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Sample records for brush border membrane vesicles

  1. Amiloride transport in rabbit renal brush-border membrane vesicles

    International Nuclear Information System (INIS)

    Rabbit renal brush-border membrane vesicles (BBMV) were used to study amiloride transport across the luminal membrane of proximal tubular cells. An outwardly directed H+ gradient (pHi 6.0; pHo 7.5) stimulated 8 microM [14C]-amiloride uptake into BBMV and supported a transient active accumulation of substrate consistent with the presence of an amiloride-H+ exchange process. Uptake was inhibited, in the presence or absence of a pH gradient, by 1 mM unlabeled amiloride or 20 mM tetraethylammonium (TEA). Amiloride transport was not directly affected by the presence of 100 mM Na+ in the extravesicular medium, suggesting that Na-H exchange did not mediate amiloride flux. Amiloride transport was a saturable process with a maximal flux (under pH gradient conditions) of 3 nmol.mg-1.min-1 and an apparent Kt of 8 microM. TEA acted as a competitive inhibitor of this process with an apparent Ki of approximately 80 microM, similar to the Kt of TEA transport via the TEA-H+ exchanger. Likewise, amiloride acted as a competitive inhibitor of TEA uptake with an apparent Ki of approximately 11 microM. Preloading BBMV with 1-2 mM TEA stimulated the rate of amiloride uptake and supported a transient active accumulation of amiloride. We conclude that amiloride and TEA are transported by a common pathway in BBMV, which involves a carrier-mediated exchange with H+ and which may play a role in the tubular secretion of these compounds

  2. Na+ and H+ transport in human jejunal brush-border membrane vesicles

    International Nuclear Information System (INIS)

    The authors have examined pH gradient-driven Na+ uptake and Na+-driven H+ transport in brush-border membrane vesicles prepared from jejunal tissue obtained from organ donors by measuring the influx of 22Na and the fluorescence quenching of acridine orange (AO). Vesicle preparation by either Ca2+ or Mg2+ precipitation showed no difference in 22Na uptake or AO fluorescence quenching and dissipation. An outward H+ gradient induced a Na+ uptake overshoot of threefold over equilibrium, whereas the absence of an H+ gradient did not produce an overshoot. The initial rate of pH-driven Na+ uptake in voltage-clamped vesicles was related to [Na+o]. Amiloride inhibited this uptake in voltage-clamped vesicles. Dissipation of AO fluorescence quench in vesicles with a preformed internal acid gradient was hastened by Na+o as well as voltage clamping in the absence of Na+. In vesicles without a pH gradient, internal Na+, as well as a diffusion potential in the absence of Na+, induced AO quenching. External Na+ and Li+, but not choline, acted to dissipate AO quenching induced by a diffusion potential, and the rate of dissipation was unaffected by the presence of Cl-. Li+ and NH+4, but not Cs+, K+, Rb+, or choline+, inhibited pH gradient-driven 22Na uptake. They conclude that human jejunal brush-border membrane vesicles contain conductive pathways for both Na+ and H+ and an Na+-H+ exchanger

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

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

    1990-01-01

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

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

    International Nuclear Information System (INIS)

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

  5. Cimetidine transport in isolated brush border membrane vesicles from bovine choroid plexus

    International Nuclear Information System (INIS)

    The purpose of this study was to elucidate the mechanisms involved in the transport of cimetidine across the brush border membrane of choroid plexus epithelium. Brush border membrane vesicles were prepared from bovine choroid plexus and the uptake of [3H]cimetidine was studied using the methods of rapid vacuum filtration and scintillation counting. Cimetidine accumulated in the vesicles with time reaching equilibrium within 2 hr. The amount of cimetidine taken up by the vesicles at equilibrium decreased with increasing extravesicular media osmolarity suggesting that cimetidine accumulates in an osmotically reactive intravesicular space. Binding of cimetidine to the membrane was estimated to be less than 18%. Michaelis-Menten studies demonstrated that cimetidine transport involved both a saturable and a nonsaturable component. The Vmax and Km (mean +/- S.E.) were 16.7 +/- 5.9 pmol/sec/mg protein and 58.1 +/- 3.1 microM, respectively, suggesting that cimetidine is transported across the choroid plexus brush border membrane with a lower affinity and a higher capacity than across the renal brush border membrane. The organic cation, quinidine (0.1 mM), and the amino acid, histidine (20 mM), both significantly reduced the initial, but not the equilibrium, uptake of cimetidine. However, high concentrations (5 mM) of more polar organic cations including tetraethylammonium, as well as of several organic anions including salicylate did not inhibit cimetidine transport. Studies with unlabeled cimetidine revealed a countertransport phenomenon. Attempts to drive the concentrative uptake of cimetidine with various ion gradients were unsuccessful. Of note was the fact that an outwardly directed proton gradient could significantly accelerate the uptake of cimetidine

  6. Brush border membrane vesicles from dipteran midgut: a tool for studies on nutrient absorption

    Directory of Open Access Journals (Sweden)

    MG Leonardi

    2006-12-01

    Full Text Available Brush border membrane vesicles (BBMV from insects midgut can be successfully used to study several membrane phenomena, including nutrient absorption, ions permeability and insecticides mode of action. Midgut BBMV, purified from Musca domestica whole larvae, were used for the functional characterization of leucine transport. The amino acid uptake was accelerated in the presence of sodium or potassium and increased significantly when the extravesicular pH was 5.0, in agreement with the luminal pH in vivo. Radiolabelled leucine uptake was significantly reduced by an excess of cold leucine, histidine, serine and glycine, suggesting that the amino acid transporter is a broad scope carrier that does not recognize proline, glutamine and the dibasic amino acids lysine and arginine.Midgut BBMV were also obtained from homogenization of M. domestica and Bactrocera oleae adults. The final preparations showed a high enrichment in the specific activity of the BBM marker enzymes aminopeptidase N and γ-glutamyl transpeptidase, and were poorly contaminated by basolateral membranes, as indicated by the low specific activities of their marker enzyme Na+/K+ ATPase. Electron microscopy of B. oleae BBM fraction showed the presence of closed vesicles. Similar SDS-PAGE patterns, with numerous distinct bands, were detected for both B. oleae and M. domestica BBMV.

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

    International Nuclear Information System (INIS)

    To investigate the pathogenetic mechanisms of interferon nephrotoxicity, we studied the effect of recombinant interferon alfa-2b on the uptake of 14C-D-glucose and 14C-L-alanine by rat renal brush-border-membrane vesicles. Interferon significantly inhibited 20 sec. sodium-dependent and 5 and 10 min. equilibrium uptake of both glucose and alanine. The inhibitory effect was dose dependent with maximum effect achieved at interferon concentration of 5 x 10-8M in the uptake media. The half-maximal inhibitory concentrations, IC50, of interferon on glucose uptake was 1.8 x 10-8M, and 5.4 x 10-9M on alanine uptake. Dixon plot analysis of uptake data was consistent with pure non-competitive inhibition. The inhibition constants, Ki, 1.5 x 10-8M for glucose uptake, and 7.3 x 10-9M for alanine uptake, derived from Dixon plots were in close agreement with the IC50s calculated from the semilog dose response curves. These observations reveal that direct interactions at the proximal tubule cell membrane are involved in the pathogenesis of interferon nephrotoxicity, and that its mechanism of nephrotoxicity is similar to that of other low molecular weight proteins

  8. Heparin blocks /sup 125/I-calmodulin internalization by isolated rat renal brush border membrane vesicles

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

    1986-03-05

    /sup 125/I-Calmodulin is internalized by isolated rat renal brush border membrane vesicles (BBV) in a time, temperature and calcium dependent manner. Internalization of /sup 125/I-calmodulin into the osmotically sensitive space of BBV was distinguished from binding of the ligand to the outer BBV surface by examining the interaction of ligand and BBV at different medium osmolarities (300-1100 mosm), uptake was inversely proportional to medium osmolarity. Internalized /sup 125/I-calmodulin was intact and Western blots of solubilized BBV with /sup 125/I-calmodulin demonstrated the presence of several calmodulin-binding proteins of 143, 118, 50, 47.5, 46.5 and 35 kilodaltons which could represent potential intravesicular binding sites for the ligand. Heparin and the related glycosaminoglycan heparin sulfate both showed a dose-dependent inhibition (0.5-50 ..mu..g/ml) of /sup 125/I-calmodulin uptake by BBV, but other sulfated and nonsulfated glycosaminoglycans including chondroitin sulfates, keratan sulfate and hyaluronic acid showed little or no inhibitory effect. Desulfation of heparin virtually abolished the inhibition of uptake while depolymerization reduced it. Heparin did not block the binding of /sup 125/I-calmodulin to BBV proteins as assessed by Western blotting technique suggesting its effect was on internalization of the ligand rather than on its association with internal membrane proteins.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-02-01

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

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

    International Nuclear Information System (INIS)

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

  11. Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers.

    Science.gov (United States)

    Alcantara, Edwin P; Aguda, Remedios M; Curtiss, April; Dean, Donald H; Cohen, Michael B

    2004-04-01

    The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins. PMID:15027071

  12. Zinc uptake in swine intestinal brush border membrane vesicles using a 65Zn/69mZn duel isotope experiment

    International Nuclear Information System (INIS)

    Supplemental zinc as ZnO is routinely added to nursery pig diets at 15 to 20 times the nutritional requirement to alleviate physiological stress and enhance performance. The mechanism by which Zn accomplishes this function is unknown, however, high concentrations of Zn are excreted in the environment at undesirable levels. To study Zn uptake, we developed a multi-stage digestion model followed by exposure to swine intestinal brush border membrane vesicles (BBMV). We report on the feasibility of using a duel label (65Zn and 69mZn) to simultaneously quantify the competitive uptake of Zn from co-existing zinc supplements using our BBMV model. (author)

  13. Relationship of membrane-bound sulfhydryl groups to vitamin D-stimulated uptake of [75Se]Selenite by the brush border membrane vesicles from chick duodenum

    International Nuclear Information System (INIS)

    The uptake of selenite by purified brush border membrane vesicles isolated from duodena of rachitic or vitamin D-treated chicks was studied by using radioactive selenite and a rapid filtration technique. Cholecalciferol treatment (500 IU at 72 h) significantly enhanced selenite uptake, a response that decreased when the vesicles were stored at room temperature for 2.5 h prior to the uptake measurement. Preincubation of the vesicles in 1.0 mmol/L H2O2 reduced [75Se]selenite uptake, indicating the involvement of oxidizable groups in the uptake reaction. Iodoacetic acid (IAA), a sulfhydryl-blocking reagent, at 1-2 mmol/L concentration eliminated the difference in selenite uptake due to cholecalciferol and had no effect on vesicles from rachitic animals. A higher concentration of IAA (10 mmol/L) enhanced selenite uptake manyfold and increased the absolute difference due to cholecalciferol treatment. Single intravenous doses of 100 IU cholecalciferol, 100 IU ergocalciferol, or 0.1 micrograms 1,25-dihydroxycholecalciferol also stimulated selenite uptake, suggesting a general response to vitamin D compounds. Normal animals given a single dose of 1,25-dihydroxycholecalciferol 12 h prior to killing also responded. Treatments that enhanced the uptake of [75Se]selenite also increased the amount of membrane-bound sulfhydryl groups, suggesting the involvement of membrane-bound sulfhydryl groups in the vitamin D response. A significant increase in selenite uptake by intravenous 1,25-dihydroxycholecalciferol occurred within 10 min. This rapid effect provides a new tool to probe early biochemical effects of vitamin D on intestinal epithelium

  14. Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1 Ac binding proteins from the midgut of Helicoverpa armigera

    Institute of Scientific and Technical Information of China (English)

    Li-Zhen Chen; Ge-Mei Liang; Brian G.Rector; Jie Zhang; Kong-Ming Wu; Yu-Yuan Guo

    2008-01-01

    Brush border membrane vesicles(BBMV)isolated from insect midguts have been widely used to study CrylA binding proteins.Sample preparation is important in two-dimensional electrophoresis(2-DE),so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE,we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).All mctllods yielded similar types and numbers of binding proteins,but in different quantifies.The Abdul.Rauf and Ellar protocol was the best of the three,but had limitations.Sufficient protein qu antity iS important for research involving limited numbers of insects,such as studies of insect resistance to Bacillus thuringiensis in the field.Consequently,we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H.armigera larval midguts.which proved suitable for 2-DE analysis.

  15. The Binding Characterization of Cry Insecticidal Proteins to the Brush Border Membrane Vesicles of Helicoverpa armigera, Spodoptera exigua, Spodoptera litura and Agrotis ipsilon

    Institute of Scientific and Technical Information of China (English)

    LU Qiong; CAO Guang-chun; ZHANG Li-li; LIANG Ge-mei; GAO Xi-wu; ZHANG Yong-jun; GUO Yu-yuan

    2013-01-01

    Cry toxins produced by Bacillus thuringiensis (Bt) are effective biological insecticides against certain insect species. However, there are potential risks of the evolved resistance of insects to Cry toxin owing to decreased binding of toxins to target sites in the brush border membranes of the larva midgut. The Cry toxins with different binding sites in the larval midgut have been considered to be a good combination to deploy in delaying resistance evolution. Bioassay results demonstrated that the toxicity of different Cry toxins ranked differently for each species. The toxicity ranking was Cry1Ac>Cry1Ab>Cry2Ab for Helicoverpa armigera, Cry1B>Cry1C>Cry2Ab for Spodoptera exigua, and Cry2Ab>Cry1B>Cry1C for S. litura. Only Cry2Ab was toxic to Agrotis ipsilon. Binding experiments were performed with 125I-Cry1Ab, 125I-Cry1Ac, 125I-Cry1B, 125I-Cry1C, 125I-Cry2Ab and the brush border membranes vesicles (BBMV) from H. armigera, S. exigua, S. litura and A. ipsilon. The binding of Cry1Ab and Cry1Ac was shown to be saturable by incubating with increasing concentrations of H. armigera BBMV (Kd=(45.00±2.01) nmol L-1 and (12.80±0.18) nmol L-1, respectively;Bmax=(54.95±1.79) ng and (55.44±0.91) ng, separately). The binding of Cry1B was shown to be saturable by incubating with increasing concentrations of S. exigua BBMV (Kd=(23.26±1.66) nmol L-1;Bmax=(65.37±1.87) ng). The binding of 125I-Cry toxins was shown to be non-saturable by incubating with increasing concentrations of S. litura and A. ipsilon BBMV. In contrast, Cry1B and Cry1C showed some combination with the BBMV of S. litura, and a certain amount of Cry2Ab could bind to the BBMV of A. ipsilon. These observations suggest that a future strategy could be devised for the focused combination of specific cry genes in transgenic crops to control target pests, widen the spectrum of insecticide effectiveness and postpone insect resistance evolution.

