmann glia were increased 2-fold and 4-fold respectively, but paired pulse ratio was substantially reduced, reversing the short-term facilitation observed under control conditions. Caffeine treatment also caused synaptic sites to depress during 1 Hz stimulation, consistent with inhibition of the usual mechanisms for replenishing vesicles at the active zone. Unexpectedly, pharmacological intervention at known targets for caffeine—intracellular calcium release, and cAMP signalling—had no impact on these effects. Conclusions We conclude that caffeine increases release probability and inhibits vesicle recovery at parallel fibre synapses, independently of known pharmacological targets. This complex effect would lead to potentiation of transmission at fibres firing at low frequencies, but depression of transmission at high frequency connections. PMID:25933382

  4. Caffeine Modulates Vesicle Release and Recovery at Cerebellar Parallel Fibre Terminals, Independently of Calcium and Cyclic AMP Signalling.

    Directory of Open Access Journals (Sweden)

    Katharine L Dobson

    Full Text Available Cerebellar parallel fibres release glutamate at both the synaptic active zone and at extrasynaptic sites-a process known as ectopic release. These sites exhibit different short-term and long-term plasticity, the basis of which is incompletely understood but depends on the efficiency of vesicle release and recycling. To investigate whether release of calcium from internal stores contributes to these differences in plasticity, we tested the effects of the ryanodine receptor agonist caffeine on both synaptic and ectopic transmission.Whole cell patch clamp recordings from Purkinje neurons and Bergmann glia were carried out in transverse cerebellar slices from juvenile (P16-20 Wistar rats.Caffeine caused complex changes in transmission at both synaptic and ectopic sites. The amplitude of postsynaptic currents in Purkinje neurons and extrasynaptic currents in Bergmann glia were increased 2-fold and 4-fold respectively, but paired pulse ratio was substantially reduced, reversing the short-term facilitation observed under control conditions. Caffeine treatment also caused synaptic sites to depress during 1 Hz stimulation, consistent with inhibition of the usual mechanisms for replenishing vesicles at the active zone. Unexpectedly, pharmacological intervention at known targets for caffeine--intracellular calcium release, and cAMP signalling--had no impact on these effects.We conclude that caffeine increases release probability and inhibits vesicle recovery at parallel fibre synapses, independently of known pharmacological targets. This complex effect would lead to potentiation of transmission at fibres firing at low frequencies, but depression of transmission at high frequency connections.

  5. Heat Profiling of Three-Dimensionally Optically Trapped Gold Nanoparticles using Vesicle Cargo Release

    DEFF Research Database (Denmark)

    Kyrsting, Anders; Bendix, Pól Martin; Stamou, Dimitrios

    2011-01-01

    Irradiated metallic nanoparticles hold great promise as heat transducers in photothermal applications such as drug delivery assays or photothermal therapy. We quantify the temperature increase of individual gold nanoparticles trapped in three dimensions near lipid vesicles exhibiting temperature ...

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

    Science.gov (United States)

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

    2015-08-01

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

  7. Mice with selective elimination of striatal acetylcholine release are lean, show altered energy homeostasis and changed sleep/wake cycle.

    Science.gov (United States)

    Guzman, Monica S; De Jaeger, Xavier; Drangova, Maria; Prado, Marco A M; Gros, Robert; Prado, Vania F

    2013-03-01

    Cholinergic neurons are known to regulate striatal circuits; however, striatal-dependent physiological outcomes influenced by acetylcholine (ACh) are still poorly under;?>stood. Here, we used vesicular acetylcholine transporter (VAChT)(D2-Cre-flox/flox) mice, in which we selectively ablated the vesicular acetylcholine transporter in the striatum to dissect the specific roles of striatal ACh in metabolic homeostasis. We report that VAChT(D) (2-Cre-flox/flox) mice are lean at a young age and maintain this lean phenotype with time. The reduced body weight observed in these mutant mice is not attributable to reduced food intake or to a decrease in growth rate. In addition, changed activity could not completely explain the lean phenotype, as only young VAChT(D) (2-Cre-flox/flox) mice showed increased physical activity. Interestingly, VAChT(D) (2-Cre-flox/flox) mice show several metabolic changes, including increased plasma levels of insulin and leptin. They also show increased periods of wakefulness when compared with littermate controls. Taken together, our data suggest that striatal ACh has an important role in the modulation of metabolism and highlight the importance of striatum cholinergic tone in the regulation of energy expenditure. These new insights on how cholinergic neurons influence homeostasis open new avenues for the search of drug targets to treat obesity. © 2012 International Society for Neurochemistry.

  8. Detection of basal acetylcholine release in the microdialysis of rat frontal cortex by high-performance liquid chromatography using a horseradish peroxidase-osmium redox polymer electrode with pre-enzyme reactor.

    Science.gov (United States)

    Kato, T; Liu, J K; Yamamoto, K; Osborne, P G; Niwa, O

    1996-06-28

    To determine the basal acetylcholine level in the dialysate of rat frontal cortex, a horseradish peroxidase-osmium redox polymer-modified glassy carbon electrode (HRP-GCE) was employed instead of the conventional platinum electrode used in high-performance liquid chromatography-electrochemical detection (HPLC-ED). In initial experiments, an oxidizable unknown compound interfered with the detection of basal acetylcholine release on HPLC-HRP-GCE. An immobilized peroxidase-choline oxidase precolumn (pre-reactor) was included in the HPLC system, to eliminate the interference from the unknown compound. This combination could detect less than 10 fmol of standard acetylcholine and basal acetylcholine levels in the dialysate from a conventional concentric design microdialysis probe, without the use of cholinesterase inhibitor, and may facilitate physiological investigation of cholinergic neuronal activity in the central nervous system.

  9. Benzodiazepine receptor agonists cause drug-specific and state-specific alterations in EEG power and acetylcholine release in rat pontine reticular formation.

    Science.gov (United States)

    Hambrecht-Wiedbusch, Viviane S; Gauthier, Elizabeth A; Baghdoyan, Helen A; Lydic, Ralph

    2010-07-01

    Benzodiazepine (BDZ) and non-benzodiazepine (NBDZ) hypnotics enhance GABAergic transmission and are widely used for the treatment of insomnia. In the pontine reticular formation (PRF), GABA inhibits rapid eye movement (REM) sleep and acetylcholine (ACh) release. No previous studies have characterized the effects of BDZ and NBDZ hypnotics on ACh release in the PRF. This study tested 2 hypotheses: (1) that microdialysis delivery of zolpidem, eszopiclone, and diazepam to rat PRF alters ACh release in PRF and electroencephalographic (EEG) delta power and (2) that intravenous (i.v.) administration of eszopiclone to non-anesthetized rat alters ACh release in the PRF, sleep, and EEG delta power. A within- and between-groups experimental design. University of Michigan. Adult male Crl:CD*(SD) (Sprague-Dawley) rats (n = 57). In vivo microdialysis of the PRF in rats anesthetized with isoflurane was used to derive the concentration-response effects of zolpidem, eszopiclone, and diazepam on ACh release. Chronically instrumented rats were used to quantify the effects of eszopiclone (3 mg/kg, i.v.) on ACh release in the PRF, sleep-wake states, and cortical EEG power. ACh release was significantly increased by microdialysis delivery to the PRF of zolpidem and eszopiclone but not diazepam. EEG delta power was increased by zolpidem and diazepam but not by eszopiclone administered to the PRF. Eszopiclone (i.v.) decreased ACh release in the PRF of both anesthetized and non-anesthetized rats. Eszopiclone (i.v.) prevented REM sleep and increased EEG delta power. The concentration-response data provide the first functional evidence that multiple GABA(A) receptor subtypes are present in rat PRF. Intravenously administered eszopiclone prevented REM sleep, decreased ACh release in the PRF, and increased EEG delta power. The effects of eszopiclone are consistent with evidence that ACh release in the PRF is lower during NREM sleep than during REM sleep, and with data showing that cholinergic

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

    Science.gov (United States)

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

    2011-01-01

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

  11. Acetylcholine release in mouse hippocampal CA1 preferentially activates inhibitory-selective interneurons via alpha4 beta2* nicotinic receptor activation

    Directory of Open Access Journals (Sweden)

    L. Andrew Bell

    2015-04-01

    Full Text Available Acetylcholine (ACh release onto nicotinic receptors directly activates subsets of inhibitory interneurons in hippocampal CA1. However, the specific interneurons activated and their effect on the hippocampal network is not completely understood. Therefore, we investigated subsets of hippocampal CA1 interneurons that respond to ACh release through the activation of nicotinic receptors and the potential downstream effects this may have on hippocampal CA1 network function. ACh was optogenetically released in mouse hippocampal slices by expressing the excitatory optogenetic protein oChIEF-tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated viral mediated transfection. The actions of optogenetically released ACh were assessed on both pyramidal neurons and different interneuron subtypes via whole cell patch clamp methods. Vasoactive intestinal peptide (VIP-expressing interneurons that selectively innervate other interneurons (VIP/IS were excited by ACh through the activation of nicotinic receptors containing alpah4 and beta2 subunits (alpha4 beta2*. ACh release onto VIP/IS was presynaptically inhibited by M2 muscarinic autoreceptors. ACh release produced spontaneous inhibitory postsynaptic current (sIPSC barrages blocked by dihydro-beta-erythroidine in interneurons but not pyramidal neurons. Optogenetic suppression of VIP interneurons did not inhibit these sIPSC barrages suggesting other interneuron-selective interneurons were also excited by 42* nicotinic receptor activation. In contrast, interneurons that innervate pyramidal neuron perisomatic regions were not activated by ACh release onto nicotinic receptors. Therefore, we propose ACh release in CA1 facilitates disinhibition through activation of 42* nicotinic receptors on interneuron-selective interneurons whereas interneurons that innervate pyramidal neurons are less affected by nicotinic receptor activation.

  12. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation

    DEFF Research Database (Denmark)

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G

    2016-01-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages...... and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD m......RNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA...

  13. Non-conducting function of the Kv2.1 channel enables it to recruit vesicles for release in neuroendocrine and nerve cells.

    Science.gov (United States)

    Feinshreiber, Lori; Singer-Lahat, Dafna; Friedrich, Reut; Matti, Ulf; Sheinin, Anton; Yizhar, Ofer; Nachman, Rachel; Chikvashvili, Dodo; Rettig, Jens; Ashery, Uri; Lotan, Ilana

    2010-06-01

    Regulation of exocytosis by voltage-gated K(+) channels has classically been viewed as inhibition mediated by K(+) fluxes. We recently identified a new role for Kv2.1 in facilitating vesicle release from neuroendocrine cells, which is independent of K(+) flux. Here, we show that Kv2.1-induced facilitation of release is not restricted to neuroendocrine cells, but also occurs in the somatic-vesicle release from dorsal-root-ganglion neurons and is mediated by direct association of Kv2.1 with syntaxin. We further show in adrenal chromaffin cells that facilitation induced by both wild-type and non-conducting mutant Kv2.1 channels in response to long stimulation persists during successive stimulation, and can be attributed to an increased number of exocytotic events and not to changes in single-spike kinetics. Moreover, rigorous analysis of the pools of released vesicles reveals that Kv2.1 enhances the rate of vesicle recruitment during stimulation with high Ca(2+), without affecting the size of the readily releasable vesicle pool. These findings place a voltage-gated K(+) channel among the syntaxin-binding proteins that directly regulate pre-fusion steps in exocytosis.

  14. Sleep and memory problems: acetylcholine in some neurodegenerative diseases, use of an extended-release formulation of galantamine

    Directory of Open Access Journals (Sweden)

    I V Litvinenko

    2012-01-01

    Full Text Available The paper reviews the literature dedicated to an association between sleep and memory problems and acetylcholine (AC levels. Moderate impairments of circadian rhythms can develop with aging; however, these changes become quite significant in dementia, which impairs the sleep-wake cycle. Low AC levels during slow-wave sleep are critical for declarative (verbal memory consolidation. An abnormal nocturnal reduction in cholinergic activity can worsen memory problems and provoke sleep deterioration. The results of the studies suggest that the type of an AC esterase inhibitor and the time of its administration are important for the development of these problems. Galantamine ensures high daytime concentrations of AC and its low nighttime levels, which enables the tone of cholinergic system to be maximally approaches physiological circadian rhythms. This may be essential to the improvement of sleep and memory in patients with dementia.

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

    Science.gov (United States)

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

    2017-05-01

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

  16. Metabolically active extracellular vesicles released from hepatocytes under drug-induced liver-damaging conditions modify serum metabolome and might affect different pathophysiological processes.

    Science.gov (United States)

    Royo, Felix; Palomo, Laura; Mleczko, Justyna; Gonzalez, Esperanza; Alonso, Cristina; Martínez, Ibon; Pérez-Cormenzana, Miriam; Castro, Azucena; Falcon-Perez, Juan M

    2017-02-15

    Hepatocytes are involved in the endogenous and drug metabolism; many of the enzymes involved in those processes are incorporated into extracellular vesicles and secreted into the bloodstream. Liver-damaging conditions modify the molecular cargo of those vesicles significantly. However, no information about the effect of these hepatic vesicles on the extracellular environment is available. Drug-induced liver damage increases the number of circulating extracellular vesicles and affects the release and content of hepatocyte-derived vesicles. In this work, we evaluated the metabolic effect of these vesicles on the composition of the serum. We performed a targeted ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics analysis of serum samples. The samples had been first incubated with hepatic extracellular vesicles from hepatocytes challenged with acetaminophen or diclofenac. The incubation affected the serum levels of 67 metabolites, such as amino acids and different species of lipids. The metabolites included various species of phosphatidylcholines and phosphatidylethanolamines. These compounds are the components of biological membranes; our observations suggest that the vesicles might take part in remodelling and maintenance of the membranes. Alterations in the levels of some other serum metabolites might have deleterious consequences, for example, the tetracosanoic acid with its cardiovascular effects. However, some of the metabolites whose levels were increased, including alpha-linoleic and tauroursodeoxycholic acids, have been reported to have a protective effect. Our targeted metabolomics analysis indicated that the hepatic extracellular vesicles act as nano-metabolic machines supplying the extracellular environment with the means to integrate diverse tissue responses. In conclusion, we show that the hepatic extracellular vesicles are metabolically active and might play a role in the physiopathological response to hepatic insults

  17. Characterization of the muscarinic receptor subtype(s) mediating contraction of the guinea-pig lung strip and inhibition of acetylcholine release in the guinea-pig trachea with the selective muscarinic receptor antagonist tripitramine

    NARCIS (Netherlands)

    Roffel, A.F; Davids, J.H; Elzinga, C.R S; Wolf, D; Zaagsma, Hans; Kilbinger, H

    1 The muscarinic receptor subtypes mediating contraction of the guinea-pig lung strip and inhibition of the release of acetylcholine from cholinergic vagus nerve endings in the guinea-pig trachea in vitro have previously been characterized as M-2-like, i.e. having antagonist affinity profiles that

  18. 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 showed a marked positive shift as compared with that for the normal immunoglobulin G (IgG isotype control. Thus, on-chip immunoelectrophoresis could sensitively detect the over-expression of CD63 glycoproteins on EVs. Moreover, to explore the applicability of on-chip immunoelectrophoresis to cancer diagnosis, EVs collected from the blood of a mouse tumor model were analyzed by this method. By comparing the zeta potential distributions of EVs after their immunochemical reaction with normal IgG, and the anti-human CD63 and anti-human CD44 (cancer stem cell marker antibodies, EVs of tumor origin circulating in blood were differentially detected in the real sample. The result indicates that the present method is potentially applicable to liquid biopsy, a promising approach to the low-invasive diagnosis of cancer.

  19. A role for cGMP during tetanus toxin blockade of acetylcholine release in the rat pheochromocytoma (PC12) cell line.

    Science.gov (United States)

    Sandberg, K; Berry, C J; Eugster, E; Rogers, T B

    1989-11-01

    In order to identify the specific molecular mechanisms involved in neurosecretion, we investigated the mechanism of action of tetanus toxin, a potent presynaptic neurotoxin, in the rat adrenal pheochromocytoma PC12 cell line. It has recently been reported that tetanus toxin is a potent inhibitor of the release of depolarization-evoked 3H-acetylcholine (ACh) from nerve growth factor-differentiated PC12 cells (Sandberg et al., 1989a). In PC12 cells, as in many neural tissue preparations, cGMP accumulation in intact cells increased 6- to 17-fold when stimulated with veratridine (200 microM), carbachol (1 mM), Ba2+ (2 mM), or K+ (30 mM). Preincubation of the cells with tetanus toxin inhibits this accumulation by greater than 95%. The toxin dose-inhibition curves for 3H-ACh release and cGMP accumulation are similar, with half-maximal doses of tetanus toxin seen at approximately 5 nM. The time courses for the development of the effects of tetanus on 3H-ACh release and on cGMP accumulation were also similar. Protocols which elevated intracellular cGMP levels reversed the action of the toxin. For example, evoked ACh release was restored in intoxicated PC12 cells by a 15 min exposure to 100 microM 8-bromo-cGMP. The half-maximal dose was observed at 50 microM nucleotide. Examination of the nucleotide specificity revealed that only cyclic guanine analogs were effective in reversing the effects of tetanus toxin. These results suggested that the inhibition of depolarization-evoked cGMP accumulation is causally related to the action of tetanus toxin on neurosecretion.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Dendrite-derived supernumerary axons on adult axotomized motor neurons possess proteins that are essential for the initiation and propagation of action potentials and synaptic vesicle release

    DEFF Research Database (Denmark)

    Meehan, Claire Francesca; MacDermid, Victoria E; Montague, Steven J

    2011-01-01

    on these processes matches the arrangement of these channels that is necessary for the initiation and conduction of action potentials. At terminal bouton-like structures they possess key proteins necessary for the release of synaptic vesicles (SV2 and synaptophysin). Thus, axon-like processes emanating from the tips...

  1. A sequential vesicle pool model with a single release sensor and a Ca(2+-dependent priming catalyst effectively explains Ca(2+-dependent properties of neurosecretion.

    Directory of Open Access Journals (Sweden)

    Alexander M Walter

    Full Text Available Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca(2+ dependence, but also upstream steps depend on Ca(2+. After deletion of the Ca(2+ sensor for fast release - synaptotagmin-1 - slower Ca(2+-dependent release components persist. These findings have provoked working models involving parallel releasable vesicle pools (Parallel Pool Models, PPM driven by alternative Ca(2+ sensors for release, but no slow release sensor acting on a parallel vesicle pool has been identified. We here propose a Sequential Pool Model (SPM, assuming a novel Ca(2+-dependent action: a Ca(2+-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP under control of synaptotagmin-1, the origins of slow release differ. In the SPM the slow release component is attributed to the Ca(2+-dependent refilling of the RRP from a Non-Releasable upstream Pool (NRP, whereas the PPM attributes slow release to a separate slowly-releasable vesicle pool. Using numerical integration we compared model predictions to data from mouse chromaffin cells. Like the PPM, the SPM explains biphasic release, Ca(2+-dependence and pool sizes in mouse chromaffin cells. In addition, the SPM accounts for the rapid recovery of the fast component after strong stimulation, where the PPM fails. The SPM also predicts the simultaneous changes in release rate and amplitude seen when mutating the SNARE-complex. Finally, it can account for the loss of fast- and the persistence of slow release in the synaptotagmin-1 knockout by assuming that the RRP is depleted, leading to slow and Ca(2+-dependent fusion from the NRP. We conclude that the elusive 'alternative Ca(2+ sensor' for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca(2+-dependent

  2. Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists.

    Science.gov (United States)

    Ambrose, Ashley R; Alsahli, Mohammed A; Kurmani, Sameer A; Goodall, Alison H

    2017-07-20

    On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific. Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70. Platelet activation triggered the release of 57-79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (r2 > 0.98; p platelets (r2 > 0.98; p < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect. These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.

  3. RIG-I activation induces the release of extracellular vesicles with antitumor activity.

    Science.gov (United States)

    Daßler-Plenker, Juliane; Reiners, Katrin S; van den Boorn, Jasper G; Hansen, Hinrich P; Putschli, Bastian; Barnert, Sabine; Schuberth-Wagner, Christine; Schubert, Rolf; Tüting, Thomas; Hallek, Michael; Schlee, Martin; Hartmann, Gunther; Pogge von Strandmann, Elke; Coch, Christoph

    2016-01-01

    Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5'-triphosphate-RNA (3pRNA) triggers antitumor immunity predominantly via NK cell activation and direct apoptosis induction in tumor cells. However, how NK cells are mobilized to attack the tumor cells remains elusive. Here, we show that RIG-I activation induced the secretion of extracellular vesicles (EVs) from melanoma cells, which by themselves revealed antitumor activity in vitro and in vivo. RIG-I-induced EVs from melanoma cells exhibited an increased expression of the NKp30-ligand (BAG6, BAT3) on their surface triggering NK cell-mediated lysis of melanoma cells via activation of the cytotoxicity NK cell-receptor NKp30. Moreover, systemic administration of RIG-I-induced melanoma-EVs showed a potent antitumor activity in a melanoma mouse model in vivo. In conclusion, our data establish a new RIG-I-dependent pathway leading to NK cell-mediated tumor cell killing.

  4. Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner.

    Science.gov (United States)

    Kakazu, Eiji; Mauer, Amy S; Yin, Meng; Malhi, Harmeet

    2016-02-01

    Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

  5. Stored platelets alter glycerophospholipid and sphingolipid species, which are differentially transferred to newly released extracellular vesicles.

    Science.gov (United States)

    Pienimaeki-Roemer, Annika; Ruebsaamen, Katharina; Boettcher, Alfred; Orsó, Evelyn; Scherer, Max; Liebisch, Gerhard; Kilalic, Dzenan; Ahrens, Norbert; Schmitz, Gerd

    2013-03-01

    Stored platelet concentrates (PLCs) for transfusion develop a platelet storage lesion (PSL), resulting in decreased platelet (PLT) viability and function. The processes leading to PSL have not been described in detail and no data describe molecular changes occurring in all three components of stored PLCs: PLTs, PLC extracellular vesicles (PLC-EVs), and plasma. Fifty PLCs from healthy individuals were stored under standard blood banking conditions for 5 days. Changes in cholesterol, glycerophospholipid, and sphingolipid species were analyzed in PLTs, PLC-EVs, and plasma by mass spectrometry and metabolic labeling. Immunoblots were performed to compare PLT and PLC-EV protein expression. During 5 days, PLTs transferred glycerophospholipids, cholesterol, and sphingolipids to newly formed PLC-EVs, which increased corresponding lipids by 30%. Stored PLTs significantly increased ceramide (Cer; +53%) and decreased sphingosine-1-phosphate (-53%), shifting sphingolipid metabolism toward Cer. In contrast, plasma accumulated minor sphingolipids. Compared to PLTs, fresh PLC-EVs were enriched in lysophosphatidic acid (60-fold) and during storage showed significant increases in cholesterol, sphingomyelin, dihydrosphingomyelin, plasmalogen, and lysophosphatidylcholine species, as well as accumulation of apolipoproteins A-I, E, and J/clusterin. This is the first detailed analysis of lipid species in all PLC components during PLC storage, which might reflect mechanisms active during in vivo PLT senescence. Stored PLTs reduce minor sphingolipids and shift sphingolipid metabolism toward Cer, whereas in the plasma fraction minor sphingolipids increase. The composition of PLC-EVs resembles that of lipid rafts and confirms their role as carriers of bioactive molecules and master regulators in vascular disease. © 2012 American Association of Blood Banks.

  6. Release of urinary extracellular vesicles in prostate cancer is associated with altered urinary N-glycosylation profile.

    Science.gov (United States)

    Vermassen, Tijl; D'Herde, Katharina; Jacobus, Dominique; Van Praet, Charles; Poelaert, Filip; Lumen, Nicolaas; Callewaert, Nico; Decaestecker, Karel; Villeirs, Geert; Hoebeke, Piet; Van Belle, Simon; Rottey, Sylvie; Delanghe, Joris

    2017-10-01

    Nowadays, extracellular vesicles are of great interest in prostate cancer (PCa) research. Asparagine (N)-linked glycosylation could play a significant role in the pathological mechanism of these vesicles. We investigated if prostatic protein N-glycosylation profiles were related to urinary vesicle-associated prostate-specific antigen (PSA) extractability and if this parameter showed diagnostic potential for PCa. Urinary extracellular vesicles were visualised using transmission electron microscopy. Urinary extracellular vesicles extraction by means of n -butanol allowed determination of urinary vesicle-associated PSA extractability. Diagnostic value was assessed between benign prostate hyperplasia (BPH; n=122) and patients with PCa (n=85). Additionally, correlation with urine N-glycosylation was assessed. Urinary extracellular vesicles with a diameter of approximately 100 nm were more abundantly present in urine of patients with PCa versus patients with BPH resulting in a higher vesicle-associated PSA extraction ratio (pvesicle-associated PSA extraction ratio was correlated to biantennary core-fucosylation (p=0.003). Finally, vesicle-associated PSA extraction ratio proved beneficial in PCa diagnosis, next to serum PSA and the urinary glycosylation marker (p=0.021). The urinary vesicle-associated PSA extraction ratio is increased in PCa which is a direct result of the abundant presence of extracellular vesicles in urine of patients with PCa. The urinary vesicle-associated PSA extraction ratio was associated with changes in N-glycoforms and showed diagnostic potential. Further research is warranted to unravel the pathological link between N-glycosylation and extracellular vesicles in cancer, as well as to assess the prognostic value of this biomarker. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  7. HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity

    Science.gov (United States)

    Sami Saribas, A; Cicalese, Stephanie; Ahooyi, Taha Mohseni; Khalili, Kamel; Amini, Shohreh; Sariyer, Ilker Kudret

    2017-01-01

    Human immunodeficiency virus-associated neurological disorders (HANDs) affect the majority of AIDS patients and are a significant problem among HIV-1-infected individuals who live longer because of combined anti-retroviral therapies. HIV-1 utilizes a number of viral proteins and subsequent cytokine inductions to unleash its toxicity on neurons. Among HIV-1 viral proteins, Nef is a small protein expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HAND. In order to explore its effect in the central nervous system, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFAs) using an adenovirus. Our results revealed that HIV-1 Nef is released in extracellular vesicles (EVs) derived from PHFA cells expressing the protein. Interestingly, HIV-1 Nef release in EVs was enriched significantly when the cells were treated with autophagy activators perifosine, tomaxifen, MG-132, and autophagy inhibitors LY294002 and wortmannin suggesting a novel role of autophagy signaling in HIV-1 Nef release from astrocytes. Next, Nef-carrying EVs were purified from astrocyte cultures and neurotoxic effects on neurons were analyzed. We observed that HIV-1 Nef-containing EVs were readily taken up by neurons as demonstrated by immunocytochemistry and immunoblotting. Furthermore, treatment of neurons with Nef-carrying EVs induced oxidative stress as evidenced by a decrease in glutathione levels. To further investigate its neurotoxic effects, we expressed HIV-1 Nef in primary neurons by adenoviral transduction. Intracellular expression of HIV-1 Nef caused axonal and neurite degeneration of neurons. Furthermore, expression of HIV-1 Nef decreased the levels of phospho-tau while enhancing total tau in primary neurons. In addition, treatment of primary neurons with Nef-carrying EVs suppressed functional neuronal action potential assessed by multielectrode array studies. Collectively, these data suggested that HIV-1 Nef can be

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

    Science.gov (United States)

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

    1999-12-01

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

  9. An Active Form of Sphingosine Kinase-1 Is Released in the Extracellular Medium as Component of Membrane Vesicles Shed by Two Human Tumor Cell Lines

    Directory of Open Access Journals (Sweden)

    Salvatrice Rigogliuso

    2010-01-01

    Full Text Available Expression of sphingosine kinase-1 (SphK-1 correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P, a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis.

  10. A sequential vesicle pool model with a single release sensor and a ca(2+)-dependent priming catalyst effectively explains ca(2+)-dependent properties of neurosecretion

    DEFF Research Database (Denmark)

    Walter, Alexander M; da Silva Pinheiro, Paulo César; Verhage, Matthijs

    2013-01-01

    Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca(2+) dependence, but also upstream steps depend on Ca(2+). After deletion of the Ca(2+) sensor for fast release - synaptot......Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca(2+) dependence, but also upstream steps depend on Ca(2+). After deletion of the Ca(2+) sensor for fast release...... identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca(2+)-dependent action: a Ca(2+)-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1...... that the elusive 'alternative Ca(2+) sensor' for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca(2+)-dependent properties of secretion without assuming parallel pools or sensors....

  11. Mildly oxidized HDL decrease agonist-induced platelet aggregation and release of pro-coagulant platelet extracellular vesicles.

    Science.gov (United States)

    Tafelmeier, M; Fischer, A; Orsó, E; Konovalova, T; Böttcher, A; Liebisch, G; Matysik, S; Schmitz, G

    2017-05-01

    Stored platelet concentrates (PLCs) for therapeutic purpose, develop a platelet storage lesion (PSL), characterized by impaired platelet (PLT) viability and function, platelet extracellular vesicle (PL-EV) release and profound lipidomic changes. Whereas oxidized low-density lipoprotein (oxLDL) activates PLTs and promotes atherosclerosis, effects linked to oxidized high-density lipoprotein (oxHDL) are poorly characterized. PLCs from blood donors were treated with native (nHDL) or mildly oxidized HDL (moxHDL) for 5days under blood banking conditions. Flow cytometry, nanoparticle tracking analysis (NTA), aggregometry, immunoblot analysis and mass spectrometry were carried out to analyze PL-EV and platelet exosomes (PL-EX) release, PLT aggregation, protein expression, and PLT and plasma lipid composition. In comparison to total nHDL, moxHDL significantly decreased PL-EV release by -36% after 5days of PLT storage and partially reversed agonist-induced PLT aggregation. PL-EV release positively correlated with PLT aggregation. MoxHDL improved PLT membrane lipid homeostasis through enhanced uptake of lysophospholipids and their remodeling to corresponding phospholipid species. This also appeared for sphingomyelin (SM) and d18:0/d18:1 sphingosine-1-phosphate (S1P) at the expense of ceramide (Cer) and hexosylceramide (HexCer) leading to reduced Cer/S1P ratio as PLT-viability indicator. This membrane remodeling was associated with increased content of CD36 and maturation of scavenger receptor-B1 (SR-B1) protein in secreted PL-EVs. MoxHDL, more potently than nHDL, improves PLT-membrane lipid homeostasis, partially antagonizes PL-EV release and agonist-induced PLT aggregation. Altogether, this may be the result of more efficient phospho- and sphingolipid remodeling mediated by CD36 and SR-B1 in the absence of ABCA1 on PLTs. As in vitro supplement in PLCs, moxHDL has the potential to improve PLC quality and to prolong storage. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. D2-dopamine receptor-mediated inhibition of intracellular Ca2+ mobilization and release of acetylcholine from guinea-pig neostriatal slices.

    Science.gov (United States)

    Fujiwara, H; Kato, N; Shuntoh, H; Tanaka, C

    1987-06-01

    The effect of dopamine receptor activation on electrically- or high K+ (30 mM)-evoked neurotransmitter release and rise in intracellular Ca2+ concentration was investigated using slices of guinea-pig neostriatum. A specific D2-dopamine receptor agonist, LY-171555 (a laevorotatory enantiomer of LY-141865: N-propyl tricyclic pyrazole) at 10(-6) M inhibited electrical stimulation- and high K+-evoked release of [3H]-acetylcholine ([3H]-ACh) to 47.7 +/- 6.0% and 54.1 +/- 5.0% of control, respectively. The maximal inhibition by LY-171555 at 10(-5) M was 54.8 +/- 5.1% reduction of the control. The half-maximal effective concentration (EC50) of LY-171555 for the inhibition of [3H]-ACh release was 2.3 X 10(-7) M. A specific D2-dopamine receptor antagonist, (-)-sulpiride (10(-7) M) reversed the inhibition of [3H]-ACh release induced by LY-171555. A specific D1-dopamine receptor agonist, SK&F 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-benzazepine) (10(-5) M) had no effect on the release of [3H]-ACh. LY-171555 (10(-6) M) also inhibited the high K+-evoked endogenous glutamate release, by 47% of control. This inhibitory effect was reversed by (-)-sulpiride (10(-7) M). We used a fluorescent, highly selective Ca2+ indicator, 'quin 2' to measure intracellular free Ca2+ concentrations ([Ca2+]i). Electrical stimulation of slices preloaded with quin 2 led to an elevation of relative fluorescence intensity and this response was reduced by the removal of Ca2+ from the bathing medium. These results indicate that the enhanced elevation in fluorescence intensity in the quin 2-loaded slices reflects the increase of intracellular free Ca2+ concentration, [Ca2+]i. The mixed D1- and D2-receptor agonist, apomorphine and LY-171555 inhibited the increase of [Ca2+]i induced by electrical stimulation or high K+ medium, in a concentration-dependent manner, while SK&F 38393 did not affect the increase of [Ca2+]i. The maximal inhibitory effect of LY-171555 at 3 X 10(-5) M was 35 +/- 3

  13. High-density lipoprotein 3 and apolipoprotein A-I alleviate platelet storage lesion and release of platelet extracellular vesicles.

    Science.gov (United States)

    Pienimaeki-Roemer, Annika; Fischer, Astrid; Tafelmeier, Maria; Orsó, Evelyn; Konovalova, Tatiana; Böttcher, Alfred; Liebisch, Gerhard; Reidel, Armin; Schmitz, Gerd

    2014-09-01

    Stored platelet (PLT) concentrates (PLCs) for transfusion develop a PLT storage lesion (PSL), decreasing PLT viability and function with profound lipidomic changes and PLT extracellular vesicle (PL-EV) release. High-density lipoprotein 3 (HDL3 ) improves PLT homeostasis through silencing effects on PLT activation in vivo. This prompted us to investigate HDL3 and apolipoprotein A-I (apoA-I) as PSL-antagonizing agents. Healthy donor PLCs were split into low-volume standard PLC storage bags and incubated with native (n)HDL3 or apoA-I from plasma ethanol fractionation (precipitate IV) for 5 days under standard blood banking conditions. Flow cytometry, Born aggregometry, and lipid mass spectrometry were carried out to analyze PL-EV release, PLT aggregation, agonist-induced PLT surface marker expression, and PLT and plasma lipid compositions. Compared to control, added nHDL3 and apoA-I significantly reduced PL-EV release by up to -62% during 5 days, correlating with the added apoA-I concentration. At the lipid level, nHDL3 and apoA-I antagonized PLT lipid loss (+12%) and decreased cholesteryl ester (CE)/free cholesterol (FC) ratios (-69%), whereas in plasma polyunsaturated/saturated CE ratios increased (+3%) and CE 16:0/20:4 ratios decreased (-5%). Administration of nHDL3 increased PLT bis(monoacylglycero)phosphate/phosphatidylglycerol (+102%) and phosphatidic acid/lysophosphatidic acid (+255%) ratios and improved thrombin receptor-activating peptide 6-induced PLT aggregation (+5%). nHDL3 and apoA-I improve PLT membrane homeostasis and intracellular lipid processing and increase CE efflux, antagonizing PSL-related reduction in PLT viability and function and PL-EV release. We suggest uptake and catabolism of nHDL3 into the PLT open canalicular system. As supplement in PLCs, nHDL3 or apoA-I from Fraction IV of plasma ethanol fractionation have the potential to improve PLC quality to prolong storage. © 2014 AABB.

  14. Shape and release control of a peptide decorated vesicle through pH sensitive orthogonal supramolecular interactions

    NARCIS (Netherlands)

    Versluis, Frank; Tomatsu, Itsuro; Kehr, Seda; Fregonese, Carlo; Tepper, Armand W. J. W.; Stuart, Marc C. A.; Ravoo, Bart Jan; Koning, Roman I.; Kros, Alexander

    2009-01-01

    A pH sensitive carrier is obtained by coating a cyclodextrin vesicle with an adamantane-terminated octapeptide through the formation of an inclusion complex. Upon lowering the pH from 7.4 to 5.0, the formation of peptide B-sheets on the vesicle surface induces a transition of the bilayer from a

  15. Exocytosis from large dense cored vesicles outside the active synaptic zones of terminals within the trigeminal subnucleus caudalis: a possible mechanism for neuropeptide release.

    Science.gov (United States)

    Zhu, P C; Thureson-Klein, A; Klein, R L

    1986-09-01

    It has been hypothesized that chemical interactions between neurons in the central nervous system can occur in the absence of well defined synaptic complexes, but morphological correlates have been difficult to find. The present study demonstrates exocytotic release from large (70-130 nm) dense cored vesicles at structurally nonspecialized areas along the plasmalemma of structurally different categories of terminals and occasionally from dendrites and axons within the neuropil of the trigeminal subnucleus caudalis. In rats, the marginal (lamina I) and substantia gelatinosa (lamina II) layers contain the central terminals of primary afferent fibers from the infraorbital nerve that supply the skin and whiskers (vibrissae). Different types of interneurons are also present and may modify the input being relayed to higher centers. While exocytotic profiles were present in control animals, they increased significantly (P less than 0.01) on the ipsilateral side 1-24 h after a unilateral skin lesion in the vibrissae area. A second increase (P less than 0.001) occurred 14-15 days after the lesion. Virtually all examples of large vesicle exocytosis were observed at structurally nonspecialized sites while those at the active synaptic zones involved small clear vesicles. Substance P-like immunofluorescence, present in controls and on the ipsilateral side during the first 6 days, subsequently declined until 4 weeks after surgery when some recovery was noted. The increase in large vesicle exocytosis and the decrease in substance P are interpreted to reflect functional adjustments of different neurons in response to the lesion. The exocytosis involving large dense cored vesicles may serve to deliver transmitters and/or neuropeptide modulators to appropriate receptors in a wider area than release into a specialized synaptic cleft would allow.

  16. Syntaxin 1B, but not syntaxin 1A, is necessary for the regulation of synaptic vesicle exocytosis and of the readily releasable pool at central synapses.

    Directory of Open Access Journals (Sweden)

    Tatsuya Mishima

    Full Text Available Two syntaxin 1 (STX1 isoforms, HPC-1/STX1A and STX1B, are coexpressed in neurons and function as neuronal target membrane (t-SNAREs. However, little is known about their functional differences in synaptic transmission. STX1A null mutant mice develop normally and do not show abnormalities in fast synaptic transmission, but monoaminergic transmissions are impaired. In the present study, we found that STX1B null mutant mice died within 2 weeks of birth. To examine functional differences between STX1A and 1B, we analyzed the presynaptic properties of glutamatergic and GABAergic synapses in STX1B null mutant and STX1A/1B double null mutant mice. We found that the frequency of spontaneous quantal release was lower and the paired-pulse ratio of evoked postsynaptic currents was significantly greater in glutamatergic and GABAergic synapses of STX1B null neurons. Deletion of STX1B also accelerated synaptic vesicle turnover in glutamatergic synapses and decreased the size of the readily releasable pool in glutamatergic and GABAergic synapses. Moreover, STX1A/1B double null neurons showed reduced and asynchronous evoked synaptic vesicle release in glutamatergic and GABAergic synapses. Our results suggest that although STX1A and 1B share a basic function as neuronal t-SNAREs, STX1B but not STX1A is necessary for the regulation of spontaneous and evoked synaptic vesicle exocytosis in fast transmission.

  17. Effects of antiplatelet therapy on platelet extracellular vesicle release and procoagulant activity in health and in cardiovascular disease.