  16. Improvement of intestinal absorption of peptides: adsorption of B1-Phe monoglucosylated insulin to rat intestinal brush-border membrane vesicles.

    Science.gov (United States)

    Hashimoto, T; Nomoto, M; Komatsu, K; Haga, M; Hayashi, M

    2000-09-01

    In a previous study we glycosylated insulin to improve its intestinal absorption. When the glycosylated product, p-(succinylamido)-phenyl-alpha-D-glucopyranoside (SAPG)-substituted insulin (SAPG-INS), was administered intra-intestinally to rats, it showed a greater hypoglycemic effect than native bovine insulin. The enhanced hypoglycemic effect of SAPG-INS was considered to be due to an increase in membrane permeability as well as an increase in resistance to enzymatic degradation. In particular, membrane permeability may be related to an interaction with the Na(+)-dependent D-glucose transporter (SGLT-1) which is located in the brush-border membrane of epithelial cells. The insulin product used in the previous study, however, comprised a mixture of mono-, di- and tri-SAPG-substituted insulin. In this study SAPG-INS with a defined substitution number and position was synthesized to examine the interaction between the transporter and glycosylated insulin in more detail. The new product was mono-SAPG-substituted insulin substituted at the B1-phenylalanine position (B1-SAPG-INS) and was selectively synthesized after protection of the A1-glycine and varepsilonB29-lysine amino acids. The hypoglycemic effect of B1-SAPG-INS in rats after an intravenous dose of 71 microg/kg was almost the same as that of native bovine insulin at a dose of 1 U/kg and B1-SAPG-INS retained about 60% of the immunoreactivity of native bovine insulin. The interaction of B1-SAPG-INS with the intestinal transporter was examined by a rapid filtration technique using (125)I-labeled B1-SAPG-INS and brush-border membrane vesicles (BBMVs) which were prepared from rat small intestine by the Mg-precipitation method. The amount of B1-SAPG-INS adsorbed or absorbed by BBMVs in the presence of an inward Na(+)-gradient into BBMVs was greater than that of native bovine insulin. This adsorption/absorption was significantly inhibited by the presence of 1 mM phloridzin. A similar inhibition was observed when Na

  17. Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles

    International Nuclear Information System (INIS)

    High-voltage discharge or one cycle of freeze-thawing are shown to transiently permeabilize rabbit ileal brush-border membrane vesicles to macromolecules. Uptake of the radiolabeled macromolecule dextran, mol wt 70,000, used as a marker for vesicle permeability, was determined by a rapid filtration technique, with uptake defined as substrate associated with the vesicle and releasable after incubation of vesicles with 0.1% saponin. Dextran added immediately after electric shock (2000 V) or at the beginning of one cycle of freeze-thawing was taken up approximately eightfold compared with control. ATP also was taken up into freeze-thawed vesicles, whereas there was no significant uptake into control vesicles. The increase in vesicle permeability was reversible, based on Na-dependent D-glucose uptake being decreased when studied 5 but not 15 min after electric shock, and was not significantly decreased after completion of one cycle of freeze-thawing. In addition, adenosine 3',5'-cyclic monophosphate and Ca2+-calmodulin-dependent protein kinase activity were similar in control vesicles and vesicles exposed to high-voltage discharge or freeze-thawing. Also, vesicles freeze-thawed with [32P]ATP demonstrated increased phosphorylation compared with nonfrozen vesicles, while freeze-thawing did not alter vesicle protein as judged by Coomassie blue staining. These techniques should allow intestinal membrane vesicles to be used for studies of intracellular control of transport processes, for instance, studies of protein kinase regulation of transport

  18. Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Donowitz, M.; Emmer, E.; McCullen, J.; Reinlib, L.; Cohen, M.E.; Rood, R.P.; Madara, J.; Sharp, G.W.G.; Murer, H.; Malmstrom, K.

    1987-06-01

    High-voltage discharge or one cycle of freeze-thawing are shown to transiently permeabilize rabbit ileal brush-border membrane vesicles to macromolecules. Uptake of the radiolabeled macromolecule dextran, mol wt 70,000, used as a marker for vesicle permeability, was determined by a rapid filtration technique, with uptake defined as substrate associated with the vesicle and releasable after incubation of vesicles with 0.1% saponin. Dextran added immediately after electric shock (2000 V) or at the beginning of one cycle of freeze-thawing was taken up approximately eightfold compared with control. ATP also was taken up into freeze-thawed vesicles, whereas there was no significant uptake into control vesicles. The increase in vesicle permeability was reversible, based on Na-dependent D-glucose uptake being decreased when studied 5 but not 15 min after electric shock, and was not significantly decreased after completion of one cycle of freeze-thawing. In addition, adenosine 3',5'-cyclic monophosphate and Ca/sup 2 +/-calmodulin-dependent protein kinase activity were similar in control vesicles and vesicles exposed to high-voltage discharge or freeze-thawing. Also, vesicles freeze-thawed with (/sup 32/P)ATP demonstrated increased phosphorylation compared with nonfrozen vesicles, while freeze-thawing did not alter vesicle protein as judged by Coomassie blue staining. These techniques should allow intestinal membrane vesicles to be used for studies of intracellular control of transport processes, for instance, studies of protein kinase regulation of transport.

  19. Library of monoclonal antibodies against brush border membrane epithelial antigens

    International Nuclear Information System (INIS)

    A purified fraction of proximal tubule brush border membranes (BBM) was prepared from dog kidney and used to immunize mice. The standard technique of hybridoma production was followed as described by Kohler and Milstein. Production of antibodies was detected by indirect immunofluorescence on dog kidney slices and by immunodot against the purified fraction on nitrocellulose. Five hybrids exhibited anti BBM activity. These were cloned twice and yielded stable cell lines producing IgG type monoclonal antibodies against BBM. They were designated A1, C7, D3, D7 and H4. As a family these five monoclonals have broad tissue specificity, i.e. positive staining of the surface mucosa of intestinal kidney proximal tubules. D3 exhibits even broader specificity for epithelium reacting with bile canaliculi and choroid plexus. The authors have verified that at least 4/5 antibodies are directed against BBM protein as revealed by immunoprecipitation of solubilized BBM and detected by Coomassie blue staining or autoradiography of lactoperoxidase labelled BBM. Most interestingly all antibodies bind to the surface of LL CPK1 cells, a continuous pig kidney cell line of undefined origin but exhibiting many characteristics of proximal tubule cells. The library of monoclonal antibodies obtained provide important probes with which to study membrane biogenesis and polarization in epithelial cells

  20. Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

    DEFF Research Database (Denmark)

    Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte;

    2011-01-01

    Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this......-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the...... brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft...

  1. Inhibition of beta-amino acid transport by diamide does not involve the brush border membrane surface

    International Nuclear Information System (INIS)

    Diamide (dicarboxylic acid bis-(N,N-dimethylamide) has been shown in previous studies to block the uptake of the beta-amino acid taurine at its high affinity transport site in rat renal cortex slices. Diamide may act by increasing the efflux of taurine from the slice. Studies performed in rat slices again indicate enhanced efflux over 8-12 minutes. The time course of reduced glutathione (GSH) depletion from renal cortex is similar, indicating a potential interaction between GSH depletion and inhibition of taurine accumulation. The effect of 9 mM diamide on the Na+ -dependent accumulation of taurine (10 and 250 microM) by brush border membrane vesicles was examined, and the taurine uptake value both initially and at equilibrium was the same in the presence and absence of diamide. Isolation of the brush border surface and subsequent transport studies of taurine are not influenced by diamide. Thus, diamide inhibition of taurine uptake does not involve physiochemical alteration of the membrane surface where active amino acid transport occurs, despite the thiol-oxidizing properties of this agent. Further, these studies suggest that diamide either acts at the basolateral surface, rather than the brush border surface of rat renal cortex or requires the presence of an intact tubule, capable of metabolism, prior to its inhibitory action

  2. Binding and degradation of /sup 125/I-insulin by isolated rat renal brush border membranes: evidence for low affinity, high capacity insulin recognition sites

    Energy Technology Data Exchange (ETDEWEB)

    Meezan, E.; Pillion, D.J.; Elgavish, A.

    1988-10-01

    The kidney plays a major role in the handling of circulating insulin in the blood, primarily via reuptake of filtered insulin at the luminal brush border membrane. 125I-insulin associated with rat renal brush border membrane vesicles (BBV) in a time- and temperature-dependent manner accompanied by degradation of the hormone to trichloroacetic acid (TCA)-soluble fragments. Both association and degradation of 125I-insulin were linearly proportional to membrane protein concentration with virtually all of the degradative activity being membrane associated. Insulin, proinsulin and desoctapeptide insulin all inhibited the association and degradation of 125I-insulin by BBV, but these processes were not appreciably affected by the insulin-like growth factors IGF-I and IGF-II or by cytochrome c and lysozyme, low molecular weight, filterable, proteins, which are known to be reabsorbed in the renal tubules by luminal endocytosis. When the interaction of 125I-insulin with BBV was studied at various medium osmolarities (300-1100 mosM) to alter intravesicular space, association of the ligand with the vesicles was unaffected, but degradation of the ligand by the vesicles decreased progressively with increasing medium osmolarity. Therefore, association of 125I-insulin to BBV represented binding of the ligand to the membrane surface and not uptake of the hormone or its degradation products into the vesicles. Attempts to crosslink 125I-insulin to a high-affinity insulin receptor using the bifunctional reagent disuccinimidyl suberate revealed only trace amounts of an 125I-insulin-receptor complex in brush border membrane vesicles in contrast to intact renal tubules where this complex was readily observed. Both binding and degradation of 125I-insulin by brush border membranes did not reach saturation even at concentrations of insulin approaching 10(-5) M.

  3. Binding and degradation of 125I-insulin by isolated rat renal brush border membranes: evidence for low affinity, high capacity insulin recognition sites

    International Nuclear Information System (INIS)

    The kidney plays a major role in the handling of circulating insulin in the blood, primarily via reuptake of filtered insulin at the luminal brush border membrane. 125I-insulin associated with rat renal brush border membrane vesicles (BBV) in a time- and temperature-dependent manner accompanied by degradation of the hormone to trichloroacetic acid (TCA)-soluble fragments. Both association and degradation of 125I-insulin were linearly proportional to membrane protein concentration with virtually all of the degradative activity being membrane associated. Insulin, proinsulin and desoctapeptide insulin all inhibited the association and degradation of 125I-insulin by BBV, but these processes were not appreciably affected by the insulin-like growth factors IGF-I and IGF-II or by cytochrome c and lysozyme, low molecular weight, filterable, proteins, which are known to be reabsorbed in the renal tubules by luminal endocytosis. When the interaction of 125I-insulin with BBV was studied at various medium osmolarities (300-1100 mosM) to alter intravesicular space, association of the ligand with the vesicles was unaffected, but degradation of the ligand by the vesicles decreased progressively with increasing medium osmolarity. Therefore, association of 125I-insulin to BBV represented binding of the ligand to the membrane surface and not uptake of the hormone or its degradation products into the vesicles. Attempts to crosslink 125I-insulin to a high-affinity insulin receptor using the bifunctional reagent disuccinimidyl suberate revealed only trace amounts of an 125I-insulin-receptor complex in brush border membrane vesicles in contrast to intact renal tubules where this complex was readily observed. Both binding and degradation of 125I-insulin by brush border membranes did not reach saturation even at concentrations of insulin approaching 10(-5) M

  4. Lipid rafts in epithelial brush borders: atypical membrane microdomains with specialized functions

    DEFF Research Database (Denmark)

    Danielsen, E Michael; Hansen, Gert H

    2003-01-01

    -linked and transmembrane digestive enzymes, most likely as an economizing effort to secure and prolong their digestive capability at the microvillar surface. However, in addition to microvilli, the brush border surface also consists of membrane invaginations between adjacent microvilli, which are the only part...... of the apical surface sterically accessible for membrane fusion/budding events. Many of these invaginations appear as pleiomorphic, deep apical tubules that extend up to 0.5-1 microm into the underlying terminal web region. Their sensitivity to methyl-beta-cyclodextrin suggests them to contain cholesterol...

  5. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    International Nuclear Information System (INIS)

    Binding studies were performed with two 125I-labeled Bacillus thuringiensis δ-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One δ-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other δ-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis δ-endotoxins active against M. sexta compete for binding of 125I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles

  6. Molecular size of the Na+-H+ antiport in renal brush border membranes, as estimated by radiation inactivation

    International Nuclear Information System (INIS)

    The radiation inactivation method was applied to brush border membrane vesicles from rat kidney, in order to estimate the molecular size of the Na+-H+ antiporter. Sodium influx (1mM) driven by an acid intravesicular pH was unaffected by the high osmolarity of the cryoprotective solution. Initial rate of influx was estimated by linear regression performed on the first 10 seconds of transport: 0.512 pmol/micrograms protein/s. There was no binding component involved. Incubation performed in the presence of 1 mM amiloride, an inhibitor of the Na+-H+ antiport gave an initial rate of only 0.071 pmol/microgram/s, an 82% inhibition. Membrane vesicles were irradiated at -78 degrees C in a Gammacel Model 220. Sodium influx was reduced, as the dose of radiation increased, but the influx remained linear for the period of time (10s) during which the initial rate was estimated, indicating no alteration of the proton driving force during this time period. Amiloride-insensitive flux remained totally unaffected by the radiation dose, indicating that the passive permeability of the membrane towards sodium was unaffected. The amiloride-sensitive pathway presented a monoexponential profile of inactivation, allowing the molecular size to be estimated at 321 kDa. Based on DCCD-binding studies suggesting the molecular size of the monomer to be around 65 kDa for rat kidney, our results suggest that the functional transporter in the membrane to be a multimer

  7. Generation of stable lipid raft microdomains in the enterocyte brush border by selective endocytic removal of non-raft membrane.

    Directory of Open Access Journals (Sweden)

    E Michael Danielsen

    Full Text Available The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces multiple deleterious agents present in the gut lumen, such as bile salts, digestive enzymes of the pancreas, and a plethora of pathogens. In the present work, we studied the constitutive endocytosis from the brush border of cultured jejunal explants of the pig, and the results indicate that this process functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs, was absent from detergent resistant membranes (DRMs, implying an association with non-raft membrane. Furthermore, neither major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM1, were not endocytosed except when cholera toxin subunit B (CTB was present. In conclusion, we propose that constitutive, selective endocytic removal of non-raft membrane acts as a sorting mechanism to enrich the brush border contents of lipid raft components, such as glycolipids and the major digestive enzymes. This sorting may be energetically driven by changes in membrane curvature when molecules move from a microvillar surface to an endocytic invagination.

  8. Native proteomic analysis of protein complexes in murine intestinal brush border membranes

    Czech Academy of Sciences Publication Activity Database

    Babušiak, M.; Man, Petr; Petrák, J.; Vyoral, D.

    2007-01-01

    Roč. 7, č. 1 (2007), s. 121-129. ISSN 1615-9853 R&D Projects: GA ČR(CZ) GD204/03/H066; GA AV ČR KJB500200612; GA MŠk LC545 Grant ostatní: GA ČR(CZ) GA303/04/0003; GA MZd(CZ) NR8930; GA MŠk(CZ) LC06044; CZ(CZ) 023736; GA MZd(CZ) NR8317 Institutional research plan: CEZ:AV0Z50200510 Keywords : blue native electrophoresis * brush border membranes * protein complexes Subject RIV: EE - Microbiology, Virology Impact factor: 5.479, year: 2007

  9. Faecal excretion of brush border membrane enzymes in patients with clostridium difficile diarrhoea

    Directory of Open Access Journals (Sweden)

    Katyal R

    2002-01-01

    Full Text Available PURPOSE: To look for the presence of intestinal brush border membrane (BBM enzymes in the faecal samples of patients with Clostridium difficile association. METHODS: One hundred faecal samples were investigated for C.difficile toxin (CDT. Simultaneous assays for faecal excretion of intestinal BBM enzymes viz., disaccharidases, alkaline phosphatase (AP and leucine aminopeptidase (LAP were also done. RESULTS: C.difficile toxin was detected in 25 (25% of the samples with a titre ranging from 10 to 160. No significant difference (p>0.05 was seen between the CDT positive and negative groups with any of the disaccharidases studied. However, significant increase (pC.difficile diarrhoea.

  10. Galectin-4 and small intestinal brush border enzymes form clusters

    DEFF Research Database (Denmark)

    Danielsen, E M; van Deurs, B

    1997-01-01

    lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed......Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin...... that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about...

  11. Generation of stable lipid raft microdomains in the enterocyte brush border by selective endocytic removal of non-raft membrane

    DEFF Research Database (Denmark)

    Danielsen, E Michael; Hansen, Gert H

    2013-01-01

    multiple deleterious agents present in the gut lumen, such as bile salts, digestive enzymes of the pancreas, and a plethora of pathogens. In the present work, we studied the constitutive endocytosis from the brush border of cultured jejunal explants of the pig, and the results indicate that this process...... functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs), was absent from detergent resistant membranes (DRMs), implying an association with non-raft membrane. Furthermore, neither...... major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM1, were not endocytosed except when cholera toxin subunit B (CTB) was present. In...

  12. Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine

    Science.gov (United States)

    Nakano, Takanari; Inoue, Ikuo; Takenaka, Yasuhiro; Ono, Hiraku; Katayama, Shigehiro; Awata, Takuya; Murakoshi, Takayuki

    2016-01-01

    Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen 3H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given 3H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given 3H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux

  13. Modulatory effect of thyroid hormones on uptake of phosphate and other solutes across luminal brush border membrane of kidney cortex

    International Nuclear Information System (INIS)

    The mechanism whereby thyroid hormones modulate the transport properties of luminal brush border membrane (BBM) of renal proximal tubules was studied in thyroparathyroidectomized rats. Administration of both T4 and T3 increased BBM capacity for Na+ gradient-dependent uptake of phosphate (P /sub i/) by BBM vesicles (BBMV). This effect of thyroid hormones was present in thyroparathyroidectomized and hypophysectomized rats, and it was not blocked by a saturating dose of propranolol. The stimulatory effect of T3 and T4 on BBM transport of P /sub i/ was dose dependent in the range of 5.2-520 nmol/100 g BW. Pretreatment of rats with inhibitors of 5'-monodeiodinase (5'-DI), iopanoic acid or ipodate, prevented the increase in serum T3 in rats injected with T4, but it did not diminish the increase in BBM transport of P /sub i/. Administration of iopanoic acid and ipodate also prevented a 5-fold increase in 5'-DI activity in renal cortical tissue elicited by T4 administration. Treatment with T3 resulted in an increase of Pi transport across BBM from kidneys of rats subjected to dietary P /sub i/ deprivation due either to total fasting or to feeding of a low P /sub i/ diet. Further, T3 administration enhanced amiloride-sensitive Na+-H+ countertransport across BBM, but the uptake of 22Na+ by BBMV in the absence of pH gradient was not changed. The Na+ gradient-dependent uptake of L-[3H]proline by BBMV was slightly decreased, but the uptake of [14C]citrate was not changed in response to T3. Administration of T3 increased P /sub i/ transport in BBMV prepared from juxtamedullary cortex, but not in BBMV from superficial cortex. Conversely, the rate of Na+-H+ countertransport was enhanced, and the enzymatic activity of alkaline phosphatase was decreased in BBMV from superficial cortex; no changes in these parameters were found in BBMV from juxtamedullary cortex

  14. Angiotensin 2 directly increases rabbit renal brush-border membrane sodium transport: Presence of local signal transduction system

    International Nuclear Information System (INIS)

    In the present study, the authors have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10(-11)-10(-7) M) was found to stimulate 22Na+ uptake by the isolated BBM vesicles directly. All did not affect the Na(+)-dependent BBM glucose uptake, and the effect of AII on BBM 22Na+ uptake was inhibited by amiloride, suggesting the involvement of Na+/H+ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na+/H+ antiport system. In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A2 (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-beta S or PTX abolished, the effects of AII on BBM PLA and 22Na+ uptake, suggesting the involvement of G-protein in AII's actions. On the other hand, inhibition of PLA by mepacrine prevented AII's effect on BBM 22Na+ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM 22Na+ uptake, suggesting the role of PLA activation in mediating AII's effect on BBM 22Na+ uptake. In summary, results of the present study show a direct stimulatory effect of AII on BBM Na+/H+ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation

  15. Dimeric assembly of enterocyte brush border enzymes

    DEFF Research Database (Denmark)

    Danielsen, E M

    1994-01-01

    appearance of the liposome-reconstituted enzyme [Norén et al. (1986) J. Biol. Chem. 261, 12306-12309], showing only the inner, membrane-anchored domains of the monomers to be in close contact with one another while the outer domains are far apart. In contrast to the other brush border enzymes studied......The noncovalent, dimeric assembly of small intestinal brush border enzymes was studied by sedimentation analysis in density gradients of extracts of pulse-labeled pig jejunal mucosal explants. Like aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48-10), aminopeptidase A (EC 3...

  16. Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion.

    Science.gov (United States)

    Saslowsky, David E; Thiagarajah, Jay R; McCormick, Beth A; Lee, Jean C; Lencer, Wayne I

    2016-04-01

    The apical brush border membrane (BBM) of intestinal epithelial cells forms a highly structured and dynamic environmental interface that serves to regulate cellular physiology and block invasion by intestinal microbes and their products. How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function. We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin. Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network. Accumulation of apical membrane ceramide is necessary and sufficient to induce the actin phenotype, and this coincides with altered membrane structure and augmented innate immune function as evidenced by resistance to invasion by Salmonella. PMID:26864627

  17. Heterogeneity of renal brush border membrane (BBM) fractions isolated by differential and density gradient centrifugation

    International Nuclear Information System (INIS)

    To differentiate BBM from proximal convoluted tubules (PCT) and from pars recta (PR), the authors isolated separately membrane fractions from superficial cortex (SC-tissue) and from juxtamedullary cortex plus outer stripe of red medulla (JM-tissue) by centrifugation in Percoll gradient. BBM separated on Percoll gradient into the higher density zone (zone A), and lower density zone (zone B). The zone B from SC-tissue membranes showed a peak with high activity of alkaline phosphatase (AP) and maltase but low activities of γ-glutamyl transferase (GGT) and leucineaminopeptidase (LAP). Membranes from JM-tissue separated into a major peak in zone A, with very high activities of GGT and LAP, markers for PR, along with lesser activity of AP. In addition, zone B showed a small peak of AP and very slight shoulder of GGT and LAP. In membranes from both SC-tissue and JM-tissue, either zone A or zone B, they found concentrative uptake of both 32Pi and L-[3H]-proline in the presence of Na+-gradient. They suggest, based on distribution of BBM marker enzymes, that membrane fraction in zone B from SC-tissue contains BBM vesicles predominantly from PCT of outer cortical nephrons. Further, BBM vesicles from JM-tissue in zone A are predominantly from PR segments and in zone B from PCT of deep nephrons

  18. Membrane proteomics characterization of brush border membrane proteins of mice intestinal mucosa : case study: cholesterol absorption

    OpenAIRE

    Tsirogianni, Eirini

    2009-01-01

    The epithelial absorbing cells of the small intestinal villi, the enterocytes, are the main protagonists for the transport of nutrients from the intestinal lumen to the interstitial fluids. The oriented flow of nutrients is carried out by different and complementary transport systems present in the apical and the basolateral domains of the enterocyte’s plasma membrane. One of the distinctive characteristics of those intestinal cells is the presence of numerous structurally distinct protrusion...

  19. Binding of E. coli heat-stable enterotoxin to rat intestinal brush borders and to basolateral membranes

    International Nuclear Information System (INIS)

    We studied the binding of E. coli heat-stable enterotoxin (STa) to rat brush borders (BB) and to basolateral membranes (BLM) using a biologically active monoiodinated radioligand [( 125I]STa) and highly enriched BB and BLM preparations free of other significant organelle contamination. Binding of [125I]STa to BB was specific; time-, temperature-, and pH-dependent; saturable; and partially reversible. Nonlabeled toxin competitively inhibited the binding of radioligand to BB in a dose-related manner. Scatchard analysis revealed a single class of receptors with an apparent affinity constant of 8.7 +/- 1.5 X 10(8) l/mol. Binding was not affected by amino acids, sugars, and lectins. Proteolytic enzymes significantly decreased binding, although several did so by modifying the radioligand. Trypsin inhibited binding without modifying the radioligand thus supporting the proteinaceous nature of the receptor. Since the enrichment in binding activity in the BB over the homogenate was significantly lower than the enrichment in sucrase activity, we concluded that binding activity is probably associated with other membranous domains, but direct examination revealed no binding activity on basolateral membranes

  20. Experimental type II diabetes and related models of impaired glucose metabolism differentially regulate glucose transporters at the proximal tubule brush border membrane.

    Science.gov (United States)

    Chichger, Havovi; Cleasby, Mark E; Srai, Surjit K; Unwin, Robert J; Debnam, Edward S; Marks, Joanne

    2016-06-01

    What is the central question of this study? Although SGLT2 inhibitors represent a promising treatment for patients suffering from diabetic nephropathy, the influence of metabolic disruption on the expression and function of glucose transporters is largely unknown. What is the main finding and its importance? In vivo models of metabolic disruption (Goto-Kakizaki type II diabetic rat and junk-food diet) demonstrate increased expression of SGLT1, SGLT2 and GLUT2 in the proximal tubule brush border. In the type II diabetic model, this is accompanied by increased SGLT- and GLUT-mediated glucose uptake. A fasted model of metabolic disruption (high-fat diet) demonstrated increased GLUT2 expression only. The differential alterations of glucose transporters in response to varying metabolic stress offer insight into the therapeutic value of inhibitors. SGLT2 inhibitors are now in clinical use to reduce hyperglycaemia in type II diabetes. However, renal glucose reabsorption across the brush border membrane (BBM) is not completely understood in diabetes. Increased consumption of a Western diet is strongly linked to type II diabetes. This study aimed to investigate the adaptations that occur in renal glucose transporters in response to experimental models of diet-induced insulin resistance. The study used Goto-Kakizaki type II diabetic rats and normal rats rendered insulin resistant using junk-food or high-fat diets. Levels of protein kinase C-βI (PKC-βI), GLUT2, SGLT1 and SGLT2 were determined by Western blotting of purified renal BBM. GLUT- and SGLT-mediated d-[(3) H]glucose uptake by BBM vesicles was measured in the presence and absence of the SGLT inhibitor phlorizin. GLUT- and SGLT-mediated glucose transport was elevated in type II diabetic rats, accompanied by increased expression of GLUT2, its upstream regulator PKC-βI and SGLT1 protein. Junk-food and high-fat diet feeding also caused higher membrane expression of GLUT2 and its upstream regulator PKC

  1. Binding kinetics of Clostridium difficile toxins A and B to intestinal brush border membranes from infant and adult hamsters

    International Nuclear Information System (INIS)

    This study was undertaken to determine if the relative resistance of neonates and infants to Clostridium difficile-associated intestinal disease can be related to age-dependent differences in intestinal receptors for C. difficile toxins A and B. Brush border membranes (BBMs) from the small intestines of adult and infant hamsters were examined for their ability to bind radiolabeled toxins A and B. [125I]toxin A bound to both infant and adult hamster BBMs at physiological temperature, whereas [125I]toxin B did not bind to the BBMs under any of the conditions examined. The number of [125I]toxin A molecules bound at saturation was approximately 4 x 10(10) per micrograms of membrane protein for adult BBMs and 1 x 10(11) per micrograms of membrane protein for infant BBMs. Scatchard plot analysis suggested the presence of a single class of toxin A binding sites on both infant and adult hamster BBMs. Maximal binding capacity and Kd values were 0.63 pmol/mg of protein and 66.7 nM, respectively, for the infant BBMs, and 0.24 pmol/mg of protein and 27 nM, respectively, for the adult BBMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of extracted BBM proteins revealed differences in the proteins of infant and adult BBMs. However, there were not any detectable differences in the protein bands which bound [125I]toxin A between infant and adult hamsters. The results from these investigations indicate that differences in the binding kinetics of toxins A and/or B to infant and adult hamster BBMs do not account for the observed differences in their susceptibility to C. difficile-associated intestinal disease