    Science.gov (United States)

    Connor, David E; Ly, Ken; Aslam, Anoosha; Boland, John; Low, Joyce; Jarvis, Susan; Muller, David W; Joseph, Joanne E

    2016-12-01

    Dual antiplatelet therapy with aspirin and clopidogrel is commonly used to prevent recurrent ischemic events in patients with cardiovascular disease. Whilst their effects on platelet reactivity are well documented, it is unclear, however, whether antiplatelet therapy inhibits platelet extracellular vesicle (EV) release. The aim of this study was to investigate the effects of antiplatelet therapy on platelet EV formation and procoagulant activity. Blood samples from 10 healthy controls not receiving antiplatelet therapy were incubated in vitro with aspirin or a P2Y12 inhibitor (MeSAMP). Blood samples from 50 patients receiving long-term dual antiplatelet therapy and undergoing coronary angiography were also studied. Platelet reactivity was assessed by Multiplate™ impedance aggregometry. Platelet EV formation and procoagulant activity of pretreated and untreated blood samples in response to arachidonic acid (AA), adenosine diphosphate (ADP), ADP+PGE1, and thrombin receptor-activating peptide (TRAP) stimulation were assessed by flow cytometry and Procoag-PL assays, respectively. Incubation of normal platelets with aspirin significantly inhibited AA-induced platelet reactivity, EV formation, and procoagulant activity, whilst MeSAMP significantly inhibited platelet reactivity and EV formation in response to AA, ADP, and TRAP, but had minimal effect on procoagulant activity. Most patients receiving dual antiplatelet therapy showed an appropriate reduction in platelet reactivity in response to their treatment; however there was not complete inhibition of increased platelet and EV procoagulant activity in response to ADP, AA, or TRAP. In addition, we could not find any correlation between platelet reactivity and procoagulant activity in patients receiving dual antiplatelet therapy.

  18. Cigarette smoke extract induces the release of extracellular vesicles by airway epithelial cells via cellular carbonyl stress

    NARCIS (Netherlands)

    Benedikter, B.J.; Volgers, C.; Haenen, G.R.M.M.; Savelkoul, P.H.M.; Wouters, E.F.M.; Rohde, G.G.U.; Weseler, A.R.; Stassen, F.R.M.

    2015-01-01

    Introduction: Secreted extracellular vesicles (EVs) participate in multiple processes by transferring proteins and RNA between cells. Yet, their contribution to chronic inflammation in the lungs is largely unexplored. We determined if exposure of airway epithelial cells (AEC) to cigarette smoke

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  20. Acetylcholine receptor antibody

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003576.htm Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood ...

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

    Science.gov (United States)

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

    2017-07-01

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

  2. Exercise and neuromodulators: choline and acetylcholine in marathon runners

    Science.gov (United States)

    Conlay, L. A.; Sabounjian, L. A.; Wurtman, R. J.

    1992-01-01

    Certain neurotransmitters (i.e., acetylcholine, catecholamines, and serotonin) are formed from dietary constituents (i.e., choline, tyrosine and tryptophan). Changing the consumption of these precursors alters release of their respective neurotransmitter products. The neurotransmitter acetylcholine is released from the neuromuscular junction and from brain. It is formed from choline, a common constituent in fish, liver, and eggs. Choline is also incorporated into cell membranes; membranes may likewise serve as an alternative choline source for acetylcholine synthesis. In trained athletes, running a 26 km marathon reduced plasma choline by approximately 40%, from 14.1 to 8.4 uM. Changes of similar magnitude have been shown to reduce acetylcholine release from the neuromuscular junction in vivo. Thus, the reductions in plasma choline associated with strenuous exercise may reduce acetylcholine release, and could thereby affect endurance or performance.

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

    Directory of Open Access Journals (Sweden)

    Carla Pérez-Cruz

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

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

    NARCIS (Netherlands)

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

    2012-01-01

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

  5. Vesicle Photonics

    Science.gov (United States)

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

    2013-07-01

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

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

    Science.gov (United States)

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

    2015-03-01

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

  7. Phencyclidine (PCP)-like inhibition of N-methyl-D-aspartate-evoked striatal acetylcholine release, /sup 3/H-TCP binding and synaptosomal dopamine uptake by metaphit, a proposed PCP receptor acylator

    Energy Technology Data Exchange (ETDEWEB)

    Snell, L.D.; Johnson, K.M.; Yi, S.J.; Lessor, R.A.; Rice, K.C.; Jacobson, A.E.

    1987-12-14

    The phencyclidine (PCP) receptor acylator, metaphit, has been reported to act as a PCP antagonist. Recent electrophysiological and behavioral assessments of metaphit action have revealed, however, that this compound can also act as a PCP-like agonist. The present study examined the effects of metaphit on the inhibition of N-methyl-D-aspartate (NMDA)-induced /sup 3/H-acetylcholine (ACh) release, /sup 3/H-TCP binding and synaptosomal /sup 3/H-dopamine (DA) uptake in the rat striatum. Preincubation of striatal slices for 10 min in the presence of metaphit, followed by a prolonged washout, produced a concentration-dependent inhibition of the ACh release evoked by 300 ..mu..M NMDA. At high concentrations, preincubation with PCP also resulted in inhibition of this measure. However, this could be reduced by extending the washout period, a procedure which had no effect on the inhibition produced by metaphit. At 10..mu..M, metaphit resulted in a 53% reduction in NMDA-evoked ACh release while PCP had no effect under identical conditions. Preincubation of slices in 10 ..mu..M PCP and metaphit reduced the metaphit inhibition by 62%. The effects of PCP and metaphit, alone or in combination, on NMDA-induced ACh release were paralleled by a loss of /sup 3/H-TCP binding sites in striatal tissue incubated under identical conditions suggesting that metaphit exerts long-lasting agonist-like actions on PCP receptors coupled to NMDA receptors. 27 references, 3 figures, 1 table.

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

    DEFF Research Database (Denmark)

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

    1993-01-01

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

  9. 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

  10. Detection of extracellular vesicles: size does matter

    NARCIS (Netherlands)

    van der Pol, E.

    2015-01-01

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

  11. Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking.

    Science.gov (United States)

    Wang, Shan Shan H; Held, Richard G; Wong, Man Yan; Liu, Changliang; Karakhanyan, Aziz; Kaeser, Pascal S

    2016-08-17

    In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near-complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Neuromuscular paralysis by the basic phospholipase A2subunit of crotoxin from Crotalus durissus terrificus snake venom needs its acid chaperone to concurrently inhibit acetylcholine release and produce muscle blockage.

    Science.gov (United States)

    Cavalcante, Walter L G; Noronha-Matos, José B; Timóteo, Maria A; Fontes, Marcos R M; Gallacci, Márcia; Correia-de-Sá, Paulo

    2017-11-01

    Crotoxin (CTX), a heterodimeric phospholipase A 2 (PLA 2 ) neurotoxin from Crotalus durissus terrificus snake venom, promotes irreversible blockade of neuromuscular transmission. Indirect electrophysiological evidence suggests that CTX exerts a primary inhibitory action on transmitter exocytosis, yet contribution of a postsynaptic action of the toxin resulting from nicotinic receptor desensitization cannot be excluded. Here, we examined the blocking effect of CTX on nerve-evoked transmitter release measured directly using radioisotope neurochemistry and video microscopy with the FM4-64 fluorescent dye. Experiments were conducted using mice phrenic-diaphragm preparations. Real-time fluorescence video microscopy and liquid scintillation spectrometry techniques were used to detect transmitter exocytosis and nerve-evoked [ 3 H]-acetylcholine ([ 3 H]ACh) release, respectively. Nerve-evoked myographic recordings were also carried out for comparison purposes. Both CTX (5μg/mL) and its basic PLA 2 subunit (CB, 20μg/mL) had biphasic effects on nerve-evoked transmitter exocytosis characterized by a transient initial facilitation followed by a sustained decay. CTX and CB reduced nerve-evoked [ 3 H]ACh release by 60% and 69%, respectively, but only the heterodimer, CTX, decreased the amplitude of nerve-evoked muscle twitches. Data show that CTX exerts a presynaptic inhibitory action on ACh release that is highly dependent on its intrinsic PLA 2 activity. Given the high safety margin of the neuromuscular transmission, one may argue that the presynaptic block caused by the toxin is not enough to produce muscle paralysis unless a concurrent postsynaptic inhibitory action is also exerted by the CTX heterodimer. Copyright © 2017. Published by Elsevier Inc.

  13. Release and cellular origin of extracellular vesicles during circulation of whole blood over adsorbent polymers for lipid apheresis.

    Science.gov (United States)

    Weiss, René; Eichhorn, Tanja; Spittler, Andreas; Mičušík, Matej; Fischer, Michael B; Weber, Viktoria

    2017-04-01

    Whole blood lipid apheresis is clinically applied in patients with familial hypercholesterolemia to reduce low density lipoprotein and other apolipoprotein B 100 containing lipoproteins. Here, the hemocompatibility of two polyacrylate-coated polyacrylamide-based polymers for lipid apheresis by evaluating the adhesion of blood cells to the adsorbent polymers, their respective activation, as well as the release of microvesicles during circulation of whole blood over the polymers was studied. Characterization of the adsorbents by scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy revealed differences with respect to their surface morphology and their surface chemical composition. Despite these differences, equivalent amounts of leukocytes and platelets adhered to both polymers during circulation of whole blood over the adsorbent columns. The release of phosphatidylserine-exposing microvesicles, in contrast, increased significantly with increasing surface roughness and with the amount of polyacrylate groups at the adsorbent surface. The majority of microvesicles generated during blood-material contact were platelet-derived, and their release was associated with enhanced thrombin generation. Microvesicles were present in free and in cell-bound form, and 75% of all monocytes, but only 0.2% and 2.3% of red blood cells and platelets, respectively, were associated with microvesicles, pointing to a role of monocytes in the clearance of released microvesicles. Taken together, microvesicles are sensitive indicators for biomaterial-induced activation of blood cells in apheresis. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 636-646, 2017. © 2015 Wiley Periodicals, Inc.

  14. Positive allosteric modulators of the α7 nicotinic acetylcholine receptor potentiate glutamate release in the prefrontal cortex of freely-moving rats

    DEFF Research Database (Denmark)

    Bortz, D M; Upton, B A; Mikkelsen, J D

    2016-01-01

    such studies have been performed in vitro. Here we test the hypothesis that PAMs’ potentiation of glutamate release in prefrontal cortex depends upon the level of endogenous cholinergic activity. NMDA stimulation of the nucleus accumbens shell (0.05–0.30 μg in 0.5 μL) increased extracellular choline (0.87 ± 0.......15 – 1.73 ± 0.31 μM) and glutamate (0.15 μg, 3.79 ± 0.87 μM) in medial prefrontal cortex, and the glutamate release was prevented by local infusions of MLA (6.75 μg, 0.19 ± 0.06 μM). The lower dose (1 mg/kg) of AVL3288 (type I) potentiated the glutamate release to a greater degree after the high dose...

  15. 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.

  16. Hemoglobin encapsulation in vesicles retards NO and CO binding and O2 release when perfused through narrow gas-permeable tubes.

    Science.gov (United States)

    Sakai, Hiromi; Okuda, Naoto; Sato, Atsushi; Yamaue, Tatsuya; Takeoka, Shinji; Tsuchida, Eishun

    2010-03-01

    Intravenous administration of cell-free Hb induces vasoconstriction and circulatory disorders, presumably because of the intrinsic affinities to endogenous nitric oxide (NO) and carbon monoxide (CO) as vasorelaxation factors and because of the facilitated O(2) release that might induce autoregulatory vasoconstriction. We examined these gas reactions when Hb-containing solutions of four kinds were perfused through artificial narrow tubes at a practical Hb concentration (10 g/dl). Purified Hb solution, polymerized bovine Hb (Poly(B)Hb), encapsulated Hb [Hb-vesicles (HbV), 279 nm], and red blood cells (RBCs) were perfused through a gas-permeable narrow tube (25 microm inner diameter) at 1 mm/s centerline velocity. The level of reactions was determined microscopically based on the visible-light absorption spectrum of Hb. When the tube was immersed in NO and CO atmospheres, both NO binding and CO binding of deoxygenated Hb (deoxy-Hb) and Poly(B)Hb in the tube was faster than those of HbV and RBCs, and HbV and RBCs showed almost identical binding rates. When the tube was immersed in a N(2) atmosphere, oxygenated Hb and Poly(B)Hb showed much faster O(2) release than did HbV and RBCs. Poly(B)Hb showed a faster reaction than Hb because of the lower O(2) affinity of Poly(B)Hb than Hb. The diffusion process of the particles was simulated using Navier-Stokes and Maxwell-Stefan equations. Results clarified that small Hb (6 nm) diffuses laterally and mixes rapidly. However, the large-dimension HbV shows no such rapid diffusion. The purely physicochemical differences in diffusivity of the particles and the resulting reactivity with gas molecules are one factor inducing biological vasoconstriction of Hb-based oxygen carriers.

  17. Synaptic vesicle proteins and active zone plasticity

    Directory of Open Access Journals (Sweden)

    Robert J Kittel

    2016-04-01

    Full Text Available Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone. The complex molecular architecture of active zones mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of active zones vary significantly, even for a given connection. Thus, there appear to be distinct active zone states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the active zone.The protein-rich cytomatrix at the active zone (CAZ provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1 and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and active zone states, which has heretofore received little attention.

  18. Extracellular Vesicles in Cardiovascular Theranostics

    OpenAIRE

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

    2017-01-01

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

  19. Oligodendroglioma cells synthesize the differentiation-specific linker histone H1˚ and release it into the extracellular environment through shed vesicles.

    Science.gov (United States)

    Schiera, Gabriella; Di Liegro, Carlo Maria; Saladino, Patrizia; Pitti, Rosario; Savettieri, Giovanni; Proia, Patrizia; Di Liegro, Italia

    2013-12-01

    Chromatin remodelling can be involved in some of the epigenetic modifications found in tumor cells. One of the mechanisms at the basis of chromatin dynamics is likely to be synthesis and incorporation of replacement histone variants, such as the H1˚ linker histone. Regulation of the expression of this protein can thus be critical in tumorigenesis. In developing brain, H1˚ expression is mainly regulated at the post-transcriptional level and RNA-binding proteins (RBPs) are involved. In the past, attention mainly focused on the whole brain or isolated neurons and little information is available on H1˚ expression in other brain cells. Even less is known relating to tumor glial cells. In this study we report that, like in maturing brain and isolated neurons, H1˚ synthesis sharply increases in differentiating astrocytes growing in a serum-free medium, while the corresponding mRNA decreases. Unexpectedly, in tumor glial cells both H1˚ RNA and protein are highly expressed, in spite of the fact that H1˚ is considered a differentiation-specific histone variant. Persistence of H1˚ mRNA in oligodendroglioma cells is accompanied by high levels of H1˚ RNA-binding activities which seem to be present, at least in part, also in actively proliferating, but not in differentiating, astrocytes. Finally, we report that oligodendroglioma cells, but not astrocytes, release H1˚ protein into the culture medium by shedding extracellular vesicles. These findings suggest that deregulation of H1˚ histone expression can be linked to tumorigenesis.

  20. Selective release of miRNAs via extracellular vesicles is associated with house-dust mite allergen-induced airway inflammation.

    Science.gov (United States)

    Gon, Y; Maruoka, S; Inoue, T; Kuroda, K; Yamagishi, K; Kozu, Y; Shikano, S; Soda, K; Lötvall, J; Hashimoto, S

    2017-12-01

    MicroRNAs (miRNAs) may facilitate cell-to-cell communication via extracellular vesicles (EVs). The biological roles of miRNAs in EVs on allergic airway inflammation are unclear. Airway-secreted EVs (AEVs) were isolated from bronchoalveolar lavage fluid (BALF) of control and house-dust mite (HDM) allergen-exposed HDM-sensitized mice. The expression of miRNAs in AEVs or miRNAs and mRNAs in lung tissue was analysed using miRNA microarray. The amount of AEV increased 8.9-fold in BALF from HDM-exposed mice compared with that from sham-control mice. HDM exposure resulted in significant changes in the expression of 139 miRNAs in EVs and 175 miRNAs in lung tissues, with 54 miRNAs being common in both samples. Expression changes of these 54 miRNAs between miRNAs in AEVs and lung tissues after HDM exposure were inversely correlated. Computational analysis revealed that 31 genes, including IL-13 and IL-5Ra, are putative targets of the miRNAs up-regulated in AEVs but down-regulated in lung tissues after HDM exposure. The amount of AEV in BALF after HDM exposure was diminished by treatment with the sphingomyelinase inhibitor GW4869. The treatment with GW4869 also decreased Th2 cytokines and eosinophil counts in BALFs and reduced eosinophil accumulation in airway walls and mucosa. These results indicate that selective sorting of miRNA including Th2 inhibitory miRNAs into AEVs and increase release to the airway after HDM exposure would be involved in the pathogenesis of allergic airway inflammation. © 2017 John Wiley & Sons Ltd.

  1. Exosome-like vesicles released from lipid-induced insulin-resistant muscles modulate gene expression and proliferation of beta recipient cells in mice.

    Science.gov (United States)

    Jalabert, Audrey; Vial, Guillaume; Guay, Claudiane; Wiklander, Oscar P B; Nordin, Joel Z; Aswad, Hala; Forterre, Alexis; Meugnier, Emmanuelle; Pesenti, Sandra; Regazzi, Romano; Danty-Berger, Emmanuelle; Ducreux, Sylvie; Vidal, Hubert; El-Andaloussi, Samir; Rieusset, Jennifer; Rome, Sophie

    2016-05-01

    The crosstalk between skeletal muscle (SkM) and beta cells plays a role in diabetes aetiology. In this study, we have investigated whether SkM-released exosome-like vesicles (ELVs) can be taken up by pancreatic beta cells and can deliver functional cargoes. Mice were fed for 16 weeks with standard chow diet (SCD) or with standard diet enriched with 20% palmitate (HPD) and ELVs were purified from quadriceps muscle. Fluorescent ELVs from HPD or SCD quadriceps were injected i.v. or intramuscularly (i.m.) into mice to determine their biodistributions. Micro (mi)RNA quantification in ELVs was determined using quantitative real-time RT-PCR (qRT-PCR)-based TaqMan low-density arrays. Microarray analyses were performed to determine whether standard diet ELVs (SD-ELVs) and high palmitate diet ELVs (HPD-ELVs) induced specific transcriptional signatures in MIN6B1 cells. In vivo, muscle ELVs were taken up by pancreas, 24 h post-injection. In vitro, both SD-ELVs and HPD-ELVs transferred proteins and miRNAs to MIN6B1 cells and modulated gene expressions whereas only HPD-ELVs induced proliferation of MIN6B1 cells and isolated islets. Bioinformatic analyses suggested that transferred HPD-ELV miRNAs may participate in these effects. To validate this, we demonstrated that miR-16, which is overexpressed in HPD-ELVs, was transferred to MIN6B1 cells and regulated Ptch1, involved in pancreas development. In vivo, islets from HPD mice showed increased size and altered expression of genes involved in development, including Ptch1, suggesting that the effect of palm oil on islet size in vivo was reproduced in vitro by treating beta cells with HPD-ELVs. Our data suggest that muscle ELVs might have an endocrine effect and could participate in adaptations in beta cell mass during insulin resistance.

  2. Ca2+ Dependence of Synaptic Vesicle Endocytosis.

    Science.gov (United States)

    Leitz, Jeremy; Kavalali, Ege T

    2016-10-01

    Ca(2+)-dependent synaptic vesicle recycling is essential for structural homeostasis of synapses and maintenance of neurotransmission. Although, the executive role of intrasynaptic Ca(2+) transients in synaptic vesicle exocytosis is well established, identifying the exact role of Ca(2+) in endocytosis has been difficult. In some studies, Ca(2+) has been suggested as an essential trigger required to initiate synaptic vesicle retrieval, whereas others manipulating synaptic Ca(2+) concentrations reported a modulatory role for Ca(2+) leading to inhibition or acceleration of endocytosis. Molecular studies of synaptic vesicle endocytosis, on the other hand, have consistently focused on the roles of Ca(2+)-calmodulin dependent phosphatase calcineurin and synaptic vesicle protein synaptotagmin as potential Ca(2+) sensors for endocytosis. Most studies probing the role of Ca(2+) in endocytosis have relied on measurements of synaptic vesicle retrieval after strong stimulation. Strong stimulation paradigms elicit fusion and retrieval of multiple synaptic vesicles and therefore can be affected by several factors besides the kinetics and duration of Ca(2+) signals that include the number of exocytosed vesicles and accumulation of released neurotransmitters thus altering fusion and retrieval processes indirectly via retrograde signaling. Studies monitoring single synaptic vesicle endocytosis may help resolve this conundrum as in these settings the impact of Ca(2+) on synaptic fusion probability can be uncoupled from its putative role on synaptic vesicle retrieval. Future experiments using these single vesicle approaches will help dissect the specific role(s) of Ca(2+) and its sensors in synaptic vesicle endocytosis. © The Author(s) 2015.

  3. Pushing synaptic vesicles over the RIM.

    Science.gov (United States)

    Kaeser, Pascal S

    2011-05-01

    In a presynaptic nerve terminal, neurotransmitter release is largely restricted to specialized sites called active zones. Active zones consist of a complex protein network, and they organize fusion of synaptic vesicles with the presynaptic plasma membrane in response to action potentials. Rab3-interacting molecules (RIMs) are central components of active zones. In a recent series of experiments, we have systematically dissected the molecular mechanisms by which RIMs operate in synaptic vesicle release. We found that RIMs execute two critical functions of active zones by virtue of independent protein domains. They tether presyanptic Ca(2+) channels to the active zone, and they activate priming of synaptic vesicles by monomerizing homodimeric, constitutively inactive Munc13. These data indicate that RIMs orchestrate synaptic vesicle release into a coherent process. In conjunction with previous studies, they suggest that RIMs form a molecular platform on which plasticity of synaptic vesicle release can operate.

  4. Extracellular Vesicles in Lung Disease.

    Science.gov (United States)

    Kubo, Hiroshi

    2018-01-01

    Accumulating evidence suggests that extracellular vesicles (EVs) play a role in the pathogenesis of lung diseases. These vesicles include exosomes, ectosomes (ie, microparticles, extracellular vesicles, microvesicles, and shedding vesicles), and apoptotic bodies. Exosomes are generated by inward budding of the membrane (endocytosis), subsequent forming of multivesicular bodies, and release by exocytosis. Ectosomes are formed by outward blebbing from the plasma membrane and are then released by proteolytic cleavage from the cell surface. Apoptotic bodies are generated on apoptotic cell shrinkage and death. Extracellular vesicles are released when the cells are activated or undergo apoptosis under inflammatory conditions. The number and types of released EVs are different according to the pathophysiological status of the disease. Therefore, EVs can be novel biomarkers for various lung diseases. EVs contain several molecules, including proteins, mRNA, microRNA, and DNA; they transfer these molecules to distant recipient cells. Circulating EVs modify the targeted cells and influence the microenvironment of the lungs. For this unique capability, EVs are expected to be a new drug delivery system and a novel therapeutic target. Copyright © 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  5. Extracellular vesicles in renal disease.

    Science.gov (United States)

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

    2017-09-01

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

  6. SMALL VESICLES, BIG VEHICLES: EXOSOMES.

    Directory of Open Access Journals (Sweden)

    Saiz-Lopez P

    2016-09-01

    Full Text Available Exosomes are small membranous vesicles released by different cell types. Since their discovery, they have evolved from being considered simple vehicles for the liberation of cellular wastes, to become one of the most promising fields in the area of biomedical research, and more specifically in oncology, since the different malignant tumors release exosomes to all biological fluids, being involved in various functions of the neoplastic process. At present, it is possible to study these vesicles by minimally invasive techniques in patients, which approach us to obtain a more detailed diagnosis and prognosis, as well as to the discovery of new antitumoral therapies

  7. Novel acetylcholine and carbamoylcholine analogues

    DEFF Research Database (Denmark)

    Hansen, Camilla Petrycer; Jensen, Anders Asbjørn; Christensen, Jeppe K.

    2008-01-01

    A series of carbamoylcholine and acetylcholine analogues were synthesized and characterized pharmacologically at neuronal nicotinic acetylcholine receptors (nAChRs). Several of the compounds displayed low nanomolar binding affinities to the alpha 4beta 2 nAChR and pronounced selectivity for this ...

  8. Cholinergic neurotransmission in human corpus cavernosum. II. Acetylcholine synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Blanco, R.; De Tejada, S.; Goldstein, I.; Krane, R.J.; Wotiz, H.H.; Cohen, R.A. (Boston Univ. Medical Center, MA (USA))

    1988-03-01

    Physiological and histochemical evidence indicates that cholinergic nerves may participate in mediating penile erection. Acetylcholine synthesis and release was studied in isolated human corporal tissue. Human corpus cavernosum incubated with ({sup 3}H)choline accumulated ({sup 3}H)choline and synthesized ({sup 3}H)acethylcholine in an concentration-dependent manner. ({sup 3}H)Acetylcholine accumulation by the tissue was inhibited by hemicholinium-3, a specific antagonist of the high-affinity choline transport in cholinergic nerves. Transmural electrical field stimulation caused release of ({sup 3}H)acetylcholine which was significantly diminished by inhibiting neurotransmission with calcium-free physiological salt solution or tetrodotoxin. These observations provide biochemical and physiological evidence for the existence of cholinergic innervation in human corpus cavernosum.

  9. Vesicle Pools: Lessons from Adrenal Chromaffin Cells

    Directory of Open Access Journals (Sweden)

    David R Stevens

    2011-02-01

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

  10. Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

    Directory of Open Access Journals (Sweden)

    R.M. Leão

    2009-01-01

    Full Text Available The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7 or older (p10-12 rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

  11. Trafficking of astrocytic vesicles in hippocampal slices

    Energy Technology Data Exchange (ETDEWEB)

    Potokar, Maja; Kreft, Marko [Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana (Slovenia); Celica Biomedical Center, Technology Park 24, 1000 Ljubljana (Slovenia); Lee, So-Young; Takano, Hajime; Haydon, Philip G. [Department of Neuroscience, Room 215, Stemmler Hall, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104 (United States); Zorec, Robert, E-mail: Robert.Zorec@mf.uni-lj.si [Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana (Slovenia); Celica Biomedical Center, Technology Park 24, 1000 Ljubljana (Slovenia)

    2009-12-25

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

  12. Extracellular Vesicles in Renal Pathophysiology.

    Science.gov (United States)

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

    2017-01-01

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

  13. A Novel Sensitive Method to Measure Catechol-O-Methyltransferase Activity Unravels the Presence of This Activity in Extracellular Vesicles Released by Rat Hepatocytes.

    Science.gov (United States)

    Casal, Enriqueta; Palomo, Laura; Cabrera, Diana; Falcon-Perez, Juan M

    2016-01-01

    There is a clear need for drug treatments to be selected according to the characteristics of an individual patient, in order to improve efficacy and reduce the number and severity of adverse drug reactions. One of the main enzymes to take into account in pharmacogenomics is catechol O-methyltransferase (COMT), which catalyzes the transfer of a methyl group from S -adenosylmethionine to catechols and catecholamines, like the neurotransmitters dopamine, epinephrine, and norepinephrine. Although, most of this enzyme is associated to intracellular vesicles, recently it has also been detected in extracellular vesicles secreted by hepatocytes and in serum circulating vesicles. COMT has implications in many neurological and psychiatric disorders like Parkinson's disease, chronic fatigue, pain response, schizophrenia, and bipolar disorders. Remarkably, genetic variations of COMT affect its activity and are associated to various human disorders from psychiatric diseases to estrogen-induced cancers. Consequently, the establishment of new methods to evaluate COMT activity is an important aspect to investigate the biology of this drug-metabolizing enzyme. Herein, we have developed a sensitive and selective method to determine COMT activity. We first optimized the activity in rat liver incubated with two different substrates; norepinephrine and dopamine. The enzymatically formed products (normetanephrine and 3-methoxytyramine, respectively) were extracted by solid-phase extraction using weak cation exchange cartridges, chromatographically separated, and detected and quantified using a mass spectrometer. The range of quantitation for both products was from 0.005 to 25 μg/mL. This methodology offers acceptable recovery for both enzymatic products (≥75%) and good accuracy and precision (≤15%). The lower limit of quantifications were 0.01 and 0.005 μM for 3-methoxytyramine and normetanephrine, respectively. Importantly, this sensitive assay was able to detect the presence of

  14. Alternative methods for characterization of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Fatemeh eMomen-Heravi

    2012-09-01

    Full Text Available Extracellular vesicles are nano-sized vesicles released by all cells in vitro as well as in vivo. Their role has been implicated mainly in cell-cell communication, but also in disease biomarkers and more recently in gene delivery. They represent a snapshot of the cell status at the moment of release and carry bioreactive macromolecules such as nucleic acids, proteins and lipids. A major limitation in this emerging new field is the availability/awareness of techniques to isolate and properly characterize Extracellular vesicles. The lack of gold standards makes comparing different studies very difficult and may potentially hinder some Extracellular vesicles -specific evidence. Characterization of Extracellular vesicles has also recently seen many advances with the use of Nanoparticle Tracking Analysis (NTA, flow cytometry, cryo-EM instruments and proteomic technologies. In this review, we discuss the latest developments in translational technologies involving characterization methods including the facts in their support and the challenges they face.

  15. Tissue-specific effects of acetylcholine in the canine heart

    DEFF Research Database (Denmark)

    Callø, Kirstine; Goodrow, Robert; Olesen, Søren-Peter

    2013-01-01

    INTRODUCTION: Acetylcholine (ACh) release from the vagus nerve slows heart rate and atrioventricular conduction. ACh stimulates a variety of receptors and channels, including an inward rectifying current (IK,ACh). The effect of ACh in ventricle is still debated. We compare the effect of ACh on ac...

  16. Influence of acetylcholine on binding of 4-[{sup 125}i]iododexetimide to muscarinic brain receptors

    Energy Technology Data Exchange (ETDEWEB)

    Weckesser, Matthias E-mail: m.weckesser@fz-juelich.de; Fixmann, Anton; Holschbach, Marcus; Mueller-Gaertner, Hans-W

    1998-11-01

    The distribution of nicotinic and muscarinic cholinergic receptors in the human brain in vivo has been successfully characterized using radiolabeled tracers and emission tomography. The effect of acetylcholine release into the synaptic cleft on receptor binding of these tracers has not yet been investigated. The present study examined the influence of acetylcholine on binding of 4-[{sup 125}I]iododexetimide to muscarinic cholinergic receptors of porcine brain synaptosomes in vitro. 4-Iododexetimide is a subtype-unspecific muscarinic receptor antagonist with high affinity. Acetylcholine competed with 4-[{sup 125}I]iododexetimide in a dose-dependent manner. A concentration of 500 {mu}M acetylcholine inhibited 50% of total specific 4-[{sup 125}I]iododexetimide binding to synaptosomes when both substances were given simultaneously. An 800 {mu}M acetylcholine solution reduced total specific 4-[{sup 125}I]iododexetimide binding by about 35%, when acetylcholine was given 60 min after incubation of synaptosomes with 4-[{sup 125}I]iododexetimide. Variations in the synaptic acetylcholine concentration might influence muscarinic cholinergic receptor imaging in vivo using 4-[{sup 123}I]iododexetimide. Conversely, 4-[{sup 123}I]iododexetimide might be an appropriate molecule to investigate alterations of acetylcholine release into the synaptic cleft in vivo using single photon emission computed tomography.

  17. Extracellular vesicles in coronary artery disease.

    Science.gov (United States)

    Boulanger, Chantal M; Loyer, Xavier; Rautou, Pierre-Emmanuel; Amabile, Nicolas

    2017-05-01

    Membrane vesicles released in the extracellular space are composed of a lipid bilayer enclosing soluble cytosolic material and nuclear components. Extracellular vesicles include apoptotic bodies, exosomes, and microvesicles (also known previously as microparticles). Originating from different subcellular compartments, the role of extracellular vesicles as regulators of transfer of biological information, acting locally and remotely, is now acknowledged. Circulating vesicles released from platelets, erythrocytes, leukocytes, and endothelial cells contain potential valuable biological information for biomarker discovery in primary and secondary prevention of coronary artery disease. Extracellular vesicles also accumulate in human atherosclerotic plaques, where they affect major biological pathways, including inflammation, proliferation, thrombosis, calcification, and vasoactive responses. Extracellular vesicles also recapitulate the beneficial effect of stem cells to treat cardiac consequences of acute myocardial infarction, and now emerge as an attractive alternative to cell therapy, opening new avenues to vectorize biological information to target tissues. Although interest in microvesicles in the cardiovascular field emerged about 2 decades ago, that for extracellular vesicles, in particular exosomes, started to unfold a decade ago, opening new research and therapeutic avenues. This Review summarizes current knowledge on the role of extracellular vesicles in coronary artery disease, and their emerging potential as biomarkers and therapeutic agents.

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

    Science.gov (United States)

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

    2014-01-01

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

  19. Impulsive behavior and nicotinic acetylcholine receptors.

    Science.gov (United States)

    Ohmura, Yu; Tsutsui-Kimura, Iku; Yoshioka, Mitsuhiro

    2012-01-01

    Higher impulsivity is thought to be a risk factor for drug addiction, criminal involvement, and suicide. Excessive levels of impulsivity are often observed in several psychiatric disorders including attention-deficit/hyperactivity disorder and schizophrenia. Previous studies have demonstrated that nicotinic acetylcholine receptors (nAChRs) are involved in impulsive behavior. Here, we introduce recent advances in this field and describe the role of the following nAChR-related brain mechanisms in modulating impulsive behavior: dopamine release in the ventral striatum; α4β2 nAChRs in the infralimbic cortex, which is a ventral part of the medial prefrontal cortex (mPFC); and dopamine release in the mPFC. We also suggest several potential therapeutic drugs to address these mechanisms in impulsivity-related disorders and explore future directions to further elucidate the roles of central nAChRs in impulsive behavior.

  20. Extracellular vesicles: new players in cardiovascular diseases.

    Science.gov (United States)

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

    2014-05-01

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

  1. Apoptotic Bodies: Selective Detection in Extracellular Vesicles.

    Science.gov (United States)

    Hauser, Paul; Wang, Sha; Didenko, Vladimir V

    2017-01-01

    Normal and dying cells release various types of membrane-bound vesicles including microvesicles, exosomes, and apoptotic bodies. These vesicles play important roles in intercellular communication and signal transduction. However, their diverse forms and subtypes fluctuate in size and other properties. In result current purification approaches do not fully discriminate between different categories of extracellular vesicles. Here, we present a fluorescence technique that specifically identifies apoptotic bodies in preparations of microvesicles, exosomes, and other extracellular vesicles.The approach exclusively labels the vesicles that contain DNA with 5'PO 4 blunt-ended DNA breaks, such as those produced by the apoptotic CAD nuclease during apoptotic DNA degradation. The technique can be useful in studies of apoptosis involving microvesicles and exosomes.

  2. Segregation of lipids near acetylcholine-receptor channels imaged by cryo-EM

    Directory of Open Access Journals (Sweden)

    Nigel Unwin

    2017-07-01

    Full Text Available Rapid communication at the chemical synapse depends on the action of ion channels residing in the postsynaptic membrane. The channels open transiently upon the binding of a neurotransmitter released from the presynaptic nerve terminal, eliciting an electrical response. Membrane lipids also play a vital but poorly understood role in this process of synaptic transmission. The present study examines the lipid distribution around nicotinic acetylcholine (ACh receptors in tubular vesicles made from postsynaptic membranes of the Torpedo ray, taking advantage of the recent advances in cryo-EM. A segregated distribution of lipid molecules is found in the outer leaflet of the bilayer. Apparent cholesterol-rich patches are located in specific annular regions next to the transmembrane helices and also in a more extended `microdomain' between the apposed δ subunits of neighbouring receptors. The particular lipid distribution can be interpreted straightforwardly in relation to the gating movements revealed by an earlier time-resolved cryo-EM study, in which the membranes were exposed briefly to ACh. The results suggest that in addition to stabilizing the protein, cholesterol may play a mechanical role by conferring local rigidity to the membrane so that there is productive coupling between the extracellular and membrane domains, leading to opening of the channel.

  3. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    Science.gov (United States)

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. UNC-18 and Tomosyn Antagonistically Control Synaptic Vesicle Priming Downstream of UNC-13 in Caenorhabditis elegans.

    Science.gov (United States)

    Park, Seungmee; Bin, Na-Ryum; Yu, Bin; Wong, Raymond; Sitarska, Ewa; Sugita, Kyoko; Ma, Ke; Xu, Junjie; Tien, Chi-Wei; Algouneh, Arash; Turlova, Ekaterina; Wang, Siyan; Siriya, Pranay; Shahid, Waleed; Kalia, Lorraine; Feng, Zhong-Ping; Monnier, Philippe P; Sun, Hong-Shuo; Zhen, Mei; Gao, Shangbang; Rizo, Josep; Sugita, Shuzo

    2017-09-06

    Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation

  5. Extracellular vesicles and blood diseases.

    Science.gov (United States)

    Nomura, Shosaku

    2017-04-01

    Extracellular vesicles (EVs) are small membrane vesicles released from many different cell types by the exocytic budding of the plasma membrane in response to cellular activation or apoptosis. EVs disseminate various bioactive effectors originating from the parent cells and transfer functional RNA and protein between cells, enabling them to alter vascular function and induce biological responses involved in vascular homeostasis. Although most EVs in human blood originate from platelets, EVs are also released from leukocytes, erythrocytes, endothelial cells, smooth muscle cells, and cancer cells. EVs were initially thought to be small particles with procoagulant activity; however, they can also evoke cellular responses in the immediate microenvironments and transport microRNAs (miRNA) into target cells. In this review, we summarize the recent literature relevant to EVs, including a growing list of clinical disorders that are associated with elevated EV levels. These studies suggest that EVs play roles in various blood diseases.

  6. Labeling Extracellular Vesicles for Nanoscale Flow Cytometry

    OpenAIRE

    Aizea Morales-Kastresana; Bill Telford; Musich, Thomas A.; Katherine McKinnon; Cassandra Clayborne; Zach Braig; Ari Rosner; Thorsten Demberg; Watson, Dionysios C.; Karpova, Tatiana S.; Freeman, Gordon J.; DeKruyff, Rosemarie H.; Pavlakis, George N.; Masaki Terabe; Marjorie Robert-Guroff

    2017-01-01

    Extracellular vesicles (EVs), including exosomes and microvesicles, are 30?800?nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be brig...

  7. Acetylcholine : Future research and perspectives

    NARCIS (Netherlands)

    Van der Zee, E. A.; Platt, B.; Riedel, G.

    2011-01-01

    Ever since the initial description of chemical transmission in the early part of the 20th century and the identification of acetylcholine (ACh) as the first such transmitter, interests grew to define the multiple facets of its functions. This multitude is only partially covered here, but even in the

  8. Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood-brain barrier.

    Science.gov (United States)

    Tominaga, Naoomi; Kosaka, Nobuyoshi; Ono, Makiko; Katsuda, Takeshi; Yoshioka, Yusuke; Tamura, Kenji; Lötvall, Jan; Nakagama, Hitoshi; Ochiya, Takahiro

    2015-04-01

    Brain metastasis is an important cause of mortality in breast cancer patients. A key event during brain metastasis is the migration of cancer cells through blood-brain barrier (BBB). However, the molecular mechanism behind the passage through this natural barrier remains unclear. Here we show that cancer-derived extracellular vesicles (EVs), mediators of cell-cell communication via delivery of proteins and microRNAs (miRNAs), trigger the breakdown of BBB. Importantly, miR-181c promotes the destruction of BBB through the abnormal localization of actin via the downregulation of its target gene, PDPK1. PDPK1 degradation by miR-181c leads to the downregulation of phosphorylated cofilin and the resultant activated cofilin-induced modulation of actin dynamics. Furthermore, we demonstrate that systemic injection of brain metastatic cancer cell-derived EVs promoted brain metastasis of breast cancer cell lines and are preferentially incorporated into the brain in vivo. Taken together, these results indicate a novel mechanism of brain metastasis mediated by EVs that triggers the destruction of BBB.