  2. Binding kinetics of Clostridium difficile toxins A and B to intestinal brush border membranes from infant and adult hamsters

    Energy Technology Data Exchange (ETDEWEB)

    Rolfe, R.D. (Texas Tech Univ. Health Sciences Center, Lubbock (USA))

    1991-04-01

    This study was undertaken to determine if the relative resistance of neonates and infants to Clostridium difficile-associated intestinal disease can be related to age-dependent differences in intestinal receptors for C. difficile toxins A and B. Brush border membranes (BBMs) from the small intestines of adult and infant hamsters were examined for their ability to bind radiolabeled toxins A and B. (125I)toxin A bound to both infant and adult hamster BBMs at physiological temperature, whereas (125I)toxin B did not bind to the BBMs under any of the conditions examined. The number of (125I)toxin A molecules bound at saturation was approximately 4 x 10(10) per micrograms of membrane protein for adult BBMs and 1 x 10(11) per micrograms of membrane protein for infant BBMs. Scatchard plot analysis suggested the presence of a single class of toxin A binding sites on both infant and adult hamster BBMs. Maximal binding capacity and Kd values were 0.63 pmol/mg of protein and 66.7 nM, respectively, for the infant BBMs, and 0.24 pmol/mg of protein and 27 nM, respectively, for the adult BBMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of extracted BBM proteins revealed differences in the proteins of infant and adult BBMs. However, there were not any detectable differences in the protein bands which bound (125I)toxin A between infant and adult hamsters. The results from these investigations indicate that differences in the binding kinetics of toxins A and/or B to infant and adult hamster BBMs do not account for the observed differences in their susceptibility to C. difficile-associated intestinal disease.

  3. Immunoelectrophoretic studies on pig intestinal brush border proteins

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael; Sjöström, H; Norén, O;

    1977-01-01

    Brush borders were prepared from pig intestinal mucosa and the membrane proteins solubilized with either Triton X-100 or papain. Proteins, thus released, were used as antigens to raise antisera in rabbits. The immunoglobulin G fractions were isolated and shown by the double layer immunofluorescence...

  4. Differential Effect of Bacillus firmus on Immune Response and Enterocyte Brush-Border Levels in BALB/c and B10.BR Mice

    Czech Academy of Sciences Publication Activity Database

    Kozáková, Hana; Mlčková, P.; Kolínská, Jiřina; Čechová, D.; Štěpánková, Renata; Řeháková, Zuzana; Prokešová, L.

    2002-01-01

    Roč. 47, č. 6 (2002), s. 759-765. ISSN 0015-5632 R&D Projects: GA ČR GA303/00/1370; GA AV ČR IAA5020101 Institutional research plan: CEZ:AV0Z5020903; CEZ:MSM 111100005 Keywords : brush-border membrane vesicles * bbacillus firmus Subject RIV: EE - Microbiology, Virology Impact factor: 0.979, year: 2002

  5. Brush border membrane vesicle purification using Andean Potato Weevil (premnotrypes vorax Hustache) epithelial midgut tissue

    OpenAIRE

    Wilson Martínez O.; Jairo Cerón S.

    2011-01-01

    La exploración del uso potencial de microorganismos como agentes de control biológico de plagas requiere estudios básicos acerca de su mecanismo de acción en el insecto blanco seleccionado. El empleo de vesículas obtenidas a partir de las microvellosidades epiteliales del intestino de los insectos (BBMVs), se ha constituido en una herramienta valiosa a nivel mundial para el estudio del mecanismo de acción de las proteínas de Bacillus thuringiensis considerado como el agente de control microbi...

  6. Molecular model of the microvillar cytoskeleton and organization of the brush border.

    Directory of Open Access Journals (Sweden)

    Jeffrey W Brown

    Full Text Available BACKGROUND: Brush border microvilli are approximately 1-microm long finger-like projections emanating from the apical surfaces of certain, specialized absorptive epithelial cells. A highly symmetric hexagonal array of thousands of these uniformly sized structures form the brush border, which in addition to aiding in nutrient absorption also defends the large surface area against pathogens. Here, we present a molecular model of the protein cytoskeleton responsible for this dramatic cellular morphology. METHODOLOGY/PRINCIPAL FINDINGS: The model is constructed from published crystallographic and microscopic structures reported by several groups over the last 30+ years. Our efforts resulted in a single, unique, self-consistent arrangement of actin, fimbrin, villin, brush border myosin (Myo1A, calmodulin, and brush border spectrin. The central actin core bundle that supports the microvillus is nearly saturated with fimbrin and villin cross-linkers and has a density similar to that found in protein crystals. The proposed model accounts for all major proteinaceous components, reproduces the experimentally determined stoichiometry, and is consistent with the size and morphology of the biological brush border membrane. CONCLUSIONS/SIGNIFICANCE: The model presented here will serve as a structural framework to explain many of the dynamic cellular processes occurring over several time scales, such as protein diffusion, association, and turnover, lipid raft sorting, membrane deformation, cytoskeletal-membrane interactions, and even effacement of the brush border by invading pathogens. In addition, this model provides a structural basis for evaluating the equilibrium processes that result in the uniform size and structure of the highly dynamic microvilli.

  7. Lipid raft organization and function in the small intestinal brush border

    DEFF Research Database (Denmark)

    Danielsen, E M; Hansen, Gert Helge

    2008-01-01

    The enterocyte brush border of the small intestine is a highly specialized membrane designed to function both as a high capacity digestive/absorptive surface of dietary nutrients and a permeability barrier towards lumenal pathogens. It is characterized by an unusually high content of glycolipids...

  8. Involvement of detergent-insoluble complexes in the intracellular transport of intestinal brush border enzymes

    DEFF Research Database (Denmark)

    Danielsen, E M

    1995-01-01

    A number of transmembrane digestive enzymes of the porcine small intestinal brush border membrane were found to be partially Triton X-100-insoluble at 0 degree C and colocalized in gradient centrifugation experiments with the GPI-anchored alkaline phosphatase in low-density, detergent......%), and lactase-phlorizin hydrolase (EC 3.2.1.23-62) was essentially fully soluble in detergent. In radioactively labeled, mucosal explants, the newly synthesized brush border enzymes began to associate with detergent-insoluble complexes while still in their transient, high mannose-glycosylated form, and...... their insolubility increased to that of the steady-state level soon after they achieved their mature, complex glycosylation, i.e., after passage through the Golgi complex. Detergent-insoluble complexes isolated by density gradient centrifugation were highly enriched in brush border enzymes, and the...

  9. Radiation inactivation studies of renal brush border water and urea transport

    International Nuclear Information System (INIS)

    Radiation inactivation was used to determine the nature and molecular weight of water and urea transport pathways in brush border membrane vesicles (BBMV) isolated from rabbit renal cortex. BBMV were frozen to -50 degrees C, irradiated with 1.5 MeV electrons, thawed, and assayed for transport or enzyme activity. The freezing process had no effect on enzyme or transport kinetics. BBMV alkaline phosphatase activity gave linear ln(activity) vs. radiation dose plots with a target size of 68 +/- 3 kDa, similar to previously reported values. Water and solute transport were measured using the stopped-flow light-scattering technique. The rates of acetamide and osmotic water transport did not depend on radiation dose (0-7 Mrad), suggesting that transport of these substances does not require a protein carrier. In contrast, urea and thiourea transport gave linear ln(activity) vs. dose curves with a target size of 125-150 kDa; 400 mM urea inhibited thiourea flux by -50% at 0 and 4.7 Mrad, showing that radiation does not affect inhibitor binding to surviving transporters. These studies suggest that BBMV urea transport requires a membrane protein, whereas osmotic water transport does not

  10. Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF Family of Adaptor Proteins with the Raft- and the Non-Raft Brush Border Membrane Fractions of NHE3

    Directory of Open Access Journals (Sweden)

    Ayesha Sultan

    2013-11-01

    Full Text Available Background/Aims: Trafficking, brush border membrane (BBM retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50, NHERF2 (E3KARP, and NHERF3 (PDZK1 with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes (“lipid rafts” were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3- mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.

  11. Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na+/H+ exchanger isoform NHE3.

    Science.gov (United States)

    Akhter, S; Nath, S K; Tse, C M; Williams, J; Zasloff, M; Donowitz, M

    1999-01-01

    Squalamine, an endogenous molecule found in the liver and other tissues of Squalus acanthias, has antibiotic properties and causes changes in endothelial cell shape. The latter suggested that its potential targets might include transport proteins that control cell volume or cell shape. The effect of purified squalamine was examined on cloned Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120 fibroblasts. Squalamine (1-h pretreatment) decreased the maximal velocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 micrograms/ml, respectively) and also increased K'[H+]i. Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitory effect of squalamine was 1) time dependent, with no effect of immediate addition and maximum effect with 1 h of exposure, and 2) fully reversible. Squalamine pretreatment of the ileum for 60 min inhibited brush-border membrane vesicle Na+/H+ activity by 51%. Further investigation into the mechanism of squalamine's effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at the concentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of the brush-border NHE isoform NHE3 and not NHE1 or NHE2, 2) acts in a nontoxic and fully reversible manner, and 3) has a delayed effect, indicating that it may influence brush-border Na+/H+ exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator. PMID:9886929

  12. Intestinal alkaline phosphatase: selective endocytosis from the enterocyte brush border during fat absorption

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Niels-Christiansen, Lise-Lotte; Immerdal, Lissi;

    2007-01-01

    explants. By immunofluorescence microscopy, fat absorption caused a translocation of IAP from the enterocyte brush border to the interior of the cell, whereas other brush-border enzymes were unaffected. By electron microscopy, the translocation occurred by a rapid (5 min) induction of endocytosis via...... clathrin-coated pits. By 60 min, IAP was seen in subapical endosomes and along membranes surrounding fat droplets. IAP is a well-known lipid raft-associated protein, and fat absorption was accompanied by a marked change in the density and morphology of the detergent-resistant membranes harboring IAP. A...... IAP and may contribute to the appearance of the enzyme in serum and surfactant-like particles....

  13. Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes

    DEFF Research Database (Denmark)

    Hansen, Gert H; Pedersen, Jens; Niels-Christiansen, Lise-Lotte;

    2003-01-01

    raft microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as...... lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized...... the resident brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to...

  14. Anti-glycosyl antibodies in lipid rafts of the enterocyte brush border: a possible host defense against pathogens

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Pedersen, Esben D K; Immerdal, Lissi;

    2005-01-01

    The pig small intestinal brush border is a glycoprotein- and glycolipid-rich membrane that functions as a digestive/absorptive surface for dietary nutrients as well as a permeability barrier for pathogens. The present work was performed to identify carbohydrate-binding (lectinlike) proteins...

  15. Endocytic trafficking from the small intestinal brush border probed with FM dye

    DEFF Research Database (Denmark)

    Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte;

    2009-01-01

    The small intestinal brush border functions as the body's main portal for uptake of dietary nutrients and simultaneously acts as the largest permeability barrier against pathogens. To enable this, the digestive enzymes of the brush border are organized in lipid raft microdomains stabilized by cross...... incorporated into the brush border, and by 15 min faint but distinct punctae were detectable about 1 m beneath the brush border, indicative of a constitutive endocytosis. The punctae represented a subpopulation of early endosomes confined to the actomyosin-rich terminal web region, and their number...... localized deeper into the cytoplasm of the enterocytes. Two major raft-associated brush border enzymes, alkaline phosphatase and aminopeptidase N, were excluded from endocytosis. We propose that the terminal web cytoskeleton, by inhibiting traffic from apical early endosomes further into the cell...

  16. Post-translational suppression of expression of intestinal brush border enzymes by fructose

    DEFF Research Database (Denmark)

    Danielsen, E M

    1989-01-01

    The two major dietary sugars, fructose and sucrose, were found to suppress effectively the biosynthetic renewal of brush border enzymes in the gut. When studied in cultured explants of pig small intestine mucosa, 10-50 mM concentrations of fructose completely prevented the expression of mature...... cotranslational glycosylation that in turn triggers a rapid proteolytic breakdown. Our findings suggest that renewal of digestive brush border enzymes is transiently suppressed during intake of fructose- or sucrose-rich meals....

  17. Effect of nonpathogenic Escherichia coli monoassociation on small intestinal brush-border glycoconjugate moieties and cytokine production after colonization in ex-germ-free rats and pigs

    Czech Academy of Sciences Publication Activity Database

    Kolínská, Jiřina; Zákostelecká, Marie; Schwarzer, Martin; Štěpánková, Renata; Hudcovic, Tomáš; Kozáková, Hana

    2010-01-01

    Roč. 2, - (2010), s. 73-84. ISSN 1179-139X R&D Projects: GA MŠk(CZ) ME10017 Grant ostatní: GA AV ČR(CZ) IAA500200710; GA ČR(CZ) GA303/09/0449 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z50200510 Keywords : nonpathogenic E. coli * glycoconjugates * brush-border vesicles Subject RIV: CE - Biochemistry

  18. Na+ uptake into colonic enterocyte membrane vesicles

    International Nuclear Information System (INIS)

    Na+ uptake was studied in colonic enterocyte membrane vesicles prepared from normal and dexamethasone-treated rats. Vesicles from rats treated with dexamethasone demonstrated a fivefold greater 22Na+ uptake compared with vesicles from normal rats. Most of the tracer uptake in membranes derived from treated rats occurred through a conductive, amiloride-blockable pathway located in vesicles with low native K+ permeability and high Cl- permeability. Kinetic analysis of the amiloride inhibition curve revealed the presence of two amiloride-blockable pathways, one with a high affinity accounting for 85% of the uptake, and one with a low affinity accounting for only 12% of the uptake. Only the low-affinity pathway was detected with vesicles from normal rats. The high sensitivity to amiloride, the dependence on dexamethasone pretreatment, and the relative permeabilities to K+ and Cl- indicate that most of the 22Na+ uptake in membranes derived from treated rats is through a Na+-specific channel located in apical membrane vesicles. Preincubation of the isolated cells from dexamethasone-treated rats at 37 degree C in Ca2+-free solutions before homogenization and membrane vesicle purification caused a 5- to 10-fold increase in amiloride-blockable 22Na+ uptake compared with vesicles derived from cells maintained at 0 degree C. The addition of Ca2+, but not of Mg2+, to the incubation solution markedly reduced this temperature-dependent enhancement in 22Na+ uptake. These results suggest that Na+ transport in colonic enterocytes from dexamethasone-treated rats is regulated by a Ca2+-dependent, temperature-sensitive process which causes a sustained change in the apical membrane

  19. Toroidal membrane vesicles in spherical confinement

    CERN Document Server

    Bouzar, Lila; Müller, Martin Michael

    2015-01-01

    We investigate the morphology of a toroidal fluid membrane vesicle confined inside a spherical container. The equilibrium shapes are assembled in a geometrical phase diagram as a function of scaled area and reduced volume of the membrane. For small area the vesicle can adopt its free form. When increasing the area, the membrane cannot avoid contact and touches the confining sphere along a circular contact line, which extends to a zone of contact for higher area. The elastic energies of the equilibrium shapes are compared to those of their confined counterparts of spherical topology to predict under which conditions a topology change is favored energetically.

  20. Toroidal membrane vesicles in spherical confinement.

    Science.gov (United States)

    Bouzar, Lila; Menas, Ferhat; Müller, Martin Michael

    2015-09-01

    We investigate the morphology of a toroidal fluid membrane vesicle confined inside a spherical container. The equilibrium shapes are assembled in a geometrical phase diagram as a function of scaled area and reduced volume of the membrane. For small area the vesicle can adopt its free form. When increasing the area, the membrane cannot avoid contact and touches the confining sphere along a circular contact line, which extends to a zone of contact for higher area. The elastic energies of the equilibrium shapes are compared to those of their confined counterparts of spherical topology to predict under which conditions a topology change is favored energetically. PMID:26465512

  1. Bacterial Outer Membrane Vesicles and Vaccine Applications

    OpenAIRE

    Acevedo, Reinaldo; Fernández, Sonsire; Zayas, Caridad; Acosta, Armando; Sarmiento, Maria Elena; Valerie A. Ferro; Rosenqvist, Einar; Campa, Concepcion; Cardoso, Daniel; Garcia, Luis; Perez, Jose Luis

    2014-01-01

    Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A...

  2. BACTERIAL OUTER MEMBRANE VESICLES AND VACCINE APPLICATIONS

    OpenAIRE

    Reinaldo eAcevedo; Sonsire eFernandez; Caridad eZayas; Armando eAcosta; Maria Elena Sarmiento; Valerie A. Ferro; Einar eRosenqvist; Concepcion eCampa; Daniel eCardoso; Luis eGarcia; Jose Luis Perez

    2014-01-01

    Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of self meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cu...

  3. The PDZ domain of the guanine nucleotide exchange factor PDZGEF directs binding to phosphatidic acid during brush border formation.

    Directory of Open Access Journals (Sweden)

    Sarah V Consonni

    Full Text Available PDZGEF is a guanine nucleotide exchange factor for the small G protein Rap. It was recently found that PDZGEF contributes to establishment of intestinal epithelial polarity downstream of the kinase Lkb1. By binding to phosphatidic acid enriched at the apical membrane, PDZGEF locally activates Rap2a resulting in induction of brush border formation via a pathway that includes the polarity players TNIK, Mst4 and Ezrin. Here we show that the PDZ domain of PDZGEF is essential and sufficient for targeting PDZGEF to the apical membrane of polarized intestinal epithelial cells. Inhibition of PLD and consequently production of phosphatidic acid inhibitis targeting of PDZGEF to the plasma membrane. Furthermore, localization requires specific positively charged residues within the PDZ domain. We conclude that local accumulation of PDZGEF at the apical membrane during establishment of epithelial polarity is mediated by electrostatic interactions between positively charged side chains in the PDZ domain and negatively charged phosphatidic acid.

  4. Effects of divalent cations on vitamin B12 adsorption to brush borders of rat intestine

    International Nuclear Information System (INIS)

    A brush border preparation from rat intestine was incubated with rat intrinsic factor-vitamin B12 complex in 0.01M Tris-HCl buffer, pH 7.4. The 57Co-B12 uptake to brush borders was proportional to the amount of protein or to alkaline phosphatase activity in the preparations. The uptake increased with time of incubation. At 370C, the uptake after incubation for 15 min. was 80-85% of that for one hr. The uptake at 4 0C was approximately 70% of that at 37 0C. There was no difference as a result of adding glucose to the incubation medium. The uptake was observed in the alkaline environment above pH 6.3. Maximum uptake occurred at pH 8.0. Brush borders washed with Krebs-Ringer bicarbonate buffer (pH 7.4) exhibited no difference in B12 uptake, whether in the presence of absence of calcium ion. But brush borders washed with ethylenediaminetetraacetate exhibited no uptake when incubated in calcium-free medium. The uptake reached a maximum by addition of calcium ion at a concentration of 0.3 mM, and was not alter up to 10 mM. Addition of magnesium ion exhibited no uptake. Calcium-dependent B12 uptake was markedly inhibited by manganese ion. Magnesium ion seemed to slightly inhibit the calcium-dependent uptake. (auth.)

  5. Brush border myosin Ia inactivation in gastric but not endometrial tumors

    NARCIS (Netherlands)

    Mazzolini, Rocco; Rodrigues, Paulo; Bazzocco, Sarah; Dopeso, Higinio; Ferreira, Ana M.; Mateo-Lozano, Silvia; Andretta, Elena; Woerner, Stefan M.; Alazzouzi, Hafid; Landolfi, Stefania; Hernandez-Losa, Javier; Macaya, Irati; Suzuki, Hiromu; Ramon y Cajal, Santiago; Mooseker, Mark S.; Mariadason, John M.; Gebert, Johannes; Hofstra, Robert M. W.; Reventos, Jaume; Yamamoto, Hiroyuki; Schwartz, Simo; Arango, Diego

    2013-01-01

    Brush border Myosin Ia (MYO1A) has been shown to be frequently mutated in colorectal tumors with microsatellite instability (MSI) and to have tumor suppressor activity in intestinal tumors. Here, we investigated the frequency of frameshift mutations in the A8 microsatellite in exon 28 of MYO1A in MS

  6. The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules

    DEFF Research Database (Denmark)

    Halberg, Kenneth A; Rainey, Stephanie M; Veland, Iben R; Neuert, Helen; Dornan, Anthony J; Klämbt, Christian; Davies, Shireen-Anne; Dow, Julian A T

    2016-01-01

    Multicellular organisms rely on cell adhesion molecules to coordinate cell-cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most...... role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border....

  7. Natural Selection at the Brush-Border: Adaptations to Carbohydrate Diets in Humans and Other Mammals

    OpenAIRE

    Pontremoli, Chiara; Mozzi, Alessandra; Forni, Diego; Cagliani, Rachele; Pozzoli, Uberto; Menozzi, Giorgia; Vertemara, Jacopo; Bresolin, Nereo; Clerici, Mario; Sironi, Manuela

    2015-01-01

    Dietary shifts can drive molecular evolution in mammals and a major transition in human history, the agricultural revolution, favored carbohydrate consumption. We investigated the evolutionary history of nine genes encoding brush-border proteins involved in carbohydrate digestion/absorption. Results indicated widespread adaptive evolution in mammals, with several branches experiencing episodic selection, particularly strong in bats. Many positively selected sites map to functional protein reg...

  8. Comparison of two pig intestinal brush border peptidases with the corresponding renal enzymes

    DEFF Research Database (Denmark)

    Norén, O; Sjöström, H; Danielsen, Erik Michael; Staun, M; Jeppesen, L; Svensson, B

    1979-01-01

    -glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic...... acid. It is suggested that the intestinal brush border peptidases, dipeptidyl peptidase IV and gamma-glutamyltransferase, are closely related to the corresponding enzymes obtained from the kidney....

  9. Exposure to Engineered Nanomaterial Results in Disruption of Brush Borders in Epithelia Models in vitro

    Science.gov (United States)

    Faust, James J.

    Engineered nanoparticles (NP; 10-9 m) have found use in a variety of consumer goods and medical devices because of the unique changes in material properties that occur when synthesized on the nanoscale. Although many definitions for nanoparticle exist, from the perspective of size, nanoparticle is defined as particles with diameters less than 100 nm in any external dimension. Examples of their use include titanium dioxide added as a pigment in products intended to be ingested by humans, silicon dioxide NPs are used in foods as an anticaking agent, and gold or iron oxide NPs can be used as vectors for drug delivery or contrast agents for specialized medical imaging. Although the intended use of these NPs is often to improve human health, it has come to the attention of investigators that NPs can have unintended or even detrimental effects on the organism. This work describes one such unintended effect of NP exposure from the perspective of exposure via the oral route. First, this Dissertation will explain an event referred to as brush border disruption that occurred after nanoparticles interacted with an in vitro model of the human intestinal epithelium. Second, this Dissertation will identify and characterize several consumer goods that were shown to contain titanium dioxide that are intended to be ingested. Third, this Dissertation shows that sedimentation due to gravity does not artifactually result in disruption of brush borders as a consequence of exposure to food grade titanium dioxide in vitro. Finally, this Dissertation will demonstrate that iron oxide nanoparticles elicited similar effects after exposure to an in vitro brush border expressing model of the human placenta. Together, these data suggest that brush border disruption is not an artifact of the material/cell culture model, but instead represents a bona fide biological response as a result of exposure to nanomaterial.

  10. Folding of intestinal brush border enzymes. Evidence that high-mannose glycosylation is an essential early event

    DEFF Research Database (Denmark)

    Danielsen, E M

    1992-01-01

    enzymes. In pulse-labeled mucosal explants, complete synthesis of the polypeptide chains of aminopeptidase N and sucrase-isomaltase required about 2 and 4 min, respectively, whereas maximal antiserum precipitation was acquired with half-times of 4-5 and 8 min, respectively. Fructose, which induces a...... posttranslational process. In the presence of fructose, not only the malglycosylated forms but also the electrophoretically normal, high-mannose-glycosylated form of the brush border enzymes were retained in the endoplasmic reticulum and proteolytically degraded. The results obtained demonstrate an intimate...... interrelationship between glycosylation and polypeptide folding in the synthesis of membrane glycoproteins and, more specifically, indicate that the timing of these two early biosynthetic events is essential for correct polypeptide folding....

  11. Bacterial outer membrane vesicles and vaccine applications.

    Science.gov (United States)

    Acevedo, Reinaldo; Fernández, Sonsire; Zayas, Caridad; Acosta, Armando; Sarmiento, Maria Elena; Ferro, Valerie A; Rosenqvist, Einar; Campa, Concepcion; Cardoso, Daniel; Garcia, Luis; Perez, Jose Luis

    2014-01-01

    Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP) process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB) using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA), serogroup W (dOMVW), and serogroup X (dOMVX) were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC), Bordetella pertussis (dOMVBP), Mycobacterium smegmatis (dOMVSM), and BCG (dOMVBCG). The immunogenicity of the OMV has been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice has shown their protective potential. dOMVB has been evaluated with non-neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin, and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates. PMID:24715891

  12. BACTERIAL OUTER MEMBRANE VESICLES AND VACCINE APPLICATIONS

    Directory of Open Access Journals (Sweden)

    Reinaldo eAcevedo

    2014-03-01

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

  13. Membrane Protrusion Coarsening and Nanotubulation within Giant Unilamellar Vesicles

    KAUST Repository

    Węgrzyn, Ilona

    2011-11-16

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

  14. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

    Science.gov (United States)

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

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  15. Electro-hydrodynamic effects on lipid membranes in giant vesicles

    Science.gov (United States)

    Staykova, Margarita; Yamamoto, Tetsuya; Lipowsky, Reinhard; Dimova, Rumiana

    2009-11-01

    Electric fields are widely applied for cell manipulation in numerous micron-scale systems. Here, we show for the first time that alternating electric fields may cause pronounced flows in the membrane of giant lipid vesicles as well as in the surrounding fluid media.^ The lipid vesicles are not only biomimetic model for the cell membrane but also have many potential biotechnological applications, e.g. as drug-delivery systems and micro-reactors. The reported effects should be considered in electric micro-manipulation procedures on cells and vesicles. They might be useful for applications in microfluidic technologies, for lipid mixing, trapping and displacement, as will be demonstrated. We also believe that our method for visualization of the lipid flows by fluorescently labeled intra-membrane domains will be helpful for studies on membrane behavior in vesicles subjected to shear or mechanical stresses.

  16. Trans-Membrane Area Asymmetry Controls the Shape of Cellular Organelles

    Directory of Open Access Journals (Sweden)

    Galina V. Beznoussenko

    2015-03-01

    Full Text Available Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature, or by intraluminal vesicles within endosomes (extraction of negative curvature controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50–60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant.

  17. Aminosilane/oleic acid vesicles as model membranes of protocells.

    Science.gov (United States)

    Douliez, Jean-Paul; Zhendre, Vanessa; Grélard, Axelle; Dufourc, Erick J

    2014-12-16

    Oleic acid vesicles represent good models of membrane protocells that could have existed in prebiotic times. Here, we report the formation, growth polymorphism, and dynamics of oleic acid spherical vesicles (1-10 μm), stable elongated vesicles (>50 μm length; 1-3 μm diameter), and chains of vesicles (pearl necklaces, >50 μm length; 1-3 μm diameter) in the presence of aminopropyl triethoxysilane and guanidine hydrochloride. These vesicles exhibit a remarkable behavior with temperature: spherical vesicles only are observed when keeping the sample at 4 °C for 2 h, and self-aggregated spherical vesicles occur upon freezing/unfreezing (-20/20 °C) samples. Rather homogeneous elongated vesicles are reformed upon heating samples at 80 °C. The phenomenon is reversible through cycles of freezing/heating or cooling/heating of the same sample. Deuterium NMR evidences a chain packing rigidity similar to that of phospholipid bilayers in cellular biomembranes. We expect these bilayered vesicles to be surrounded by a layer of aminosilane oligomers, offering a variant model for membrane protocells. PMID:25420203

  18. Apical Invasion of Intestinal Epithelial Cells by Salmonella typhimurium Requires Villin to Remodel the Brush Border Actin Cytoskeleton

    OpenAIRE

    Lhocine, Nouara; Arena, Ellen T.; Bomme, Perrine; Ubelmann, Florent; Prévost, Marie-Christine; Robine, Sylvie; Sansonetti, Philippe J.