  9. Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood–brain barrier

    Science.gov (United States)

    Tominaga, Naoomi; Kosaka, Nobuyoshi; Ono, Makiko; Katsuda, Takeshi; Yoshioka, Yusuke; Tamura, Kenji; Lötvall, Jan; Nakagama, Hitoshi; Ochiya, Takahiro

    2015-01-01

    Brain metastasis is an important cause of mortality in breast cancer patients. A key event during brain metastasis is the migration of cancer cells through blood–brain barrier (BBB). However, the molecular mechanism behind the passage through this natural barrier remains unclear. Here we show that cancer-derived extracellular vesicles (EVs), mediators of cell–cell communication via delivery of proteins and microRNAs (miRNAs), trigger the breakdown of BBB. Importantly, miR-181c promotes the destruction of BBB through the abnormal localization of actin via the downregulation of its target gene, PDPK1. PDPK1 degradation by miR-181c leads to the downregulation of phosphorylated cofilin and the resultant activated cofilin-induced modulation of actin dynamics. Furthermore, we demonstrate that systemic injection of brain metastatic cancer cell-derived EVs promoted brain metastasis of breast cancer cell lines and are preferentially incorporated into the brain in vivo. Taken together, these results indicate a novel mechanism of brain metastasis mediated by EVs that triggers the destruction of BBB. PMID:25828099

  10. Enzyme-linked DNA dendrimer nanosensors for acetylcholine

    Science.gov (United States)

    Walsh, Ryan; Morales, Jennifer M.; Skipwith, Christopher G.; Ruckh, Timothy T.; Clark, Heather A.

    2015-10-01

    It is currently difficult to measure small dynamics of molecules in the brain with high spatial and temporal resolution while connecting them to the bigger picture of brain function. A step towards understanding the underlying neural networks of the brain is the ability to sense discrete changes of acetylcholine within a synapse. Here we show an efficient method for generating acetylcholine-detecting nanosensors based on DNA dendrimer scaffolds that incorporate butyrylcholinesterase and fluorescein in a nanoscale arrangement. These nanosensors are selective for acetylcholine and reversibly respond to levels of acetylcholine in the neurophysiological range. This DNA dendrimer architecture has the potential to overcome current obstacles to sensing in the synaptic environment, including the nanoscale size constraints of the synapse and the ability to quantify the spatio-temporal fluctuations of neurotransmitter release. By combining the control of nanosensor architecture with the strategic placement of fluorescent reporters and enzymes, this novel nanosensor platform can facilitate the development of new selective imaging tools for neuroscience.

  11. 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.

  12. Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis.

    Science.gov (United States)

    Lee, Jaewook; Kim, Si-Hyun; Choi, Dong-Sic; Lee, Jong Seok; Kim, Dae-Kyum; Go, Gyeongyun; Park, Seon-Min; Kim, Si Hyun; Shin, Jeong Hwan; Chang, Chulhun L; Gho, Yong Song

    2015-10-01

    The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC-MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 (http://proteomecentral.proteomexchange.org/dataset/PXD001160). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Transcriptome and long noncoding RNA sequencing of three extracellular vesicle subtypes released from the human colon cancer LIM1863 cell line

    Science.gov (United States)

    Chen, Maoshan; Xu, Rong; Ji, Hong; Greening, David W.; Rai, Alin; Izumikawa, Keiichi; Ishikawa, Hideaki; Takahashi, Nobuhiro; Simpson, Richard J.

    2016-01-01

    Previously we reported that LIM1863 colorectal cancer (CRC) cells secrete three distinct extracellular vesicle subtypes – two subpopulations of exosomes (apical EpCAM-Exos and basolateral A33-Exos) and shed microvesicles (sMVs) – with distinct protein and miRNA signatures. Here, we extend our omics approach to understand the fundamental role of LIM1863-derived EVs by performing a comprehensive analysis of their mRNAs and long non-coding RNAs (lncRNAs) using RNA-Seq. We show that 2,389 mRNAs, 317 pseudogene transcripts, 1,028 lncRNAs and 206 short non-coding RNAs selectively distributed to (i.e., are enriched in) LIM1863 EVs, relative to the parent cell. An Ensembl/UniProtKB analysis revealed 1,937 mRNAs encode canonical proteins, 348 isoforms (including splice-variant proteins), and 119 ‘missing proteins’ (i.e., annotated in Ensembl but not UniProtKB). Further dissection of our protein/RNA data revealed that 6/151 observed RNA binding proteins have the potential to interact with ~75% of EV-enriched RNAs. Intriguingly, the co-existence of U1 and U2 ribonucleoproteins and their cognate snRNAs in LIM1863 EVs suggests a possible association of CRC EVs with recipient cell splicing events. Our data reveal several potential lncRNA CRC biomarkers and novel splicing/fusion genes that, collectively, will advance our understanding of EV biology in CRC and accelerate the development of EV-based diagnostics and therapeutics. PMID:27917920

  14. Extracellular vesicles as emerging intercellular communicasomes.

    Science.gov (United States)

    Yoon, Yae Jin; Kim, Oh Youn; Gho, Yong Song

    2014-10-01

    All living cells release extracellular vesicles having pleiotropic functions in intercellular communication. Mammalian extracellular vesicles, also known as exosomes and microvesicles, are spherical bilayered proteolipids composed of various bioactive molecules, including RNAs, DNAs, proteins, and lipids. Extracellular vesicles directly and indirectly control a diverse range of biological processes by transferring membrane proteins, signaling molecules, mRNAs, and miRNAs, and activating receptors of recipient cells. The active interaction of extracellular vesicles with other cells regulates various physiological and pathological conditions, including cancer, infectious diseases, and neurodegenerative disorders. Recent developments in high-throughput proteomics, transcriptomics, and lipidomics tools have provided ample data on the common and specific components of various types of extracellular vesicles. These studies may contribute to the understanding of the molecular mechanism involved in vesicular cargo sorting and the biogenesis of extracellular vesicles, and, further, to the identification of disease-specific biomarkers. This review focuses on the components, functions, and therapeutic and diagnostic potential of extracellular vesicles under various pathophysiological conditions.

  15. Structure of Amphiphilic Terpolymer Raspberry Vesicles

    Directory of Open Access Journals (Sweden)

    Yingying Guo

    2017-07-01

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

  16. 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.

  17. Deep sequencing of RNA from three different extracellular vesicle (EV subtypes released from the human LIM1863 colon cancer cell line uncovers distinct miRNA-enrichment signatures.

    Directory of Open Access Journals (Sweden)

    Hong Ji

    Full Text Available Secreted microRNAs (miRNAs enclosed within extracellular vesicles (EVs play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863--shed microvesicles (sMVs and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes. Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs--miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32 of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.

  18. 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.

  19. IN-VITRO FUSION OF RETICULOCYTE ENDOCYTIC VESICLES WITH LIPOSOMES

    NARCIS (Netherlands)

    VIDAL, M; HOEKSTRA, D

    1995-01-01

    Since reticulocytes have a high demand for iron, which is required for heme biosynthesis, these cells are highly specialized in the endocytosis of the iron carrier transferrin (Tf). From the resulting endocytic vesicles (EVs), iron is released and the vesicles rapidly return to the cell membrane

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

    NARCIS (Netherlands)

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

    2012-01-01

    Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These

  1. Cyclic nucleotides of canine antral smooth muscle. Effects of acetylcholine, catecholamines and gastrin.

    Science.gov (United States)

    Baur, S; Grant, B; Wooton, J

    1981-01-07

    1. The effects of acetylcholine, catecholamines and gastrin on the intracellular content of cyclic AMP and cyclic GMP in antral circular muscle have been determined. 2. Acetylcholine results in a significant but transient increase in intracellular cyclic GMP. 3. Isoproterenol and norepinephrine increase intracellular cyclic AMP. Based on half-maximal effective doses, isoproterenol is 2.7-times more effective than norepinephrine. The increase in intracellular cyclic AMP by both agents is inhibited by propranolol but not phentolamine, indicating that both agents act on the muscle cell by a beta-receptor-coupled mechanism. 4. Gastrin has no demonstrable effect on either cyclic AMP or cyclic GMP. This suggests that while gastrin and acetylcholine can produce a like myoelectric response in the muscle cell, the action of gastrin is mediated by a separate receptor, presumably on the muscle cell, and not by a release of acetylcholine.

  2. A Network of Three Types of Filaments Organizes Synaptic Vesicles for Storage, Mobilization, and Docking.

    Science.gov (United States)

    Cole, Andy A; Chen, Xiaobing; Reese, Thomas S

    2016-03-16

    Synaptic transmission between neurons requires precise management of synaptic vesicles. While individual molecular components of the presynaptic terminal are well known, exactly how the molecules are organized into a molecular machine serving the storage and mobilization of synaptic vesicles to the active zone remains unclear. Here we report three filament types associated with synaptic vesicles in glutamatergic synapses revealed by electron microscope tomography in unstimulated, dissociated rat hippocampal neurons. One filament type, likely corresponding to the SNAREpin complex, extends from the active zone membrane and surrounds docked vesicles. A second filament type contacts all vesicles throughout the active zone and pairs vesicles together. On the third filament type, vesicles attach to side branches extending from the long filament core and form vesicle clusters that are distributed throughout the vesicle cloud and along the active zone membrane. Detailed analysis of presynaptic structure reveals how each of the three filament types interacts with synaptic vesicles, providing a means to traffic reserved and recycled vesicles from the cloud of vesicles into the docking position at the active zone. The formation and release of synaptic vesicles has been extensively investigated. Explanations of the release of synaptic vesicles generally begin with the movement of vesicles from the cloud into the synaptic active zone. However, the presynaptic terminal is filled with filamentous material that would appear to limit vesicular diffusion. Here, we provide a systematic description of three filament types connecting synaptic vesicles. A picture emerges illustrating how the cooperative attachment and release of these three filament types facilitate the movement of vesicles to the active zone to become docked in preparation for release. Copyright © 2016 the authors 0270-6474/16/363222-09$15.00/0.

  3. RNA in extracellular vesicles.

    Science.gov (United States)

    Kim, Kyoung Mi; Abdelmohsen, Kotb; Mustapic, Maja; Kapogiannis, Dimitrios; Gorospe, Myriam

    2017-07-01

    Cells release a range of membrane-enclosed extracellular vesicles (EVs) into the environment. Among them, exosomes and microvesicles (collectively measuring 40-1000 nm in diameter) carry proteins, signaling lipids, and nucleic acids from donor cells to recipient cells, and thus have been proposed to serve as intercellular mediators of communication. EVs transport cellular materials in many physiologic processes, including differentiation, stem cell homeostasis, immune responses, and neuronal signaling. EVs are also increasingly recognized as having a direct role in pathologies such as cancer and neurodegeneration. Accordingly, EVs have been the focus of intense investigation as biomarkers of disease, prognostic indicators, and even therapeutic tools. Here, we review the classes of RNAs present in EVs, both coding RNAs (messenger RNAs) and noncoding RNAs (long noncoding RNAs, microRNAs, and circular RNAs). The rising attention to EV-resident RNAs as biomarkers stems from the fact that RNAs can be detected at extremely low quantities using a number of methods. To illustrate the interest in EV biology, we discuss EV RNAs in cancer and neurodegeneration, two major age-associated disease processes. WIREs RNA 2017, 8:e1413. doi: 10.1002/wrna.1413 For further resources related to this article, please visit the WIREs website. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

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

    Directory of Open Access Journals (Sweden)

    Ajay Kumar Botcha

    2014-01-01

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

  5. Gram-negative and Gram-positive bacterial extracellular vesicles.

    Science.gov (United States)

    Kim, Ji Hyun; Lee, Jaewook; Park, Jaesung; Gho, Yong Song

    2015-04-01

    Like mammalian cells, Gram-negative and Gram-positive bacteria release nano-sized membrane vesicles into the extracellular environment either in a constitutive manner or in a regulated manner. These bacterial extracellular vesicles are spherical bilayered proteolipids enriched with bioactive proteins, lipids, nucleic acids, and virulence factors. Recent progress in this field supports the critical pathophysiological functions of these vesicles in both bacteria-bacteria and bacteria-host interactions. This review provides an overview of the current understanding on Gram-negative and Gram-positive bacterial extracellular vesicles, especially regarding the biogenesis, components, and functions in poly-species communities. We hope that this review will stimulate additional research in this emerging field of bacterial extracellular vesicles and contribute to the development of extracellular vesicle-based diagnostic tools and effective vaccines against pathogenic Gram-negative and Gram-positive bacteria. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Synaptic Vesicle Endocytosis

    Science.gov (United States)

    Saheki, Yasunori; De Camilli, Pietro

    2012-01-01

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

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

    Science.gov (United States)

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

    2015-09-15

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

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

    Science.gov (United States)

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

    2013-01-01

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

  9. 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.

  10. Preparation of large monodisperse vesicles.

    Directory of Open Access Journals (Sweden)

    Ting F Zhu

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

  11. Fusion of Nonionic Vesicles

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  12. Outer membrane vesicles and soluble factors released by probiotic Escherichia coli Nissle 1917 and commensal ECOR63 enhance barrier function by regulating expression of tight junction proteins in intestinal epithelial cells

    Directory of Open Access Journals (Sweden)

    Carina Shianya Alvarez

    2016-12-01

    Full Text Available The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Escherichia coli Nissle 1917 (EcN is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the

  13. Deletion of muscarinic type 1 acetylcholine receptors alters splenic lymphocyte functions and splenic noradrenaline concentration.

    Science.gov (United States)

    Hainke, Susanne; Wildmann, Johannes; Del Rey, Adriana

    2015-11-01

    The existence of interactions between the immune and the sympathetic nervous systems is well established. Noradrenaline can promote or inhibit the immune response, and conversely, the immune response itself can affect noradrenaline concentration in lymphoid organs, such as the spleen. It is also well known that acetylcholine released by pre-ganglionic neurons can modulate noradrenaline release by the postsynaptic neuron. The spleen does not receive cholinergic innervation, but it has been reported that lymphocytes themselves can produce acetylcholine, and express acetylcholine receptors and acetylcholinesterase. We found that the spleen of not overtly immunized mice in which muscarinic type 1 acetylcholine receptors have been knocked out (M1KO) has higher noradrenaline concentrations than that of the wildtype mice, without comparable alterations in the heart, in parallel to a decreased number of IgG-producing B cells. Splenic lymphocytes from M1KO mice displayed increased in vitro-induced cytotoxicity, and this was observed only when CD4(+) T cells were present. In contrast, heterozygous acetylcholinesterase (AChE+/-) mice, had no alterations in splenic noradrenaline concentration, but the in vitro proliferation of AChE+/- CD4(+) T cells was increased. It is theoretically conceivable that reciprocal effects between neuronally and non-neuronally derived acetylcholine and noradrenaline might contribute to the results reported. Our results emphasize the need to consider the balance between the effects of these mediators for the final immunoregulatory outcome. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Mechanics of post-fusion exocytotic vesicle.

    Science.gov (United States)

    Stephens, Thomas; Wu, Zhanghan; Liu, Jian

    2017-05-23

    Exocytosis is an important cellular process controlled by metabolic signaling. It involves vesicle fusion to the plasma membrane, followed by the opening of a fusion pore, and the subsequent release of the vesicular lumen content into the extracellular space. While most modeling efforts focus on the events leading to membrane fusion, how the vesicular membrane remodels after fusing to plasma membrane remains unclear. This latter event dictates the nature and the efficiency of exocytotic vesicular secretions, and is thus critical for exocytotic function. We provide a generic membrane mechanical model to systematically study the fate of post-fusion vesicles. We show that while membrane stiffness favors full-collapse vesicle fusion into the plasma membrane, the intravesicular pressure swells the vesicle and causes the fusion pore to shrink. Dimensions of the vesicle and its associated fusion pore further modulate this mechanical antagonism. We systematically define the mechanical conditions that account for the full spectrum of the observed vesicular secretion modes. Our model therefore can serve as a unified theoretical framework that sheds light on the elaborate control mechanism of exocytosis.

  15. Mechanics of post-fusion exocytotic vesicle

    Science.gov (United States)

    Stephens, Thomas; Wu, Zhanghan; Liu, Jian

    2017-06-01

    Exocytosis is an important cellular process controlled by metabolic signaling. It involves vesicle fusion to the plasma membrane, followed by the opening of a fusion pore, and the subsequent release of the vesicular lumen content into the extracellular space. While most modeling efforts focus on the events leading to membrane fusion, how the vesicular membrane remodels after fusing to plasma membrane remains unclear. This latter event dictates the nature and the efficiency of exocytotic vesicular secretions, and is thus critical for exocytotic function. We provide a generic membrane mechanical model to systematically study the fate of post-fusion vesicles. We show that while membrane stiffness favors full-collapse vesicle fusion into the plasma membrane, the intravesicular pressure swells the vesicle and causes the fusion pore to shrink. Dimensions of the vesicle and its associated fusion pore further modulate this mechanical antagonism. We systematically define the mechanical conditions that account for the full spectrum of the observed vesicular secretion modes. Our model therefore can serve as a unified theoretical framework that sheds light on the elaborate control mechanism of exocytosis.

  16. Characterization of extracellular vesicles in whole blood: Influence of pre-analytical parameters and visualization of vesicle-cell interactions using imaging flow cytometry.

    Science.gov (United States)

    Fendl, Birgit; Weiss, René; Fischer, Michael B; Spittler, Andreas; Weber, Viktoria

    2016-09-09

    Extracellular vesicles are central players in intercellular communication and are released from the plasma membrane under tightly regulated conditions, depending on the physiological and pathophysiological state of the producing cell. Their heterogeneity requires a spectrum of methods for isolation and characterization, where pre-analytical parameters have profound impact on vesicle analysis, particularly in blood, since sampling, addition of anticoagulants, as well as post-sampling vesicle generation may influence the outcome. Here, we characterized microvesicles directly in whole blood using a combination of flow cytometry and imaging flow cytometry. We assessed the influence of sample agitation, anticoagulation, and temperature on post-sampling vesicle generation, and show that vesicle counts remained stable over time in samples stored without agitation. Storage with gentle rolling mimicking agitation, in contrast, resulted in strong release of platelet-derived vesicles in blood anticoagulated with citrate or heparin, whereas vesicle counts remained stable upon anticoagulation with EDTA. Using imaging flow cytometry, we could visualize microvesicles adhering to blood cells and revealed an anticoagulant-dependent increase in vesicle-cell aggregates over time. We demonstrate that vesicles adhere preferentially to monocytes and granulocytes in whole blood, while no microvesicles could be visualized on lymphocytes. Our data underscore the relevance of pre-analytical parameters in vesicle analysis and demonstrate that imaging flow cytometry is a suitable tool to study the interaction of extracellular vesicles with their target cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Decomposition of acetylcholine with ethylene formation in vitro. Possible free radical mechanism of acetylcholine action.

    Science.gov (United States)

    Kurchii, V M; Kurchii, B A

    2000-01-01

    Experiments were designed to investigate the effect of different buffered solutions, Fenton reagent and hydrogen peroxide on acetylcholine decomposition with ethylene formation. The data of the present study suggests that acetylcholine is decomposed in vitro to form ethylene by interacting with the free radicals or in the Hofmann's splitting reaction. It is found that free radicals are required for the fast decomposition of acetylcholine to form ethylene. A general mechanism to explain the rapid biological effects that can be influenced by the free radicals was proposed. We have concluded that endogenous metabolic free radicals can be involved in the decomposition of acetylcholine as well in the biological activation of formed ethylene in vivo.

  18. 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.

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Lilian Chiang

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

  1. Extracellular Vesicles in Cardiovascular Theranostics.

    Science.gov (United States)

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

    2017-01-01

    Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells play essential roles in pathophysiological processes such as cardiac hypertrophy, cardiomyocyte survival and apoptosis, cardiac fibrosis, and angiogenesis in relation to CVDs. In this review, we will first outline the current knowledge about the physical characteristics, biological contents, and isolation methods of EVs. We will then focus on the functional roles of cardiovascular EVs and their pathophysiological effects in CVDs, as well as summarize the potential of EVs as therapeutic agents and biomarkers for CVDs. Finally, we will discuss the specific application of EVs as a novel drug delivery system and the utility of EVs in the field of regenerative medicine.

  2. Extracellular Vesicles Released from Human Umbilical Cord-Derived Mesenchymal Stromal Cells Prevent Life-Threatening Acute Graft-Versus-Host Disease in a Mouse Model of Allogeneic Hematopoietic Stem Cell Transplantation.

    Science.gov (United States)

    Wang, Li; Gu, Zhenyang; Zhao, Xiaoli; Yang, Nan; Wang, Feiyan; Deng, Ailing; Zhao, Shasha; Luo, Lan; Wei, Huaping; Guan, Lixun; Gao, Zhe; Li, Yonghui; Wang, Lili; Liu, Daihong; Gao, Chunji

    2016-12-15

    Mesenchymal stromal cells (MSCs) are attractive agents for the prophylaxis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, safety concerns remain about their clinical application. In this study, we explored whether extracellular vesicles released from human umbilical cord-derived MSCs (hUC-MSC-EVs) could prevent aGVHD in a mouse model of allo-HSCT. hUC-MSC-EVs were intravenously administered to recipient mice on days 0 and 7 after allo-HSCT, and the prophylactic effects of hUC-MSC-EVs were assessed by observing the in vivo manifestations of aGVHD, histologic changes in target organs, and recipient mouse survival. We evaluated the effects of hUC-MSC-EVs on immune cells and inflammatory cytokines by flow cytometry and ProcartaPlex™ Multiplex Immunoassays, respectively. The in vitro effects of hUC-MSC-EVs were determined by mitogen-induced proliferation assays. hUC-MSC-EVs alleviated the in vivo manifestations of aGVHD and the associated histologic changes and significantly reduced the mortality of the recipient mice. Recipients treated with hUC-MSC-EVs had significantly lower frequencies and absolute numbers of CD3+CD8+ T cells; reduced serum levels of IL-2, TNF-α, and IFN-γ; a higher ratio of CD3+CD4+ and CD3+CD8+ T cells; and higher serum levels of IL-10. An in vitro experiment demonstrated that hUC-MSC-EVs inhibited the mitogen-induced proliferation of splenocytes in a dose-dependent manner, and the cytokine changes were similar to those observed in vivo. This study indicated that hUC-MSC-EVs can prevent life-threatening aGVHD by modulating immune responses. These data provide the first evidence that hUC-MSC-EVs represent an ideal alternative in the prophylaxis of aGVHD after allo-HSCT.

  3. Vesicles containing ion channels on crystalline surfaces-An FTIR and surface enhanced FTIR spectroscopic study

    Science.gov (United States)

    Fischer, W. B.; Unverricht, I.; Kuhne, Ch.; Steiner, G.; Schrattenholz, A.; Maelicke, A.; Salzer, R.

    1998-06-01

    The kinetics of the adsorption of native vesicles containing the nicotinic acetylcholine receptor (nAChR) is monitored by ATR-FTIR and SEIRA spectroscopy. The membrane vesicles are adsorbed on Ge crystals. Experiments are done with neat Ge and with Ge crystals covered with a thin layer of silver clusters in order to obtain the enhancement effect of infrared adsorption. The nAChR shows β-sheet/turn structures at the interface. These results give evidence for the existence of these structures in the extracellular domains of the receptor. The potential for SEIRA in the investigation of proteins at interfaces and membrane processes is outlined.

  4. Concurrent imaging of synaptic vesicle recycling and calcium dynamics.

    Directory of Open Access Journals (Sweden)

    Haiyan eLi

    2011-11-01

    Full Text Available Synaptic transmission involves the calcium-dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2, and a presynaptically-localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3 with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Re-acidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real-time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released.

  5. Role of extracellular vesicles in de novo mineralization: an additional novel mechanism of cardiovascular calcification.

    Science.gov (United States)

    New, Sophie E P; Aikawa, Elena

    2013-08-01

    Extracellular vesicles are membrane micro/nanovesicles secreted by many cell types into the circulation and the extracellular milieu in physiological and pathological conditions. Evidence suggests that extracellular vesicles, known as matrix vesicles, play a role in the mineralization of skeletal tissue, but emerging ultrastructural and in vitro studies have demonstrated their contribution to cardiovascular calcification as well. Cells involved in the progression of cardiovascular calcification release active vesicles capable of nucleating hydroxyapatite on their membranes. This review discusses the role of extracellular vesicles in cardiovascular calcification and elaborates on this additional mechanism of calcification as an alternative pathway to the currently accepted mechanism of biomineralization via osteogenic differentiation.

  6. Proteomic Analysis of Blood Extracellular Vesicles in Cardiovascular Disease by LC-MS/MS Analysis.

    Science.gov (United States)

    Baldan-Martin, Montserrat; de la Cuesta, Fernando; Alvarez-Llamas, Gloria; Ruiz-Hurtado, Gema; Ruilope, Luis M; Barderas, Maria G

    2017-01-01

    Extracellular vesicles are membrane vesicles related to cell communication. These vesicles consist of proteins, RNA, and microRNA and are an interesting and important tool to understand the processes taking place in the secreting cell, especially in diseases in which its release is often enhanced. The used of blood extracellular vesicles in cardiovascular disease as a low invasive, easily accessible source of circulating markers could give us important information related to pathological process even more with the use of proteomic analysis. In this chapter, we describe a protocol to isolate and proteomic analyze extracellular vesicles from blood associated with cardiovascular disease.

  7. Placenta-derived extracellular vesicles: their cargo and possible functions.

    Science.gov (United States)

    Familari, Mary; Cronqvist, Tina; Masoumi, Zahra; Hansson, Stefan R

    2017-03-01

    The literature on extracellular vesicles consists of rapidly expanding and often contradictory information. In this paper we attempt to review what is currently known regarding extracellular vesicles released specifically from human placental syncytiotrophoblast cells with a focus on the common but complex pregnancy-associated syndrome pre-eclampsia, where the level of syncytiotrophoblast extracellular vesicle release is significantly increased. We review common methods for syncytiotrophoblast extracellular vesicle derivation and isolation and we discuss the cargo of syncytiotrophoblast extracellular vesicles including proteins, RNA and lipids and their possible functions. A meta-analysis of available trophoblast-derived extracellular vesicle proteomic datasets revealed only three proteins in common: albumin, fibronectin-1 and plasminogen activator inhibitor-1, suggesting some variability in vesicle cargo, most likely reflecting stage and cell type of origin. We discuss the possible sources of variability that may have led to the low number of common markers, which has led us to speculate that markers and density in common use may not be strict criteria for identifying and isolating placenta-derived exosomes.

  8. Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation.

    Science.gov (United States)

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

    2017-08-01

    Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs. EVs were quantified and analyzed in the sera of patients after an acute stroke (inflammation in immune cells. © 2017 American Heart Association, Inc.

  9. 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

  10. 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...... vesicles. Pulse-chase and intravital imaging experiments suggested EVs liberated by exercise have a propensity to localize in the liver and can transfer their protein cargo. Moreover, by employing arteriovenous balance studies across the contracting human limb, we identified several novel candidate...

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced...... by hypertonic solutions. We show that complexinI/II deficiency or phorbol ester stimulation indeed affects responses to hypertonic solution in a supralinear manner. An additive vs multiplicative relationship between activation energy and fusion rate provides a novel explanation for previously observed non...

  12. TC299423, a Novel Agonist for Nicotinic Acetylcholine Receptors

    Directory of Open Access Journals (Sweden)

    Teagan R. Wall

    2017-09-01

    Full Text Available (E-5-(Pyrimidin-5-yl-1,2,3,4,7,8-hexahydroazocine (TC299423 is a novel agonist for nicotinic acetylcholine receptors (nAChRs. We examined its efficacy, affinity, and potency for α6β2∗ (α6β2-containing, α4β2∗, and α3β4∗ nAChRs, using [125I]-epibatidine binding, whole-cell patch-clamp recordings, synaptosomal 86Rb+ efflux, [3H]-dopamine release, and [3H]-acetylcholine release. TC299423 displayed an EC50 of 30–60 nM for α6β2∗ nAChRs in patch-clamp recordings and [3H]-dopamine release assays. Its potency for α6β2∗ in these assays was 2.5-fold greater than that for α4β2∗, and much greater than that for α3β4∗-mediated [3H]-acetylcholine release. We observed no major off-target binding on 70 diverse molecular targets. TC299423 was bioavailable after intraperitoneal or oral administration. Locomotor assays, measured with gain-of-function, mutant α6 (α6L9′S nAChR mice, show that TC299423 elicits α6β2∗ nAChR-mediated responses at low doses. Conditioned place preference assays show that low-dose TC299423 also produces significant reward in α6L9′S mice, and modest reward in WT mice, through a mechanism that probably involves α6(non-α4β2∗ nAChRs. However, TC299423 did not suppress nicotine self-administration in rats, indicating that it did not block nicotine reinforcement in the dosage range that was tested. In a hot-plate test, TC299423 evoked antinociceptive responses in mice similar to those of nicotine. TC299423 and nicotine similarly inhibited mouse marble burying as a measure of anxiolytic effects. Taken together, our data suggest that TC299423 will be a useful small-molecule agonist for future in vitro and in vivo studies of nAChR function and physiology.

  13. 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.

  14. How pure are your vesicles?

    Science.gov (United States)

    Webber, Jason; Clayton, Aled

    2013-01-01

    We propose a straightforward method to estimate the purity of vesicle preparations by comparing the ratio of nano-vesicle counts to protein concentration, using tools such as the increasingly available NanoSight platform and a colorimetric protein assay such as the BCA-assay. Such an approach is simple enough to apply to every vesicle preparation within a given laboratory, assisting researchers as a routine quality control step. Also, the approach may aid in comparing/standardising vesicle purity across diverse studies, and may be of particular importance in evaluating vesicular biomarkers. We herein propose some criteria to aid in the definition of pure vesicles. PMID:24009896

  15. The Ghosts of Acetylcholine : Structure- activity relationships of ...

    African Journals Online (AJOL)

    Adele

    . This presentation attempts to see the structure of acetylcholine in all muscle relaxants that are clinically useful. It begins with the structure of acetylcholine itself , then progresses to suxamethonium and incorporates all nondepolarising agents.

  16. Docking to flexible nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Sander, Tommy; Bruun, Anne T; Balle, Thomas

    2010-01-01

    Computational docking to nicotinic acetylcholine receptors (nAChRs) and other members of the Cys-loop receptor family is complicated by the flexibility of the so-called C-loop. As observed in the large number of published crystal structures of the acetylcholine binding protein (AChBP), a structural...... surrogate and homology modeling template for the nAChRs, the conformation of this loop is controlled by the ligand present in the binding pocket. As part of the development of a protocol for unbiased docking to the nAChRs, we here present the results of docking of ligands with known binding modes to an ACh...

  17. Fornix deep brain stimulation enhances acetylcholine levels in the hippocampus.

    Science.gov (United States)

    Hescham, Sarah; Jahanshahi, Ali; Schweimer, Judith V; Mitchell, Stephen N; Carter, Guy; Blokland, Arjan; Sharp, Trevor; Temel, Yasin

    2016-11-01

    Deep brain stimulation (DBS) of the fornix has gained interest as a potential therapy for advanced treatment-resistant dementia, yet the mechanism of action remains widely unknown. Previously, we have reported beneficial memory effects of fornix DBS in a scopolamine-induced rat model of dementia, which is dependent on various brain structures including hippocampus. To elucidate mechanisms of action of fornix DBS with regard to memory restoration, we performed c-Fos immunohistochemistry in the hippocampus. We found that fornix DBS induced a selective activation of cells in the CA1 and CA3 subfields of the dorsal hippocampus. In addition, hippocampal neurotransmitter levels were measured using microdialysis before, during and after 60 min of fornix DBS in a next experiment. We observed a substantial increase in the levels of extracellular hippocampal acetylcholine, which peaked 20 min after stimulus onset. Interestingly, hippocampal glutamate levels did not change compared to baseline. Therefore, our findings provide first experimental evidence that fornix DBS activates the hippocampus and induces the release of acetylcholine in this region.

  18. Extracellular Vesicles Produced by the Gram-positive Bacterium Bacillus subtilis are Disrupted by the Lipopeptide Surfactin

    Science.gov (United States)

    Brown, Lisa; Kessler, Anne; Cabezas-Sanchez, Pablo; Luque-Garcia, Jose L.; Casadevall, Arturo

    2014-01-01

    Summary Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harboring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin. PMID:24826903

  19. ATP: The crucial component of secretory vesicles.

    Science.gov (United States)

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

    2016-07-12

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

  20. Reconstitution of lipid vesicles associated with HVJ (Sendai virus) sikes. Purification and some properties of vesicles containing nontoxic fragment A of diphtheria toxin

    Science.gov (United States)

    1979-01-01

    A mixture of HVJ (Sendai virus) spike proteins, the nontoxic fragment A of diphtheria toxin, lecithin, and cholesterol was solubilized in sucrose solution containing a nonionic neutral detergent. The liposomal vesicles which formed on removal of the detergent by dialysis were purified by gel filtration and centrifugation on a sucrose gradient. The resulting purified vesicles had hemagglutinating activity, hemolytic activity and, after solubilization, the enzymic activity of fragment A. The vesicles had no cell fusion activity. Electron microscopy showed that both the outside and inside of membranes of the vesicles were associated with the spikes. When the vesicles were freeze- fractured, no large aggregates of particles were seen on either face. Such fragment A-containing lipid vesicles (liposomes) with HVJ spikes bound to mamalian cell membrane and released their fragment A into the cytoplasm causing cell death. Neither fragment A-containing liposomes without spikes nor empty liposomes with spikes were toxic. PMID:217880

  1. Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors.

    Science.gov (United States)

    Rozman, Klara Bulc; Araoz, Romulo; Sepcić, Kristina; Molgo, Jordi; Suput, Dusan

    2010-09-06

    Nicotinic acetylcholine receptors are implicated in different nervous system-related disorders, and their modulation could improve existing therapy of these diseases. Parazoanthoxanthin A (ParaA) is a fluorescent pigment of the group of zoanthoxanthins. Since it is a potent acetylcholinesterase inhibitor, it may also bind to nicotinic acetylcholine receptors (nAChRs). For this reason its effect on Torpedo nAChR (alpha1(2)betagammadelta) transplanted to Xenopus laevis oocytes was evaluated, using the voltage-clamp technique. ParaA dose-dependently reduced the acetylcholine-induced currents. This effect was fully reversible only at lower concentrations. ParaA also reduced the Hill coefficient and the time to peak current, indicating a channel blocking mode of action. On the other hand, the combined effect of ParaA and d-tubocurarine (d-TC) on acetylcholine-induced currents exhibited only partial additivity, assuming a competitive mode of action of ParaA on nAChR. These results indicate a dual mode of action of ParaA on the Torpedo AChR. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  2. 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

  3. Acetylcholine affects osteocytic MLO-Y4 cells via acetylcholine receptors.

    Science.gov (United States)

    Ma, Yuanyuan; Li, Xianxian; Fu, Jing; Li, Yue; Gao, Li; Yang, Ling; Zhang, Ping; Shen, Jiefei; Wang, Hang

    2014-03-25

    The identification of the neuronal control of bone remodeling has become one of the many significant recent advances in bone biology. Cholinergic activity has recently been shown to favor bone mass accrual by complex cellular regulatory networks. Here, we identified the gene expression of the muscarinic and nicotinic acetylcholine receptors (m- and nAChRs) in mice tibia tissue and in osteocytic MLO-Y4 cells. Acetylcholine, which is a classical neurotransmitter and an osteo-neuromediator, not only influences the mRNA expression of the AChR subunits but also significantly induces the proliferation and viability of osteocytes. Moreover, acetylcholine treatment caused the reciprocal regulation of RANKL and OPG mRNA expression, which resulted in a significant increase in the mRNA ratio of RANKL:OPG in osteocytes via acetylcholine receptors. The expression of neuropeptide Y and reelin, which are two neurogenic markers, was also modulated by acetylcholine via m- and nAChRs in MLO-Y4 cells. These results indicated that osteocytic acetylcholine receptors might be a new valuable mediator for cell functions and even for bone remodeling. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. Extracellular Vesicles in Luminal Fluid of the Ovine Uterus

    Science.gov (United States)

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

    2014-01-01

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

  5. UNC-41/stonin functions with AP2 to recycle synaptic vesicles in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Gregory P Mullen

    Full Text Available The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2 have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.

  6. The toolbox of vesicle sidedness determination

    NARCIS (Netherlands)

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

    2012-01-01

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

  7. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya (Stanford-MED); (Kyoto); (Gakushuin); (Kyushu)

    2012-03-15

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  8. New insights in the composition of extracellular vesicles from pancreatic cancer cells: implications for biomarkers and functions.

    Science.gov (United States)

    Klein-Scory, Susanne; Tehrani, Mahnaz Moradian; Eilert-Micus, Christina; Adamczyk, Kamila A; Wojtalewicz, Nathalie; Schnölzer, Martina; Hahn, Stephan A; Schmiegel, Wolff; Schwarte-Waldhoff, Irmgard

    2014-01-01

    Pancreatic cancer development is associated with characteristic alterations like desmoplastic reaction and immune escape which are mediated by the cell-cell communication mechanism and by the microenvironment of the cells. The whole of released components are important determinants in these processes. Especially the extracellular vesicles released by pancreatic cancer cells play a role in cell communication and modulate cell growth and immune responses. Here, we present the proteomic description of affinity purified extracellular vesicles from pancreatic tumour cells, compared to the secretome, defined as the whole of the proteins released by pancreatic cancer cells. The proteomic data provide comprehensive catalogues of hundreds of proteins, and the comparison reveals a special proteomic composition of pancreatic cancer cell derived extracellular vesicles. The functional analysis of the protein composition displayed that membrane proteins, glycoproteins, small GTP binding proteins and a further, heterogeneous group of proteins are enriched in vesicles, whereas proteins derived from proteasomes and ribosomes, as well as metabolic enzymes, are not components of the vesicles. Furthermore proteins playing a role in carcinogenesis and modulators of the extracellular matrix (ECM) or cell-cell interactions are components of affinity purified extracellular vesicles. The data deepen the knowledge of extracellular vesicle composition by hundreds of proteins that have not been previously described as vesicle components released by pancreatic cancer cells. Extracellular vesicles derived from pancreatic cancer cells show common proteins shared with other vesicles as well as cell type specific proteins indicating biomarker candidates and suggesting functional roles in cancer cell stroma interactions.

  9. An allosteric enhancer of M(4) muscarinic acetylcholine receptor function inhibits behavioral and neurochemical effects of cocaine

    DEFF Research Database (Denmark)

    Nielsen, Ditte Dencker; Weikop, Pia; Sørensen, Gunnar

    2012-01-01

    The mesostriatal dopamine system plays a key role in mediating the reinforcing effects of psychostimulant drugs like cocaine. The muscarinic M(4) acetylcholine receptor subtype is centrally involved in the regulation of dopamine release in striatal areas. Consequently, striatal M(4) receptors could...... be a novel target for modulating psychostimulant effects of cocaine....

  10. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.P.

    1978-01-01

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

  11. Vesicles and vesicle gels - structure and dynamics of formation

    CERN Document Server

    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 ...