    2015-01-01

    Summary Salmonella invasion of intestinal epithelial cells requires extensive, though transient, actin modifications at the site of bacterial entry. The actin-modifying protein villin is present in the brush border where it participates in the constitution of microvilli and in epithelial restitution after damage through its actin-severing activity. We investigated a possible role for villin in Salmonella invasion. The absence of villin, which is normally located at the bacterial entry site, l...

  19. Effect of monoassociation with probiotic strain Bifidobacterium bifidum on enterocyte brush-border enzymes in gnotobiotic mice

    Czech Academy of Sciences Publication Activity Database

    Kozáková, Hana; Řeháková, Zuzana; Kolínská, Jiřina; Tlaskalová, Helena

    2002-01-01

    Roč. 88, č. 1 (2002), s. 113. ISSN 0007-1145. [ Probiotic s and Health /2001./. London, 13.09.2002-14.09.2002] R&D Projects: GA ČR GA306/99/1383; GA ČR GA310/01/0933; GA AV ČR IAA5020101 Keywords : enterocyte * brush * border Subject RIV: EE - Microbiology, Virology Impact factor: 2.491, year: 2002

  20. Natural Selection at the Brush-Border: Adaptations to Carbohydrate Diets in Humans and Other Mammals.

    Science.gov (United States)

    Pontremoli, Chiara; Mozzi, Alessandra; Forni, Diego; Cagliani, Rachele; Pozzoli, Uberto; Menozzi, Giorgia; Vertemara, Jacopo; Bresolin, Nereo; Clerici, Mario; Sironi, Manuela

    2015-09-01

    Dietary shifts can drive molecular evolution in mammals and a major transition in human history, the agricultural revolution, favored carbohydrate consumption. We investigated the evolutionary history of nine genes encoding brush-border proteins involved in carbohydrate digestion/absorption. Results indicated widespread adaptive evolution in mammals, with several branches experiencing episodic selection, particularly strong in bats. Many positively selected sites map to functional protein regions (e.g., within glucosidase catalytic crevices), with parallel evolution at SI (sucrase-isomaltase) and MGAM (maltase-glucoamylase). In human populations, five genes were targeted by positive selection acting on noncoding variants within regulatory elements. Analysis of ancient DNA samples indicated that most derived alleles were already present in the Paleolithic. Positively selected variants at SLC2A5 (fructose transporter) were an exception and possibly spread following the domestication of specific fruit crops. We conclude that agriculture determined no major selective event at carbohydrate metabolism genes in humans, with implications for susceptibility to metabolic disorders. PMID:26319403

  1. Natural Selection at the Brush-Border: Adaptations to Carbohydrate Diets in Humans and Other Mammals

    Science.gov (United States)

    Pontremoli, Chiara; Mozzi, Alessandra; Forni, Diego; Cagliani, Rachele; Pozzoli, Uberto; Menozzi, Giorgia; Vertemara, Jacopo; Bresolin, Nereo; Clerici, Mario; Sironi, Manuela

    2015-01-01

    Dietary shifts can drive molecular evolution in mammals and a major transition in human history, the agricultural revolution, favored carbohydrate consumption. We investigated the evolutionary history of nine genes encoding brush-border proteins involved in carbohydrate digestion/absorption. Results indicated widespread adaptive evolution in mammals, with several branches experiencing episodic selection, particularly strong in bats. Many positively selected sites map to functional protein regions (e.g., within glucosidase catalytic crevices), with parallel evolution at SI (sucrase-isomaltase) and MGAM (maltase-glucoamylase). In human populations, five genes were targeted by positive selection acting on noncoding variants within regulatory elements. Analysis of ancient DNA samples indicated that most derived alleles were already present in the Paleolithic. Positively selected variants at SLC2A5 (fructose transporter) were an exception and possibly spread following the domestication of specific fruit crops. We conclude that agriculture determined no major selective event at carbohydrate metabolism genes in humans, with implications for susceptibility to metabolic disorders. PMID:26319403

  2. Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

    OpenAIRE

    Friedberg, I.; Kaback, H R

    1980-01-01

    Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+...

  3. Phospholipid flippases: building asymmetric membranes and transport vesicles

    OpenAIRE

    Sebastian, Tessy T.; Baldridge, Ryan D.; Xu, Peng; Graham, Todd R.

    2011-01-01

    Phospholipid flippases in the type IV P-type ATPase family (P4-ATPases) are essential components of the Golgi, plasma membrane and endosomal system that play critical roles in membrane biogenesis. These pumps flip phospholipid across the bilayer to create an asymmetric membrane structure with substrate phospholipids, such as phosphatidylserine and phosphatidylethanolamine, enriched within the cytosolic leaflet. The P4-ATPases also help form transport vesicles that bud from Golgi and endosomal...

  4. Membrane-enhanced surface acoustic wave analysis of grafted polymer brushes

    Science.gov (United States)

    Brass, David A.; Shull, Kenneth R.

    2008-04-01

    An analysis is developed for the frequency response of a quartz crystal resonator (often referred to as a quartz crystal microbalance) that is modified with a grafted solvent-swollen polymer brush and placed in contact with a membrane capping layer. The shear wave generated at the resonator surface couples into the membrane layer with an efficiency that is strongly dependent on the thickness of the swollen brush layer. As a result, the resonant frequency changes by a maximum amount that is closely approximated by the Sauerbrey shift for the capping layer. The calculated shift substantially decreases for increases in the brush thickness of approximately 10nm, which gives a net frequency response that is extremely sensitive to the degree of swelling of the polymer brush. An optimum capping layer thickness is determined by balancing the Sauerbrey shift against dissipative effects that weaken the crystal resonance. This optimum membrane thickness depends only weakly on the properties of the membrane material and is in the micron range. Detailed multilayer calculations are presented for the specific case of a poly(ethylene glycol) brush swollen with water and brought into contact with an elastomeric water-permeable membrane. These calculations confirm that the method is sensitive to the properties of the brush layer in the experimentally relevant thickness regime. Connections are also made to conceptually simpler two and three layer models of the acoustic impedance of the material systems that are brought into contact with the resonator.

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

    Directory of Open Access Journals (Sweden)

    Jong Suk Jin

    2011-12-01

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

  6. Hyperthermophilic archaea produce membrane vesicles that can transfer DNA

    NARCIS (Netherlands)

    Gaudin, M.; Gauliard, E.; Schouten, S.; Houel-Renault, L.; Lenormand, P.; Marguet, E.; Forterre, P.

    2013-01-01

    Thermococcales are hyperthermophilic archaea found in deep-sea hydrothermal vents. They have been recently reported to produce membrane vesicles (MVs) into their culture medium. Here, we have characterized the mode of production and determined the biochemical composition of MVs from two species of T

  7. Aggregation state of melittin in lipid vesicle membranes

    OpenAIRE

    John, Edgar; Jähnig, Fritz

    1991-01-01

    We have performed time-resolved fluorescence energy transfer measurements using melittin as donor and a modified melittin as acceptor. The melittin molecules were bound to fluid vesicle membranes of dimyristoylphosphatidylcholine. Analysis of the temporal decay of the energy transfer and of its variation with the donor and acceptor concentrations led to the conclusion that melittin in fluid membranes is usually monomeric. Only at the high melittin/lipid molar ratio of 1/200 and high ionic str...

  8. Giant vesicles functionally expressing membrane receptors for an insect pheromone.

    Science.gov (United States)

    Hamada, Satoshi; Tabuchi, Masashi; Toyota, Taro; Sakurai, Takeshi; Hosoi, Tomohiro; Nomoto, Tomonori; Nakatani, Kei; Fujinami, Masanori; Kanzaki, Ryohei

    2014-03-18

    To date, biochemical approaches to membrane receptors have been limited to the following methods: knockout or overexpression of membrane receptors by gene introduction and genome engineering or extraction of membrane receptor-surfactant complexes from innate cells and their introduction into model biomembranes. Here, we describe the development of a third method involving gene expression using cell-free in situ protein synthesis inside model biomembrane capsules. We verified this method by synthesizing olfactory receptors from the silkmoth Bombyx mori inside giant vesicles and found that they were excited in the presence of their ligand the Bombyx mori sex pheromone. PMID:24509495

  9. Control of biofouling on reverse osmosis polyamide membranes modified with biocidal nanoparticles and antifouling polymer brushes

    KAUST Repository

    Rahaman, Md. Saifur

    2014-01-01

    Thin-film composite (TFC) polyamide reverse osmosis (RO) membranes are prone to biofouling due to their inherent physicochemical surface properties. In order to address the biofouling problem, we have developed novel surface coatings functionalized with biocidal silver nanoparticles (AgNPs) and antifouling polymer brushes via polyelectrolyte layer-by-layer (LBL) self-assembly. The novel surface coating was prepared with polyelectrolyte LBL films containing poly(acrylic acid) (PAA) and poly(ethylene imine) (PEI), with the latter being either pure PEI or silver nanoparticles coated with PEI (Ag-PEI). The coatings were further functionalized by grafting of polymer brushes, using either hydrophilic poly(sulfobetaine) or low surface energy poly(dimethylsiloxane) (PDMS). The presence of both LBL films and sulfobetaine polymer brushes at the interface significantly increased the hydrophilicity of the membrane surface, while PDMS brushes lowered the membrane surface energy. Overall, all surface modifications resulted in significant reduction of irreversible bacterial cell adhesion. In microbial adhesion tests with E. coli bacteria, a normalized cell adhesion in the range of only 4 to 16% on the modified membrane surfaces was observed. Modified surfaces containing silver nanoparticles also exhibited strong antimicrobial activity. Membranes coated with LBL films of PAA/Ag-PEI achieved over 95% inactivation of bacteria attached to the surface within 1 hour of contact time. Both the antifouling and antimicrobial results suggest the potential of using these novel surface coatings in controlling the fouling of RO membranes. © The Royal Society of Chemistry 2014.

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

    Science.gov (United States)

    Gill, S.; Forterre, P.

    2016-01-01

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

  11. Ion induced changes in the structure of bordered pit membranes

    Directory of Open Access Journals (Sweden)

    Jinkee eLee

    2012-03-01

    Full Text Available Xylem hydraulic resistance varies with ion concentration in sap solution. It is assumed that this variation in resistance results from hydrogel like properties of pectins located in bordered pit membranes separating adjacent vessels. Although kinetics of the resistance change suggests swelling/deswelling behavior of the pectins, there is no direct evidence of this activity. In this report we provide evidence of structural changes in bordered pit membranes responding to variation in ionic concentration of solute around it using atomic force microscopy (AFM. AFM revealed bordered pit membranes as relatively smooth, soft and lacking any sharp edges surface when submerged in de-ionized water, in contrast to pictures from scanning electron microscope (SEM or AFM performed on air dry material. Exposure of the bordered pit membranes to 50 mM KCl solution resulted in significant changes in both surface physical properties with and elevation features as bordered pit membrane became harder, with visible edges of fibers and collapsed, while no change in porosity was observed. Analysis suggests a need for a major shift in our understanding to the physical bases of variable xylem resistance from change in porosity to change in pathway length. Findings support the role of actuating properties of hybrid hydrogel-cellulose materials in water redistribution and embolism resistance.

  12. Protective role of E. coli outer membrane vesicles against antibiotics.

    Science.gov (United States)

    Kulkarni, Heramb M; Nagaraj, R; Jagannadham, Medicharla V

    2015-12-01

    The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics. PMID:26640046

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

    Science.gov (United States)

    Barrett, John W.; Garcke, Harald; Nürnberg, Robert

    2015-11-01

    Vesicles and many biological membranes are made of two monolayers of lipid molecules and form closed lipid bilayers. The dynamical behavior of vesicles is very complex and a variety of forms and shapes appear. Lipid bilayers can be considered as a surface fluid and hence the governing equations for the evolution include the surface (Navier-)Stokes equations, which in particular take the membrane viscosity into account. The evolution is driven by forces stemming from the curvature elasticity of the membrane. In addition, the surface fluid equations are coupled to bulk (Navier-)Stokes equations. We introduce a parametric finite-element method to solve this complex free boundary problem and present the first three-dimensional numerical computations based on the full (Navier-)Stokes system for several different scenarios. For example, the effects of the membrane viscosity, spontaneous curvature, and area difference elasticity (ADE) are studied. In particular, it turns out, that even in the case of no viscosity contrast between the bulk fluids, the tank treading to tumbling transition can be obtained by increasing the membrane viscosity. Besides the classical tank treading and tumbling motions, another mode (called the transition mode in this paper, but originally called the vacillating-breathing mode and subsequently also called trembling, transition, and swinging mode) separating these classical modes appears and is studied by us numerically. We also study how features of equilibrium shapes in the ADE and spontaneous curvature models, like budding behavior or starfish forms, behave in a shear flow.