  12. Routes and mechanisms of extracellular vesicle uptake

    Directory of Open Access Journals (Sweden)

    Laura Ann Mulcahy

    2014-08-01

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

  13. Role of extracellular vesicles in autoimmune diseases.

    Science.gov (United States)

    Turpin, Delphine; Truchetet, Marie-Elise; Faustin, Benjamin; Augusto, Jean-François; Contin-Bordes, Cécile; Brisson, Alain; Blanco, Patrick; Duffau, Pierre

    2016-02-01

    Extracellular vesicles (EVs) consist of exosomes released upon fusion of multivesicular bodies with the cell plasma membrane and microparticles shed directly from the cell membrane of many cell types. EVs can mediate cell-cell communication and are involved in many processes including inflammation, immune signaling, angiogenesis, stress response, senescence, proliferation, and cell differentiation. Accumulating evidence reveals that EVs act in the establishment, maintenance and modulation of autoimmune processes among several others involved in cancer and cardiovascular complications. EVs could also present biomedical applications, as disease biomarkers and therapeutic targets or agents for drug delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Biogenesis and function of ESCRT-dependent extracellular vesicles.

    Science.gov (United States)

    Juan, Thomas; Fürthauer, Maximilian

    2018-02-01

    From bacteria to humans, cells secrete a large variety of membrane-bound extracellular vesicles. Only relatively recently has it however started to become clear that the exovesicular transport of proteins and RNAs is important for normal physiology and numerous pathological conditions. Extracellular vesicles can be formed through the release of the intralumenal vesicles of multivesicular endosomes as so-called exosomes, or through direct, ectosomal, budding from the cell surface. Through their ability to promote the bending of membranes away from the cytoplasm, the components of the Endosomal Sorting Complex Required for Transport (ESCRT) have been implicated in both exo- and ectosomal biogenesis. Studies of the ESCRT machinery may therefore provide important insights into the formation and function of extracellular vesicles. In the present review, we first describe the cell biological mechanisms through which ESCRT components contribute to the biogenesis of different types of extracellular vesicles. We then discuss how recent functional studies have started to uncover important roles of ESCRT-dependent extracellular vesicles in a wide variety of processes, including the transport of developmental signaling molecules and embryonic morphogenesis, the regulation of social behavior and host-pathogen interactions, as well as the etiology and progression of neurodegenerative pathologies and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Preeclampsia and Extracellular Vesicles.

    Science.gov (United States)

    Gilani, Sarwat I; Weissgerber, Tracey L; Garovic, Vesna D; Jayachandran, Muthuvel

    2016-09-01

    Preeclampsia is a hypertensive pregnancy disorder characterized by development of hypertension and proteinuria after 20 weeks of gestation that remains a leading cause of maternal and neonatal morbidity and mortality. While preeclampsia is believed to result from complex interactions between maternal and placental factors, the proximate pathophysiology of this syndrome remains elusive. Cell-to-cell communication is a critical signaling mechanism for feto-placental development in normal pregnancies. One mechanism of cellular communication relates to activated cell-derived sealed membrane vesicles called extracellular vesicles (EVs). The concentrations and contents of EVs in biological fluids depend upon their cells of origin and the stimuli which trigger their production. Research on EVs in preeclampsia has focused on EVs derived from the maternal vasculature (endothelium, vascular smooth muscle) and blood (erythrocytes, leukocytes, and platelets), as well as placental syncytiotrophoblasts. Changes in the concentrations and contents of these EVs may contribute to the pathophysiology of preeclampsia by accentuating the pro-inflammatory and pro-coagulatory states of pregnancy. This review focuses on possible interactions among placental- and maternal-derived EVs and their contents in the initiation and progression of the pathogenesis of preeclampsia. Understanding the contributions of EVs in the pathogenesis of preeclampsia may facilitate their use as diagnostic and prognostic biomarkers.

  16. The Role of Extracellular Vesicles: An Epigenetic View of the Cancer Microenvironment.

    Science.gov (United States)

    Qian, Zhongrun; Shen, Qi; Yang, Xi; Qiu, Yongming; Zhang, Wenbin

    2015-01-01

    Exosomes, microvesicles, and other extracellular vesicles are released by many cell types, including cancer cells and cancer-related immune cells. Extracellular vesicles can directly or indirectly facilitate the transfer of bioinformation to recipient cells or to the extracellular environment. In cancer, exosomes have been implicated in tumor initiation, proliferation, and metastasis. Extracellular vesicles can transmit proteins and nucleic acids that participate in DNA methylation, histone modification, and posttranscriptional regulation of RNA. Factors transmitted by extracellular vesicles reflect the donor cell status, and extracellular vesicles derived from tumor cells may be also responsible for altering expression of tumor promoting and tumor suppressing genes in recipient cells. Thus, circulating extracellular vesicles may act as biomarkers of cancer, and detection of these biomarkers may be applied to diagnosis or assessment of prognosis in patients with cancer.

  17. 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.

  18. Dimensional characterization of extracellular vesicles using atomic force microscopy

    NARCIS (Netherlands)

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

    2017-01-01

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

  19. T-2: PRO-COAGULANT POTENTIAL OF PLATELET-DERIVED EXTRACELLULAR VESICLES

    National Research Council Canada - National Science Library

    Tripisciano, C; Weiss, R; Eichhorn, T; Linsberger, I; Fischer, M.B; Weber, V

    2015-01-01

    INTRODUCTION:Extracellular vesicles (EVs) released from blood cells in response to injury or inflammation are broadly classified into exosomes (EX; 30–150 nm) and microvesicles (MV; 100–1000 nm...

  20. Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper

    NARCIS (Netherlands)

    Lener, Thomas; Gimona, Mario; Aigner, Ludwig; Börger, Verena; Buzas, Edit; Camussi, Giovanni; Chaput, Nathalie; Chatterjee, Devasis; Court, Felipe A; Del Portillo, Hernando A; O'Driscoll, Lorraine; Fais, Stefano; Falcon-Perez, Juan M; Felderhoff-Mueser, Ursula; Fraile, Lorenzo; Gho, Yong Song; Görgens, André; Gupta, Ramesh C; Hendrix, An; Hermann, Dirk M; Hill, Andrew F; Hochberg, Fred; Horn, Peter A; de Kleijn, Dominique; Kordelas, Lambros; Kramer, Boris W; Krämer-Albers, Eva-Maria; Laner-Plamberger, Sandra; Laitinen, Saara; Leonardi, Tommaso; Lorenowicz, Magdalena J; Lim, Sai Kiang; Lötvall, Jan; Maguire, Casey A; Marcilla, Antonio; Nazarenko, Irina; Ochiya, Takahiro; Patel, Tushar; Pedersen, Shona; Pocsfalvi, Gabriella; Pluchino, Stefano; Quesenberry, Peter; Reischl, Ilona G; Rivera, Francisco J; Sanzenbacher, Ralf; Schallmoser, Katharina; Slaper-Cortenbach, Ineke; Strunk, Dirk; Tonn, Torsten; Vader, Pieter; van Balkom, Bas W M; Wauben, Marca|info:eu-repo/dai/nl/112675735; Andaloussi, Samir El; Théry, Clotilde; Rohde, Eva; Giebel, Bernd

    2015-01-01

    Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information

  1. 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

  2. [The role of cholesterol in exo- and endocytosis of the synaptic vesicles at the frog motor nerve terminal].

    Science.gov (United States)

    Petrov, A M; kasimov, M R; Giniatullin, A R; Tarakanova, O I; Zefirov, A L

    2009-07-01

    In experiments on the frog neuro-muscular preparations using electrophysiological (two electrode fixing of potential) and optical (fluorescent endocytic dye FM1-43) methods, the value of surface cholestertol for exo-endocytic cycle of synaptic vesicles at the prolonged rhythmic activity (20 Hz--3 minutes) was investigated. It is shown that extraction of cholesterol from surface membranes by methyl-betta-cyclodextrin (1 mM MCD) leads to the expressed shifts in recycling of synaptic vesicles. Exocytosis of vesicles is decreased, and oppression of processes leading to restoration of the number of vesicles of ready releasable pool is observed. Cholesterol replacement from external membranes and membranes of recycling synaptic vesicles in addition to above described effects breaks processes of endocytosis and recycle of synaptic vesicles. Thus, in the processes of exocytosis, the key role is played by cholesterol of plasmatic membranes, and endocytosis critically depends on the amount of cholesterol in the membranes of synaptic vesicles.

  3. Chronic early life lead (Pb2+) exposure alters presynaptic vesicle pools in hippocampal synapses.

    Science.gov (United States)

    Guariglia, Sara Rose; Stansfield, Kirstie H; McGlothan, Jennifer; Guilarte, Tomas R

    2016-11-02

    Lead (Pb2+) exposure has been shown to impair presynaptic neurotransmitter release in both in vivo and in vitro model systems. The mechanism by which Pb2+ impairs neurotransmitter release has not been fully elucidated. In previous work, we have shown that Pb2+ exposure inhibits vesicular release and reduces the number of fast-releasing sites in cultured hippocampal neurons. We have also shown that Pb2+ exposure inhibits vesicular release and alters the distribution of presynaptic vesicles in Shaffer Collateral - CA1 synapses of rodents chronically exposed to Pb2+ during development. In the present study, we used transmission electron microscopy to examine presynaptic vesicle pools in Mossy Fiber-CA3 synapses and in Perforant Path-Dentate Gyrus synapses of rats to determine if in vivo Pb2+ exposure altered presynaptic vesicle distribution in these hippocampal regions. Data were analyzed using T-test for each experimental endpoint. We found that Pb2+ exposure significantly reduced the number of vesicles in the readily releasable pool and recycling pool in Mossy Fiber-CA3 terminals. In both Mossy Fiber-CA3 terminals and in Perforant Path-Dentate Gyrus terminals, Pb2+ exposure significantly increased vesicle nearest neighbor distance in all vesicular pools (Rapidly Releasable, Recycling and Resting). We also found a reduction in the size of the postsynaptic densities of CA3 dendrites in the Pb2+ exposed group. In our previous work, we have demonstrated that Pb2+ exposure impairs vesicular release in Shaffer Collateral - CA1 terminals of the hippocampus and that the number of docked vesicles in the presynaptic active zone was reduced. Our current data shows that Pb2+ exposure reduces the number of vesicles that are in proximity to release sites in Mossy Fiber- CA3 terminals. Furthermore, Pb2+ exposure causes presynaptic vesicles to be further from one another, in both Mossy Fiber- CA3 terminals and in Perforant Pathway - Dentate Gyrus terminals, which may interfere with

  4. 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

  5. Structural Studies of Nicotinic Acetylcholine Receptors

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette

    2016-01-01

    -resolution structure of a nAChR is yet to be determined, structural studies are to a large extent based on acetylcholine binding proteins (AChBPs) that despite low overall sequence identity display high degree of conservation of overall structure and amino acids at the ligand-binding site. Further, AChBPs reproduce...... relative binding affinities of ligands at nAChRs. Over the past decade, AChBPs have been used extensively as models for nAChRs and have aided the understanding of drug receptor interactions at nAChRs significantly. This article is protected by copyright. All rights reserved....

  6. 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.

  7. Extracellular Vesicles in Brain Tumors and Neurodegenerative Diseases

    Science.gov (United States)

    Ciregia, Federica; Urbani, Andrea; Palmisano, Giuseppe

    2017-01-01

    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. PMID:28912682

  8. Quantitative analysis of vesicle recycling at the calyx of Held synapse

    Science.gov (United States)

    Qiu, Xufeng; Zhu, Qianwen; Sun, Jianyuan

    2015-01-01

    Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ∼80% of vesicles (∼270,000 out of ∼330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ∼47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (∼0.6%), the readily priming pool (∼46%), the premature pool (∼33%), and the resting pool (∼20%). PMID:25825725

  9. Enhanced Detection of Cancer Biomarkers in Blood-Borne Extracellular Vesicles Using Nanodroplets and Focused Ultrasound.

    Science.gov (United States)

    Paproski, Robert J; Jovel, Juan; Wong, Gane Ka-Shu; Lewis, John D; Zemp, Roger J

    2017-01-01

    The feasibility of personalized medicine approaches will be greatly improved by the development of noninvasive methods to interrogate tumor biology. Extracellular vesicles shed by solid tumors into the bloodstream have been under recent investigation as a source of tumor-derived biomarkers such as proteins and nucleic acids. We report here an approach using submicrometer perfluorobutane nanodroplets and focused ultrasound to enhance the release of extracellular vesicles from specific locations in tumors into the blood. The released extracellular vesicles were enumerated and characterized using micro flow cytometry. Only in the presence of nanodroplets could ultrasound release appreciable levels of tumor-derived vesicles into the blood. Sonication of HT1080-GFP tumors did not increase the number of circulating tumor cells or the metastatic burden in the tumor-bearing embryos. A variety of biological molecules were successfully detected in tumor-derived extracellular vesicles, including cancer-associated proteins, mRNAs, and miRNAs. Sonication of xenograft HT1080 fibrosarcoma tumors released extracellular vesicles that contained detectable RAC1 mRNA with the highly tumorigenic N92I mutation known to exist in HT1080 cells. Deep sequencing serum samples of embryos with sonicated tumors allowed the identification of an additional 13 known heterozygous mutations in HT1080 cells. Applying ultrasound to HT1080 tumors increased tumor-derived DNA in the serum by two orders of magnitude. This work is the first demonstration of enhanced extracellular vesicle release by ultrasound stimulation and suggests that nanodroplets/ultrasound offers promise for genetic profiling of tumor phenotype and aggressiveness by stimulating the release of extracellular vesicles. Cancer Res; 77(1); 3-13. ©2016 AACR. ©2016 American Association for Cancer Research.

  10. Immunotherapeutic Potential of Extracellular Vesicles

    OpenAIRE

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

    2014-01-01

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

  11. Preparation and Evaluation of Nano-vesicles of Brimonidine Tartrate as an Ocular Drug Delivery System.

    Science.gov (United States)

    Prabhu, P; Nitish, Kumar R; Koland, M; Harish, Nm; Vijayanarayan, K; Dhondge, G; Charyulu, Rn

    2010-10-01

    The objective of the present investigation was to design a vesicular formulation of brimonidine tartrate and evaluate its ability to reduce the dosing frequency and improve the therapeutic efficacy of the drug. Nano-vesicles of brimonidine tartrate were prepared by film hydration method. The prepared vesicles were evaluated for photomicroscopic characteristics, entrapment efficiency, in vitro, and ex-in vitro drug release and in vivo intraocular pressure (IOP) lowering activity. The methods employed for preparation of vesicles produced nano vesicles of acceptable shape and size. The in vitro, and ex-in vitro drug release studies showed that there was slow and prolonged release of the drug, which followed zero-order kinetics. The IOP-lowering activity of nano vesicles was determined and compared with that of pure drug solution and showed that the IOP-lowering action of nano-vesicles sustained for a longer period of time. Stability studies revealed that the vesicle formulations were stable at the temperature range of 2-8°C, with no change in shape and drug content. The results of the study indicate that it is possible to develop a safe and physiologically effective topical formulation that is also convenient for patients.

  12. Acetylcholine facilitates recovery of episodic memory after brain damage.

    Science.gov (United States)

    Croxson, Paula L; Browning, Philip G F; Gaffan, David; Baxter, Mark G

    2012-10-03

    Episodic memory depends on a network of interconnected brain structures including the inferior temporal cortex, hippocampus, fornix, and mammillary bodies. We have previously shown that a moderate episodic memory impairment in monkeys with transection of the fornix is exacerbated by prior depletion of acetylcholine from inferotemporal cortex, despite the fact that depletion of acetylcholine from inferotemporal cortex on its own has no effect on episodic memory. Here we show that this effect occurs because inferotemporal acetylcholine facilitates recovery of function following structural damage within the neural circuit for episodic memory. Episodic memory impairment caused by lesions of the mammillary bodies, like fornix transection, was exacerbated by prior removal of temporal cortical acetylcholine. However, removing temporal cortical acetylcholine after the lesion of the fornix or mammillary bodies did not increase the severity of the impairment. This lesion order effect suggests that acetylcholine within the inferior temporal cortex ordinarily facilitates functional recovery after structural lesions that impair episodic memory. In the absence of acetylcholine innervation to inferotemporal cortex, this recovery is impaired and the amnesia caused by the structural lesion is more severe. These results suggest that humans with loss of cortical acetylcholine function, for example in Alzheimer's disease, may be less able to adapt to memory impairments caused by structural neuronal damage to areas in the network important for episodic memory.

  13. Models of Acetylcholine and Dopamine Signals Differentially Improve Neural Representations

    Science.gov (United States)

    Holca-Lamarre, Raphaël; Lücke, Jörg; Obermayer, Klaus

    2017-01-01

    Biological and artificial neural networks (ANNs) represent input signals as patterns of neural activity. In biology, neuromodulators can trigger important reorganizations of these neural representations. For instance, pairing a stimulus with the release of either acetylcholine (ACh) or dopamine (DA) evokes long lasting increases in the responses of neurons to the paired stimulus. The functional roles of ACh and DA in rearranging representations remain largely unknown. Here, we address this question using a Hebbian-learning neural network model. Our aim is both to gain a functional understanding of ACh and DA transmission in shaping biological representations and to explore neuromodulator-inspired learning rules for ANNs. We model the effects of ACh and DA on synaptic plasticity and confirm that stimuli coinciding with greater neuromodulator activation are over represented in the network. We then simulate the physiological release schedules of ACh and DA. We measure the impact of neuromodulator release on the network's representation and on its performance on a classification task. We find that ACh and DA trigger distinct changes in neural representations that both improve performance. The putative ACh signal redistributes neural preferences so that more neurons encode stimulus classes that are challenging for the network. The putative DA signal adapts synaptic weights so that they better match the classes of the task at hand. Our model thus offers a functional explanation for the effects of ACh and DA on cortical representations. Additionally, our learning algorithm yields performances comparable to those of state-of-the-art optimisation methods in multi-layer perceptrons while requiring weaker supervision signals and interacting with synaptically-local weight updates. PMID:28690509

  14. Expression and function of nicotinic acetylcholine receptors in stem cells

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  15. Extracellular vesicles in obesity and diabetes mellitus.

    Science.gov (United States)

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

    2017-11-24

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

  16. Dopamine neurons release transmitter via a flickering fusion pore.

    Science.gov (United States)

    Staal, Roland G W; Mosharov, Eugene V; Sulzer, David

    2004-04-01

    A key question in understanding mechanisms of neurotransmitter release is whether the fusion pore of a synaptic vesicle regulates the amount of transmitter released during exocytosis. We measured dopamine release from small synaptic vesicles of rat cultured ventral midbrain neurons using carbon fiber amperometry. Our data indicate that small synaptic vesicle fusion pores flicker either once or multiple times in rapid succession, with each flicker releasing approximately 25-30% of vesicular dopamine. The incidence of events with multiple flickers was reciprocally regulated by phorbol esters and staurosporine. Thus, dopamine neurons regulate the amount of neurotransmitter released by small synaptic vesicles by controlling the number of fusion pore flickers per exocytotic event. This mode of exocytosis is a potential mechanism whereby neurons can rapidly reuse vesicles without undergoing the comparatively slow process of recycling.

  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. The origin, function, and diagnostic potential of RNA within extracellular vesicles present in human biological fluids

    Science.gov (United States)

    Taylor, Douglas D.; Gercel-Taylor, Cicek

    2013-01-01

    We have previously demonstrated that tumor cells release membranous structures into their extracellular environment, which are termed exosomes, microvesicles or extracellular vesicles depending on specific characteristics, including size, composition and biogenesis pathway. These cell-derived vesicles can exhibit an array of proteins, lipids and nucleic acids derived from the originating tumor. This review focuses of the transcriptome (RNA) of these extracellular vesicles. Based on current data, these vesicular components play essential roles as conveyers of intercellular communication and mediators of many of the pathological conditions associated with cancer development, progression and therapeutic failures. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, signal pathway activation through growth factor/receptor transfer, chemoresistance, and genetic exchange. These tumor-derived extracellular vesicles not only to represent a central mediator of the tumor microenvironment, but their presence in the peripheral circulation may serve as a surrogate for tumor biopsies, enabling real-time diagnosis and disease monitoring. PMID:23908664

  19. Extracellular Vesicles and Their Convergence with Viral Pathways

    Directory of Open Access Journals (Sweden)

    Thomas Wurdinger

    2012-01-01

    Full Text Available Extracellular vesicles (microvesicles, such as exosomes and shed microvesicles, contain a variety of molecules including proteins, lipids, and nucleic acids. Microvesicles appear mostly to originate from multivesicular bodies or to bud from the plasma membrane. Here, we review the convergence of microvesicle biogenesis and aspects of viral assembly and release pathways. Herpesviruses and retroviruses, amongst others, recruit several elements from the microvesicle biogenesis pathways for functional virus release. In addition, noninfectious pleiotropic virus-like vesicles can be released, containing viral and cellular components. We highlight the heterogeneity of microvesicle function during viral infection, addressing microvesicles that can either block or enhance infection, or cause immune dysregulation through bystander action in the immune system. Finally, endogenous retrovirus and retrotransposon elements deposited in our genomes millions of years ago can be released from cells within microvesicles, suggestive of a viral origin of the microvesicle system or perhaps of an evolutionary conserved system of virus-vesicle codependence. More research is needed to further elucidate the complex function of the various microvesicles produced during viral infection, possibly revealing new therapeutic intervention strategies.

  20. Dysregulations of Synaptic Vesicle Trafficking in Schizophrenia.

    Science.gov (United States)

    Egbujo, Chijioke N; Sinclair, Duncan; Hahn, Chang-Gyu

    2016-08-01

    Schizophrenia is a serious psychiatric illness which is experienced by about 1 % of individuals worldwide and has a debilitating impact on perception, cognition, and social function. Over the years, several models/hypotheses have been developed which link schizophrenia to dysregulations of the dopamine, glutamate, and serotonin receptor pathways. An important segment of these pathways that have been extensively studied for the pathophysiology of schizophrenia is the presynaptic neurotransmitter release mechanism. This set of molecular events is an evolutionarily well-conserved process that involves vesicle recruitment, docking, membrane fusion, and recycling, leading to efficient neurotransmitter delivery at the synapse. Accumulated evidence indicate dysregulation of this mechanism impacting postsynaptic signal transduction via different neurotransmitters in key brain regions implicated in schizophrenia. In recent years, after ground-breaking work that elucidated the operations of this mechanism, research efforts have focused on the alterations in the messenger RNA (mRNA) and protein expression of presynaptic neurotransmitter release molecules in schizophrenia and other neuropsychiatric conditions. In this review article, we present recent evidence from schizophrenia human postmortem studies that key proteins involved in the presynaptic release mechanism are dysregulated in the disorder. We also discuss the potential impact of dysfunctional presynaptic neurotransmitter release on the various neurotransmitter systems implicated in schizophrenia.

  1. Bioinformatics Tools for Extracellular Vesicles Research.

    Science.gov (United States)

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

    2017-01-01

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

  2. The α7 nicotinic acetylcholine receptor complex

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2012-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of diseases ranging from schizophrenia and Alzheimer's disease to chronic pain and inflammatory diseases. Focusing on the central nervous system, we describe how endogenous and experimental compounds...... in diseases such as schizophrenia and Alzheimer's disease. Furthermore, α7 nAChR agonists and allosteric modulators differentially alter expression and functionality of the α7 nAChR with repeated administration, which suggests that there may be fundamentally different outcomes of long-term administration...... with these different types of compounds. Finally, we describe the special case of Aβ1-42 binding to the α7 nAChR, which may pose a unique challenge to drug development of α7 nAChR-specific ligands for Alzheimer's disease. Hopefully, a greater knowledge of the many factors influencing α7 nAChR function as well...

  3. Structural model of nicotinic acetylcholine receptor isotypes bound to acetylcholine and nicotine

    Directory of Open Access Journals (Sweden)

    Abagyan Ruben

    2002-01-01

    Full Text Available Abstract Background Nicotine is a psychoactive drug presenting a diverse array of biological activities, some positive, such as enhancement of cognitive performances, others negative, such as addiction liability. Ligands that discriminate between the different isotypes of nicotinic acetylcholine receptors (nAChRs could present improved pharmacology and toxicity profile. Results Based on the recent crystal structure of a soluble acetylcholine binding protein from snails, we have built atomic models of acetylcholine and nicotine bound to the pocket of four different human nAChR subtypes. The structures of the docked ligands correlate with available biochemical data, and reveal that the determinants for isotype selectivity are relying essentially on four residues, providing diversity of the ligand binding pocket both in terms of Van der Waals boundary, and electrostatic potential. We used our models to screen in silico a large compound database and identify a new ligand candidate that could display subtype selectivity. Conclusion The nAChR-agonist models should be useful for the design of nAChR agonists with diverse specificity profiles.

  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

    Protein Interacting with C Kinase 1 (PICK1) is a Bin/Amphiphysin/Rvs (BAR) domain protein involved in AMPA receptor trafficking. Here, we identify a selective role for PICK1 in the biogenesis of large, dense core vesicles (LDCVs) in mouse chromaffin cells. PICK1 colocalized with syntaxin-6......, 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.......e., reduced quantal size). This was paralleled by a reduction in the mean single-vesicle capacitance, estimated by averaging time-locked capacitance traces. EM confirmed that LDCVs were fewer and of markedly reduced size in the PICK1 KO, demonstrating that all phenotypes can be explained by reductions...

  5. Understanding the biosynthesis of platelets-derived extracellular vesicles.

    Science.gov (United States)

    Antwi-Baffour, Samuel; Adjei, Jonathan; Aryeh, Claudia; Kyeremeh, Ransford; Kyei, Foster; Seidu, Mahmood A

    2015-09-01

    Platelet-derived extracellular vesicles (PEVs) are described as sub-cellular vesicles released into circulation upon platelets shear stress, activation, injury, or apoptosis. They are considered as universal biomarkers in a wide range of physiological and pathological processes. They are of tremendous significance for the prediction, diagnosis, and observation of the therapeutic success of many diseases. Understanding their biosynthesis and therefore functional properties would contribute to a better understanding of the pathological mechanisms leading to various diseases in which their levels are raised and they are implicated. The review takes a critical look at the historical background of PEVs, their structural components, the mechanism of their formation, physiological, and exogenous stimuli inducing their release and their detection. It concludes by highlighting on the importance of undertaking in-depth studies into PEVs biosynthesis and subsequently gaining a better understanding of their biological role in general.

  6. 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 complex and inhibiting membrane fusion. However, several other studies also suggest that CPXs are positive regulators of neurotransmitter release. Thus, whether CPXs are positive or negative regulators of exocytosis is not known, much less the stage in the vesicle life cycle at which they function. Here......, we systematically dissect the vesicle stages leading up to exocytosis using a knockout-rescue strategy in a mammalian model system. We show that adrenal chromaffin cells from CPX II knockout mice exhibit markedly diminished releasable vesicle pools (comprising the readily and slowly releasable pools...

  7. 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.

  8. Cystadenoma of the seminal vesicle

    Directory of Open Access Journals (Sweden)

    Gil Antônio O.

    2003-01-01

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

  9. Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering

    DEFF Research Database (Denmark)

    Mohrmann, Ralf; de Wit, Heidi; Connell, Emma

    2013-01-01

    that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle...... ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused...

  10. 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.

  11. When to biopsy seminal vesicles.

    Science.gov (United States)

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

    2015-05-01

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

  12. Plasma-derived extracellular vesicles contain predictive biomarkers and potential therapeutic targets for Myocardial Ischemic (MI) injury

    NARCIS (Netherlands)

    Cheow, Esther Sok Hwee; Cheng, Woo Chin; Lee, Chuen Neng; De Kleijn, Dominique; Sorokin, Vitaly; Sze, Siu Kwan

    2016-01-01

    Myocardial infarction (MI) triggers a potent inflammatory response via the release of circulatory mediators, including extracellular vesicles (EVs) by damaged cardiac cells, necessary for myocardial healing. Timely repression of inflammatory response are critical to prevent and minimize cardiac

  13. Vesicles and vesicle fusion: coarse-grained simulations

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2010-01-01

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

  14. Extracellular vesicles in physiological and pathological conditions

    NARCIS (Netherlands)

    Yuana, Yuana; Sturk, Auguste; Nieuwland, Rienk

    2013-01-01

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

  15. 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 ...

  16. Extracellular vesicles during Herpes Simplex Virus type 1 infection: an inquire.

    Science.gov (United States)

    Kalamvoki, Maria; Deschamps, Thibaut

    2016-04-05

    Extracellular vesicles are defined as a heterogeneous group of vesicles that are released by prokaryotic to higher eukaryotic cells and by plant cells in an evolutionary conserved manner. The significance of these vesicles lies in their capacity to transfer selected cargo composed of proteins, lipids and nucleic acids to both recipient and parent cells and to influence various physiological and pathological functions. Microorganisms such as parasites, fungi and protozoa and even single cell organisms such as bacteria generate extracellular vesicles. In addition, several viruses have evolved strategies to hijack the extracellular vesicles for egress or to alter the surrounding environment. The thesis of this article is that: a) during HSV-1 infection vesicles are delivered from infected to uninfected cells that influence the infection; b) the cargo of these vesicles consists of viral and host transcripts (mRNAs, miRNAs and non-coding RNAs) and proteins including innate immune components, such as STING; and c) the viral vesicles carry the tetraspanins CD9, CD63 and CD81, which are considered as markers of exosomes. Therefore, we assume that the STING-carrying vesicles, produced during HSV-1 infection, are reminiscent to exosomes. The presumed functions of the exosomes released from HSV-1 infected cells include priming the recipient cells and accelerating antiviral responses to control the dissemination of the virus. This may be one strategy used by the virus to prevent the elimination by the host and establish persistent infection. In conclusion, the modification of the cargo of exosomes appears to be part of the strategy that HSV-1 has evolved to establish lifelong persistent infections into the human body to ensure successful dissemination between individuals.

  17. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration.

    Science.gov (United States)

    Ma, Liang; Li, Ying; Peng, Junya; Wu, Danni; Zhao, Xiaoxin; Cui, Yitong; Chen, Lilian; Yan, Xiaojun; Du, Yanan; Yu, Li

    2015-01-01

    Cells communicate with each other through secreting and releasing proteins and vesicles. Many cells can migrate. In this study, we report the discovery of migracytosis, a cell migration-dependent mechanism for releasing cellular contents, and migrasomes, the vesicular structures that mediate migracytosis. As migrating cells move, they leave long tubular strands, called retraction fibers, behind them. Large vesicles, which contain numerous smaller vesicles, grow on the tips and intersections of retraction fibers. These fibers, which connect the vesicles with the main cell body, eventually break, and the vesicles are released into the extracellular space or directly taken up by surrounding cells. Since the formation of these vesicles is migration-dependent, we named them "migrasomes". We also found that cytosolic contents can be transported into migrasomes and released from the cell through migrasomes. We named this migration-dependent release mechanism "migracytosis".

  18. The role of extracellular vesicles in neurodegenerative diseases.

    Science.gov (United States)

    Quek, Camelia; Hill, Andrew F

    2017-02-19

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

  19. The interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in mouse corpus cavernosum.

    Science.gov (United States)

    Aydinoglu, Fatma; Dalkir, Fatma Tugce; Demirbag, Hatice Oruc; Ogulener, Nuran

    2017-11-01

    The aim of this study was to investigate the possible interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in the mouse corpus cavernosum (CC). l-cysteine (endogenous H2S substrate; 10-6-10-3 M), sodium hydrogen sulfide (NaHS; exogenous H2S; 10-6-10-3 M) and acetylcholine (10-9-10-4 M) produced concentration-dependent relaxation in isolated mouse CC tissues. Relaxations to endogenous and exogenous H2S were reduced by non-selective mAChR antagonist atropine (5 × 10-5 M), selective M1 mAChR antagonist pirenzepine (5 × 10-5 M) and selective M3 mAChR antagonist 4-DAMP (10-7 M) but not by selective M2 mAChR antagonist AF-DX 116 (10-6 M). Also, acetylcholine-induced relaxations were reduced by atropine, pirenzepine, 4-DAMP and AF-DX 116, confirming the selective effects of mAChR antagonists. Furthermore, acetylcholine-induced relaxations were attenuated by cystathionine-gamma-lyase (CSE) inhibitor d,l-propargylglycine (PAG, 10-2 M) and cystathionine-β-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10-3 M). l-nitroarginine, nitric oxide synthase inhibitor, augmented the inhibitory effects of mAChR antagonists and H2S enzyme inhibitors on acetylcholine-induced relaxations. In addition, the existence and localization of CSE, CBS and 3-MST were demonstrated in mouse CC. Furthermore, tissue acetylcholine release was significantly increased by l-cysteine but not by exogenous H2S. The increase in acetylcholine level was completely inhibited by AOAA and PAG. These results suggest that M1 and M3 mAChRs contributes to relaxant effect mediated by endogenous H2S but at same time l-cysteine triggers acetylcholine release from cavernosal tissue. Also, the role of NO in the interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) could not be excluded. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Purification of a vesicle-vacuole fraction functionally linked to aflatoxin synthesis in Aspergillus parasiticus.

    Science.gov (United States)

    Chanda, Anindya; Roze, Ludmila V; Pastor, Alicia; Frame, Melinda K; Linz, John E

    2009-07-01

    Current studies in our laboratory demonstrate a functional link between vesicles, vacuoles and aflatoxin biosynthesis in the filamentous fungus, Aspergillus parasiticus. Under aflatoxin inducing conditions in liquid yeast-extract sucrose medium, A. parasiticus undergoes a shift from vacuole biogenesis to accumulation of an enhanced number of vesicles which exhibit significant heterogeneity in size and density. As a first step in conducting a detailed analysis of the role of these organelles in aflatoxin synthesis, we developed a novel method to purify the vesicle and vacuole fraction using protoplasts prepared from cells harvested during aflatoxin synthesis. The method includes the following steps: 1] preparation of protoplasts from mycelia grown for 36 h under aflatoxin inducing conditions; 2] release of vesicles and vacuoles from purified protoplasts in the presence of Triton X-100; and 3] fractionation of the vesicles and vacuoles using a "one-step high density cushion". The vesicle-vacuole fraction showed a 35 fold enrichment in alpha-mannosidase activity (vacuole marker) and non-detectable succinate dehydrogenase and lactate dehydrogenase activities (mitochondrial and cytoplasmic markers, respectively). Confocal laser scanning microscopy with the vacuole dyes MDY-64 and CMAC demonstrated that the fraction contained pure vesicles and vacuoles and was devoid of membranous debris. Transmission electron microscopy (TEM) confirmed that no mitochondria or unbroken protoplasts contaminated the purified fraction. The purified organelles exhibited significant size heterogeneity with a range of sizes similar to that observed in whole cells and protoplasts.

  1. Extracellular vesicles: structure, function, and potential clinical uses in renal diseases

    Directory of Open Access Journals (Sweden)

    F.T. Borges

    2013-10-01

    Full Text Available Interest in the role of extracellular vesicles in various diseases including cancer has been increasing. Extracellular vesicles include microvesicles, exosomes, apoptotic bodies, and argosomes, and are classified by size, content, synthesis, and function. Currently, the best characterized are exosomes and microvesicles. Exosomes are small vesicles (40-100 nm involved in intercellular communication regardless of the distance between them. They are found in various biological fluids such as plasma, serum, and breast milk, and are formed from multivesicular bodies through the inward budding of the endosome membrane. Microvesicles are 100-1000 nm vesicles released from the cell by the outward budding of the plasma membrane. The therapeutic potential of extracellular vesicles is very broad, with applications including a route of drug delivery and as biomarkers for diagnosis. Extracellular vesicles extracted from stem cells may be used for treatment of many diseases including kidney diseases. This review highlights mechanisms of synthesis and function, and the potential uses of well-characterized extracellular vesicles, mainly exosomes, with a special focus on renal functions and diseases.

  2. CD47-dependent immunomodulatory and angiogenic activities of extracellular vesicles produced by T cells.

    Science.gov (United States)

    Kaur, Sukhbir; Singh, Satya P; Elkahloun, Abdel G; Wu, Weiwei; Abu-Asab, Mones S; Roberts, David D

    2014-07-01

    Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell-cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Extracellular Vesicles and Autophagy in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Tianyang Gao

    2016-01-01

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

  4. Extracellular Vesicles and Autophagy in Osteoarthritis

    Science.gov (United States)

    Guo, Weimin; Chen, Mingxue; Huang, Jingxiang; Yuan, Zhiguo; Zhang, Yu; Wang, Mingjie; Li, Penghao; Wang, Aiyuan; Wang, Yu; Sui, Xiang; Zhang, Li; Xu, Wenjing; Lu, Shibi

    2016-01-01

    Osteoarthritis (OA) is a type of chronic joint disease that is characterized by the degeneration and loss of articular cartilage and hyperplasia of the synovium and subchondral bone. There is reasonable knowledge about articular cartilage physiology, biochemistry, and chondrocyte metabolism. However, the etiology and pathogenesis of OA remain unclear and need urgent clarification to guide the early diagnosis and treatment of OA. Extracellular vesicles (EVs) are small membrane-linking particles that are released from cells. In recent decades, several special biological properties have been found in EV, especially in terms of cartilage. Autophagy plays a critical role in the regulation of cellular homeostasis. Likewise, more and more research has gradually focused on the effect of autophagy on chondrocyte proliferation and function in OA. The synthesis and release of EV are closely associated with autophagy. At the same time, both EV and autophagy play a role in OA development. Based on the mechanism of EV and autophagy in OA development, EV may be beneficial in the early diagnosis of OA; on the other hand, the combination of EV and autophagy-related regulatory drugs may provide insight into possible OA therapeutic strategies. PMID:28078284

  5. In vitro toxicology studies of extracellular vesicles.

    Science.gov (United States)

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

    2017-03-01

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

  6. Differential effects of caffeine on dopamine and acetylcholine transmission in brain areas of drug-naive and caffeine-pretreated rats.

    Science.gov (United States)

    Acquas, Elio; Tanda, Gianluigi; Di Chiara, Gaetano

    2002-08-01

    The effects of caffeine on extracellular dopamine and acetylcholine have been studied in freely moving rats implanted with concentric microdialysis probes in the nucleus accumbens shell and core and in the medial prefrontal cortex. Intravenous administration of caffeine (0.25, 0.5, 1.0, 2.5 and 5.0 mg/kg) dose-dependently increased dopamine and acetylcholine dialysate concentrations in the medial prefrontal cortex, while it did not affect dialysate dopamine in the shell and core of the nucleus accumbens. Intraperitoneal administration of caffeine (1.5, 3, 10, 30 mg/kg) also failed to affect DA in the shell and core of the nucleus accumbens. Such effects were duplicated by intravenous administration of DPCPX, a selective antagonist of adenosine A1 receptors, and of SCH 58261, an antagonist of A2a receptors. The effect of caffeine on prefrontal dopamine and acetylcholine transmission was also studied in rats chronically administered with caffeine (25 mg/kg, twice a day for seven days). At the end of this treatment rats became tolerant to the locomotor stimulating effects of a dose of 1 and 2.5 mg/kg i.v. of caffeine; these doses, however, still increased dialysate acetylcholine but did not affect dopamine in the prefrontal cortex. Therefore, in rats made tolerant to the locomotor stimulant effects of caffeine, tolerance developed to the dopamine stimulant but not to the acetylcholine stimulant effect of caffeine in the prefrontal cortex. The lack of acute stimulation of dopamine release in the nucleus accumbens shell by caffeine is relevant to the issue of its addictive properties and of the role of DA in drug- and substance-addiction. On the other hand, the dissociation between tolerance to the locomotor effects of caffeine and stimulation of acetylcholine release in the prefrontal cortex suggests that this effect might be correlated to the arousing effects of caffeine as distinct from its locomotor stimulant properties.

  7. Amyloid precursor protein knockout diminishes synaptic vesicle proteins at the presynaptic active zone in mouse brain.

    Science.gov (United States)

    Laßek, Melanie; Weingarten, Jens; Acker-Palmer, Amparo; Bajjalieh, Sandra M; Muller, Ulrike; Volknandt, Walter

    2014-01-01

    The amyloid precursor protein (APP) has previously been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a presynaptic active zone-localized pool. By analyzing homozygous APP knockout mice we evaluated the impact of APP on synaptic vesicle protein abundance at synaptic release sites. Following immunopurification of synaptic vesicles and the attached presynaptic plasma membrane, individual proteins were subjected to quantitative Western blot analysis. We demonstrate that APP deletion in knockout animals reduces the abundance of the synaptic vesicle proteins synaptophysin, synaptotagmin-1, and SV2A at the presynaptic active zone. Conversely, deletion of the additional APP family members, APLP1 and APLP2 resulted in an increase in synaptophysin, synaptogamin-1, and SV2A abundance. When transmembrane APP is lacking in APPsα-KI/APLP2-KO mice synaptic vesicle protein abundance corresponds to that in APP -KO mice. Deletion of the synaptic vesicle protein 2 (SV2) A and B had no effect on APP and synaptophysin abundance but decreased synaptotagmin-1. Our data suggest that APP controls the abundance of synaptic vesicle proteins at the presynaptic release sites and thus impacts synaptic transmission.

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

    Directory of Open Access Journals (Sweden)

    Manuela eBasso

    2016-03-01

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

  9. DSCR1/RCAN1 regulates vesicle exocytosis and fusion pore kinetics: implications for Down syndrome and Alzheimer's disease.

    Science.gov (United States)

    Keating, Damien J; Dubach, Daphne; Zanin, Mark P; Yu, Yong; Martin, Katherine; Zhao, Yu-Feng; Chen, Chen; Porta, Sílvia; Arbonés, Maria L; Mittaz, Laureane; Pritchard, Melanie A

    2008-04-01

    Genes located on chromosome 21, over-expressed in Down syndrome (DS) and Alzheimer's disease (AD) and which regulate vesicle trafficking, are strong candidates for involvement in AD neuropathology. Regulator of calcineurin activity 1 (RCAN1) is one such gene. We have generated mutant mice in which RCAN1 is either over-expressed (RCAN1(ox)) or ablated (Rcan1-/-) and examined whether exocytosis from chromaffin cells, a classic cellular model of neuronal exocytosis, is altered using carbon fibre amperometry. We find that Rcan1 regulates the number of vesicles undergoing exocytosis and the speed at which the vesicle fusion pore opens and closes. Cells from both Rcan1-/- and RCAN1(ox) mice display reduced levels of exocytosis. Changes in single-vesicle fusion kinetics are also evident resulting in the less catecholamine released per vesicle with increasing Rcan1 expression. Acute calcineurin inhibition did not replicate the effect of RCAN1 overexpression. These changes are not due to alterations in Ca2+ entry or the readily releasable vesicle pool size. Thus, we illustrate a novel regulator of vesicle exocytosis, Rcan1, which influences both exocytotic rate and vesicle fusion kinetics. If Rcan1 functions similarly in neurons then overexpression of this protein, as occurs in DS and AD brains, will reduce both the number of synaptic vesicles undergoing exocytosis and the amount of neurotransmitter released per fusion event. This has direct implications for the pathogenesis of these diseases as sufficient levels of neurotransmission are required for synaptic maintenance and the prevention of neurodegeneration and vesicle trafficking defects are the earliest hallmark of AD neuropathology.

  10. Vesicle self-assembly by tetrathiafulvalene derivatives in both polar and nonpolar solvents and pseudo-rotaxane mediated vesicle-to-microtube transformation.

    Science.gov (United States)

    Zhang, Kang-Da; Wang, Gui-Tao; Zhao, Xin; Jiang, Xi-Kui; Li, Zhan-Ting

    2010-05-18

    This paper reports the self-assemblies of vesicles from two tetrathiafulvalene (TTF) derivatives (T1 and T2), that bear four or two amphiphilic side chains, in both polar and nonpolar solvents. The formation of vesicles is evidenced by scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) experiments, while the microstructural aspects of the vesicles are investigated by UV-vis, (1)H NMR, and high resolution TEM, which support a monolayer model for the vesicles. It is revealed that the formation of vesicles is driven by the combination of multiple noncovalent interactions, including pi-pi stacking, hydrogen-bonding, van der Waals force, and S...S interactions. It is also found that, in the presence of electron-deficient cyclobis(paraquat-p-phenylene) tetracation cyclophane, vesicles of T2 can transform into microtubes as a result of the formation of the pseudo[2]rotaxane between the TTF unit of T2 and the cyclophane. This process can be reversed by introducing pristine TTF into the solution of microtubes, due to release of T2 from the pseudo[2]rotaxane through the formation of a more stable complex between pristine TTF and tetracation cyclophane.

  11. 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.

  12. Dynamics of endocytic vesicle creation.

    Science.gov (United States)

    Perrais, David; Merrifield, Christien J

    2005-11-01

    Clathrin-mediated endocytosis is the main path for receptor internalization in metazoans and is essential for controlling cell integrity and signaling. It is driven by a large array of protein and lipid interactions that have been deciphered mainly by biochemical and genetic means. To place these interactions into context, and ultimately build a fully operative model of endocytosis at the molecular level, it is necessary to know the kinetic details of the role of each protein in this process. In this review, we describe the recent efforts made, by using live cell imaging, to define clear steps in the formation of endocytic vesicles and to observe the recruitment of key proteins during membrane invagination, the scission of a newly formed vesicle, and its movement away from the plasma membrane.

  13. 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.

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

    Science.gov (United States)

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

    2016-02-06

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

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

    Directory of Open Access Journals (Sweden)

    Andrew I. Yao

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jordi Rovira

    2017-03-01

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

  17. Extracellular Vesicles as Therapeutic Agents in Systemic Lupus Erythematosus.

    Science.gov (United States)

    Perez-Hernandez, Javier; Redon, Josep; Cortes, Raquel

    2017-03-28

    Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease that affects multiple organs. Currently, therapeutic molecules present adverse side effects and are only effective in some SLE patient subgroups. Extracellular vesicles (EV), including exosomes, microvesicles and apoptotic bodies, are released by most cell types, carry nucleic acids, proteins and lipids and play a crucial role in cell-to-cell communication. EVs can stimulate or suppress the immune responses depending on the context. In SLE, EVs can work as autoadjuvants, enhance immune complex formation and maintaining inflammation state. Over the last years, EVs derived from mesenchymal stem cells and antigen presenting cells have emerged as cell-free therapeutic agents to treat autoimmune and inflammatory diseases. In this review, we summarize the current therapeutic applications of extracellular vesicles to regulate immune responses and to ameliorate disease activity in SLE and other autoimmune disorders.

  18. Production and Characterization of Extracellular Vesicles in Malaria.

    Science.gov (United States)

    Mbagwu, Smart; Walch, Michael; Filgueira, Luis; Mantel, Pierre-Yves

    2017-01-01

    Growing attention is drawn toward the role of extracellular vesicles (EVs) in infectious diseases. EVs, which are small vesicles released by cells, are involved in cellular communication, immune regulation, and pathogenesis. EVs act as messenger carrying functional cargoes, including RNA, DNA, lipids and proteins from a donor cell to regulate the function of a recipient cell. In malaria, EVs play a key role in regulating the progression from the blood to the transmission stage by promoting the switch between asexual and sexual stages that are taken up by mosquitoes. In addition to their role in parasite communication, EVs modulate the immune system and regulate endothelial cell function.In this chapter, we describe protocols to isolate, purify and characterize EVs derived from Plasmodium falciparum infected red blood cell culture.

  19. Immunotherapeutic potential of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Bin eZhang

    2014-10-01

    Full Text Available Extracellular vesicles or EVs is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized endosome-derived vesicles secreted by many cell types and their immunomodulatory potential is independent of their cell source. Besides immune cells such as dendritic cells, macrophages and T cells, cancer and stem cells also secrete immunologically active exosomes that could influence both physiological and pathological processes. The immunological activities of exosomes affect both innate and adaptive immunity and include antigen presentation, T cell activation, T cell polarisation to Tregs, immune suppression and anti-inflammation. As such, exosomes carry much immunotherapeutic potential as a therapeutic agent and a therapeutic target.

  20. Acetylcholine Attenuates Hypoxia/ Reoxygenation-Induced Mitochondrial and Cytosolic ROS Formation in H9c2 Cells via M2 Acetylcholine Receptor

    Directory of Open Access Journals (Sweden)

    Yi Miao

    2013-02-01

    Full Text Available Background: The anti-infammatory and cardioprotective effect of acetylcholine (ACh has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R-induced oxidative stress remains obscure. Methods: In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS by measuring mitochondrial ROS (mtROS, mitochondrial DNA (mtDNA copy number, xanthine oxidase (XO and NADPH oxidase (NOX activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR expression. Results: 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group. Conclusions: ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.

  1. Endocannabinoids mediate muscarinic acetylcholine receptor-dependent long-term depression in the adult medial prefrontal cortex

    Directory of Open Access Journals (Sweden)

    Henry Giles Stratten Martin

    2015-12-01

    Full Text Available Cholinergic inputs into the prefrontal cortex (PFC are associated with attention and cognition; however there is evidence that acetylcholine also has a role in PFC dependent learning and memory. Muscarinic acetylcholine receptors (mAChR in the PFC can induce synaptic plasticity, but the underlying mechanisms remain either opaque or unresolved. We have characterized a form of mAChR mediated long-term depression (LTD at glutamatergic synapses of layer 5 principal neurons in the adult medial PFC. This mAChR LTD is induced with the mAChR agonist carbachol and inhibited by selective M1 mAChR antagonists. In contrast to other cortical regions, we find that this M1 mAChR mediated LTD is coupled to endogenous cannabinoid (eCB signaling. Inhibition of the principal eCB CB1 receptor blocked carbachol induced LTD in both rats and mice. Furthermore, when challenged with a sub-threshold carbachol application, LTD was induced in slices pretreated with the monoacylglycerol lipase inhibitor JZL184, suggesting that the eCB 2-arachidonylglyerol (2-AG mediates M1 mAChR LTD. Yet, when endogenous acetylcholine was released from local cholinergic afferents in the PFC using optogenetics, it failed to trigger eCB-LTD. However coupling patterned optical and electrical stimulation to generate local synaptic signaling allowed the reliable induction of LTD. The light – electrical pairing induced LTD was M1 mAChR and CB1 receptor mediated. This shows for the first time that connecting excitatory synaptic activity with coincident endogenously released acetylcholine controls synaptic gain via eCB signaling. Together these results shed new light on the mechanisms of synaptic plasticity in the adult PFC and expand on the actions of endogenous cholinergic signaling.

  2. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting

    Directory of Open Access Journals (Sweden)

    Oscar P. B. Wiklander

    2015-04-01

    Full Text Available Extracellular vesicles (EVs have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs.

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

    Science.gov (United States)

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

    2017-12-01

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

  4. Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions.

    NARCIS (Netherlands)

    Nolte-'t Hoen, E.N.M.|info:eu-repo/dai/nl/261632175; Buermans, H.P.; Waasdorp, M.; Stoorvogel, W.|info:eu-repo/dai/nl/074352385; Wauben, M.H.M.|info:eu-repo/dai/nl/112675735; `t Hoen, P.A.C.

    2012-01-01

    Cells release RNA-carrying vesicles and membrane-free RNA/protein complexes into the extracellular milieu. Horizontal vesicle-mediated transfer of such shuttle RNA between cells allows dissemination of genetically encoded messages, which may modify the function of target cells. Other studies used

  5. 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

  6. Extracellular Vesicles: Evolving Contributors in Autoimmunity

    OpenAIRE

    Katsiougiannis, Stergios

    2015-01-01

    Extracellular vesicles, including microvesicles, exosomes and apoptotic bodies are recognized as carriers of pathogen-associated molecules with direct involvement in immune signaling and inflammation. Those observations have enforced the way these membranous vesicles are being considered as promising immunotherapeutic targets. In this review, we discuss the emerging roles of extracellular vesicles in autoimmunity and highlights their potential use as disease biomarkers as well as targets for ...

  7. Exosomes: secreted vesicles and intercellular communications

    OpenAIRE

    Théry, Clotilde

    2011-01-01

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

  8. Topographic Studies of Torpedo Acetylcholine Receptor Subunits as a Transmembrane Complex

    Science.gov (United States)

    Strader, Catherine D.; Raftery, Michael A.

    1980-10-01

    The exposure of the four subunits of the acetylcholine receptor from Torpedo californica on both the extracellular and cytoplasmic faces of the postsynaptic membranes of the electroplaque cells has been investigated. Sealed membrane vesicles containing no protein components other than the receptor were isolated and were shown to have 95% of their synaptic surfaces facing the medium. The susceptibility of the four receptor subunits in these preparations to hydrolysis by trypsin both from the external and from the internal medium was used to investigate the exposure of the subunits on the synaptic and cytoplasmic surfaces of the membrane. It was shown by sodium dodecyl sulfate gel electrophoresis of the tryptic products that all four subunits are exposed on the extracellular surface to a similar degree. All four subunits are also exposed on the internal surface of the membrane, but the apparent degree of exposure varies with the subunit size, the larger subunits being more exposed. The results are discussed in terms of a possible topographic model of the receptor as a transmembrane protein complex.

  9. Acetylcholine chloride 1% usage for intraoperative cataract surgery miosis

    Directory of Open Access Journals (Sweden)

    Fernando Macei Drudi

    Full Text Available Abstract Objective: To test the efficacy of Acetylcholine chloride use in obtaining intraoperative miosis on phacoemulsification cataract surgery. Methods: Patients with cataract diagnosis and elected for surgical phacoemulsification procedure were selected. All patients underwent conventional phacoemulsification procedure performed by a single surgeon and all patients had 0.2 ml of Acetylcholine chloride 1% irrigated in the anterior chamber at the end of the surgery. The pupillary diameter was measured immediately before the beginning of surgery, immediately before and two minutes after the use of acetylcholine chloride 1%. Results: A total of 30 eyes from 30 patients were included in the study. 18 were female, and mean age was of 69.5 years with a 7.2y standard deviation on the population study. The mean pupillary diameter immediately before the beginning of surgery was 7.5 mm with a standard deviation of 0.56 mm; the mean pupillary diameter immediately before the acetylcholine chloride 1% use (after the intraocular lens im-plantation was 7.1 mm with a standard deviation of 0.57 mm. The mean pupillary diameter two minutes after the use of acetylcholine chloride 1% in the anterior chamber was 3.4 mm with standard deviation of 0.66 mm. The mean maximum action time of ACH chloride 1% was 64 seconds, with a standard deviation of 8 seconds. The mean intraocular pressure on the first postoperative day was 19.1 mmHg with a standard deviation of 2.45 mmHg. Conclusion: We conclude that acetylcholine chloride 1% is an important drug to obtaining intraoperative miosis in cataract surgery.

  10. Programmed release triggered by osmotic gradients in multicomponent vesicles

    Science.gov (United States)

    Dong, Ruo-Yu; Jang, Hyun-Sook; Granick, Steve

    Polymersomes, a good candidate for encapsulation and delivery of active ingredients, can be constructed with inter-connected multiple compartments. These so-called multisomes on the one hand enable the spatial separation of various incompatible contents or processes, and on the other hand provide an efficient route for inter-compartment communication via the interface semipermeable membrane. Here we show that by establishing osmotic imbalances between different compartments, interesting synergetic morphology changes of the multisomes can be observed. And by further carefully adjusting the osmotic gradients and the arrangement of compartments, we can realize a cascade rupture of these individual units, which may be a new step towards controlled mixing and timed sequences of chemical reactions.

  11. Reversibly formed bilayer vesicles: Energetics and polydispersity

    DEFF Research Database (Denmark)

    Bergstöm, M.

    1997-01-01

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

  12. Extracellular vesicles in cardiovascular homeostasis and disease.

    Science.gov (United States)

    Hutcheson, Joshua D; Aikawa, Elena

    2018-02-19

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

  13. Extracellular vesicles in cartilage homeostasis and osteoarthritis.

    Science.gov (United States)

    Miyaki, Shigeru; Lotz, Martin K

    2018-01-01

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

  14. Cells release subpopulations of exosomes with distinct molecular and biological properties

    NARCIS (Netherlands)

    Willms, Eduard; Johansson, Henrik J.; Mäger, Imre; Lee, Yi; Blomberg, K. Emelie M.; Sadik, Mariam; Alaarg, Amr Muhmed Sabry Abdelhakeem; Smith, C.I. Edvard; Lehtio, Janne; El Andaloussi, Samir; Wood, Matthew J.A.; Vader, Pieter

    2016-01-01

    Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a

  15. Branched nanotrees with immobilized acetylcholine esterase for nanobiosensor applications

    DEFF Research Database (Denmark)

    Risveden, Klas; Dick, Kimberly A; Bhand, Sunil

    2010-01-01

    A novel lab-on-a-chip nanotree enzyme reactor is demonstrated for the detection of acetylcholine. The reactors are intended for use in the RISFET (regional ion sensitive field effect transistor) nanosensor, and are constructed from gold-tipped branched nanorod structures grown on SiN(x)-covered w......A novel lab-on-a-chip nanotree enzyme reactor is demonstrated for the detection of acetylcholine. The reactors are intended for use in the RISFET (regional ion sensitive field effect transistor) nanosensor, and are constructed from gold-tipped branched nanorod structures grown on Si...

  16. Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: Application to the nicotinic acetylcholine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Perez-Ramirez, B.; Martinez-Carrion, M. (Univ. of Missouri, Kansas City (USA))

    1989-06-13

    A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with ({sup 3}H)pyridoxine 5-phosphate (({sup 3}H)PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of ({sup 3}H)PNP at 37{degree}C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the {alpha}-subunit, internal surface labeling by PLP was greatest for the highest molecular weight ({delta}) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion that is proportional to their subunit molecular weight. Yet, the labeling data do not fit well to any of the models proposed for AcChR subunit folding. The method described can be used for selective labeling of the cytoplasmic domains of transmembrane proteins in sealed membrane vesicles.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  18. Complexin synchronizes primed vesicle exocytosis and regulates fusion pore dynamics

    Science.gov (United States)

    Dhara, Madhurima; Yarzagaray, Antonio; Schwarz, Yvonne; Dutta, Soumyajit; Grabner, Chad; Moghadam, Paanteha K.; Bost, Anneka; Schirra, Claudia; Rettig, Jens; Reim, Kerstin; Brose, Nils; Mohrmann, Ralf

    2014-01-01

    ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca2+-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca2+ concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca2+-triggered exocytosis by increasing the Ca2+ affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca2+-triggered release apparatus. PMID:24687280

  19. Isolation and characterization of platelet-derived extracellular vesicles.

    Science.gov (United States)

    Aatonen, Maria T; Ohman, Tiina; Nyman, Tuula A; Laitinen, Saara; Grönholm, Mikaela; Siljander, Pia R-M

    2014-01-01

    Platelet-derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS) or Ca(2+) ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA) and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. The main EV populations were 100-250 nm and over 90% were vesicle subpopulations. Although platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100-250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As the EV subpopulations could not be distinguished and large vesicle populations may be lost by differential centrifugation, novel methods are required for the isolation and the differentiation of all EVs.

  20. 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.

  1. Nicotine Accelerates Atherosclerosis in Apolipoprotein E-Deficient Mice by Activating α7 Nicotinic Acetylcholine Receptor on Mast Cells.

    Science.gov (United States)

    Wang, Chen; Chen, Han; Zhu, Wei; Xu, Yinchuan; Liu, Mingfei; Zhu, Lianlian; Yang, Fan; Zhang, Ling; Liu, Xianbao; Zhong, Zhiwei; Zhao, Jing; Jiang, Jun; Xiang, Meixiang; Yu, Hong; Hu, Xinyang; Lu, Hong; Wang, Jian'an

    2017-01-01

    Cigarette smoking is an independent risk factor for atherosclerosis. Nicotine, the addictive component of cigarettes, induces mast cell (MC) release and contributes to atherogenesis. The purpose of this study was to determine whether nicotine accelerates atherosclerosis through MC-mediated mechanisms and whether MC stabilizer prevents this pathological process. Nicotine administration increased the size of atherosclerotic lesions in apolipoprotein E-deficient (Apoe-/-) mice fed a fat-enriched diet. This was accompanied by enhanced intraplaque macrophage content and lipid deposition but reduced collagen and smooth muscle cell contents. MC deficiency in Apoe-/- mice (Apoe-/-KitW-sh/W-sh) diminished nicotine-induced atherosclerosis. Nicotine activated bone marrow-derived MCs in vitro, which was inhibited by a MC stabilizer disodium cromoglycate or a nonselective nicotinic acetylcholine receptor blocker mecamylamine. Further investigation revealed that α7 nicotinic acetylcholine receptor was a target for nicotine activation in MCs. Nicotine did not change atherosclerotic lesion size of Apoe-/-KitW-sh/W-sh mice reconstituted with MCs from Apoe-/-α7nAChR-/- animals. Activation of α7 nicotinic acetylcholine receptor on MCs is a mechanism by which nicotine enhances atherosclerosis. © 2016 American Heart Association, Inc.

  2. 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.

  3. 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...

  4. Sequential interactions with Sec23 control the direction of vesicle traffic.

    Science.gov (United States)

    Lord, Christopher; Bhandari, Deepali; Menon, Shekar; Ghassemian, Majid; Nycz, Deborah; Hay, Jesse; Ghosh, Pradipta; Ferro-Novick, Susan

    2011-05-12

    How the directionality of vesicle traffic is achieved remains an important unanswered question in cell biology. The Sec23p/Sec24p coat complex sorts the fusion machinery (SNAREs) into vesicles as they bud from the endoplasmic reticulum (ER). Vesicle tethering to the Golgi begins when the tethering factor TRAPPI binds to Sec23p. Where the coat is released and how this event relates to membrane fusion is unknown. Here we use a yeast transport assay to demonstrate that an ER-derived vesicle retains its coat until it reaches the Golgi. A Golgi-associated kinase, Hrr25p (CK1δ orthologue), then phosphorylates the Sec23p/Sec24p complex. Coat phosphorylation and dephosphorylation are needed for vesicle fusion and budding, respectively. Additionally, we show that Sec23p interacts in a sequential manner with different binding partners, including TRAPPI and Hrr25p, to ensure the directionality of ER-Golgi traffic and prevent the back-fusion of a COPII vesicle with the ER. These events are conserved in mammalian cells.

  5. A Preferentially Segregated Recycling Vesicle Pool of Limited Size Supports Neurotransmission in Native Central Synapses

    Science.gov (United States)

    Marra, Vincenzo; Burden, Jemima J.; Thorpe, Julian R.; Smith, Ikuko T.; Smith, Spencer L.; Häusser, Michael; Branco, Tiago; Staras, Kevin

    2012-01-01

    Summary At small central synapses, efficient turnover of vesicles is crucial for stimulus-driven transmission, but how the structure of this recycling pool relates to its functional role remains unclear. Here we characterize the organizational principles of functional vesicles at native hippocampal synapses with nanoscale resolution using fluorescent dye labeling and electron microscopy. We show that the recycling pool broadly scales with the magnitude of the total vesicle pool, but its average size is small (∼45 vesicles), highly variable, and regulated by CDK5/calcineurin activity. Spatial analysis demonstrates that recycling vesicles are preferentially arranged near the active zone and this segregation is abolished by actin stabilization, slowing the rate of activity-driven exocytosis. Our approach reveals a similarly biased recycling pool distribution at synapses in visual cortex activated by sensory stimulation in vivo. We suggest that in small native central synapses, efficient release of a limited pool of vesicles relies on their favored spatial positioning within the terminal. PMID:23141069

  6. Fibronectin-Containing Extracellular Vesicles Protect Melanocytes against Ultraviolet Radiation-Induced Cytotoxicity.

    Science.gov (United States)

    Bin, Bum-Ho; Kim, Dae-Kyum; Kim, Nan-Hyung; Choi, Eun-Jeong; Bhin, Jinhyuk; Kim, Sung Tae; Gho, Yong Song; Lee, Ai-Young; Lee, Tae Ryong; Cho, Eun-Gyung

    2016-05-01

    Skin melanocytes are activated by exposure to UV radiation to secrete melanin-containing melanosomes to protect the skin from UV-induced damage. Despite the continuous renewal of the epidermis, the turnover rate of melanocytes is very slow, and they survive for long periods. However, the mechanisms underlying the survival of melanocytes exposed to UV radiation are not known. Here, we investigated the role of melanocyte-derived extracellular vesicles in melanocyte survival. Network analysis of the melanocyte extracellular vesicle proteome identified the extracellular matrix component fibronectin at a central node, and the release of fibronectin-containing extracellular vesicles was increased after exposure of melanocytes to UVB radiation. Using an anti-fibronectin neutralizing antibody and specific inhibitors of extracellular vesicle secretion, we demonstrated that extracellular vesicles enriched in fibronectin were involved in melanocyte survival after UVB radiation. Furthermore, we observed that in the hyperpigmented lesions of patients with melasma, the extracellular space around melanocytes contained more fibronectin compared with normal skin, suggesting that fibronectin is involved in maintaining melanocytes in pathological conditions. Collectively, our findings suggest that melanocytes secrete fibronectin-containing extracellular vesicles to increase their survival after UVB radiation. These data provide important insight into how constantly stimulated melanocytes can be maintained in pathological conditions such as melasma. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. AHNAK enables mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility.

    Science.gov (United States)

    Silva, Thaiomara A; Smuczek, Basílio; Valadão, Iuri C; Dzik, Luciana M; Iglesia, Rebeca P; Cruz, Mário C; Zelanis, André; de Siqueira, Adriane S; Serrano, Solange M T; Goldberg, Gary S; Jaeger, Ruy G; Freitas, Vanessa M

    2016-08-02

    Extracellular vesicles play important roles in tumor development. Many components of these structures, including microvesicles and exosomes, have been defined. However, mechanisms by which extracellular vesicles affect tumor progression are not fully understood. Here, we investigated vesicular communication between mammary carcinoma cells and neighboring nontransformed mammary fibroblasts. Nonbiased proteomic analysis found that over 1% of the entire proteome is represented in these vesicles, with the neuroblast differentiation associated protein AHNAK and annexin A2 being the most abundant. In particular, AHNAK was found to be the most prominent component of these vesicles based on peptide number, and appeared necessary for their formation. In addition, we report here that carcinoma cells produce vesicles that promote the migration of recipient fibroblasts. These data suggest that AHNAK enables mammary carcinoma cells to produce and release extracellular vesicles that cause disruption of the stroma by surrounding fibroblasts. This paradigm reveals fundamental mechanisms by which vesicular communication between carcinoma cells and stromal cells can promote cancer progression in the tumor microenvironment.

  8. Extracellular Vesicles in Cardiovascular Disease: Potential Applications in Diagnosis, Prognosis, and Epidemiology.

    Science.gov (United States)

    Jansen, Felix; Nickenig, Georg; Werner, Nikos

    2017-05-12

    Extracellular vesicles originate from diverse subcellular compartments and are released in the extracellular space. By transferring their cargoes into target cells and tissues, they now emerge as novel regulators of intercellular communication between adjacent and remote cells. Because vesicle composition and biological content are specific signatures of cellular activation and injury, their potential as diagnostic and prognostic biomarkers has raised significant interest in cardiovascular diseases. Characterization of circulating vesicles- or nonvesicles-bound nucleic acids represents a valuable tool for diagnosing and monitoring cardiovascular diseases, recently referred to as a liquid biopsy. Circulating extracellular vesicles offer a noninvasive and almost continuous access to circulating information on the disease state in epidemiological investigations. Finally, genetic engineering and cell-specific application of extracellular vesicles could display a novel therapeutic option for the treatment of cardiovascular diseases. In this review, we summarize the current knowledge about extracellular vesicles as diagnostic and prognostic biomarkers, as well as their potential applications for longitudinal epidemiological studies in cardiovascular diseases. © 2017 American Heart Association, Inc.

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

    Science.gov (United States)

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

    2011-03-01

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

  10. A New Enzyme-linked Sorbent Assay (ELSA) to Quantify Syncytiotrophoblast Extracellular Vesicles in Biological Fluids

    NARCIS (Netherlands)

    Goehner, Claudia; Weber, Maja; Tannetta, Dionne S.; Groten, Tanja; Ploesch, Torsten; Faas, Marijke M.; Scherjon, Sicco A.; Schleussner, Ekkehard; Markert, Udo R.; Fitzgerald, Justine S.

    ProblemThe pregnancy-associated disease preeclampsia is related to the release of syncytiotrophoblast extracellular vesicles (STBEV) by the placenta. To improve functional research on STBEV, reliable and specific methods are needed to quantify them. However, only a few quantification methods are

  11. Human adipocyte extracellular vesicles in reciprocal signaling between adipocytes and macrophages

    NARCIS (Netherlands)

    Kranendonk, Mariëtte E G; Visseren, Frank L J; van Balkom, Bas W M; Nolte-'t Hoen, Esther N M; van Herwaarden, Joost A; de Jager, Wilco; Schipper, Henk S; Brenkman, Arjan B; Verhaar, Marianne C; Wauben, Marca H M; Kalkhoven, Eric

    2014-01-01

    OBJECTIVE: Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined.

  12. Applying extracellular vesicles based therapeutics in clinical trials – an ISEV position paper

    OpenAIRE

    Lener, Thomas; Gimona, Mario; Aigner, Ludwig; Börger, Verena; Buzas, Edit; Camussi, Giovanni; Chaput, Nathalie; Chatterjee, Devasis; Court, Felipe A.; del Portillo, Hernando A.; O'Driscoll, Lorraine; Fais, Stefano; Falcon-Perez, Juan M.; Felderhoff-Mueser, Ursula; Fraile, Lorenzo

    2015-01-01

    Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs ...

  13. Histamine stimulates secretion of extracellular vesicles with nucleotidase activity in rat submandibular gland.

    Science.gov (United States)

    González, Débora Alejandra; Barbieri van Haaster, Martín Matías; Quinteros Villarruel, Emmanuel; Brandt, Macarena; Benítez, María Belén; Stranieri, Graciela Mabel; Orman, Betina

    2018-01-01

    Extracellular vesicles released by different cells have been isolated from diverse fluids including saliva. We previously reported that rat submandibular glands secrete nanovesicles that catalyze hydrolysis of ATP, ADP and AMP, which are actors of the purinergic signaling system along with adenosine. Extracellular nucleotides like ATP and adenosine are involved in the regulation of inflammatory processes and apoptosis. Histamine, a widely distributed biogenic amine, is involved in inflammatory response. To test if activation of histamine receptors in rat submandibular gland promotes changes in the release of vesicles with nucleotidase activity that could modulate purinergic signaling. Rat submandibular glands were incubated in the absence or presence of histamine and JNJ7777120, an antagonist for H 4 receptors. Extracellular vesicles were isolated from incubation media by differential centrifugation. Vesicular nucleotidase activity was measured following Pi release by 3mM MgATP, MgADP or MgAMP. Histamine increased the release of vesicles with nucleotidase activity in a concentration dependent manner. JNJ7777120 significantly reduced this effect. Vesicular nucleotidases obtained in the absence or presence of histamine promoted Pi production from ATP, ADP and AMP. The results show a relationship between histamine and the regulation of purinergic signaling, which could be important in the modulation of inflammatory processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper

    NARCIS (Netherlands)

    Mateescu, Bogdan; Kowal, Emma J K; van Balkom, Bas W M; Bartel, Sabine; Bhattacharyya, Suvendra N; Buzás, Edit I; Buck, Amy H; de Candia, Paola; Chow, Franklin W N; Das, Saumya; Driedonks, Tom A P; Fernández-Messina, Lola; Haderk, Franziska; Hill, Andrew F; Jones, Jennifer C; Van Keuren-Jensen, Kendall R; Lai, Charles P; Lässer, Cecilia; Liegro, Italia di; Lunavat, Taral R; Lorenowicz, Magdalena J; Maas, Sybren L N; Mäger, Imre; Mittelbrunn, Maria; Momma, Stefan; Mukherjee, Kamalika; Nawaz, Muhammed; Pegtel, D Michiel; Pfaffl, Michael W; Schiffelers, Raymond M|info:eu-repo/dai/nl/212909509; Tahara, Hidetoshi; Théry, Clotilde; Tosar, Juan Pablo; Wauben, Marca H M|info:eu-repo/dai/nl/112675735; Witwer, Kenneth W; Nolte-'t Hoen, Esther N M

    2017-01-01

    The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to

  15. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - An ISEV position paper

    NARCIS (Netherlands)

    Mateescu, Bogdan; Kowal, Emma J K; van Balkom, Bas W M|info:eu-repo/dai/nl/256594783; Bartel, Sabine; Bhattacharyya, Suvendra N.; Buzás, Edit I.; Buck, Amy H.; de Candia, Paola; Chow, Franklin W N; Das, Saumya; Driedonks, Tom A P; Fernández-Messina, Lola; Haderk, Franziska; Hill, Andrew F.; Jones, Jennifer C.; Van Keuren-Jensen, Kendall R.; Lai, Charles P.; Lässer, Cecilia; di Liegro, Italia; Lunavat, Taral R.; Lorenowicz, Magdalena J.; Maas, Sybren L N; Mäger, Imre; Mittelbrunn, Maria; Momma, Stefan; Mukherjee, Kamalika; Nawaz, Muhammed; Pegtel, D. Michiel; Pfaffl, Michael W.; Schiffelers, Raymond M.|info:eu-repo/dai/nl/212909509; Tahara, Hidetoshi; Théry, Clotilde; Tosar, Juan Pablo; Wauben, Marca H M; Witwer, Kenneth W.; Nolte-'t Hoen, Esther N M

    2017-01-01

    The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to

  16. Peptide-mediated ‘miniprep’ isolation of extracellular vesicles is suitable for high-throughput proteomics

    OpenAIRE

    Jaco C. Knol; Inge de Reus; Tim Schelfhorst; Robin Beekhof; Meike de Wit; Sander R. Piersma; Thang V. Pham; Egbert F. Smit; Henk M.W. Verheul; Connie R. Jiménez

    2016-01-01

    Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since EVs are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, they represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation are laborious, time-consuming, and require high inputs. Ghosh and co-workers recently described an isolation method utilizi...

  17. Acetylcholine receptors in the retinas of the α7 nicotinic acetylcholine receptor knockout mouse.

    Science.gov (United States)

    Smith, Marci L; Souza, Fred G Oliveira; Bruce, Kady S; Strang, Christianne E; Morley, Barbara J; Keyser, Kent T

    2014-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse. To examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes. In the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO

  18. Illuminating the physiology of extracellular vesicles

    OpenAIRE

    Choi, Hongyoon; Lee, Dong Soo

    2016-01-01

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

  19. 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.

  20. 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.

  1. Urinary extracellular vesicles: biomarkers and beyond

    NARCIS (Netherlands)

    M. Salih (Mahdi)

    2017-01-01

    markdownabstractExtracellular vesicles have been isolated in various body fluids including urine. The cargo of urinary extracellular vesicles (uEVs) is composed of proteins and nucleic acids reflecting the physiological and possibly the pathophysiological state of cells lining the nephron. Because

  2. Insect nicotinic acetylcholine receptors (nAChRs): Important amino ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-29

    Dec 29, 2008 ... neonicotinoid insecticides affinity remarkably, but showed little effects on insect nAChRs normal function. Key words: Nicotinic acetylcholine receptor, neonicotinoid insecticides, selectivity, resistance. INTRODUCTION. Most commercially important insecticides are neurotoxins that act on ion channels, ...

  3. Cholinergic modulation of dopamine pathways through nicotinic acetylcholine receptors.

    NARCIS (Netherlands)

    de Kloet, S.F.; Mansvelder, H.D.; de Vries, T.J.

    2015-01-01

    Nicotine addiction is highly prevalent in current society and is often comorbid with other diseases. In the central nervous system, nicotine acts as an agonist for nicotinic acetylcholine receptors (nAChRs) and its effects depend on location and receptor composition. Although nicotinic receptors are

  4. Insect nicotinic acetylcholine receptors (nAChRs): Important amino ...

    African Journals Online (AJOL)

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The great abundance of nAChRs within the insect central nervous system has led to the development of insecticides targeting these receptors, such as ...

  5. Measurement of anti- acetylcholine receptor auto-antibodies in ...

    African Journals Online (AJOL)

    myasthenia gravis. K. J. Steenkamp, W. Duim, M. s. Myer,. S. C. K. Malfeld, R. Anderson. Two different acetylcholine receptor (AChR) preparations derived from amputated human muscle (AChRAMP) and from the human rhabdomyosarcoma cell line TE671 (AChRTE67,) were compared in radio-immunoprecipitation assays ...

  6. Functional partial agonism at cloned human muscarinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Ebert, B; Brann, M R

    1996-01-01

    of maximal response, depending on the molar ratio of agonist and antagonist used. Using recombinant human muscarinic acetylcholine receptors (m1 and m5) and the functional assay, receptor selection and amplification technology (R-SAT), we have now shown that co-administration of the full agonist, carbachol...

  7. Dimensional characterization of extracellular vesicles using atomic force microscopy

    Science.gov (United States)

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

    2017-03-01

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

  8. Whole-Retina Reduced Electrophysiological Activity in Mice Bearing Retina-Specific Deletion of Vesicular Acetylcholine Transporter.

    Science.gov (United States)

    Bedore, Jake; Martyn, Amanda C; Li, Anson K C; Dolinar, Eric A; McDonald, Ian S; Coupland, Stuart G; Prado, Vania F; Prado, Marco A; Hill, Kathleen A

    2015-01-01

    Despite rigorous characterization of the role of acetylcholine in retinal development, long-term effects of its absence as a neurotransmitter are unknown. One of the unanswered questions is how acetylcholine contributes to the functional capacity of mature retinal circuits. The current study investigates the effects of disrupting cholinergic signalling in mice, through deletion of vesicular acetylcholine transporter (VAChT) in the developing retina, pigmented epithelium, optic nerve and optic stalk, on electrophysiology and structure of the mature retina. A combination of electroretinography, optical coherence tomography imaging and histological evaluation assessed retinal integrity in mice bearing retina- targeted (embryonic day 12.5) deletion of VAChT (VAChTSix3-Cre-flox/flox) and littermate controls at 5 and 12 months of age. VAChTSix3-Cre-flox/flox mice did not show any gross changes in nuclear layer cellularity or synaptic layer thickness. However, VAChTSix3-Cre-flox/flox mice showed reduced electrophysiological response of the retina to light stimulus under scotopic conditions at 5 and 12 months of age, including reduced a-wave, b-wave, and oscillatory potential (OP) amplitudes and decreased OP peak power and total energy. Reduced a-wave amplitude was proportional to the reduction in b-wave amplitude and not associated with altered a-wave 10%-90% rise time or inner and outer segment thicknesses. This study used a novel genetic model in the first examination of function and structure of the mature mouse retina with disruption of cholinergic signalling. Reduced amplitude across the electroretinogram wave form does not suggest dysfunction in specific retinal cell types and could reflect underlying changes in the retinal and/or extraretinal microenvironment. Our findings suggest that release of acetylcholine by VAChT is essential for the normal electrophysiological response of the mature mouse retina.

  9. Whole-Retina Reduced Electrophysiological Activity in Mice Bearing Retina-Specific Deletion of Vesicular Acetylcholine Transporter.