  14. Bacterial Outer Membrane Vesicles Induce Plant Immune Responses.

    Science.gov (United States)

    Bahar, Ofir; Mordukhovich, Gideon; Luu, Dee Dee; Schwessinger, Benjamin; Daudi, Arsalan; Jehle, Anna Kristina; Felix, Georg; Ronald, Pamela C

    2016-05-01

    Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions. PMID:26926999

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

    DEFF Research Database (Denmark)

    Ciofu, O; Beveridge, T J; Kadurugamuwa, J; Walther-Rasmussen, J; Høiby, N

    2000-01-01

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

  16. Ciprofloxacin encapsulation into giant unilamellar vesicles: membrane binding and release.

    Science.gov (United States)

    Kaszás, Nóra; Bozó, Tamás; Budai, Marianna; Gróf, Pál

    2013-02-01

    This study aimed at investigating some respects of binding and interaction between water-soluble drugs and liposomal carrier systems depending on their size and lamellarity. As model substance, ciprofloxacin hydrochloride (CPFX) was incorporated into giant unilamellar vesicles (GUVs) to study their CPFX encapsulation/binding capacity. To characterize molecular interactions of various CPFX microspecies with lipid bilayer, zeta potential and electron paramagnetic resonance (EPR) spectroscopy measurements were performed. The increase of the zeta potential at pH 5.4 but no change at pH 7.2 was interpreted in terms of the CPFX microspecies' distribution at the two pH values. EPR observations showed an increased fluidity because of CPFX binding to GUVs. We worked out and applied a three-compartment dialysis model to separately determine the rate of drug diffusion through the liposomal membrane. Equilibrium dialysis showed (a) different permeation of CPFX through the membranes of GUVs and multilamellar vesicles (MLVs), with characteristic half-lives of 54.4 and 18.1 h, respectively; and (b) increased retention of CPFX in case of GUVs with released amounts of 70% compared with about 97% in case of MLVs. Our results may provide further details for efficient design of liposomal formulations incorporating water-soluble drugs. PMID:23233199

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

    Science.gov (United States)

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

    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. PMID:27517914

  18. 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.

  19. Mechanisms of equinatoxin II-induced transport through the membrane of a giant phospholipid vesicle.

    OpenAIRE

    Mally, M; Majhenc, J; Svetina, S; Zeks, B.

    2002-01-01

    Protein equinatoxin II from sea anemone Actinia equina L. was used to form pores in phospholipid membranes. We studied the effect of these pores on the net transmembrane transport of sucrose and glucose by observing single giant (cell-size) vesicles under the phase contrast microscope. Sugar composition in the vesicle was determined by measuring the width of the halo, which appears around the vesicle in the phase contrast image. The transport of sugars was induced when a vesicle, filled with ...

  20. Determining membrane permeability of giant phospholipid vesicles from a series of videomicroscopy images

    CERN Document Server

    Peterlin, Primoz; Pisanski, Tomaz

    2008-01-01

    A technique for determining the permeability of a phospholipid membrane from a sequence of videomicrographs is described. A single giant unilamellar vesicle (GUV) is transferred using a micropipette from a solution of an impermeable solute (e.g., glucose or sucrose) into an iso-osmolar solution of a solute with a higher membrane permeability (e.g., glycerol). Upon the transfer, the vesicle swells until it reaches the tensile strength of the membrane, when the membrane breaks and a fraction of the vesicle volume is ejected, sufficient for the membrane to return to its relaxed value. The swelling-burst cycle repeats itself until the composition of the solution in the vesicle interior equilibrates with the external solution. A sequence of ~10.000 image frames is obtained from a CCD camera mounted on the optical microscope, documenting the process. On each frame, the vesicle radius is determined, and from the rate of swelling the membrane permeability can be obtained.

  1. Massive Formation of Intracellular Membrane Vesicles in Escherichia coli by a Monotopic Membrane-bound Lipid Glycosyltransferase*

    OpenAIRE

    Eriksson, Hanna M.; Wessman, Per; Ge, Changrong; Edwards, Katarina; Wieslander, Åke

    2009-01-01

    The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualiz...

  2. Borders as membranes :metaphors and models for improved policy in border regions.

    Energy Technology Data Exchange (ETDEWEB)

    Malczynski, Leonard A.; Passell, Howard David; Forster, Craig B. (University of Utah, Salt Lake City, UT); Cockerill, Kristan (Cockerill Consulting, Boone, NC)

    2005-10-01

    Political borders are controversial and contested spaces. In an attempt to better understand movement along and through political borders, this project applied the metaphor of a membrane to look at how people, ideas, and things ''move'' through a border. More specifically, the research team employed this metaphor in a system dynamics framework to construct a computer model to assess legal and illegal migration on the US-Mexico border. Employing a metaphor can be helpful, as it was in this project, to gain different perspectives on a complex system. In addition to the metaphor, the multidisciplinary team utilized an array of methods to gather data including traditional literature searches, an experts workshop, a focus group, interviews, and culling expertise from the individuals on the research team. Results from the qualitative efforts revealed strong social as well as economic drivers that motivate individuals to cross the border legally. Based on the information gathered, the team concluded that legal migration dynamics were of a scope we did not want to consider hence, available demographic models sufficiently capture migration at the local level. Results from both the quantitative and qualitative data searches were used to modify a 1977 border model to demonstrate the dynamic nature of illegal migration. Model runs reveal that current US-policies based on neo-classic economic theory have proven ineffective in curbing illegal migration, and that proposed enforcement policies are also likely to be ineffective. We suggest, based on model results, that improvement in economic conditions within Mexico may have the biggest impact on illegal migration to the U.S. The modeling also supports the views expressed in the current literature suggesting that demographic and economic changes within Mexico are likely to slow illegal migration by 2060 with no special interventions made by either government.

  3. Membrane vesicles: A simplified system for studying auxin transport

    Energy Technology Data Exchange (ETDEWEB)

    Goldsmith, M.H.M.

    1989-01-01

    Indoleacetic acid (IAA), the auxin responsible for regulation of growth, is transported polarly in plants. Several different models have been suggested to account for IAA transport by cells and its accumulation by membrane vesicles. One model sees diffusion of IAA driven by a pH gradient. The anion of a lipophilic weak acid like IAA or butyrate accumulates in an alkaline compartment in accord with the size of the pH gradient The accumulation of IAA may be diminished by the permeability of its lipophilic anion. This anion leak may be blocked by NPA. With anion efflux blocked, a gradient of two pH units would support an IAA accumulation of less than 50-fold at equilibrium (2) Another model sees diffusion of IAA in parallel with a saturable symport (IAA[sup [minus

  4. Na+-independent D-glucose transport in rabbit renal basolateral membranes

    International Nuclear Information System (INIS)

    To define the mechanism by which glucose is transported across the basolateral membrane of the renal proximal tubular cell, we measured D-[14C]glucose uptake in basolateral membrane vesicles from rabbit kidney. Na+-dependent D-glucose transport, demonstrable in brush-border vesicles, could not be demonstrated in basolateral membrane vesicles. In the absence of Na+, the uptake of D-[14C]glucose in basolateral vesicles was more rapid than that of L-[3H]glucose over a concentration range of 1-50 mM. Subtraction of the latter from the former uptakes revealed a saturable process with apparent Km of 9.9 mM and Vmax of 0.80 nmol.mg protein-1.s-1. To characterize the transport component of D-glucose uptake in basolateral vesicles, we measured trans stimulation of 2 mM D-[14C]glucose entry in the absence of Na+. Trans stimulation could be effected by preloading basolateral vesicles with D-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose, but not with L-glucose or alpha-methyl-D-glucoside. Trans-stimulated D-[14C]glucose uptake was inhibited by 0.1 mM phloretin or cytochalasin B but not phlorizin. In contrast, Na+-dependent D-[14C]glucose transport in brush-border vesicles was inhibited by phlorizin but not phloretin or cytochalasin B. Our findings are consistent with the presence of a Na+-independent D-glucose transporter in the proximal tubular basolateral membrane with characteristics similar to those of transporters present in nonepithelial cells

  5. The Lethal Cargo of Myxococcus xanthus Outer Membrane Vesicles

    Directory of Open Access Journals (Sweden)

    JamesEBerleman

    2014-09-01

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

  6. Ultrathin, freestanding, stimuli-responsive, porous membranes from polymer hydrogel-brushes

    Science.gov (United States)

    Kang, Chengjun; Ramakrishna, Shivaprakash N.; Nelson, Adrienne; Cremmel, Clement V. M.; Vom Stein, Helena; Spencer, Nicholas D.; Isa, Lucio; Benetti, Edmondo M.

    2015-07-01

    The fabrication of freestanding, sub-100 nm-thick, pH-responsive hydrogel membranes with controlled nano-morphology, based on modified poly(hydroxyethyl methacrylate) (PHEMA) is presented. Polymer hydrogel-brush films were first synthesized by surface-initiated atom transfer radical polymerization (SI-ATRP) and subsequently detached from silicon substrates by UV-induced photo-cleavage of a specially designed linker within the initiator groups. The detachment was also assisted by pH-induced osmotic forces generated within the films in the swollen state. The mechanical properties and morphology of the freestanding films were studied by atomic force microscopy (AFM). Inclusion of nanopores of controlled diameter was accomplished by performing SI-ATRP from initiator-coated surfaces that had previously been patterned with polystyrene nanoparticles. Assembly parameters and particle sizes could be varied, in order to fabricate nanoporous hydrogel-brush membranes with tunable pore coverage and characteristics. Additionally, due to the presence of weak polyacid functions within the hydrogel, the membranes exhibited pH-dependent thickness in water and reversible opening/closing of the pores.The fabrication of freestanding, sub-100 nm-thick, pH-responsive hydrogel membranes with controlled nano-morphology, based on modified poly(hydroxyethyl methacrylate) (PHEMA) is presented. Polymer hydrogel-brush films were first synthesized by surface-initiated atom transfer radical polymerization (SI-ATRP) and subsequently detached from silicon substrates by UV-induced photo-cleavage of a specially designed linker within the initiator groups. The detachment was also assisted by pH-induced osmotic forces generated within the films in the swollen state. The mechanical properties and morphology of the freestanding films were studied by atomic force microscopy (AFM). Inclusion of nanopores of controlled diameter was accomplished by performing SI-ATRP from initiator-coated surfaces that had

  7. Galectin-2 at the enterocyte brush border of the small intestine

    DEFF Research Database (Denmark)

    Thomsen, Martha Kampp; Hansen, Gert H; Danielsen, E Michael

    2009-01-01

    boundary. Together with the membrane glycolipids these lectins form stable lipid raft microdomains that also harbour several of the major digestive microvillar enzymes. In the present work, we identified a lactose-sensitive 14-kDa protein enriched in a microvillar detergent resistant fraction as galectin-2...... enzymes and pathogens....

  8. NMR studies of 5-hydroxytryptamine transport through large unilamellar vesicle membranes.

    OpenAIRE

    Viscio, D B; Prestegard, J H

    1981-01-01

    Nuclear magnetic resonance techniques developed to study membrane permeability in closed membrane systems have been used to investigate transport of 5-hydroxytryptamine across the phospholipid membranes of large unilamellar vesicles. The vesicles, modeling the 5-hydroxytryptamine storage organelles of blood platelets, contained a high internal level of ATP buffered at a pH low relative to the external solution. The resultant pH gradient drove accumulation of 5-hydroxytryptamine to a level con...

  9. Size-dependent, stochastic nature of lipid exchange between nano-vesicles and model membranes

    Science.gov (United States)

    Tabaei, Seyed R.; Gillissen, Jurriaan J. J.; Vafaei, Setareh; Groves, Jay T.; Cho, Nam-Joon

    2016-07-01

    The interaction of nanoscale lipid vesicles with cell membranes is of fundamental importance for the design and development of vesicular drug delivery systems. Here, we introduce a novel approach to study vesicle-membrane interactions whereby we are able to probe the influence of nanoscale membrane properties on the dynamic adsorption, exchange, and detachment of vesicles. Using total internal reflection fluorescence (TIRF) microscopy, we monitor these processes in real-time upon the electrostatically tuned attachment of individual, sub-100 nm vesicles to a supported lipid bilayer. The observed exponential vesicle detachment rate depends strongly on the vesicle size, but not on the vesicle charge, which suggests that lipid exchange occurs during a single stochastic event, which is consistent with membrane stalk formation. The fluorescence microscopy assay developed in this work may enable measuring of the probability of stalk formation in a controlled manner, which is of fundamental importance in membrane biology, offering a new tool to understand nanoscale phenomena in the context of biological sciences.The interaction of nanoscale lipid vesicles with cell membranes is of fundamental importance for the design and development of vesicular drug delivery systems. Here, we introduce a novel approach to study vesicle-membrane interactions whereby we are able to probe the influence of nanoscale membrane properties on the dynamic adsorption, exchange, and detachment of vesicles. Using total internal reflection fluorescence (TIRF) microscopy, we monitor these processes in real-time upon the electrostatically tuned attachment of individual, sub-100 nm vesicles to a supported lipid bilayer. The observed exponential vesicle detachment rate depends strongly on the vesicle size, but not on the vesicle charge, which suggests that lipid exchange occurs during a single stochastic event, which is consistent with membrane stalk formation. The fluorescence microscopy assay developed

  10. A method to investigate protein association with intact sealed mycobacterial membrane vesicles.

    Science.gov (United States)

    D'Lima, Nadia G; Teschke, Carolyn M

    2015-09-15

    In mycobacteria, probing the association of cytoplasmic proteins with the membrane itself, as well as with integral or peripheral membrane proteins, is limited by the difficulty in extracting intact sealed membrane vesicles due to the complex cell wall structure. Here we tested the association of Mycobacterium tuberculosis SecA1 and SecA2 proteins with intact membrane vesicles by a flotation assay using iodixanol density gradients. These protocols have wide applications for studying the association of other mycobacterial cytoplasmic proteins with the membrane and membrane-associated proteins. PMID:26099936

  11. Evidence that coated vesicles transport acetylcholine receptors to the surface membrane of chick myotubes

    OpenAIRE

    1984-01-01

    Coated vesicles are present in the myoplasm of embryonic chick myotubes grown in vitro. They are most numerous beneath regions of the surface membrane that contain a high density of acetylcholine receptors (AChR). Prolonged exposure of myotubes to saline extract of chick brain increases the number of intracellular AChR and the number of coated vesicles. This suggests that coated vesicles contain AChR, and this hypothesis was tested with horseradish peroxidase-alpha-bungarotoxin (HRP-alpha BTX...

  12. Outer membrane vesicles – offensive weapons or good Samaritans?

    Directory of Open Access Journals (Sweden)

    Ingar Olsen

    2015-04-01

    Full Text Available Outer membrane vesicles (OMVs from Gram-negative bacteria were first considered as artifacts and were followed with disbelief and bad reputation. Later, their existence was accepted and they became characterized as bacterial bombs, virulence bullets, and even decoys. Today, we know that OMVs also can be involved in cell–cell signaling/communication and be mediators of immune regulation and cause disease protection. Furthermore, OMVs represent a distinct bacterial secretion pathway selecting and protecting their cargo, and they can even be good Samaritans providing nutrients to the gut microbiota maintaining commensal homeostasis beneficial to the host. The versatility in functions of these nanostructures is remarkable and includes both defense and offense. The broad spectrum of usability does not stop with that, as it now seems that OMVs can be used as vaccines and adjuvants or vehicles engineered for drug treatment of emerging and new diseases not only caused by bacteria but also by virus. They may even represent new ways of selective drug treatment.