    Directory of Open Access Journals (Sweden)

    Jake Bedore

    Full Text Available Despite rigorous characterization of the role of acetylcholine in retinal development, long-term effects of its absence as a neurotransmitter are unknown. One of the unanswered questions is how acetylcholine contributes to the functional capacity of mature retinal circuits. The current study investigates the effects of disrupting cholinergic signalling in mice, through deletion of vesicular acetylcholine transporter (VAChT in the developing retina, pigmented epithelium, optic nerve and optic stalk, on electrophysiology and structure of the mature retina.A combination of electroretinography, optical coherence tomography imaging and histological evaluation assessed retinal integrity in mice bearing retina- targeted (embryonic day 12.5 deletion of VAChT (VAChTSix3-Cre-flox/flox and littermate controls at 5 and 12 months of age. VAChTSix3-Cre-flox/flox mice did not show any gross changes in nuclear layer cellularity or synaptic layer thickness. However, VAChTSix3-Cre-flox/flox mice showed reduced electrophysiological response of the retina to light stimulus under scotopic conditions at 5 and 12 months of age, including reduced a-wave, b-wave, and oscillatory potential (OP amplitudes and decreased OP peak power and total energy. Reduced a-wave amplitude was proportional to the reduction in b-wave amplitude and not associated with altered a-wave 10%-90% rise time or inner and outer segment thicknesses.This study used a novel genetic model in the first examination of function and structure of the mature mouse retina with disruption of cholinergic signalling. Reduced amplitude across the electroretinogram wave form does not suggest dysfunction in specific retinal cell types and could reflect underlying changes in the retinal and/or extraretinal microenvironment. Our findings suggest that release of acetylcholine by VAChT is essential for the normal electrophysiological response of the mature mouse retina.

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

    Science.gov (United States)

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

    2013-07-01

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

  11. Pharmacology of neurotransmitter release: measuring exocytosis.

    Science.gov (United States)

    Khvotchev, Mikhail; Kavalali, Ege T

    2008-01-01

    Neurotransmission in the nervous system is initiated at presynaptic terminals by fusion of synaptic vesicles with the plasma membrane and subsequent exocytic release of chemical transmitters. Currently, there are multiple methods to detect neurotransmitter release from nerve terminals, each with their own particular advantages and disadvantages. For instance, most commonly employed methods monitor actions of released chemical substances on postsynaptic receptors or artificial substrates such as carbon fibers. These methods are closest to the physiological setting because they have a rapid time resolution and they measure the action of the endogenous neurotransmitters rather than the signals emitted by exogenous probes. However, postsynaptic receptors only indirectly report neurotransmitter release in a form modified by the properties of receptors themselves, which are often nonlinear detectors of released substances. Alternatively, released chemical substances can be detected biochemically, albeit on a time scale slower than electrophysiological methods. In addition, in certain preparations, where presynaptic terminals are accessible to whole cell recording electrodes, fusion of vesicles with the plasma membrane can be monitored using capacitance measurements. In the last decade, in addition to electrophysiological and biochemical methods, several fluorescence imaging modalities have been introduced which report synaptic vesicle fusion, endocytosis, and recycling. These methods either take advantage of styryl dyes that can be loaded into recycling vesicles or exogenous expression of synaptic vesicle proteins tagged with a pH-sensitive GFP variant at regions facing the vesicle lumen. In this chapter, we will provide an overview of these methods with particular emphasis on their relative strengths and weaknesses and discuss the types of information one can obtain from them.

  12. [Transvesical Removal of Seminal Vesicle Cystadenoma].

    Science.gov (United States)

    Takayasu, Kenta; Harada, Jiro; Kawa, Gen; Ota, Syuichi; Sakurai, Takanori

    2015-07-01

    Primary tumors of the seminal vesicles are extremely rare. There have been 25 reports of this tumor from overseas and most cases are cystadenoma. We report a case of seminal vesicle cystadenoma in a 70-year-old man who presented with lower abdominal pain and urinary frequency. A digital rectal examination detected a projecting and hard mass in the right side of the prostate. Magnetic resonance imaging (MRI) showed a 15 cm multiple cystic mass continuous with the right seminal vesicle. A transrectal needle biopsy revealed benign tissue. The tumor was resected using an open transvesical approach that enabled full exposure of the seminal vesicle without damaging the nerves and blood supply of the bladder. Pathology was consistent with a benign seminal vesicle cystadenoma. We describe the natural history, pathology,and surgical approach in this case.

  13. Supramolecular Vesicles Coassembled from Disulfide-Linked Benzimidazolium Amphiphiles and Carboxylate-Substituted Pillar[6]arenes that Are Responsive to Five Stimuli.

    Science.gov (United States)

    Jiang, Long; Huang, Xuan; Chen, Dong; Yan, Hua; Li, Xueyuan; Du, Xuezhong

    2017-03-01

    Novel supramolecular vesicles based on host-guest systems were coassembled from carboxylate-substituted pillar[6]arene (CPA[6]) and disulfide-linked benzimidazolium amphiphiles, and the microstructures of the CPA-based supramolecular vesicles were clearly elaborated. The supramolecular vesicles showed controlled drug release in response to five stimuli, with glutathione, pH, CO2 , Zn2+ ions, and hexanediamine, leading to cleavage of the disulfide bonds, protonation of the carboxylate groups, metal chelation, and competitive binding. This is the first case of a smart pillararene-based supramolecular vesicle being integrated with five stimuli-responsive functions to meet the diverse requirements of controlled drug release. Importantly, each of the five stimuli is closely related to microenvironments of tumors and diseases of the human body. The smart stimuli-responsive supramolecular vesicles have promising applications in drug therapy of tumors and relevant diseases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Differential role of ventral tegmental area acetylcholine and N-Methyl-D-Aspartate receptors in cocaine-seeking

    Science.gov (United States)

    Solecki, Wojciech; Wickham, Robert J.; Behrens, Shay; Wang, Jie; Zwerling, Blake; Mason, Graeme F.; Addy, Nii A.

    2013-01-01

    Exposure to drug-associated cues evokes drug-seeking behavior and is regarded as a major cause of relapse. Cues evoke burst firing of ventral tegmental area (VTA) dopamine (DA) neurons and phasic DA release in the nucleus accumbens (NAc). Cholinergic and glutamatergic input to the VTA is suggested to gate phasic DA activity. However, the role of VTA cholinergic and glutamatergic receptors in regulating phasic dopamine release and cue-induced drug-seeking in cocaine experienced subjects is not known. In male Sprague-Dawley rats, we found that VTA inactivation strongly inhibited, while VTA stimulation promoted, cocaine-seeking behavior during early withdrawal. Blockade of phasic activated D1 receptors in the NAc core also strongly inhibited cue-induced cocaine-seeking - suggesting an important role of phasic DA activity in the VTA to NAc core circuit. Next, we examined the role of VTA acetylcholine receptors (AChRs) and N-methyl-D-aspartate receptors (NMDARs) in regulating both NAc core phasic DA release and cue-induced cocaine-seeking. In cocaine naïve subjects, VTA infusion of the nicotinic acetylcholine receptor (AChR) antagonist mecamylamine, the muscarinic AChR antagonist scopolamine, or the NMDAR antagonist AP-5, led to robust attenuation of phasic DA release in the NAc core. During early cocaine withdrawal, VTA infusion of AP-5 had limited effects on NAc phasic DA release and cue-induced cocaine-seeking while VTA infusion of mecamylamine or scopolamine robustly inhibited both phasic DA release and cocaine-seeking. The results demonstrate that VTA AChRs, but not NMDARs, strongly regulate cue-induced cocaine-seeking and phasic DA release during early cocaine withdrawal. PMID:23850572

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

    National Research Council Canada - National Science Library

    M A Zoccoli; G E Lienhard

    1977-01-01

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

  16. Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells.

    Science.gov (United States)

    Akopova, Irina; Tatur, Sabina; Grygorczyk, Mariusz; Luchowski, Rafał; Gryczynski, Ignacy; Gryczynski, Zygmunt; Borejdo, Julian; Grygorczyk, Ryszard

    2012-03-01

    Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.

  17. 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.

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

    Science.gov (United States)

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

    2008-07-01

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

  19. Association of Extracellular Membrane Vesicles with Cutaneous Wound Healing

    Directory of Open Access Journals (Sweden)

    Uyen Thi Trang Than

    2017-05-01

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

  20. Extracellular Vesicles from Ovarian Carcinoma Cells Display Specific Glycosignatures

    Directory of Open Access Journals (Sweden)

    Joana Gomes

    2015-08-01

    Full Text Available Cells release vesicles to the extracellular environment with characteristic nucleic acid, protein, lipid, and glycan composition. Here we have isolated and characterized extracellular vesicles (EVs and total cell membranes (MBs from ovarian carcinoma OVMz cells. EVs were enriched in specific markers, including Tsg101, CD63, CD9, annexin-I, and MBs contained markers of cellular membrane compartments, including calnexin, GRASP65, GS28, LAMP-1, and L1CAM. The glycoprotein galectin-3 binding protein (LGALS3BP was strongly enriched in EVs and it contained sialylated complex N-glycans. Lectin blotting with a panel of lectins showed that EVs had specific glycosignatures relative to MBs. Furthermore, the presence of glycoproteins bearing complex N-glycans with α2,3-linked sialic acid, fucose, bisecting-GlcNAc and LacdiNAc structures, and O-glycans with the T-antigen were detected. The inhibition of N-glycosylation processing from high mannose to complex glycans using kifunensine caused changes in the composition of EVs and induced a decrease of several glycoproteins. In conclusion, the results showed that glycosignatures of EVs were specific and altered glycosylation within the cell affected the composition and/or dynamics of EVs release. Furthermore, the identified glycosignatures of EVs could provide novel biomarkers for ovarian cancer.

  1. RIM determines Ca2+ channel density and vesicle docking at the presynaptic active zone

    Science.gov (United States)

    Han, Yunyun; Kaeser, Pascal S.; Südhof, Thomas C.; Schneggenburger, Ralf

    2012-01-01

    At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca2+ channels close to docked vesicles. The mechanisms that enrich Ca2+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. Here, we have established a Cre-lox based conditional knock-out approach at a presynaptically accessible CNS synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca2+ channel density, revealing a new role of RIM proteins in Ca2+ channel targeting. Removal of RIMs also reduced the readily-releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca2+ channel - vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release: enabling a high presynaptic Ca2+ channel density, and vesicle docking at the active zone. PMID:21262468

  2. Pulmonary Extracellular Vesicles as Mediators of Local and Systemic Inflammation.

    Science.gov (United States)

    Wahlund, Casper J E; Eklund, Anders; Grunewald, Johan; Gabrielsson, Susanne

    2017-01-01

    Cells of the airways are constantly exposed to environmental hazards including cigarette smoke, irritants, pathogens, and mechanical insults. Maintaining barrier integrity is vital, and mounting responses to threats depends on intercellular communication. Extracellular vesicles (EVs), including exosomes and microvesicles, are major signal mediators between cells, shuttling cargo in health and disease. Depending on the state of the originating cells, EVs are capable of inducing proinflammatory effects including antigen presentation, cellular migration, apoptosis induction, and inflammatory cytokine release. Cells of the airways release EVs, which can be found in bronchoalveolar lavage fluid. EVs of the airways can support inflammation in the lung, but may also exit into the circulation and carry a cocktail of pro-inflammatory molecules to recipient cells in distant organs. In this review, we discuss the possibility that EVs originating from the airways contribute to dissemination of inflammation in both lung disorders and systemic inflammatory conditions.

  3. Pulmonary Extracellular Vesicles as Mediators of Local and Systemic Inflammation

    Directory of Open Access Journals (Sweden)

    Susanne Gabrielsson

    2017-04-01

    Full Text Available Cells of the airways are constantly exposed to environmental hazards including cigarette smoke, irritants, pathogens, and mechanical insults. Maintaining barrier integrity is vital, and mounting responses to threats depends on intercellular communication. Extracellular vesicles (EVs, including exosomes and microvesicles, are major signal mediators between cells, shuttling cargo in health and disease. Depending on the state of the originating cells, EVs are capable of inducing proinflammatory effects including antigen presentation, cellular migration, apoptosis induction, and inflammatory cytokine release. Cells of the airways release EVs, which can be found in bronchoalveolar lavage fluid. EVs of the airways can support inflammation in the lung, but may also exit into the circulation and carry a cocktail of pro-inflammatory molecules to recipient cells in distant organs. In this review, we discuss the possibility that EVs originating from the airways contribute to dissemination of inflammation in both lung disorders and systemic inflammatory conditions.

  4. Altered acetylcholine metabolism of brain in uremia: role of secondary hyperparathyroidism.

    Science.gov (United States)

    Smogorzewski, Miroslaw J; Massry, Shaul G

    2008-01-01

    Cholinergic system and its neurotransmitter, acetylcholine (ACh), play a major role in both behavior and motor function of the nervous system. Cholinergic neurons synthesize ACh from choline and acetyl-CoA by choline acetyltransferase in the nerve ending. The release of ACh in response to nerve impulses is dependent on the intracellular calcium ([Ca2+]i) concentration and its gradient. The regulation of a synthesis of ACh after depolarization and ACh release is controlled by mass-action effect on choline acetyltransferase equilibrium. Behavioral and motor changes in uremia may be due in part to derangements in ACh metabolism and such possible abnormalities may be related to the state of secondary hyperparathyroidism of chronic renal failure (CRF). We studied ACh and choline content, choline release, choline kinase activity in brain synaptosomes of CRF with and without secondary hyperparathyroidism and in CRF rats treated with verapamil which normalize [Ca2+]i in brain synaptosomes of CRF rats. The content of ACh of brain synaptosomes increased progressively with the duration of CRF from 3 to 6 weeks. ACh and choline release as well as choline uptake were significantly higher in CRF rats at all time intervals studied. Choline content and the activity of choline kinase of brain synaptosomes were deceased after 3 weeks of CRF and were significantly lower than in synaptosomes of normal. Normalization of ACh and choline content as well as ACh release and the activity of choline kinase by parathyroidectomy or treatment with verapamil but these maneuvers did not prevent the rise in choline uptake and choline release. Resting levels of cytosolic calcium of brain synaptosomes in rats with CRF were significantly higher (437 +/- 9 nM) as compared to normal rats (345 +/- 9 nM). This rise in [Ca2+]i was prevented either by parathyroidectomy prior induction of CRF or by treatment of CRF rats with calcium channel blocker verapamil.

  5. Illuminating the physiology of extracellular vesicles.

    Science.gov (United States)

    Choi, Hongyoon; Lee, Dong Soo

    2016-04-16

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

  6. Microfluidics fabrication of monodisperse biocompatible phospholipid vesicles for encapsulation and delivery of hydrophilic drug or active compound.

    Science.gov (United States)

    Kong, Feng; Zhang, Xu; Hai, Mingtan

    2014-04-08

    We encapsulate the hydrophilic anti-cancer drug doxurubicin hydrochloride (DOX) with about 94% drug encapsulation efficiency, either alone or with nanomagnetite, in monodisperse biocompatible phospholipid vesicles. Glass capillary microfluidics is used to generate monodisperse water in oil in water (w/o/w) double-emulsion templates with a core-shell structure by using a mixture of liquid unsaturated phospholipids and powdered saturated phospholipid. This combination would overcome the low transition temperature of unsaturated powdered phospholipid and the solubility limitation of saturated phospholipid, as well as improving the fabrication of stable monodisperse phospholipid vesicles. The double-emulsion droplet is controlled from 50 to 200 μm according to different flow rates, and the final phospholipid vesicles are retained after a solvent removal step by dewetting. DOX-loaded phospholipid vesicles show sustained release compared with free DOX water solution. The in vitro cell viability of 100 μg/mL phospholipid vesicles on HeLa or MCF-7 cells after 24 h incubation at 310 K is above 90%, confirming the excellent biocompatibility of the phospholipid vesicles. These biocompatible phospholipid vesicles are promising oral drug delivery vehicles for biomedical applications and magnetic resonance imaging contrast agents for biomedical diagnosis.

  7. Glioblastoma stem-like cells secrete the pro-angiogenic VEGF-A factor in extracellular vesicles.

    Science.gov (United States)

    Treps, Lucas; Perret, Raul; Edmond, Sébastien; Ricard, Damien; Gavard, Julie

    2017-01-01

    Glioblastoma multiforme (GBM) are mortifying brain tumours that contain a subpopulation of tumour cells with stem-like properties, termed glioblastoma stem-like cells (GSCs). GSCs largely contribute to tumour initiation, propagation and resistance to current anti-cancer therapies. GSCs are situated in perivascular niches, closely associated with brain microvascular endothelial cells, thereby involved in bidirectional molecular and cellular interactions. Moreover, extracellular vesicles are suspected to carry essential information that can adapt the microenvironment to the tumour's needs, including tumour-induced angiogenesis. In GBM, extracellular vesicles produced by differentiated tumour cells and GSCs were demonstrated to disseminate locally and at distance. Here, we report that the pro-angiogenic pro-permeability factor VEGF-A is carried in extracellular vesicles secreted from ex vivo cultured patient-derived GSCs. Of note, extracellular vesicle-derived VEGF-A contributes to the in vitro elevation of permeability and angiogenic potential in human brain endothelial cells. Indeed, VEGF-A silencing in GSCs compromised in vitro extracellular vesicle-mediated increase in permeability and angiogenesis. From a clinical standpoint, extracellular vesicles isolated from circulating blood of GBM patients present higher levels of VEGF-A, as compared to healthy donors. Overall, our results suggest that extracellular vesicle-harboured VEGF-A targets brain endothelial cells and might impact their ability to form new vessels. Thus, tumour-released EV cargo might emerge as an instrumental part of the tumour-induced angiogenesis and vascular permeability modus operandi in GBM.

  8. A low nicotine concentration augments vesicle motion and exocytosis triggered by K(+) depolarisation of chromaffin cells.

    Science.gov (United States)

    de Diego, Antonio M G; Tapia, Laura; Alvarez, Rocío M; Mosquera, Marta; Cortés, Lorena; López, Inmaculada; Gutiérrez, Luis M; Gandía, Luis; García, Antonio G

    2008-11-19

    Tobacco smokers have an increased risk of cardiovascular disease; this is likely associated to an enhanced catecholamine release by circulating nicotine. Here, we have explored how low concentrations of nicotine in the range of those found in the blood of tobacco smokers, might affect the release of catecholamines in bovine chromaffin cells. We have combined patch-clamp and Ca(2+) imaging techniques to study cell excitability, cytosolic Ca(2+) transients, vesicle movement, and secretory responses. We found that low concentrations of nicotine (1.5-3 microM) did not enhance catecholamine release by themselves. However, they drastically augmented the catecholamine release response triggered by a supramaximal K(+) depolarising pulse. Furthermore, low nicotine concentrations caused slight depolarisation with superimposed action potentials, a transient elevation of [Ca(2+)](c) and augmented Ca(2+)-dependent vesicle motion underneath the plasmalemma. We suggest that low nicotine concentrations overload the secretory machinery with secretory vesicles, which cause chromaffin cells to respond with an exaggerated adrenaline release into the circulation during stress. This might contribute to the higher cardiovascular risk of tobacco smokers.

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

    OpenAIRE

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

    2016-01-01

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

  10. 3D estimation of synaptic vesicle distributions in serial section transmission electron microscopy

    DEFF Research Database (Denmark)

    Khanmohammadi, Mahdieh; Darkner, Sune; Nava, Nicoletta

    To transfer information between neurons, synaptic vesicles move toward the presynaptic membrane, called the active zone, and fuse with it, releasing neurotransmitters into the synaptic cleft. Thus, the shortest distance from vesicles to the active zone affects the speed of signal transportation...... brains. We demonstrate that ssTEM images have an additive bias field, which is well modelled by a quadratic polynomial. ssTEM images make a 3D study on very high-resolution images possible. However, due to the physical cutting of a section from a 3D embedded tissue, the relations between sections...... are lost. To reconstruct the 3D data we register the images in a common coordinate system. The traditional method to measure the distribution of the vesicles is to measure the distance independently of neighbouring sections. This is biased depending on the slope of the active zone with respect...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-07-01

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

  12. 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.

  13. Nicotinic Acetylcholine Receptors in the Pathophysiology of Alzheimer's Disease

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Andreasen T., Jesper; Arvaniti, Maria

    2016-01-01

    Nicotinic acetylcholine receptors (nAChRs) have been pursued for decades as potential molecular targets to treat cognitive dysfunction in Alzheimer's disease (AD) due to their positioning within regions of the brain critical in learning and memory, such as the prefrontal cortex and hippocampus......, and their demonstrated role in processes underlying cognition such as synaptic facilitation, and theta and gamma wave activity. Historically, activity at these receptors is facilitated in AD by use of drugs that increase the levels of their endogenous agonist acetylcholine, and more recently nAChR selective ligands have...... interactions to modify nAChR function adds a new level of complexity to cholinergic signaling in the brain that may be specifically altered in AD. It is currently not known to what degree current nAChR ligands affect these interactions, and it is possible that the difference in the clinical effect of n...

  14. Demonstration of two distributions of vesicle radius in the dopamine neuron of Planorbis corneus from electrochemical data.

    Science.gov (United States)

    Anderson, B B; Chen, G; Gutman, D A; Ewing, A G

    1999-05-01

    An electrochemical model to calculate the relative size and neurotransmitter concentration of individual nerve cell vesicles is presented to examine potentially different types of vesicles in Planorbis corneus. Amperometric current transients resulting from individual exocytosis events detected from single cells contain the information necessary to quantify vesicular neurotransmitter amount and to estimate other important cellular properties such as vesicular neurotransmitter concentration and vesicle radius. Use of a simplifying assumption that the cross-sectional area of the contents of each release event is the apparent electroactive area of the electrode and that the shape of the decreasing phase of each current transient follows Cottrell-like behavior, the Cottrell equation and Faraday's law can be combined to yield expressions for relative vesicle radius and neurotransmitter concentration. This analysis has been applied to data obtained from the cell body of the giant dopamine neuron of the pond snail P. corneus. The histogram of vesicular dopamine concentration reveals a single wide distribution and the histogram of vesicle radius reveals a bimodal radius distribution. These data strongly suggest two distinct classes of vesicle radius in the P. corneus neuron lead to the bimodal distribution of amount released reported earlier.

  15. Mechanisms of Action of Anticholinesterases and Oximes on Acetylcholine Receptors

    Science.gov (United States)

    1988-07-23

    psychotropic, antipsychotic, opiate, antidepressant, antibiotic, antiviral, and antiarrhythmic S drugs . Molec. Pharmacol. 22:72-81 (1982). 25. Kohanski, R.A...physostigmine as a pretreatment drug for protection of rats from organosphate poisoning. Fund. Appl. Tox. 6:566-77 (1986). 8. Seifert, S.A. and M.E...Eldefrawi. Affinity of myasthenia drugs to acetylcholinesterase and acetylcholine receptor. Biochem. Med. 10:258-265 (1974). 9. Carpenter, D.O., L.A

  16. Activation and allosteric modulation of a muscarinic acetylcholine receptor

    OpenAIRE

    Kruse, Andrew C.; Ring, Aaron M.; Manglik, Aashish; Hu, Jianxin; Hu, Kelly; Eitel, Katrin; Hübner, Harald; Pardon, Els; Valant, Celine; Sexton, Patrick M.; Christopoulos, Arthur; Felder, Christian C.; Gmeiner, Peter; Steyaert, Jan; Weis, William I.

    2013-01-01

    Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display....

  17. Stability of Spherical Vesicles in Electric Fields

    Science.gov (United States)

    2010-01-01

    The stability of spherical vesicles in alternating (ac) electric fields is studied theoretically for asymmetric conductivity conditions across their membranes. The vesicle deformation is obtained from a balance between the curvature elastic energies and the work done by the Maxwell stresses. The present theory describes and clarifies the mechanisms for the four types of morphological transitions observed experimentally on vesicles exposed to ac fields in the frequency range from 500 to 2 × 107 Hz. The displacement currents across the membranes redirect the electric fields toward the membrane normal to accumulate electric charges by the Maxwell−Wagner mechanism. These accumulated electric charges provide the underlying molecular mechanism for the morphological transitions of vesicles as observed on the micrometer scale. PMID:20575588

  18. Kinetic regulation of coated vesicle secretion

    CERN Document Server

    Foret, Lionel

    2008-01-01

    The secretion of vesicles for intracellular transport often rely on the aggregation of specialized membrane-bound proteins into a coat able to curve cell membranes. The nucleation and growth of a protein coat is a kinetic process that competes with the energy-consuming turnover of coat components between the membrane and the cytosol. We propose a generic kinetic description of coat assembly and the formation of coated vesicles, and discuss its implication to the dynamics of COP vesicles that traffic within the Golgi and with the Endoplasmic Reticulum. We show that stationary coats of fixed area emerge from the competition between coat growth and the recycling of coat components, in a fashion resembling the treadmilling of cytoskeletal filaments. We further show that the turnover of coat components allows for a highly sensitive switching mechanism between a quiescent and a vesicle producing membrane, upon a slowing down of the exchange kinetics. We claim that the existence of this switching behaviour, also tri...

  19. A novel synaptic vesicle fusion path in the rat cerebral cortex: the "saddle" point hypothesis.

    Science.gov (United States)

    Zampighi, Guido A; Serrano, Raul; Vergara, Julio L

    2014-01-01

    We improved freeze-fracture electron microscopy to study synapses in the neuropil of the rat cerebral cortex at ∼2 nm resolution and in three-dimensions. In the pre-synaptic axon, we found that "rods" assembled from short filaments protruding from the vesicle and the plasma membrane connects synaptic vesicles to the membrane of the active zone. We equated these "connector rods" to protein complexes involved in "docking" and "priming" vesicles to the active zone. Depending on their orientation, the "rods" define two synaptic vesicle-fusion paths: When parallel to the plasma membrane, the vesicles hemi-fuse anywhere ("randomly") in the active zone following the conventional path anticipated by the SNARE hypothesis. When perpendicular to the plasma membrane, the vesicles hemi-fuse at the base of sharp crooks, called "indentations," that are spaced 75-85 nm center-to-center, arranged in files and contained within gutters. They result from primary and secondary membrane curvatures that intersect at stationary inflection ("saddle") points. Computer simulations indicate that this novel vesicle-fusion path evokes neurotransmitter concentration domains on the post-synaptic spine that are wider, shallower, and that reach higher average concentrations than the more conventional vesicle fusion path. In the post-synaptic spine, large (∼9× ∼15 nm) rectangular particles at densities of 72±10/ µm2 (170-240/spine) match the envelopes of the homotetrameric GluR2 AMPA-sensitive receptor. While these putative receptors join clusters, called the "post-synaptic domains," the overwhelming majority of the rectangular particles formed bands in the "non-synaptic" plasma membrane of the spine. In conclusion, in the neuropil of the rat cerebral cortex, curvatures of the plasma membrane define a novel vesicle-fusion path that preconditions specific regions of the active zone for neurotransmitter release. We hypothesize that a change in the hybridization of the R-SNARE synaptobrevin

  20. Mutations in Synaptojanin Disrupt Synaptic Vesicle Recycling

    OpenAIRE

    Harris, Todd W.; Hartwieg, Erika; Horvitz, H. Robert; Jorgensen, Erik M.

    2000-01-01

    Synaptojanin is a polyphosphoinositide phosphatase that is found at synapses and binds to proteins implicated in endocytosis. For these reasons, it has been proposed that synaptojanin is involved in the recycling of synaptic vesicles. Here, we demonstrate that the unc-26 gene encodes the Caenorhabditis elegans ortholog of synaptojanin. unc-26 mutants exhibit defects in vesicle trafficking in several tissues, but most defects are found at synaptic termini. Specifically, we observed defects in ...

  1. Concentration-Independent Spontaneously Forming Biomimetric Vesicles

    Science.gov (United States)

    Nieh, M.-P.; Harroun, T. A.; Raghunathan, V. A.; Glinka, C. J.; Katsaras, J.

    2003-10-01

    In this Letter we present small-angle neutron scattering data from a biomimetic system composed of the phospholipids dimyristoyl and dihexanoyl phosphorylcholine (DMPC and DHPC, respectively). Doping DMPC-DHPC multilamellar vesicles with either the negatively charged lipid dimyristoyl phosphorylglycerol (DMPG, net charge -1) or the divalent cation, calcium (Ca2+), leads to the spontaneous formation of energetically stabilized monodisperse unilamellar vesicles whose radii are concentration independent and in contrast with previous experimental observations.

  2. Cellular Phenotype and Extracellular Vesicles: Basic and Clinical Considerations

    OpenAIRE

    Quesenberry, Peter J.; Goldberg, Laura R.; Aliotta, Jason M.; Mark S Dooner; Pereira, Mandy G.; Wen, Sicheng; Camussi, Giovanni

    2014-01-01

    Early work on platelet and erythrocyte vesicles interpreted the phenomena as a discard of material from cells. Subsequently, vesicles were studied as possible vaccines and, most recently, there has been a focus on the effects of vesicles on cell fate. Recent studies have indicated that extracellular vesicles, previously referred to as microvesicles or exosomes, have the capacity to change the phenotype of neighboring cells. Extensive work has shown that vesicles derived from either the lung o...

  3. Nicotinic acetylcholine receptor and the structural basis of neuromuscular transmission: insights from Torpedo postsynaptic membranes.

    Science.gov (United States)

    Unwin, Nigel

    2013-11-01

    The nicotinic acetylcholine (ACh) receptor, at the neuromuscular junction, is a neurotransmitter-gated ion channel that has been fine-tuned through evolution to transduce a chemical signal into an electrical signal with maximum efficiency and speed. It is composed from three similar and two identical polypeptide chains, arranged in a ring around a narrow membrane pore. Central to the design of this assembly is a hydrophobic gate in the pore, more than 50 Å away from sites in the extracellular domain where ACh binds. Although the molecular properties of the receptor have been explored intensively over the last few decades, only recently have structures emerged revealing its complex architecture and illuminating how ACh entering the binding sites opens the distant gate. Postsynaptic membranes isolated from the (muscle-derived) electric organ of the Torpedo ray have underpinned most of the structural studies: the membranes form tubular vesicles having receptors arranged on a regular surface lattice, which can be imaged directly in frozen physiological solutions. Advances in electron crystallographic techniques have also been important, enabling analysis of the closed- and open-channel forms of the receptor in unreacted tubes or tubes reacted briefly with ACh. The structural differences between these two forms show that all five subunits participate in a concerted conformational change communicating the effect of ACh binding to the gate, but that three of them (αγ, β and δ) play a dominant role. Flexing of oppositely facing pore-lining α-helices is the principal motion determining the closed/open state of the gate. These results together with the findings of biochemical, biophysical and other structural studies allow an integrated description of the receptor and of its mode of action at the synapse.

  4. Hierarchical unilamellar vesicles of controlled compositional heterogeneity.

    Directory of Open Access Journals (Sweden)

    Maik Hadorn

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

  5. Elastic energy of polyhedral bilayer vesicles.

    Science.gov (United States)

    Haselwandter, Christoph A; Phillips, Rob

    2011-06-01

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

  6. Endothelial Extracellular Vesicles-Promises and Challenges.

    Science.gov (United States)

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

    2017-01-01

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

  7. Extracellular vesicles: Pharmacological modulators of the peripheral and central signals governing obesity.

    Science.gov (United States)

    Milbank, Edward; Martinez, M Carmen; Andriantsitohaina, Ramaroson

    2016-01-01

    Obesity and its metabolic resultant dysfunctions such as insulin resistance, hyperglycemia, dyslipidemia and hypertension, grouped as the "metabolic syndrome", are chronic inflammatory disorders that represent one of the most severe epidemic health problems. The imbalance between energy intake and expenditure, leading to an excess of body fat and an increase of cardiovascular and diabetes risks, is regulated by the interaction between central nervous system (CNS) and peripheral signals in order to regulate behavior and finally, the metabolism of peripheral organs. At present, pharmacological treatment of obesity comprises actions in both CNS and peripheral organs. In the last decades, the extracellular vesicles have emerged as participants in many pathophysiological regulation processes. Whether used as biomarkers, targets or even tools, extracellular vesicles provided some promising effects in the treatment of a large variety of diseases. Extracellular vesicles are released by cells from the plasma membrane (microvesicles) or from multivesicular bodies (exosomes) and contain lipids, proteins and nucleic acids, such as DNA, protein coding, and non-coding RNAs. Owing to their composition, extracellular vesicles can (i) activate receptors at the target cell and then, the subsequent intracellular pathway associated to the specific receptor; (ii) transfer molecules to the target cells and thereby change their phenotype and (iii) be used as shuttle of drugs and, thus, to carry specific molecules towards specific cells. Herein, we review the impact of extracellular vesicles in modulating the central and peripheral signals governing obesity. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation

    Science.gov (United States)

    Bobrie, Angélique; Colombo, Marina; Krumeich, Sophie; Raposo, Graça; Théry, Clotilde

    2012-01-01

    Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane. Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate. Here, using Rab27a inhibition to modulate exosome secretion, we show the existence of at least 2 distinct populations of vesicles after purification by classical ultracentrifugation from mouse tumor cell conditioned medium. Rab27a inhibition lead to decreased vesicular secretion of some conventional markers of exosomes (CD63, Tsg101, Alix and Hsc70) but did not affect secretion of others (CD9 and Mfge8). By electron microscopy, CD9 was observed on vesicles of various sizes, ranging from 30 nm to more than 150 nm in diameter. Flotation onto sucrose gradients showed different proportions of CD63, CD9 and Mfge8 not only in fractions of densities classically described for exosomes (around 1.15 g/ml) but also in fractions of densities over 1.20 g/ml, indicating the presence of heterogenous vesicle populations. CD9 and Mfge8 were also found in large vesicles pelleted at low speed and can thus not be considered as specific components of endosome-derived vesicles. We propose that the most commonly used protocols for exosome preparations co-purify vesicles from endosomal and other origins, possibly the plasma membrane. Future work will be required to improve techniques for accurate purification and characterization of the different populations of extracellular vesicles. PMID:24009879

  9. Phospholipase C activity affinity purifies with the Torpedo nicotinic acetylcholine receptor.

    Science.gov (United States)

    Labriola, Jonathan M; daCosta, Corrie J B; Wang, Shuzhi; Figeys, Daniel; Smith, Jeffrey C; Sturgeon, R Michel; Baenziger, John E

    2010-04-02

    Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function.

  10. Isolation and characterization of platelet-derived extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Maria T. Aatonen

    2014-08-01

    Full Text Available Background: Platelet-derived extracellular vesicles (EVs participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Methods: Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS or Ca2+ ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. Results: The main EV populations were 100–250 nm and over 90% were <500 nm irrespective of the activation. However, activation pathways differentially regulated the quantity and the quality of EVs, which also formed constitutively. Thrombogenic activation was the most potent physiological EV-generator. LPS was a weak inducer of EVs, which had a selective protein content from the thrombogenic EVs. Ca2+ ionophore generated a large population of protein-poor and unselectively packed EVs. By proteomic analysis, EVs were highly heterogeneous after the different activations and between the vesicle subpopulations. Conclusions: Although platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100–250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As

  11. 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.

  12. A Stem Cell-Derived Platform for Studying Single Synaptic Vesicles in Dopaminergic Synapses.

    Science.gov (United States)

    Gu, Haigang; Lazarenko, Roman M; Koktysh, Dmitry; Iacovitti, Lorraine; Zhang, Qi

    2015-08-01

    The exocytotic release of dopamine is one of the most characteristic but also one of the least appreciated processes in dopaminergic neurotransmission. Fluorescence imaging has yielded rich information about the properties of synaptic vesicles and the release of neurotransmitters in excitatory and inhibitory neurons. In contrast, imaging-based studies for in-depth understanding of synaptic vesicle behavior in dopamine neurons are lagging largely because of a lack of suitable preparations. Midbrain culture has been one of the most valuable preparations for the subcellular investigation of dopaminergic transmission; however, the paucity and fragility of cultured dopaminergic neurons limits their use for live cell imaging. Recent developments in stem cell technology have led to the successful production of dopamine neurons from embryonic or induced pluripotent stem cells. Although the dopaminergic identity of these stem cell-derived neurons has been characterized in different ways, vesicle-mediated dopamine release from their axonal terminals has been barely assessed. We report a more efficient procedure to reliably generate dopamine neurons from embryonic stem cells, and it yields more dopamine neurons with more dopaminergic axon projections than midbrain culture does. Using a collection of functional measurements, we show that stem cell-derived dopamine neurons are indistinguishable from those in midbrain culture. Taking advantage of this new preparation, we simultaneously tracked the turnover of hundreds of synaptic vesicles individually using pH-sensitive quantum dots. By doing so, we revealed distinct fusion kinetics of the dopamine-secreting vesicles, which is consistent within both preparations. ©AlphaMed Press.

  13. Cholesterol regulates multiple forms of vesicle endocytosis at a mammalian central synapse.

    Science.gov (United States)

    Yue, Hai-Yuan; Xu, Jianhua

    2015-07-01

    Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from non-specific effects after cholesterol manipulation. Furthermore, it remains unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase or methyl-β-cyclodextrin impaired three different forms of endocytosis, including slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca(2+) channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of methyl-β-cyclodextrin reduced exocytosis, mainly by decreasing the readily releasable pool and the vesicle replenishment after readily releasable pool depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses. © 2015 International Society for Neurochemistry.

  14. Extracellular Vesicles in Metabolic Syndrome.

    Science.gov (United States)

    Martínez, M Carmen; Andriantsitohaina, Ramaroson

    2017-05-12

    Metabolic syndrome defines a cluster of interrelated risk factors for cardiovascular disease and diabetes mellitus. These factors include metabolic abnormalities, such as hyperglycemia, elevated triglyceride levels, low high-density lipoprotein cholesterol levels, high blood pressure, and obesity, mainly central adiposity. In this context, extracellular vesicles (EVs) may represent novel effectors that might help to elucidate disease-specific pathways in metabolic disease. Indeed, EVs (a terminology that encompasses microparticles, exosomes, and apoptotic bodies) are emerging as a novel mean of cell-to-cell communication in physiology and pathology because they represent a new way to convey fundamental information between cells. These microstructures contain proteins, lipids, and genetic information able to modify the phenotype and function of the target cells. EVs carry specific markers of the cell of origin that make possible monitoring their fluctuations in the circulation as potential biomarkers inasmuch their circulating levels are increased in metabolic syndrome patients. Because of the mixed components of EVs, the content or the number of EVs derived from distinct cells of origin, the mode of cell stimulation, and the ensuing mechanisms for their production, it is difficult to attribute specific functions as drivers or biomarkers of diseases. This review reports recent data of EVs from different origins, including endothelial, smooth muscle cells, macrophages, hepatocytes, adipocytes, skeletal muscle, and finally, those from microbiota as bioeffectors of message, leading to metabolic syndrome. Depicting the complexity of the mechanisms involved in their functions reinforce the hypothesis that EVs are valid biomarkers, and they represent targets that can be harnessed for innovative therapeutic approaches. © 2017 American Heart Association, Inc.

  15. Secretion of goblet cell serine proteinase, ingobsin, is stimulated by vasoactive intestinal polypeptide and acetylcholine

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Nexø, Ebba

    1987-01-01

    Ingobsin is localized to the intestinal goblet cells in the rat and in man. In the present study, we investigated the effect of vasoactive intestinal polypeptide (VIP) and acetylcholine on the secretion of ingobsin from the proximal duodenum. Intravenous infusion of VIP or acetylcholine increased...... the concentration of ingobsin in duodenal secretion, while the concentration in the duodenum was unchanged. Simultaneous infusion of VIP and acetylcholine increased the concentration of ingobsin in duodenal secretion and decreased the concentration of ingobsin in the duodenum. This study demonstrates that secretion...... of ingobsin from the proximal duodenum is exocrine and can be stimulated by VIP and acetylcholine....

  16. Back to the future: Rational maps for exploring acetylcholine receptor space and time.

    Science.gov (United States)

    Tessier, Christian J G; Emlaw, Johnathon R; Cao, Zhuo Qian; Pérez-Areales, F Javier; Salameh, Jean-Paul J; Prinston, Jethro E; McNulty, Melissa S; daCosta, Corrie J B

    2017-11-01

    Global functions of nicotinic acetylcholine receptors, such as subunit cooperativity and compatibility, likely emerge from a network of amino acid residues distributed across the entire pentameric complex. Identification of such networks has stymied traditional approaches to acetylcholine receptor structure and function, likely due to the cryptic interdependency of their underlying amino acid residues. An emerging evolutionary biochemistry approach, which traces the evolutionary history of acetylcholine receptor subunits, allows for rational mapping of acetylcholine receptor sequence space, and offers new hope for uncovering the amino acid origins of these enigmatic properties. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Piccolo Promotes Vesicle Replenishment at a Fast Central Auditory Synapse

    Directory of Open Access Journals (Sweden)

    Tanvi Butola

    2017-10-01

    Full Text Available Piccolo and Bassoon are the two largest cytomatrix of the active zone (CAZ proteins involved in scaffolding and regulating neurotransmitter release at presynaptic active zones (AZs, but have long been discussed as being functionally redundant. We employed genetic manipulation to bring forth and segregate the role of Piccolo from that of Bassoon at central auditory synapses of the cochlear nucleus—the endbulbs of Held. These synapses specialize in high frequency synaptic transmission, ideally poised to reveal even subtle deficits in the regulation of neurotransmitter release upon molecular perturbation. Combining semi-quantitative immunohistochemistry, electron microscopy, and in vitro and in vivo electrophysiology we first studied signal transmission in Piccolo-deficient mice. Our analysis was not confounded by a cochlear deficit, as a short isoform of Piccolo (“Piccolino” present at the upstream ribbon synapses of cochlear inner hair cells (IHC, is unaffected by the mutation. Disruption of Piccolo increased the abundance of Bassoon at the AZs of endbulbs, while that of RIM1 was reduced and other CAZ proteins remained unaltered. Presynaptic fiber stimulation revealed smaller amplitude of the evoked excitatory postsynaptic currents (eEPSC, while eEPSC kinetics as well as miniature EPSCs (mEPSCs remained unchanged. Cumulative analysis of eEPSC trains indicated that the reduced eEPSC amplitude of Piccolo-deficient endbulb synapses is primarily due to a reduced readily releasable pool (RRP of synaptic vesicles (SV, as was corroborated by a reduction of vesicles at the AZ found on an ultrastructural level. Release probability seemed largely unaltered. Recovery from short-term depression was slowed. We then performed a physiological analysis of endbulb synapses from mice which, in addition to Piccolo deficiency, lacked one functional allele of the Bassoon gene. Analysis of the double-mutant endbulbs revealed an increase in release probability

  18. A two phase field model for tracking vesicle-vesicle adhesion.

    Science.gov (United States)

    Gu, Rui; Wang, Xiaoqiang; Gunzburger, Max

    2016-11-01

    A multi-phase-field model for simulating the adhesion between two vesicles is constructed. Two phase field functions are introduced to simulate each of the two vesicles. An energy model is defined which accounts for the elastic bending energy of each vesicle and the contact potential energy between the two vesicles; the vesicle volume and surface area constraints are imposed using a penalty method. Numerical results are provided to verify the efficacy of our model and to provide visual illustrations of the different types of contact. The method can be adjusted to solve endocytosis problems by modifying the bending rigidity coefficients of the two elastic bending energies. The method can also be extended to simulate multi-cell adhesions, one example of which is erythrocyte rouleaux. A comparison with laboratory observations demonstrates the effectiveness of the multi-phase field approach.

  19. Insights into the self-reproduction of oleate vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Stano, P [' Enrico Fermi' Centre, Compendio Viminale, 00184 Rome (Italy); Wehrli, E [Electron Microscopy Centre (EMEZ), Applied Physics Institute, ETH Hoenggerberg, 8093 Zurich (Switzerland); Luisi, P L [Biology Department, University of RomaTre, Viale Marconi 446, 00146 Rome (Italy)

    2006-08-23

    In view of the importance of vesicles as models for early cells, several groups have started work looking for conditions under which vesicles can undergo growth and division. Evidence for growth and division has been obtained with the help of ferritin-labelled vesicles; furthermore, it has been shown that in such processes the vesicle size distribution is largely conserved. In both cases, the data suggest that the process under study is mainly characterized by vesicle growth and eventually division into daughter vesicles. However, direct evidence for vesicle division has not been obtained. In this paper, mostly based on freeze-fracture electron microscopy, we describe conditions under which for the first time division intermediates can be trapped in the form of twin vesicles. This finding, together with supporting dynamic light scattering and fluorescence investigations, permits us to establish some additional points in the mechanism of vesicle self-reproduction.

  20. Nucleus accumbens core acetylcholine is preferentially activated during acquisition of drug- vs food-reinforced behavior.

    Science.gov (United States)

    Crespo, Jose A; Stöckl, Petra; Zorn, Katja; Saria, Alois; Zernig, Gerald

    2008-12-01

    Acquisition of drug-reinforced behavior is accompanied by a systematic increase of release of the neurotransmitter acetylcholine (ACh) rather than dopamine, the expected prime reward neurotransmitter candidate, in the nucleus accumbens core (AcbC), with activation of both muscarinic and nicotinic ACh receptors in the AcbC by ACh volume transmission being necessary for the drug conditioning. The present findings suggest that the AcbC ACh system is preferentially activated by drug reinforcers, because (1) acquisition of food-reinforced behavior was not paralleled by activation of ACh release in the AcbC whereas acquisition of morphine-reinforced behavior, like that of cocaine or remifentanil (tested previously), was, and because (2) local intra-AcbC administration of muscarinic or nicotinic ACh receptor antagonists (atropine or mecamylamine, respectively) did not block the acquisition of food-reinforced behavior whereas acquisition of drug-reinforced behavior had been blocked. Interestingly, the speed with which a drug of abuse distributed into the AcbC and was eliminated from the AcbC determined the size of the AcbC ACh signal, with the temporally more sharply delineated drug stimulus producing a more pronounced AcbC ACh signal. The present findings suggest that muscarinic and nicotinic ACh receptors in the AcbC are preferentially involved during reward conditioning for drugs of abuse vs sweetened condensed milk as a food reinforcer.

  1. Noble gases released by vacuum crushing of EETA 79001 glass

    Science.gov (United States)

    Wiens, R. C.

    1988-01-01

    An EETA 79001 glass sample was crushed in a vacuum to observe the gases released. About 15 pct of the total gas concentrations were a mixture of a small amount of SPB-type gas with larger proportions of another air-like component. Less than 5 pct of the SPB gas was released by crushing, while 36-40 pct of the EETV (indigenous) gas was crush-released. The results are consistent with a siting of the EETV component in 10-100 micron vesicles seen in the glass. It is suggested that the SPB component is either in vesicles less than 6 microns in diameter or is primarily sited elsewhere.

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

    Science.gov (United States)

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

    2017-10-01

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

  3. Extracellular vesicles as therapeutic tools in cardiovascular diseases

    Directory of Open Access Journals (Sweden)

    Audrey eFleury

    2014-08-01

    Full Text Available Extracellular vesicles (EVs, including microvesicles (MVs and exosomes, are small vesicles secreted from a wide variety of cells. Whereas MVs are particles released by the outward budding of the plasma membrane, exosomes are derived from endocytic compartments. Secretion of EVs can be enhanced by specific stimuli, and increased plasma circulating levels of EVs have been correlated with pathophysiological situations.MVs, already present in the blood of healthy individuals, are considerably elevated in several cardiovascular diseases associated with inflammation, suggesting that they can mediate deleterious effects such as endothelial dysfunction or thrombosis. Nonetheless, very recent studies also demonstrate that MVs may act as biological information vectors transferring proteins or genetic material to maintain cell homeostasis, favor cell repair or even promote angiogenesis. Additionally, exosomes have also been shown to have proangiogenic and cardioprotective properties. These beneficial effects therefore reveal the potential therapeutical use of EVs in the field of cardiovascular medicine and regenerative therapy.In this review, we will provide an update of cellular processes modulated by EVs of specific interest in the treatment of cardiovascular pathologies. A special focus will be made on the morphogen sonic hedgehog (Shh associated with EVs (EVsShh+, which have been shown to mediate many pro-angiogenic effects. In addition to offer a potential source of cardiovascular markers, therapeutical potential of EVs reveal exciting opportunities to deliver specific agents by non-immunogenic means to cardiovascular system.

  4. Facile preparation of salivary extracellular vesicles for cancer proteomics

    Science.gov (United States)

    Sun, Yan; Xia, Zhijun; Shang, Zhi; Sun, Kaibo; Niu, Xiaomin; Qian, Liqiang; Fan, Liu-Yin; Cao, Cheng-Xi; Xiao, Hua

    2016-04-01

    Extracellular vesicles (EVs) are membrane surrounded structures released by cells, which have been increasingly recognized as mediators of intercellular communication. Recent reports indicate that EVs participate in important biological processes and could serve as potential source for cancer biomarkers. As an attractive EVs source with merit of non-invasiveness, human saliva is a unique medium for clinical diagnostics. Thus, we proposed a facile approach to prepare salivary extracellular vesicles (SEVs). Affinity chromatography column combined with filter system (ACCF) was developed to efficiently remove the high abundant proteins and viscous interferences of saliva. Protein profiling in the SEVs obtained by this strategy was compared with conventional centrifugation method, which demonstrated that about 70% more SEVs proteins could be revealed. To explore its utility for cancer proteomics, we analyzed the proteome of SEVs in lung cancer patients and normal controls. Shotgun proteomic analysis illustrated that 113 and 95 proteins have been identified in cancer group and control group, respectively. Among those 63 proteins that have been consistently discovered only in cancer group, 12 proteins are lung cancer related. Our results demonstrated that SEVs prepared through the developed strategy are valuable samples for proteomics and could serve as a promising liquid biopsy for cancer.

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

    Science.gov (United States)

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

    2012-01-01

    Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These 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. PMID:23271954

  6. 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.

  7. Plasmonic Vesicles of Amphiphilic Nanocrystals: Optically Active Multifunctional Platform for Cancer Diagnosis and Therapy.

    Science.gov (United States)

    Song, Jibin; Huang, Peng; Duan, Hongwei; Chen, Xiaoyuan

    2015-09-15

    Vesicular structures with compartmentalized, water-filled cavities, such as liposomes of natural and synthetic amphiphiles, have tremendous potential applications in nanomedicine. When block copolymers self-assemble, the result is polymersomes with tailored structural properties and built-in releasing mechanisms, controlled by stimuli-responsive polymer building blocks. More recently, chemists are becoming interested in multifunctional hybrid vesicles containing inorganic nanocrystals with unique optical, electronic, and magnetic properties. In this Account, we review our recent progress in assembling amphiphilic plasmonic nanostructures to create a new class of multifunctional hybrid vesicles and applying them towards cancer diagnosis and therapy. Localized surface plasmon resonance (LSPR) gives plasmonic nanomaterials a unique set of optical properties that are potentially useful for both biosensing and nanomedicine. For instance, the strong light scattering at their LSPR wavelength opens up the applications of plasmonic nanostructures in single particle plasmonic imaging. Their superior photothermal conversion properties, on the other hand, make them excellent transducers for photothermal ablation and contrast agents for photoacoustic imaging. Of particular note for ultrasensitive detection is that the confined electromagnetic field resulting from excitation of LSPR can give rise to highly efficient surface enhanced Raman scattering (SERS) for molecules in close proximity. We have explored several ways to combine well-defined plasmonic nanocrystals with amphiphilic polymer brushes of diverse chemical functionalities. In multiple systems, we have shown that the polymer grafts impart amphiphilicity-driven self-assembly to the hybrid nanoparticles. This has allowed us to synthesize well-defined vesicles in which we have embedded plasmonic nanocrystals in the shell of collapsed hydrophobic polymers. The hydrophilic brushes extend into external and interior aqueous

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

    Science.gov (United States)

    Wang, Xi

    peptide activities. The study of vesicle rupture mechanics and mechanical properties provide a means of understanding triggered release of internal payload from vesicular structures. POPC vesicles were also deposited on graphene; a transparent and highly conductive electrode. A combination method of diffusion bonding and template-stripping was used to prepare metal surfaces for graphene growth without concerns of outgassing, thermal and chemical compatibility. Continuous LBM formed on graphene-single crystal Cu, while tubular features with 120°C patterns formed on graphene-Cu foil, indicating the step edge of Cu below graphene may also guide the assembly of tubular LBM features on graphene.

  9. Astrocytic Vesicle Mobility in Health and Disease

    Directory of Open Access Journals (Sweden)

    Robert Zorec

    2013-05-01

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

  10. EXTRACELLULAR VESICLES: CLASSIFICATION, FUNCTIONS AND CLINICAL RELEVANCE

    Directory of Open Access Journals (Sweden)

    A. V. Oberemko

    2014-12-01

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

  11. Acetylcholine synthesis and possible functions during sea urchin development

    Directory of Open Access Journals (Sweden)

    C Angelini

    2009-06-01

    Full Text Available Cholinergic neurotransmitter system molecules were found to play a role during fertilisation and early cell cycles of a large number of invertebrate and vertebrate organisms. In this study, we investigated the presence and possible function of choline acetyltransferase (ChAT, the biosynthetic enzyme of acetylcholine in gametes of the sea urchin, Paracentrotus lividus, through localisation and functional studies. ChAT-like molecules were detected in oocytes, mature eggs and zygotes with indirect immunofluorescence methods. Positive immunoreactivity was found in the ovarian egg cytoplasm and surface as well as at the zygote surface. This suggests the eggs' capacity to autonomously synthesise acetylcholine (ACh, the signal molecule of the cholinergic system. Acetylcholinesterase (AChE, the lytic enzyme of acetylcholine was also found in ovarian eggs, with a similar distribution; however, it disappeared after fertilisation. Ultrastructural ChAT localisation in sperms, which was carried out with the immuno-gold method, showed immunoreactivity in the acrosome of unreacted sperms and at the head surface of reacted sperms. In order to verify a functional role of ACh during fertilization and sea urchin development, in vivo experiments were performed. Exposure of the eggs before fertilisation to 1 mM ACh + 1 ?M eserine caused an incomplete membrane depolarisation and consequently enhanced polyspermy, while lower concentrations of ACh caused developmental anomalies. The exposure of zygotes to 0,045 AChE Units/mL of sea water caused developmental anomalies as well, in 50% of the embryos. Altogether, these findings and other previously obtained results, suggest that the cholinergic system may subserve two different tasks during development, according to which particular type of ACh receptor is active during each temporal window. The first function, taking place in the course of fertilisation is a result of autonomously synthesised ACh in sperms, while the

  12. PGE2/EP4Signaling Controls the Transfer of the Mammary Stem Cell State by Lipid Rafts in Extracellular Vesicles.

    Science.gov (United States)

    Lin, Meng-Chieh; Chen, Shih-Yin; Tsai, Ho-Min; He, Pei-Lin; Lin, Yen-Chun; Herschman, Harvey; Li, Hua-Jung

    2017-02-01

    Prostaglandin E 2 (PGE 2 )-initiated signaling contributes to stem cell homeostasis and regeneration. However, it is unclear how PGE 2 signaling controls cell stemness. This study identifies a previously unknown mechanism by which PGE 2 /prostaglandin E receptor 4 (EP 4 ) signaling regulates multiple signaling pathways (e.g., PI3K/Akt signaling, TGFβ signaling, Wnt signaling, EGFR signaling) which maintain the basal mammary stem cell phenotype. A shift of basal mammary epithelial stem cells (MaSCs) from a mesenchymal/stem cell state to a non-basal-MaSC state occurs in response to prostaglandin E receptor 4 (EP 4 ) antagonism. EP 4 antagonists elicit release of signaling components, by controlling their trafficking into extracellular vesicles/exosomes in a lipid raft/caveolae-dependent manner. Consequently, EP 4 antagonism indirectly inactivates, through induced extracellular vesicle/exosome release, pathways required for mammary epithelial stem cell homeostasis, e.g. canonical/noncanonical Wnt, TGFβ and PI3K/Akt pathways. EP 4 antagonism causes signaling receptors and signaling components to shift from non-lipid raft fractions to lipid raft fractions, and to then be released in EP 4 antagonist-induced extracellular vesicles/exosomes, resulting in the loss of the stem cell state by mammary epithelial stem cells. In contrast, luminal mammary epithelial cells can acquire basal stem cell properties following ingestion of EP 4 antagonist-induced stem cell extracellular vesicles/exosomes, and can then form mammary glands. These findings demonstrate that PGE 2 /EP 4 signaling controls homeostasis of mammary epithelial stem cells through regulating extracellular vesicle/exosome release. Reprogramming of mammary epithelial cells can result from EP 4 -mediated stem cell property transfer by extracellular vesicles/exosomes containing caveolae-associated proteins, between mammary basal and luminal epithelial cells. Stem Cells 2017;35:425-444. © 2016 The Authors STEM CELLS

  13. 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

    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...... 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...... 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...

  14. Functional interaction between Lypd6 and nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Arvaniti, Maria; Jensen, Majbrit M; Soni, Neeraj

    2016-01-01

    Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain-containing 6 (Lypd6) with n...... findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain, and that Lypd6 is dysregulated by nicotine exposure during early development. Regulatory proteins of the Lynx family modulate the function of nicotinic receptors (nAChRs). We report for the first time that the Lynx...

  15. Structures of acetylcholine picrate and methoxycarbonylcholine picrate hemihydrate

    DEFF Research Database (Denmark)

    Frydenvang, Karla Andrea; Grønborg, L; Jensen, B

    1988-01-01

    Acetylcholine picrate, C7H16NO2+.C6H2N3-O7-, Mr = 374.3, orthorhombic, Pbca, at 105 K: a = 18.799 (4), b = 7.726 (2), c = 22.878 (4) A, V = 3323 (2) A3, Z = 8, Dm(295 K, flotation) = 1.44, D chi(105 K) = 1.496 Mg m-3, mu(Mo K alpha) = 0.120 mm-1, F(000) = 1568, m.p. (hot-stage microscope) 381-382...

  16. Nicotinic acetylcholine receptors: specific antibodies and functions in humoral immunity

    Directory of Open Access Journals (Sweden)

    M. V. Skok,

    2013-12-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are ligand-gated ion channels initially discovered in muscles and neurons and further found in many non-excitable cells. The present review summarizes the results of studies performed in the Department of Molecular Immunology during the last decade and concerning the structure and functions of nAChRs in B lymphocytes and in mitochondria, as well as the role of nAChR-specific antibodies in the develop­ment of neurodegenerative disorders like Alzheimer disease.

  17. Functionally polymerized surfactant vesicles: synthesis and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Tundo, P.; Kippenberger, D.J.; Klahn, P.L.; Prieto, N.E.; Fendler, J.H.

    1982-01-27

    Bis(2-(10-undecenoyloxycarbony bromide, bis(2-(10-undecenoyloxycarbony (2-hydroxyethyl)methylammonium bromide, bis(2-(10-undecenoyloxycarbony acid, bis(2-(10-undecenoyloxycarbony allylbis(2-dodecanoyloxycarbon bromide, and dimethyl-n-hexadecyl (10-(p-vin decyl)ammonium bromide have been synthesized. The predominantly single compartment bilayer vesicles formed from these surfactants could be polymerized either by exposure to ultraviolet irradiation or by the use of azoisobutyronitrile as an initiator. The presence of vesicles (unpolymerized and polymeric) has been demonstrated by electron micrography, H/sup 1/ NMR, gel filtration, phase transition, turbidity changes, substrate entrapment, and permeability. Polymerized vesicles are considerably more stable and less permeable and have reduced rates of turbidity changes compared to their unpolymerized counterparts. 19 references.

  18. Directed vesicle transport by diffusio-osmosis

    Science.gov (United States)

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

    2015-04-01

    We present a study on surfactant vesicles that spontaneously move towards an oil droplet that is deposited on a glass substrate. Tracer particles in the surfactant solution show that the motion is not self-propelled: the vesicles are entrained by a macroscopic hydrodynamic flow. Measurements of the flow velocity suggest that the flow is of diffusio-osmotic nature. The surfactant is observed to move into the oil phase which creates a gradient in ion concentration in the vicinity of the droplet. As the diffusion coefficients of the surfactant's co- and counter-ions differ, a charge separation takes place and an electric field arises. This electric field then generates a hydrodynamic flow along the charged glass substrate in which the vesicles are entrained.

  19. 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.

  20. Mesenchymal stem cell-derived extracellular vesicles for renal repair

    Science.gov (United States)

    Nargesi, Arash Aghajani; Lerman, Lilach O.; Eirin, Alfonso

    2017-01-01

    Transplantation of autologous mesenchymal stem cells (MSCs) has been shown to attenuate renal injury and dysfunction in several animal models, and its efficacy is currently being tested in clinical trials for patients with renal disease. Accumulating evidence indicates that MSCs release extracellular vesicles (EVs) that deliver genes, microRNAs and proteins to recipient cells, acting as mediators of MSC paracrine actions. In this context, it is critical to characterize the MSC-derived EV cargo to elucidate their potential contribution to renal repair. In recent years, researchers have performed high-throughput sequencing and proteomic analysis to detect and identify genes, microRNAs, and proteins enriched in MSC-derived EVs. The present review summarizes the current knowledge of the MSC-derived EV secretome to shed light into the mechanisms mediating MSC renal repair, and discusses preclinical and clinical studies testing the efficacy of MSC-derived EVs for treating renal disease. PMID:28403795

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

    Directory of Open Access Journals (Sweden)

    Campbell Kevin P

    2007-08-01

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

  2. Electrohydrodynamics of a compound vesicle under an AC electric field.

    Science.gov (United States)

    Sinha, Kumari Priti; Thaokar, Rochish M

    2017-07-12

    Compound vesicles are relevant as simplified models for biological cells as well as in technological applications such as drug delivery. Characterization of these compound vesicles, especially the inner vesicle, remains a challenge. Similarly their response to electric field assumes importance in light of biomedical applications such as electroporation. Fields lower than that required for electroporation cause electrodeformation in vesicles and can be used to characterize their mechanical and electrical properties. A theoretical analysis of the electrohydrodynamics of a compound vesicle with outer vesicle of radius R o and an inner vesicle of radius [Formula: see text], is presented. A phase diagram for the compound vesicle is presented and elucidated using detailed plots of electric fields, free charges and electric stresses. The electrohydrodynamics of the outer vesicle in a compound vesicle shows a prolate-sphere and prolate-oblate-sphere shape transitions when the conductivity of the annular fluid is greater than the outer fluid, and vice-versa respectively, akin to single vesicle electrohydrodynamics reported in the literature. The inner vesicle in contrast shows sphere-prolate-sphere and sphere-prolate-oblate-sphere transitions when the inner fluid conductivity is greater and smaller than the annular fluid, respectively. Equations and methodology are provided to determine the bending modulus and capacitance of the outer as well as the inner membrane, thereby providing an easy way to characterize compound vesicles and possibly biological cells.

  3. Electrohydrodynamics of a compound vesicle under an AC electric field

    Science.gov (United States)

    Priti Sinha, Kumari; Thaokar, Rochish M.

    2017-07-01

    Compound vesicles are relevant as simplified models for biological cells as well as in technological applications such as drug delivery. Characterization of these compound vesicles, especially the inner vesicle, remains a challenge. Similarly their response to electric field assumes importance in light of biomedical applications such as electroporation. Fields lower than that required for electroporation cause electrodeformation in vesicles and can be used to characterize their mechanical and electrical properties. A theoretical analysis of the electrohydrodynamics of a compound vesicle with outer vesicle of radius R o and an inner vesicle of radius λ {{R}o} , is presented. A phase diagram for the compound vesicle is presented and elucidated using detailed plots of electric fields, free charges and electric stresses. The electrohydrodynamics of the outer vesicle in a compound vesicle shows a prolate-sphere and prolate-oblate-sphere shape transitions when the conductivity of the annular fluid is greater than the outer fluid, and vice-versa respectively, akin to single vesicle electrohydrodynamics reported in the literature. The inner vesicle in contrast shows sphere-prolate-sphere and sphere-prolate-oblate-sphere transitions when the inner fluid conductivity is greater and smaller than the annular fluid, respectively. Equations and methodology are provided to determine the bending modulus and capacitance of the outer as well as the inner membrane, thereby providing an easy way to characterize compound vesicles and possibly biological cells.

  4. A "release" protocol for isothermal titration calorimetry.

    Science.gov (United States)

    Heerklotz, H H; Binder, H; Epand, R M

    1999-05-01

    Isothermal titration calorimetry (ITC) has become a standard method for investigating the binding of ligands to receptor molecules or the partitioning of solutes between water and lipid vesicles. Accordingly, solutes are mixed with membranes (or ligands with receptors), and the subsequent heats of incorporation (or binding) are measured. In this paper we derive a general formula for modeling ITC titration heats in both binding and partitioning systems that allows for the modeling of the classic incorporation or binding protocols, as well as of new protocols assessing the release of solute from previously solute-loaded vesicles (or the dissociation of ligand/receptor complexes) upon dilution. One major advantage of a simultaneous application of the incorporation/binding and release protocols is that it allows for the determination of whether a ligand is able to access the vesicle interior within the time scale of the ITC experiment. This information cannot be obtained from a classical partitioning experiment, but it must be known to determine the partition coefficient (or binding constant and stochiometry) and the transfer enthalpy. The approach is presented using the partitioning of the nonionic detergent C12EO7 to palmitoyloleoylphosphatidylcholine vesicles. The release protocol could also be advantageous in the case of receptors that are more stable in the ligand-saturated rather than the ligand-depleted state.

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

    OpenAIRE

    Allan, Nick D.; Beveridge, Terry J.

    2003-01-01

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

  6. Platelet extracellular vesicles induce a pro-inflammatory smooth muscle cell phenotype

    OpenAIRE

    Vajen, Tanja; Benedikter, Birke J.; Heinzmann, Alexandra C. A.; Vasina, Elena M; Henskens, Yvonne; PARSONS, Martin; Maguire, Patricia B.; Stassen, Frank R.; Heemskerk, Johan W.M.; Schurgers, Leon J; Koenen, Rory R.

    2017-01-01

    ABSTRACT Extracellular vesicles (EVs) are mediators of cell communication during health and disease, and abundantly released by platelets upon activation or during ageing. Platelet EVs exert modulatory effects on immune and vascular cells. Platelet EVs may modulate the function of vascular smooth muscle cells (SMC). Platelet EVs were isolated from platelet-rich plasma and incubated with SMC in order to assess binding, proliferation, migration and pro-inflammatory phenotype of the cells. Plate...

  7. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - An ISEV position paper

    OpenAIRE

    Mateescu, Bogdan; Kowal, Emma J K; van Balkom, Bas W. M.; Bartel, Sabine; Bhattacharyya, Suvendra N.; Buzás, Edit I.; Buck, Amy H; de Candia, Paola; Chow, Franklin W. N.; Das, Saumya; Driedonks, Tom A. P.; Fernández-Messina, Lola; Haderk, Franziska; Hill, Andrew F.; Jones, Jennifer C.

    2017-01-01

    ABSTRACT The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, ...

  8. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper

    OpenAIRE

    Mateescu, Bogdan; Kowal, Emma; Balkom, Bastiaan Wilhelmus Maria van; Bartel, Sabine; Bhattacharyya, Suvendra N.; Buzás, Edit I.; Buck, Amy H.; de-Candia, Paola; Chow, Franklin W. N.; Das, Saumya; Driedonks, Tom A. P.; Fernández-Messina, Lola; Haderk, Franziska; Hill, Andrew F.; Jones, Jennifer C.

    2017-01-01

    The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and indus...

  9. Modulation of tissue tropism and biological activity of exosomes and other extracellular vesicles : New nanotools for cancer treatment

    NARCIS (Netherlands)

    Kooijmans, Sander A A|info:eu-repo/dai/nl/413754170; Schiffelers, Raymond M.|info:eu-repo/dai/nl/212909509; Zarovni, Natasa; Vago, Riccardo

    2016-01-01

    Exosomes are naturally secreted nanovesicles that have recently aroused a great interest in the scientific and clinical community for their roles in intercellular communication in almost all physiological and pathological processes. These 30–100 nm sized vesicles are released from the cells into the

  10. Imaging of evoked dense-core-vesicle exocytosis in hippocampal neurons reveals long latencies and kiss-and-run fusion events

    OpenAIRE

    Xia, Xiaofeng; Lessmann, Volkmar; Martin, Thomas F. J.

    2008-01-01

    Evoked neuropeptide secretion in the central nervous system occurs slowly, but the basis for slow release is not fully understood. Whereas exocytosis of single synaptic vesicles in neurons and of dense-core vesicles (DCVs) in endocrine cells have been directly visualized, single DCV exocytic events in neurons of the central nervous system have not been previously studied. We imaged DCV exocytosis in primary cultured hippocampal neurons using fluorescent propeptide carg...

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

    Science.gov (United States)

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

    2016-08-01

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

  12. Change in desensitization of cat muscle acetylcholine receptor caused by coexpression of Torpedo acetylcholine receptor subunits in Xenopus oocytes.

    OpenAIRE

    Sumikawa, K; Miledi, R

    1989-01-01

    Cat muscle acetylcholine receptors (AcChoR) expressed in Xenopus oocytes desensitized more slowly than Torpedo electric organ AcChoRs, also expressed in oocytes. To examine the bases for the different degrees of desensitization, cat-Torpedo AcChoR hybrids were formed by injecting oocytes with cat denervated muscle mRNA mixed with a large excess of cloned Torpedo AcChoR subunit mRNAs. Hybrid AcChoRs formed by coinjection of cat muscle mRNA with the Torpedo beta or delta subunit mRNAs desensiti...

  13. Endothelial Cell-derived Extracellular Vesicles Size-dependently Exert Procoagulant Activity Detected by Thromboelastometry.

    Science.gov (United States)

    Holnthoner, Wolfgang; Bonstingl, Cornelia; Hromada, Carina; Muehleder, Severin; Zipperle, Johannes; Stojkovic, Stefan; Redl, Heinz; Wojta, Johann; Schöchl, Herbert; Grillari, Johannes; Weilner, Sylvia; Schlimp, Christoph J

    2017-06-16

    Endothelial cells (ECs) are major modulators of hemostasis by expressing and releasing pro- and anticoagulant mediators into the circulation. Previous studies showed that cultured ECs release procoagulant mediators into cell culture supernatants as evidenced by the reduction of viscoelastic clotting time. This effect was reversed with an anti-tissue factor antibody. Here, we aimed to investigate whether tissue factor (TF) was released by endothelial-derived extracellular vesicles (EVs) and which portion of the released vesicles displays the most prominent procoagulant properties. After stimulation of ECs with tumor-necrosis factor-α (TNF-α) the supernatants of EC cultures were subjected to differential centrifugation steps to collect larger and smaller EVs which were then characterised by nanoparticle tracking analysis (NTA) and flow cytometry. Mixed with fresh human blood and analysed by thromboelastometry EVs exerted a significant procoagulant stimulus, which could be partly reversed by addition of an anti-TF antibody. Moreover, TF activity was confirmed in the centrifuged fractions. In summary, our results provide evidence of the procoagulant potential of smaller and larger endothelial-derived EV fractions detected by thromboelastometry. The observed effect is most likely due to the release of TF-bearing EVs of different dimensions, which are released upon TNF-α stimulation of endothelial cell cultures.

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

    Science.gov (United States)

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

    2017-12-08

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

  15. RIM1α SUMOylation Is Required for Fast Synaptic Vesicle Exocytosis

    Directory of Open Access Journals (Sweden)

    Fatima Girach

    2013-12-01

    Full Text Available The rapid, activity-dependent quantal presynaptic release of neurotransmitter is vital for brain function. The complex process of vesicle priming, fusion, and retrieval is very precisely controlled and requires the spatiotemporal coordination of multiple protein-protein interactions. Here, we show that posttranslational modification of the active zone protein Rab3-interacting molecule 1α (RIM1α by the small ubiquitin-like modifier 1 (SUMO-1 functions as a molecular switch to direct these interactions and is essential for fast synaptic vesicle exocytosis. RIM1α SUMOylation at lysine residue K502 facilitates the clustering of CaV2.1 calcium channels and enhances the Ca2+ influx necessary for vesicular release, whereas non-SUMOylated RIM1α participates in the docking/priming of synaptic vesicles and maintenance of active zone structure. These results demonstrate that SUMOylation of RIM1α is a key determinant of rapid, synchronous neurotransmitter release, and the SUMO-mediated “switching” of RIM1α between binding proteins provides insight into the mechanisms underpinning synaptic function and dysfunction.

  16. Recruitment of resting vesicles into recycling pools supports NMDA receptor-dependent synaptic potentiation in cultured hippocampal neurons

    Science.gov (United States)

    Ratnayaka, Arjuna; Marra, Vincenzo; Bush, Daniel; Burden, Jemima J; Branco, Tiago; Staras, Kevin

    2012-01-01

    Most presynaptic terminals in the central nervous system are characterized by two functionally distinct vesicle populations: a recycling pool, which supports action potential-driven neurotransmitter release via vesicle exocytosis, and a resting pool. The relative proportions of these two pools are highly variable between individual synapses, prompting speculation on their specific relationship, and on the possible functions of the resting pool. Using fluorescence imaging of FM-styryl dyes and synaptophysinI-pHluorin (sypHy) as well as correlative electron microscopy approaches, we show here that Hebbian plasticity-dependent changes in synaptic strength in rat hippocampal neurons can increase the recycling pool fraction at the expense of the resting pool in individual synaptic terminals. This recruitment process depends on NMDA-receptor activation, nitric oxide signalling and calcineurin and is accompanied by an increase in the probability of neurotransmitter release at individual terminals. Blockade of actin-mediated intersynaptic vesicle exchange does not prevent recycling pool expansion demonstrating that vesicle recruitment is intrasynaptic. We propose that the conversion of resting pool vesicles to the functionally recycling pool provides a rapid mechanism to implement long-lasting changes in presynaptic efficacy. PMID:22271866

  17. Extracellular vesicle-mediated transfer of long non-coding RNA ROR modulates chemosensitivity in human hepatocellular cancer.

    Science.gov (United States)

    Takahashi, Kenji; Yan, Irene K; Kogure, Takayuki; Haga, Hiroaki; Patel, Tushar

    2014-01-01

    Hepatocellular cancers (HCC) are highly resistant to chemotherapy. TGFβ has been associated with chemoresistance in some human cancers but the mechanisms involved are unknown. We explored how TGFβ might contribute to altered responses to therapy by assessing the involvement and mechanistic contribution of extracellular vesicle long non-coding RNA (lncRNA) in mediating TGFβ-dependent chemoresistance. TGFβ reduced the sensitivity of HCC cells to sorafenib or doxorubicin and altered the release of both extracellular vesicles and of selected lncRNA within these vesicles. Amongst these, lincRNA-ROR (linc-ROR), a stress-responsive lncRNA was highly expressed in HCC cells and enriched within extracellular vesicles derived from tumor cells. Incubation with HCC-derived extracellular vesicles increased linc-ROR expression and reduced chemotherapy-induced cell death in recipient cells. Sorafenib increased linc-ROR expression in both tumor cells and extracellular vesicles, whereas siRNA to linc-ROR increased chemotherapy-induced apoptosis and cytotoxicity. Tumor-initiating cells that express CD133 have an increased resistance to therapy. TGFβ increased expression of CD133+ cells and colony growth in limiting dilution assays, both of which were attenuated by linc-ROR knockdown. These data provide mechanistic insights into primary chemoresistance in HCC by showing that: (a) TGFβ selectively enriches linc-RoR within extracellular vesicles, which has a potential role in intercellular signaling in response to TGFβ; (b) expression and enrichment of linc-ROR during chemotherapeutic stress plays a functional role in chemoresistance; and (c) the effects of TGFβ on chemoresistance in HCC may involve linc-RoR-dependent effects on tumor-initiating cells. These findings implicate extracellular vesicle lncRNA as mediators of the chemotherapeutic response, and support targeting linc-ROR to enhance chemosensitivity in HCC.

  18. Conformationally restrained carbamoylcholine homologues. Synthesis, pharmacology at neuronal nicotinic acetylcholine receptors and biostructural considerations

    DEFF Research Database (Denmark)

    de la Fuente Revenga, M; Balle, Thomas; Jensen, Anders A.

    2015-01-01

    Exploration of small selective ligands for the nicotinic acetylcholine receptors (nAChRs) based on acetylcholine (ACh) has led to the development of potent agonists with clear preference for the α4β2 nAChR, the most prevalent nAChR subtype in the central nervous system. In this work we present th...

  19. Effects of antihistamines on the function of human α7-nicotinic acetylcholine receptors.

    Science.gov (United States)

    Sadek, Bassem; Khanian, Seyedeh Soha; Ashoor, Abrar; Prytkova, Tatiana; Ghattas, Mohammad A; Atatreh, Noor; Nurulain, Syed M; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Oz, Murat

    2015-01-05

    Effects of the histamine H₁ receptor (H1R) antagonists (antihistamines), promethazine (PMZ), orphenadrine (ORP), chlorpheniramine (CLP), pyrilamine (PYR), diphenhydramine (DPH), citerizine (CTZ), and triprolidine (TRP) on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes were investigated. Antihistamines inhibited the α7-nicotinic acetylcholine receptor in the order PYR>CLP>TRP>PMZ>ORP≥DPH≥CTZ. Among the antihistamines, PYR showed the highest reversible inhibition of acetylcholine (100 µM)-induced responses with IC₅₀ of 6.2 µM. PYR-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. Specific binding of [¹²⁵I] α-bungarotoxin, a selective antagonist for α7-nicotinic acetylcholine receptor, was not changed in the presence of PYR suggesting a non-competitive inhibition of nicotinic receptors. In line with functional experiments, docking studies indicated that PYR can potentially bind allosterically with the α7 transmembrane domain. Our results indicate that the H₂-H₄ receptor antagonists tested in this study (10 µM) showed negligible inhibition of α7-nicotinic acetylcholine receptors. On the other hand, H₁ receptor antagonists inhibited the function of human α7-nicotinic acetylcholine receptor, with varying potencies. These results emphasize the importance of α7-nicotinic acetylcholine receptor for future pharmacological/toxicological profiling. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Fluorescent agonists for the Torpedo nicotinic acetylcholine receptor.

    Science.gov (United States)

    Krieger, Florian; Mourot, Alexandre; Araoz, Romulo; Kotzyba-Hibert, Florence; Molgó, Jordi; Bamberg, Ernst; Goeldner, Maurice

    2008-05-05

    We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment-sensitive fluorophores 7-(diethylamino)coumarin-3-carbonyl (DEAC) or N-(7-nitrobenz-2-oxa-1,3-diazol-yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR-rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker. One particular analogue, DEAC-Gly-C6-choline, showed partial agonist behavior with about half of the maximum response of acetylcholine, which is at least 20 times higher than those observed with previously described fluorescent dansyl- and NBD-acylcholine analogues. Binding of DEAC-Gly-C6-choline to Torpedo nAChR induces a strong enhancement of fluorescence intensity. Association and displacement kinetic experiments revealed dissociation constants of 0.5 nM for the alphadelta-binding site and 15.0 nM for the alphagamma-binding site. Both the pharmacological and the spectroscopic properties of this agonist show great promise for characterizing the allosteric mechanism behind the function of the Torpedo nAChR, as well as for drug-screening studies.

  1. Effects of acetylcholine on neuronal properties in entorhinal cortex

    Directory of Open Access Journals (Sweden)

    James G Heys

    2012-07-01

    Full Text Available The entorhinal cortex receives prominent cholinergic innervation from the medial septum and the vertical limb of the diagonal band of Broca (MSDB. To understand how cholinergic neurotransmission can modulate behavior, research has been directed towards identification of the specific cellular mechanisms in entorhinal cortex that can be modulated through cholinergic activity. This review focuses on intrinsic cellular properties of neurons in entorhinal cortex that may underlie functions such as working memory, spatial processing and episodic memory. In particular, the study of stellate cells in medial entorhinal has resulted in discovery of correlations between physiological properties of these neurons and properties of the unique spatial representation that is demonstrated through unit recordings of neurons in medial entorhinal cortex from awake-behaving animals. A separate line of investigation has demonstrated persistent firing behavior among neurons in entorhinal cortex that is enhanced by cholinergic activity and could underlie working memory. There is also evidence that acetylcholine plays a role in modulation of synaptic transmission that could also enhance mnemonic function in entorhinal cortex. Finally, the local circuits of entorhinal cortex demonstrate a variety of interneuron physiology, which is also subject to cholinergic modulation. Together these effects alter the dynamics of entorhinal cortex to underlie the functional role of acetylcholine in memory.

  2. Nicotine increases GABAergic input on rat dorsal raphe serotonergic neurons through alpha7 nicotinic acetylcholine receptor.

    Science.gov (United States)

    Hernández-Vázquez, F; Chavarría, K; Garduño, J; Hernández-López, S; Mihailescu, S P

    2014-12-15

    The dorsal raphe nucleus (DRN) contains large populations of serotonergic (5-HT) neurons. This nucleus receives GABAergic inhibitory afferents from many brain areas and from DRN interneurons. Both GABAergic and 5-HT DRN neurons express functional nicotinic acetylcholine receptors (nAChRs). Previous studies have demonstrated that nicotine increases 5-HT release and 5-HT DRN neuron discharge rate by stimulating postsynaptic nAChRs and by increasing glutamate and norepinephrine release inside DRN. However, the influence of nicotine on the GABAergic input to 5-HT DRN neurons was poorly investigated. Therefore, the aim of this work was to determine the effect of nicotine on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of 5-HT DRN neurons and the subtype of nAChR(s) involved in this response. Experiments were performed in coronal slices obtained from young Wistar rats. GABAergic sIPSCs were recorded from post hoc-identified 5-HT DRN neurons with the whole cell voltage patch-clamp technique. Administration of nicotine (1 μM) increased sIPSC frequency in 72% of identified 5-HT DRN neurons. This effect was not reproduced by the α4β2 nAChR agonist RJR-2403 and was not influenced by TTX (1 μM). It was mimicked by the selective agonist for α7 nAChR, PNU-282987, and exacerbated by the positive allosteric modulator of the same receptor, PNU-120596. The nicotine-induced increase in sIPSC frequency was independent on voltage-gated calcium channels and dependent on Ca(2+)-induced Ca(2+) release (CICR). These results demonstrate that nicotine increases the GABAergic input to most 5-HT DRN neurons, by activating α7 nAChRs and producing CICR in DRN GABAergic terminals. Copyright © 2014 the American Physiological Society.

  3. Pemphigus vulgaris antibodies target the mitochondrial nicotinic acetylcholine receptors that protect keratinocytes from apoptolysis.

    Science.gov (United States)

    Chernyavsky, Alex; Chen, Yumay; Wang, Ping H; Grando, Sergei A

    2015-11-01

    The mechanism of detachment and death of keratinocytes in pemphigus vulgaris (PV) involves pro-apoptotic action of constellations of autoantibodies determining disease severity and response to treatment. The presence of antibodies to nicotinic acetylcholine receptors (nAChRs) and the therapeutic efficacy of cholinomimetics in PV is well-established. Recently, adsorption of anti-mitochondrial antibodies abolished the ability of PVIgGs to cause acantholysis, demonstrating their pathophysiological significance. Since, in addition to cell membrane, nAChRs are also present on the mitochondrial outer membrane, wherein they act to prevent activation of intrinsic (mitochondrial apoptosis), we hypothesized that mitochondrial (mt)-nAChRs might be targeted by PVIgGs. To test this hypothesis, we employed the immunoprecipitation-western blot assay of keratinocyte mitochondrial proteins that visualized the α3, α5, α7, α9, α10, β2 and β4 mt-nAChR subunits precipitated by PV IgGs, suggesting that functions of mt-nAChRs are compromised in PV. To pharmacologically counteract the pro-apoptotic action of anti-mitochondrial antibodies in PV, we exposed naked keratinocyte mitochondria to PVIgGs in the presence of the nicotinic agonist nicotine ± antagonists, and measured cytochrome c (CytC) release. Nicotine abolished PVIgG-dependent CytC release, showing a dose-dependent effect, suggesting that protection of mitochondria can be a novel mechanism of therapeutic action of nicotinic agonists in PV. The obtained results indicated that the mt-nAChRs targeted by anti-mitochondrial antibodies produced by PV patients are coupled to inhibition of CytC release, and that nicotinergic stimulation can abolish PVIgG-dependent activation of intrinsic apoptosis in KCs. Future studies should determine if and how the distinct anti-mt-nAChR antibodies penetrate KCs and correlate with disease severity. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2017-06-09

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

  5. Organophosphate acetylcholine esterase inhibitor poisoning from a home-made shampoo.

    Science.gov (United States)

    Sadaka, Yair; Broides, Arnon; Tzion, Raffi Lev; Lifshitz, Matitiahu

    2011-07-01

    Organophosphate acetylcholine esterase inhibitor poisoning is a major health problem in children. We report an unusual cause of organophosphate acetylcholine esterase inhibitor poisoning. Two children were admitted to the pediatric intensive care unit due to organophosphate acetylcholine esterase inhibitor poisoning after exposure from a home-made shampoo that was used for the treatment of head lice. Owing to no obvious source of poisoning, the diagnosis of organophosphate acetylcholine esterase inhibitor poisoning in one of these patients was delayed. Both patients had an uneventful recovery. Organophosphate acetylcholine esterase inhibitor poisoning from home-made shampoo is possible. In cases where the mode of poisoning is unclear, direct questioning about the use of home-made shampoo is warranted, in these cases the skin and particularly the scalp should be rinsed thoroughly as soon as possible.

  6. Characterization of nicotinic receptors involved in the release of noradrenaline from the hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Vizi, E.S. [Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67, H-1450 Budapest (Hungary); Lajtha, A. [Center of Neurochemistry, The Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY (United States); Balla, A. [Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67, H-1450 Budapest (Hungary); Sershen, H. [Center of Neurochemistry, The Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY (United States)

    1997-01-06

    The pharmacological features of putative nicotinic acetylcholine receptor sites involved in the release of [{sup 3}H]noradrenaline were assessed in rat hippocampus. The effect of nicotinic agonists to induce [{sup 3}H]noradrenaline release was examined in superfused slices. The nicotinic agonists (-)-epibatidine, (+)-anatoxin-a, dimethylphenylpiperazinium, (-)-nicotine and (-)-lobeline released [{sup 3}H]noradrenaline. The dose-response curves to nicotinic agonists were bell shaped, and indicated that their functional efficacies and potency vary across agonists. Maximal efficacy was seen with dimethylphenylpiperazinium and lobeline (E{sub max} values two to three times higher than other agonists). The rank order of potency for the agonists to release [{sup 3}H]noradrenaline was (-)-epibatidine (+)-anatoxin-a dimethylphenylpiperazinium cytisine nicotine (-)-lobeline. The nicotinic acetylcholine receptor antagonists [n-bungarotoxin (+)-tubocurarine hexamethonium>>{alpha}-bungarotoxin=dihydro-{beta}-erythroidine] and tetrodotoxin antagonized the effect of dimethylphenylpiperazinium to release [{sup 3}H]noradrenaline. The results, based on these pharmacological profiles, suggest the possible involvement of nicotinic acetylcholine receptor {alpha}3 and {beta}2 nicotinic acetylcholine receptor subunits in the control of [{sup 3}H]noradrenaline release from hippocampal slices. The absence of effect of {alpha}-bungarotoxin and {alpha}-conotoxin-IMI excludes the possible involvement of nicotinic acetylcholine receptors containing the {alpha}7 subunit. The release of [{sup 3}H]noradrenaline by dimethylphenylpiperazinium was Ca{sup 2+} dependent. Nifedipine failed to prevent the dimethylphenylpiperazinium-induced release of [{sup 3}H]noradrenaline, but Cd{sup 2+}, {omega}-conotoxin and Ca{sup 2+}-free conditions significantly reduced the dimethylphenylpiperazinium-induced release, suggesting that N-type voltage-sensitive Ca{sup 2+} channels are involved in the nicotinic

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

    Indian Academy of Sciences (India)

    Administrator

    In the present study, the interaction between an intact DOPC vesicle and the hydropho- bic surface is mainly through van der Waals interac- tion. In presence of increasing concentrations of electrolytes, counter ions are present in the vicinity of the DOPC vesicle. As the vesicle approaches the solid substrate, the counter ions ...

  8. Role of Outer Membrane Vesicles of Bacteria

    Indian Academy of Sciences (India)

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

  9. 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.

  10. Towards traceable size determination of extracellular vesicles

    NARCIS (Netherlands)

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

    2014-01-01

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

  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. Characterization of Extracellular Vesicles using Raman Spectroscopy

    NARCIS (Netherlands)

    Lee, Wooje; Nanou, Afroditi; Terstappen, Leonardus Wendelinus Mathias Marie; Rho, Hoon Suk; le Gac, Severine; Offerhaus, Herman L.

    2017-01-01

    In this research, we aim to characterize extracellular vesicles(EVs) with Confocal Raman spectroscopy to reveal relevant spectral lines that signify differences between EVs derived from different cell lines. In the first stage we performed confocal Raman measurements on various EV samples. For these

  13. ADF/Cofilin Controls Synaptic Actin Dynamics and Regulates Synaptic Vesicle Mobilization and Exocytosis.

    Science.gov (United States)

    Wolf, Michael; Zimmermann, Anika-Maria; Görlich, Andreas; Gurniak, Christine B; Sassoè-Pognetto, Marco; Friauf, Eckhard; Witke, Walter; Rust, Marco B

    2015-09-01

    Actin is a regulator of synaptic vesicle mobilization and exocytosis, but little is known about the mechanisms that regulate actin at presynaptic terminals. Genetic data on LIMK1, a negative regulator of actin-depolymerizing proteins of the ADF/cofilin family, suggest a role for ADF/cofilin in presynaptic function. However, synapse physiology is fully preserved upon genetic ablation of ADF in mice, and n-cofilin mutant mice display defects in postsynaptic plasticity, but not in presynaptic function. One explanation for this phenomenon is overlapping functions of ADF and n-cofilin in presynaptic physiology. Here, we tested this hypothesis and genetically removed ADF together with n-cofilin from synapses. In double mutants for ADF and n-cofilin, synaptic actin dynamics was impaired and more severely affected than in single mutants. The resulting cytoskeletal defects heavily affected the organization, mobilization, and exocytosis of synaptic vesicles in hippocampal CA3-CA1 synapses. Our data for the first time identify overlapping functions for ADF and n-cofilin in presynaptic physiology and vesicle trafficking. We conclude that n-cofilin is a limiting factor in postsynaptic plasticity, a function which cannot be substituted by ADF. On the presynaptic side, the presence of either ADF or n-cofilin is sufficient to control actin remodeling during vesicle release. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Cholesterol Regulates Multiple Forms of Vesicle Endocytosis at a Mammalian Central Synapse

    Science.gov (United States)

    Yue, Hai-Yuan; Xu, Jianhua

    2015-01-01

    Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from nonspecific effects after cholesterol manipulation. Furthermore, it is unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase (COase) or methyl-β-cyclodextrin (MCD) impaired three different forms of endocytosis, i.e., slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca2+ channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of MCD reduced exocytosis, mainly by decreasing the readily releasable pool (RRP) and the vesicle replenishment after RRP depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses. PMID:25893258

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

    Science.gov (United States)

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

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

  16. 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

  17. Ethanol decreases agrin-induced acetylcholine receptor clustering in C2C12 myotube culture.

    Science.gov (United States)

    Owen, David B; Chamberlain, Kevin T; Shishido, Sonia; Grow, Wade A

    2010-03-01

    We investigated the effect of ethanol on skeletal muscle development using C2C12 cell culture. The ethanol concentrations of 10mM, 25mM, and 100mM, were tested because plasma samples of alcohol-dependent individuals fall within this range. We assessed two specific events in skeletal muscle development, the fusion of myoblasts to form myotubes and the acetylcholine receptor (AChR) clustering associated with neuromuscular synapse formation. We report that ethanol does not effect myotube formation or the viability of myoblasts or myotubes in C2C12 cell culture. However, ethanol does effect AChR clustering on C2C12 myotubes. As motor neurons approach skeletal muscle during development, agrin is released by motor neurons and induces AChR clustering on muscle fibers. In our experiments, agrin was applied to cell cultures during the period when myoblasts fuse to form myotubes. In cell cultures exposed to ethanol during myotube formation, agrin-induced AChR clustering was decreased compared to untreated cultures. In cell cultures exposed to ethanol during myoblast proliferation, with ethanol removed during myotube formation, agrin-induced AChR clustering was unaffected. We conclude that exposure to a physiologically relevant concentration of ethanol during the specific period of myotube formation decreases agrin-induced AChR clustering. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2018-01-01

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

  19. Acetylcholine Encodes Long-Lasting Presynaptic Plasticity at Glutamatergic Synapses in the Dorsal Striatum after Repeated Amphetamine Exposure

    Science.gov (United States)

    Wang, Wengang; Darvas, Martin; Storey, Granville P.; Bamford, Ian J.; Gibbs, Jeffrey T.; Palmiter, Richard D.

    2013-01-01

    Locomotion and cue-dependent behaviors are modified through corticostriatal signaling whereby short-term increases in dopamine availability can provoke persistent changes in glutamate release that contribute to neuropsychiatric disorders, including Parkinson's disease and drug dependence. We found that withdrawal of mice from repeated amphetamine treatment caused a chronic presynaptic depression (CPD) in glutamate release that was most pronounced in corticostriatal terminals with a low probability of release and lasted >50 d in treated mice. An amphetamine challenge reversed CPD via a dopamine D1-receptor-dependent paradoxical presynaptic potentiation (PPP) that increased corticostriatal activity in direct pathway medium spiny neurons. This PPP was correlated with locomotor responses after a drug challenge, suggesting that it may underlie the sensitization process. Experiments in brain slices and in vivo indicated that dopamine regulation of acetylcholine release from tonically active interneurons contributes to CPD, PPP, locomotor sensitization, and cognitive ability. Therefore, a chronic decrease in corticostriatal activity during withdrawal is regulated around a new physiological range by tonically active interneurons and returns to normal upon reexposure to amphetamine, suggesting that this paradoxical return of striatal activity to a more stable, normalized state may represent an additional source of drug motivation during abstinence. PMID:23785153

  20. 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....

  1. Loading of Vesicles into Soft Amphiphilic Nanotubes using Osmosis.

    Science.gov (United States)

    Erne, Petra M; van Bezouwen, Laura S; Štacko, Peter; van Dijken, Derk Jan; Chen, Jiawen; Stuart, Marc C A; Boekema, Egbert J; Feringa, Ben L

    2015-12-07

    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 flexible bilayers. When a hyperosmotic gradient is applied to these vesicle-capped nanotubes, the closed system loses water and the more flexible vesicle bilayer is pulled inwards. This leads to inclusion of vesicles inside the nanotubes without affecting the tube structure, showing controlled reorganization of the self-assembled multicomponent system upon a simple osmotic stimulus. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Targeting nicotinic acetylcholine receptor to treat smoking-related periodontitis.

    Science.gov (United States)

    Liu, Ying-Feng; Ge, Xin; Wen, Ling-Ying; Wang, Xiao-Jing

    2011-02-01

    Tobacco smoking is considered to be one of the major risk factors for periodontitis. Nicotine, the major component in tobacco smoke, has been considered playing an important role in tobacco-related morbidity by acting through the nicotinic acetylcholine receptors (nAChRs) expressed by non-neuronal cells. Recently studies found that nAChRs could be expressed on oral gingival and periodontal tissues. We hypothesize that nicotine may act on periodontal tissues directly and specifically through nAChRs to affect periodontitis activity, and that nicotine-induced periodontitis could be prevented by tissue-selective nAChR inhibitors targeting periodontal nAChRs. Thus, periodontal nAChRs may provide to be novel molecular targets to treat smoking-related periodontitis, effectively blocking of periodontal nAChRs may offer an optimistic outlook for the therapy of smoking- related periodontitis. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Cholinergic modulation of dopamine pathways through nicotinic acetylcholine receptors.

    Science.gov (United States)

    de Kloet, Sybren F; Mansvelder, Huibert D; De Vries, Taco J

    2015-10-15

    Nicotine addiction is highly prevalent in current society and is often comorbid with other diseases. In the central nervous system, nicotine acts as an agonist for nicotinic acetylcholine receptors (nAChRs) and its effects depend on location and receptor composition. Although nicotinic receptors are found in most brain regions, many studies on addiction have focused on the mesolimbic system and its reported behavioral correlates such as reward processing and reinforcement learning. Profound modulatory cholinergic input from the pedunculopontine and laterodorsal tegmentum to dopaminergic midbrain nuclei as well as local cholinergic interneuron projections to dopamine neuron axons in the striatum may play a major role in the effects of nicotine. Moreover, an indirect mesocorticolimbic feedback loop involving the medial prefrontal cortex may be involved in behavioral characteristics of nicotine addiction. Therefore, this review will highlight current understanding of the effects of nicotine on the function of mesolimbic and mesocortical dopamine projections in the mesocorticolimbic circuit. Copyright © 2015. Published by Elsevier Inc.

  4. Interaction of insulin with SDS/CTAB catanionic Vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Tah, Bidisha; Pal, Prabir; Talapatra, G.B., E-mail: spgbt@iacs.res.in

    2014-01-15

    In the present study, a novel method was used for entrapping the protein, insulin into the catanionic SDS/CTAB vesicle membrane. The anionic SDS and cationic CTAB formed catanionic vesicles at particular concentration (35:65 by volume). In this study, vesicle membrane can be considered as model membrane. The vesicle formation and entrapment efficiency depend on the pH of the aqueous solution. The insulin molecules have attached with the vesicular membrane at pH 7.0. However, at acidic pH, the vesicles were ruptured and the insulin did not entrap into the vesicle membrane, whereas at alkaline pH insulin became fibriller. The scanning electron microscope (SEM), Dynamic light scattering (DLS), and Zeta potential studies established the self-assembled structure formation of insulin and catanionic vesicles. To know the protein confirmations, Circular dichroism (CD) was also employed. The temperature dependent steady state and time resolved emission spectroscopy show that at room temperature (25 °C), apart from the 305 nm tyrosine fluorescence, a new emission peak at 450 nm was observed only in case of insulin-vesicle system, and was assigned as the tyrosine phosphorescence. This phosphorescence peak is the signature of the entrapment of insulin into the vesicle membrane. Highlights: • SDS-CTAB based catanionic vesicle has been fabricated. • Insulin has been successfully immobilized on these vesicles. • Immobilized insulin shows room temperature phosphorescence.

  5. Methamphetamine exposure during brain development alters the brain acetylcholine system in adolescent mice.

    Science.gov (United States)

    Siegel, Jessica A; Park, Byung S; Raber, Jacob

    2011-10-01

    Children exposed to methamphetamine during brain development as a result of maternal drug use have long-term hippocampus-dependent cognitive impairments, but the mechanisms underlying these impairments are not understood. The acetylcholine system plays an important role in cognitive function and potential methamphetamine-induced acetylcholine alterations may be related to methamphetamine-induced cognitive impairments. In this study, we investigated the potential long-term effects of methamphetamine exposure during hippocampal development on the acetylcholine system in adolescence mice on postnatal day 30 and in adult mice on postnatal day 90. Methamphetamine exposure increased the density of acetylcholine neurons in regions of the basal forebrain and the area occupied by acetylcholine axons in the hippocampus in adolescent female mice. In contrast, methamphetamine exposure did not affect the density of GABA cells or total neurons in the basal forebrain. Methamphetamine exposure also increased the number of muscarinic acetylcholine receptors in the hippocampus of adolescent male and female mice. Our results demonstrate for the first time that methamphetamine exposure during hippocampal development affects the acetylcholine system in adolescent mice and that these changes are more profound in females than males. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  6. Serotoninergic dorsal raphe neurons possess functional postsynaptic nicotinic acetylcholine receptors.

    Science.gov (United States)

    Galindo-Charles, Luis; Hernandez-Lopez, Salvador; Galarraga, Elvira; Tapia, Dagoberto; Bargas, José; Garduño, Julieta; Frías-Dominguez, Carmen; Drucker-Colin, René; Mihailescu, Stefan

    2008-08-01

    Very few neurons in the telencephalon have been shown to express functional postsynaptic nicotinic acetylcholine receptors (nAChRs), among them, the noradrenergic and dopaminergic neurons. However, there is no evidence for postsynaptic nAChRs on serotonergic neurons. In this study, we asked if functional nAChRs are present in serotonergic (5-HT) and nonserotonergic (non-5-HT) neurons of the dorsal raphe nucleus (DRN). In rat midbrain slices, field stimulation at the tegmental pedunculopontine (PPT) nucleus evoked postsynaptic currents (eEPSCs) with different components in DRN neurons. After blocking the glutamatergic and GABAergic components, the remaining eEPSCs were blocked by mecamylamine and reduced by either the selective alpha7 nAChR antagonist methyllycaconitine (MLA) or the selective alpha4beta2 nAChR antagonist dihydro-beta-eritroidine (DHbetaE). Simultaneous addition of MLA and DHbetaE blocked all eEPSCs. Integrity of the PPT-DRN pathway was assessed by both anterograde biocytin tracing and antidromic stimulation from the DRN. Inward currents evoked by the direct application of acetylcholine (ACh), in the presence of atropine and tetrodotoxin, consisted of two kinetically different currents: one was blocked by MLA and the other by DHbetaE; in both 5-HT and non-5-HT DR neurons. Analysis of spontaneous (sEPSCs) and evoked (eEPSCs) synaptic events led to the conclusion that nAChRs were located at the postsynaptic membrane. The possible implications of these newly described nAChRs in various physiological processes and behavioral events, such as the wake-sleep cycle, are discussed.

  7. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jürgen; Kobilka, Brian K. (Stanford); (NIH); (D.E. Shaw); (Hanyang); (UTSMC)

    2012-03-01

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  8. Combined α7 nicotinic acetylcholine receptor agonism and partial serotonin transporter inhibition produce antidepressant-like effects in the mouse forced swim and tail suspension tests

    DEFF Research Database (Denmark)

    Andreasen, Jesper T; Redrobe, John P; Nielsen, Elsebet Ø

    2012-01-01

    Emerging evidence points to an involvement of nicotinic acetylcholine receptors (nAChRs) in major depression. Nicotine improves symptoms of depression in humans and shows antidepressant-like effects in rodents. Monoamine release is facilitated by nAChR stimulation, and nicotine-evoked serotonin (5...... represents a compound displaying the synergistic effect of α7 nAChR agonism combined with partial 5-HT reuptake inhibition previously described. The addition of α7 nAChR agonism to classical monoamine-based mechanisms may represent a novel option for the improved treatment of major depression....

  9. Soft vesicles in the synthesis of hard materials.

    Science.gov (United States)

    Dong, Renhao; Liu, Weimin; Hao, Jingcheng

    2012-04-17

    Vesicles of surfactants in aqueous solution have received considerable attention because of their use as simple model systems for biological membranes and their applications in various fields including colloids, pharmaceuticals, and materials. Because of their architecture, vesicles could prove useful as "soft" templates for the synthesis of "hard materials". The vesicle phase, however, has been challenging and difficult to work with in the construction of hard materials. In the solution-phase synthesis of various inorganic or macromolecular materials, templating methods provide a powerful strategy to control the size, morphology, and composition of the resulting micro- and nanostructures. In comparison with hard templates, soft templates are generally constructed using amphiphilic molecules, especially surfactants and amphiphilic polymers. These types of compounds offer advantages including the wide variety of available templates, simple fabrication processes under mild conditions, and easy removal of the templates with less damage to the final structures. Researchers have used many ordered molecular aggregates such as vesicles, micelles, liquid crystals, emulsion droplets, and lipid nanotubes as templates or structure-directing agents to control the synthesis or assembly hard micro- and nanomaterials composed from inorganic compounds or polymers. In addition to their range of sizes and morphologies, vesicles present unique structures that can simultaneously supply different microenvironments for the growth and assembly of hard materials: the inner chamber of vesicles, the outer surface of the vesicles, and the space between bilayers. Two main approaches for applying vesicles in the field of hard materials have been explored: (i) in situ synthesis of micro- or nanomaterials within a specific microenvironment by vesicle templating and (ii) the assembly or incorporation of guest materials during the formation of vesicles. This Account provides an in-depth look at

  10. Complexin regulates the closure of the fusion pore during regulated vesicle exocytosis.

    Science.gov (United States)

    Archer, Deborah A; Graham, Margaret E; Burgoyne, Robert D

    2002-05-24

    Membrane fusion during exocytosis and throughout the cell is believed to involve members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) family of proteins. The assembly of these proteins into a four-helix bundle may be part of the driving force for bilayer fusion. Regulated exocytosis in neurons and related cell types is specialized to be fast and Ca(2+)-dependent suggesting the involvement of other regulatory proteins specific for regulated exocytosis. Among these are the complexins, two closely related proteins that bind only to the assembled SNARE complex. We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using carbon fiber amperometry. These cells express complexin II, and overexpression of this protein modified the kinetics of vesicle release events so that their time course was shortened. This effect depended on complexin interaction with the SNARE complex as introduction of a mutation of Arg-59, a residue that interacts with synaptobrevin in the SNARE complex, abolished its effects. The data are consistent with a function for complexin in stabilizing an intermediate of the SNARE complex to allow kiss-and-run recycling of the exocytosed vesicle.

  11. LL-37 Triggers Formation of Streptococcus pyogenes Extracellular Vesicle-Like Structures with Immune Stimulatory Properties.

    Science.gov (United States)

    Uhlmann, Julia; Rohde, Manfred; Siemens, Nikolai; Kreikemeyer, Bernd; Bergman, Peter; Johansson, Linda; Norrby-Teglund, Anna

    2016-01-01

    Reports have shown that the antimicrobial peptide LL-37 is abundantly expressed but has limited bactericidal effect in Streptococcus pyogenes infections. At sub-inhibitory concentrations, LL-37 has been reported to alter virulence gene expression. Here, we explored the interaction of S. pyogenes strains with LL-37, focusing on bacterial growth, cell surface alterations and pro-inflammatory responses. Bioscreen turbidity measurements of strain 5448 cultured in the presence or absence of LL-37 confirmed the poor antimicrobial effect, and revealed a significant increase in turbidity of bacterial cultures exposed to sub-inhibitory concentrations of LL-37. However, this was not linked to increased bacterial counts. Electron microscopy of LL-37-exposed bacteria revealed the presence of vesicle-like structures on the bacterial surface. The vesicles stained positive for LL-37 and were released from the bacterial surface. Concentrated supernatants enriched in these structures had a broader protein content, including several virulence factors, compared to supernatants from untreated bacteria. The supernatants from LL-37-exposed bacteria were pro-inflammatory and elicited resistin and myeloperoxidase release from neutrophils. This is the first report on S. pyogenes extracellular vesicle-like structures formed at the bacterial surface in response to LL-37. The associated increased pro-inflammatory activity further implicates LL-37 as a potential factor involved in S. pyogenes pathogenesis. © 2015 S. Karger AG, Basel.

  12. Retroviral-mediated gene transfer of the porcine choline acetyltransferase: a model to study the synthesis and secretion of acetylcholine in mammalian cells.

    Science.gov (United States)

    Xi, X G; Horellou, P; Leroy, C; Mallet, J

    1993-05-01

    We have constructed a recombinant retrovirus that expresses choline acetyltransferase (ChAT) by placing the porcine enzyme cDNA under the control of the 5' long terminal repeat of the retroviral vector pMMuLV. Using retrovirus-mediated gene transfer, we have expressed ChAT in astroglial (STR-SVLT) and neuroendocrine (RIN) cell lines. Both genetically modified cell types synthesize acetylcholine (ACh). ACh is also present in the culture medium at a low concentration relative to that found in the modified cells. This result suggests that the synthesized ACh is retained within the cells and released by these two cell types. Release of ACh is not increased in the presence of the calcium ionophore A23187 or by depolarizing concentrations of potassium in either STR-SVLT or in RIN cells. The implications of these studies for understanding ACh release mechanisms are discussed.

  13. From glioblastoma to endothelial cells through extracellular vesicles: messages for angiogenesis.

    Science.gov (United States)

    Giusti, Ilaria; Delle Monache, Simona; Di Francesco, Marianna; Sanità, Patrizia; D'Ascenzo, Sandra; Gravina, Giovanni Luca; Festuccia, Claudio; Dolo, Vincenza

    2016-09-01

    Glioblastoma has one of the highest mortality rates among cancers, and it is the most common and malignant form of brain cancer. Among the typical features of glioblastoma tumors, there is an aberrant vascularization: all gliomas are among the most vascularized/angiogenic tumors. In recent years, it has become clear that glioblastoma cells can secrete extracellular vesicles which are spherical and membrane-enclosed particles released, in vitro or in vivo, by both normal and tumor cells; they are involved in the regulation of both physiological and pathological processes; among the latter, cancer is the most widely studied. Extracellular vesicles from tumor cells convey messages to other tumor cells, but also to normal stromal cells in order to create a microenvironment that supports cancer growth and progression and are implicated in drug resistance, escape from immunosurveillance and from apoptosis, as well as in metastasis formation; they are also involved in angiogenesis stimulation, inducing endothelial cells proliferation, and other pro-angiogenic activities. To this aim, the present paper assesses in detail the extracellular vesicles phenomenon in the human glioblastoma cell line U251 and evaluates extracellular vesicles ability to promote the processes required to achieve the formation of new blood vessels in human brain microvascular endothelial cells, highlighting that they stimulate proliferation, motility, and tube formation in a dose-response manner. Moreover, a molecular characterization shows that extracellular vesicles are fully equipped for angiogenesis stimulation in terms of proteolytic enzymes (gelatinases and plasminogen activators), pro-angiogenic growth factors (VEGF and TGFβ), and the promoting-angiogenic CXCR4 chemokine receptor.

  14. Micro- and Nano-vesicles from First Trimester Human Placentae Carry Flt-1 and Levels Are Increased in Severe Preeclampsia

    Directory of Open Access Journals (Sweden)

    Mancy Tong

    2017-07-01

    Full Text Available Background/objectivesPreeclampsia is a life-threatening hypertensive disease affecting 3–5% of pregnancies. While the pathogenesis of preeclampsia remains unclear, it is known that placenta-derived factors trigger the disease by activating maternal endothelial cells prior to the onset of clinical symptoms. Extracellular vesicles (EVs of different sizes extruded by the placenta may be one factor. The truncated/secreted form of Flt-1 (sFlt-1 has also been implicated in the pathogenesis of preeclampsia. We investigated whether placental EV production is altered in preeclampsia such that they induce endothelial cell activation, and whether (sFlt-1 is involved.MethodsMacro-, micro-, and nano-vesicles were collected from normal and preeclamptic (PE placental explants, and separated by differential centrifugation. The number and size of micro- and nano-vesicles was measured by nanoparticle tracking analysis and their ability to activate endothelial cells was quantified by endothelial cell intercellular adhesion molecule 1 expression and monocyte adhesion. The levels of Flt-1 were measured by western blots and ELISA.ResultsPE placentae extruded significantly more micro- and nano-vesicles than control placentae and the extruded micro-vesicles were larger than those from control placentae. Micro- and nano-vesicles from both first trimester and term human placentae carried Flt-1 and levels were significantly increased in EVs from severe, but not mild, PE compared to normotensive placentae. All fractions of EVs from PE placentae activated endothelial cells, and for micro- and nano-vesicles, activation was reduced in the presence of exogenous vascular endothelial growth factor (VEGF, a Flt-1 neutralizing antibody, or by pre-treatment with VEGF. While EV-bound VEGF constituted over 20% of the total detected VEGF secreted by PE and normotensive placentae, EV-bound Flt-1 did not significantly contribute to the total level of sFlt-1/Flt-1 released by human

  15. Signaling by Extracellular Vesicles Advances Cancer Hallmarks.

    Science.gov (United States)

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

    2016-02-01

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

  16. Seminal vesicle cystadenoma: a rare clinical perspective.

    Science.gov (United States)

    Lorber, Gideon; Pizov, Galina; Gofrit, Ofer N; Pode, Dov

    2011-08-01

    A 52-yr-old man presented with severe obstructive urinary symptoms. Ten years earlier, a digital rectal examination disclosed a small mass above the prostate, and a computed tomography (CT) scan showed a 3.5-cm cystic tumor of the right seminal vesicle. He had been followed conservatively elsewhere. Reevaluation of the mass with a CT scan and magnetic resonance imaging showed that the mass had grown to a maximal diameter of 14 cm. A transabdominal needle biopsy revealed benign fibromuscular tissue. The tumor was then resected by an open transvesical approach. Pathology was consistent with a benign seminal vesicle cystadenoma. The natural history, pathology, and surgical approach are described. Copyright © 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  17. Docking of secretory vesicles is syntaxin dependent.

    Directory of Open Access Journals (Sweden)

    Heidi de Wit

    Full Text Available Secretory vesicles dock at the plasma membrane before they undergo fusion. Molecular docking mechanisms are poorly defined but believed to be independent of SNARE proteins. Here, we challenged this hypothesis by acute deletion of the target SNARE, syntaxin, in vertebrate neurons and neuroendocrine cells. Deletion resulted in fusion arrest in both systems. No docking defects were observed in synapses, in line with previous observations. However, a drastic reduction in morphologically docked secretory vesicles was observed in chromaffin cells. Syntaxin-deficient chromaffin cells showed a small reduction in total and plasma membrane staining for the docking factor Munc18-1, which appears insufficient to explain the drastic reduction in docking. The sub-membrane cortical actin network was unaffected by syntaxin deletion. These observations expose a docking role for syntaxin in the neuroendocrine system. Additional layers of regulation may have evolved to make syntaxin redundant for docking in highly specialized systems like synaptic active zones.

  18. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label.

    Science.gov (United States)

    Middleton, R E; Cohen, J B

    1991-07-16

    The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sites on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]nicotine binds at equilibrium with Keq = 0.6 microM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the alpha- and gamma-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the alpha-subunit was labeled via both agonist sites but the gamma-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Within the alpha-subunit, 93% of the labeling was contained within a 20-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Ser-173. Sequence analysis of this peptide indicated that approximately 80% of the incorporation was into Tyr-198, approximately 13% was into Cys-192, and approximately 7% was into Tyr-190. Chymotryptic digestion of the alpha-subunit confirmed that Tyr-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radio-sequencing strategy employing omicron-phthalaldehyde, since the efficiency of photolabeling was low (approximately 1.0%) and the labeled chymotryptic peptide was not isolated in sufficient quantity to be identified by mass. [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.

  19. Vitrification of Germinal Vesicle Stage Oocytes

    OpenAIRE

    ABE, Yasuyuki; AONO, Nobuya; Hara, Kenshiro; Matsumoto, Hiromichi; BAKHTIYARI, Mehrdad; Sasada, Hiroshi; Sato, Eimei

    2004-01-01

    In order to cryopreserve germinal vesicle (GV) stage oocytes, we first need to develop a novel container for keeping large quantities of GV oocytes, because of collecting them as cumulus oocytes complexes (COCs) that have bigger size and larger volume than oocytes themselves, and second modify a protocol for optimizing vitrification of them. In this mini-review, we describe our recent progress for attaining these objectives. When 65 bovine COCs having GV oocytes could be placed on a sheet of ...

  20. A readily retrievable pool of synaptic vesicles

    OpenAIRE

    Hua, Y; Sinha, R.; Thiel, C.; Schmidt, R.; Hueve, J.; Martens, H.; Hell, S.; Egner, A.; Klingauf, J.

    2011-01-01

    Abstract Although clathrin-mediated endocytosis (CME) is thought to be the predominant mechanism of synaptic vesicle (SV) recycling, it seems to be too slow for fast recycling. Therefore, it was suggested that a pre-sorted and pre-assembled pool of SV proteins on the presynaptic membrane might support a first wave of fast CME. In this study we monitored the temporal dynamics of such a 'readily retrievable pool' of SV proteins in rat hippocampal neurons using a novel probe. Applying...

  1. Vesicle-to-micelle transition in aqueous solutions of amphiphilic calixarene derivatives

    Science.gov (United States)

    Micali, Norberto; Villari, Valentina; Consoli, Grazia M. L.; Cunsolo, Francesca; Geraci, Corrada

    2006-05-01

    Structure and conformation of spontaneous self-assembled calix[8]arenes derivatives are studied by means of static and dynamic light scattering and electrophoretic mobility. These amphiphilic molecules are in the aggregated form in aqueous solution, in a wide range of pH ; they take a vesicle structure in neutral and basic pH environment, but, in relatively strong acidic conditions (below pH=4.5 ), a transition from vesicle to micelle occurs. The structural change is driven by the surface charge density. At neutral pH calix[8]arenes take a negative surface charge, which prevents coagulation and ensures stability; at acidic pH the surface charge tend to become positive because of the protonation of the hydrophilic head. These pH -responsive aggregates, able to release an encapsulated hydrophilic guest, are promising systems for application as nanocarriers.

  2. SMN requirement for synaptic vesicle, active zone and microtubule postnatal organization in motor nerve terminals.

    Directory of Open Access Journals (Sweden)

    Laura Torres-Benito

    Full Text Available Low levels of the Survival Motor Neuron (SMN protein produce Spinal Muscular Atrophy (SMA, a severe monogenetic disease in infants characterized by muscle weakness and impaired synaptic transmission. We report here severe structural and functional alterations in the organization of the organelles and the cytoskeleton of motor nerve terminals in a mouse model of SMA. The decrease in SMN levels resulted in the clustering of synaptic vesicles (SVs and Active Zones (AZs, reduction in the size of the readily releasable pool (RRP, and the recycling pool (RP of synaptic vesicles, a decrease in active mitochondria and limiting of neurofilament and microtubule maturation. We propose that SMN is essential for the normal postnatal maturation of motor nerve terminals and that SMN deficiency disrupts the presynaptic organization leading to neurodegeneration.

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

    Science.gov (United States)

    Landsmann, Steve; Luka, Martin; Polarz, Sebastian

    2012-01-01

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

  4. Stem cell-extracellular vesicles as drug delivery systems: New frontiers for silk/curcumin nanoparticles.

    Science.gov (United States)

    Perteghella, Sara; Crivelli, Barbara; Catenacci, Laura; Sorrenti, Milena; Bruni, Giovanna; Necchi, Vittorio; Vigani, Barbara; Sorlini, Marzio; Torre, Maria Luisa; Chlapanidas, Theodora

    2017-03-30

    The aim of this work was to develop a novel carrier-in-carrier system based on stem cell-extracellular vesicles loaded of silk/curcumin nanoparticles by endogenous technique. Silk nanoparticles were produced by desolvation method and curcumin has been selected as drug model because of its limited water solubility and poor bioavailability. Nanoparticles were stable, with spherical geometry, 100nm in average diameter and the drug content reached about 30%. Cellular uptake studies, performed on mesenchymal stem cells (MSCs), showed the accumulation of nanoparticles in the cytosol around the nuclear membrane, without cytotoxic effects. Finally, MSCs were able to release extracellular vesicles entrapping silk/curcumin nanoparticles. This combined biological-technological approach represents a novel class of nanosystems, combining beneficial effects of both regenerative cell therapies and pharmaceutical nanomedicine, avoiding the use of viable replicating stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Detection of platelet vesicles by flow cytometry.

    Science.gov (United States)

    Nolan, John P; Jones, Jennifer C

    2017-05-01

    The comp