  13. Liquid general anesthetics lower critical temperatures in plasma membrane vesicles

    CERN Document Server

    Gray, Ellyn; Machta, Benjamin B; Veatch, Sarah L

    2013-01-01

    A large and diverse array of small hydrophobic molecules induce general anesthesia. Their efficacy as anesthetics has been shown to correlate both with their affinity for a hydrophobic environment and with their potency in inhibiting certain ligand gated ion channels. Here we explore the effects that n-alcohols and other liquid anesthetics have on the two-dimensional miscibility critical point observed in cell derived giant plasma membrane vesicles (GPMVs). We show that anesthetics depress the critical temperature (Tc) of these GPMVs without strongly altering the ratio of the two liquid phases found below Tc. The magnitude of this affect is consistent across n-alcohols when their concentration is rescaled by the median anesthetic concentration (AC50) for tadpole anesthesia, but not when plotted against the overall concentration in solution. At AC50 we see a 4{\\deg}C downward shift in Tc, much larger than is typically seen in the main chain transition at these anesthetic concentrations. GPMV miscibility critic...

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

    Science.gov (United States)

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

    2016-04-01

    Outer membrane vesicles (OMV) are critical elements in many host-cell/microbe interactions. Previous studies of the symbiotic association between Euprymna scolopes and Vibrio fischeri had shown that within 12 h of colonizing crypts deep within the squid's light organ, the symbionts trigger an irreversible programme of tissue development in the host. Here, we report that OMV produced by V. fischeri are powerful contributors to this process. The first detectable host response to the OMV is an increased trafficking of macrophage-like cells called haemocytes into surface epithelial tissues. We showed that exposing the squid to other Vibrio species fails to induce this trafficking; however, addition of a high concentration of their OMV, which can diffuse into the crypts, does. We also provide evidence that tracheal cytotoxin released by the symbionts, which can induce haemocyte trafficking, is not part of the OMV cargo, suggesting two distinct mechanisms to induce the same morphogenesis event. By manipulating the timing and localization of OMV signal delivery, we showed that haemocyte trafficking is fully induced only when V. fischeri, the sole species able to reach and grow in the crypts, succeeds in establishing a sustained colonization. Further, our data suggest that the host's detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis. PMID:26399913

  15. Calibration of Membrane Viscosity of the Reconstituted Vesicles by Measurement of the Rotational Diffusion of Bacteriorhodopsin

    Institute of Scientific and Technical Information of China (English)

    王敖金; 胡坤生

    2002-01-01

    Membrane viscosity of the reconstituted vesicles was calibrated by rotational diffusion of bacteriorhodopsin (BR) in dimyristoylphosphatidylcholine (DMPC) and egg phosphatidylcholine (PC) vesicles. Rotational diffusion of BR in the vesicles was measured by flash-induced absorption anisotropy decay. BR was, for the first time, reconstituted successfully into DMPC and egg PC vesicles. From the measurement of flash-induced absorption anisotropy decay of BR, the value of rotational diffusion coefficient D was obtained from each curve fitting by a global fitting procedure and, in turn, membrane viscosity η was estimated from D. The results have shown that membrane viscosity is temperature-dependent. It was decreased as temperature increased, but a transition occurred in the region of the respective phase transition of DMPC and egg PC, respectively. The decrease of η was fast near the phase transition for DMPC and egg PC. Few effects of lipid/BR ratio and glycerol or sucrose in suspension medium on membrane viscosity were found.

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

    DEFF Research Database (Denmark)

    Bagatolli, Luis; Needham, David

    2014-01-01

    This manuscript discusses basic methodological aspects of optical microscopy and micromanipulation methods to study membranes and reviews methods to generate giant unilamellar vesicles (GUVs). In particular, we focus on the use of fluorescence microscopy and micropipette manipulation techniques to...... experimentation. For instance, using different fluorescent reporters, fluorescence microscopy allows strategies to study membrane lateral structure/dynamics at the level of single vesicles of diverse compositions. The micropipette manipulation technique on the other hand, uses Hoffman modulation contrast...

  17. Outer membrane vesicles of Lysobacter sp. XL1: biogenesis, functions, and applied prospects.

    Science.gov (United States)

    Kudryakova, Irina V; Shishkova, Nina A; Vasilyeva, Natalia V

    2016-06-01

    Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have been intensively investigated in recent times. Vesicle formation models have been proposed, some factors affecting the process were established, and important roles vesicles play in vital activities of their producing cells were determined. Studies of pathogenic bacterial vesicles contribute to understanding the causes of acute infection and developing drugs on their basis. Despite intensive research, issues associated with the understanding of vesicle biogenesis, the mechanisms of bacterium-bacterium and pathogen-host interactions with participation of vesicles, still remain unresolved. This review discusses some results obtained in the research into OMVs of Lysobacter sp. XL1 VKM B-1576. This bacterium secretes into the environment a spectrum of bacteriolytic enzymes that hydrolyze peptidoglycan of competing bacteria, thus leading to their lysis. One of these enzymes, lytic endopeptidase L5, has been shown not only to be secreted by means of vesicles but also to be involved in their formation. As part of vesicles, the antimicrobial potential of L5 enzyme has been found to be considerably expanded. Vesicles have been shown to have a therapeutic effect in respect of anthrax infection and staphylococcal sepsis modelled in mice. The scientific basis for constructing liposomal antimicrobial preparations from vesicle phospholipids and recombinant bacteriolytic enzyme L5 has been formed. PMID:27098257

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    exosomes and microvesicles. These vesicles contain various types of RNAs and proteins, suggested to transfer health-promoting messages from mother to offspring. However, the variety of the vesicles in milk is less understood and, additionally, complicated by the complexity of more pronounced milk...... components. Here we present a novel strategy for a short, gentle and non-denaturing isolation of skim milk-derived membrane vesicles. Methods: Untreated fresh bovine milk was defatted to remove milk fat globules. The resulting skim milk was subjected to ultracentrifugation. The resulting ochre......-coloured soluble fraction above the casein pellet were further isolated from casein remnants by size-exclusion chromatography. Isolated membrane vesicles were investigated by electron microscopy, sucrose density centrifugation, western blotting and particle size analysis. Results: A crude phospholipid membrane...

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

    DEFF Research Database (Denmark)

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

    exosomes and microvesicles. These vesicles contain various types of RNAs and proteins, suggested to transfer health-promoting messages from mother to offspring. However, the variety of the vesicles in milk is less understood and, additionally, complicated by the complexity of more pronounced milk...... components. Here we present a novel strategy for a short, gentle and non-denaturing isolation of skim milk-derived membrane vesicles. Methods: Untreated fresh bovine milk was defatted to remove milk fat globules. The resulting skim milk was subjected to ultracentrifugation. The resulting ochre......-coloured soluble fraction above the casein pellet were further isolated from casein remnants by size-exclusion chromatography. Isolated membrane vesicles were investigated by electron microscopy, sucrose density centrifugation, western blotting and particle size analysis. Results: A crude phospholipid membrane...

  20. Extracellular Membrane Vesicles and Phytopathogenicity of Acholeplasma laidlawii PG8

    Directory of Open Access Journals (Sweden)

    Vladislav M. Chernov

    2012-01-01

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

  1. A model for membrane patchiness: lateral diffusion in the presence of barriers and vesicle traffic.

    OpenAIRE

    Gheber, L A; Edidin, M

    1999-01-01

    Patches (lateral heterogeneities) of cell surface membrane proteins and lipids have been imaged by a number of different microscopy techniques. This patchiness has been taken as evidence for the organization of membranes into domains whose composition differs from the average for the entire membrane. However, the mechanism and specificity of patch formation are not understood. Here we show how vesicle traffic to and from a cell surface membrane can create patches of molecules of the size obse...

  2. Towards fully automated Identification of Vesicle-Membrane Fusion Events in TIRF Microscopy

    Science.gov (United States)

    Vallotton, Pascal; James, David E.; Hughes, William E.

    2007-11-01

    Total Internal Reflection Fluorescence Microscopy (TIRFM) is imposing itself as the tool of choice for studying biological activity in close proximity to the plasma membrane. For example, the exquisite selectivity of TIRFM allows monitoring the diffusion of GFP-phogrin vesicles and their recruitment to the plasma membrane in pancreatic β-cells. We present a novel computer vision system for automatically identifying the elusive fusion events of GFP-phogrin vesicles with the plasma membrane. Our method is based on robust object tracking and matched filtering. It should accelerate the quantification of TIRFM data and allow the extraction of more biological information from image data to support research in diabetes and obesity.

  3. Formation of polyhedral vesicles and polygonal membrane tubes induced by banana-shaped proteins

    Science.gov (United States)

    Noguchi, Hiroshi

    2015-12-01

    The shape transformations of fluid membranes induced by curved protein rods are studied using meshless membrane simulations. The rod assembly at low rod density induces a flat membrane tube and oblate vesicle. It is found that the polyhedral shapes are stabilized at high rod densities. The discrete shape transition between triangular and buckled discoidal tubes is obtained and their curvature energies are analyzed by a simple geometric model. For vesicles, triangular hosohedron and elliptic-disk shapes are formed in equilibrium, whereas tetrahedral and triangular prism shapes are obtained as metastable states.

  4. Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles

    Science.gov (United States)

    Kristjansson, H.; Hochstein, L. I.

    1986-01-01

    Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.

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

    Directory of Open Access Journals (Sweden)

    Smith Pete J

    2002-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Bartosz Różycki

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

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

    CERN Document Server

    Walani, Nikhil; Agrawal, Ashutosh

    2015-01-01

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

  8. Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components

    OpenAIRE

    Ellen, Albert F.; Albers, Sonja-Verena; Huibers, Wim; Pitcher, Angela; Hobel, Cedric F. V.; Schwarz, Heinz; Folea, Mihaela; Schouten, Stefan; Boekema, Egbert J.; Poolman, Bert; Driessen, Arnold J. M.

    2009-01-01

    The crenarchaea Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii, release membrane vesicles into the medium. These membrane vesicles consist of tetraether lipids and are coated with an S-layer. A proteomic analysis reveals the presence of proteins homologous to subunits of the eukaryotic endosomal sorting complex required for transport (ESCRT). Immunodetection of one of these homologs suggest a cell surface localization in intact cells. These data suggest that the membrane vesicles ...

  9. Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

    International Nuclear Information System (INIS)

    Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

  10. Studies on resistance mechanism of Bombyx mori against Bacillus thuringiensis insecticidal Cry1Ac toxin-Role of Cry1Ac binding proteins localizing on peritrophic and epithelial cell membranes of midgut-

    OpenAIRE

    Shitomi, Yasuyuki; 蔀, 泰幸

    2006-01-01

    Bombyx mori, hybrid Shunrei x Shogetsu, is susceptible to Cry1Aa and insensitive to Cry1Ac. Toxicity of Cry toxin can be correlated with the presence of a specific receptor in midgut epithelial cell membrane in insect. In surface plasmon resonance (SPR) and ligand blot analysis, however, many kinds of brush border membrane vesicle (BBMV) proteins were shown to bind to Cry1Ac with almost equal intensity in both resistance and susceptible insects. This suggests that majority of the bindings bet...

  11. Active uptake of tetracycline by membrane vesicles from susceptible Escherichia coli.

    OpenAIRE

    McMurry, L M; Cullinane, J C; Petrucci, R E; Levy, S. B.

    1981-01-01

    A major portion of tetracycline accumulation by susceptible bacterial cells is energy dependent. Inner membrane vesicles prepared from susceptible Escherichia coli cells concentrated tetracycline 2.5 to 5 times above the external concentration when the electron transport substrate D-lactate or reduced phenazine methosulfate was added. This stimulation was reversed by cyanide, 2,4-dinitrophenol, and carbonyl cyanide m-chlorophenyl hydrazone. These vesicles data showed that proton motive force ...

  12. Growth Conditions and Cell Cycle Phase Modulate Phase Transition Temperatures in RBL-2H3 Derived Plasma Membrane Vesicles.

    Directory of Open Access Journals (Sweden)

    Erin M Gray

    Full Text Available Giant plasma membrane vesicle (GPMV isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12 h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune

  13. ACTIVE CALCIUM TRANSPORT IN PLASMA MEMBRANE VESICLES FROM DEVELOPING COTYLEDONS OF COMMON BEAN

    Institute of Scientific and Technical Information of China (English)

    黄建中; 陈子元

    1995-01-01

    Plasma membrane vesicles were prepared from the developing cotyledons of common bean (Phaseolus vulgaris L cv Diyundou)by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes.The putative plasma membrane fraction was minimalyy contaminated by membranes other than plasma membrane and hence was of high purity,It exhibited a Ca2+-dependent ATPase activity,which was inhibited by 1umol/L EB and promoted by calcium ionophore A23187.Such an activity was responsible for the observed ATP dependent 45Ca2+ uptake into inside-out plasma membrane vesicles.This process was stimulated by 0.5μmol/L CaM and 20μmol/L IAA but inhibited by 2μmol/L ABA and abolished by A23187,Possible role of cytoplasmic Ca2+ in mediating phytohormones activity is discussed.

  14. Active calcium transport in plasma membrane vesicles from developing cotyledons of common bean

    International Nuclear Information System (INIS)

    Plasma membrane vesicles were prepared from the developing cotyledons of common bean (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes. The putative plasma membrane fraction was minimally contaminated by membranes other than plasma membrane and hence was of high purity. It exhibited a Ca2+-dependent ATPase activity, which was inhibited by 1 μmol/L EB and promoted by calcium ionophore A23187. Such an activity was responsible for the observed ATP-dependent 45Ca2+ uptake into inside-out plasma membrane vesicles. This process was stimulated by 0.6 μmol/L CaM and 20 μmol/L IAA but inhibited by 2 μmol/L ABA and abolished by A23187. Possible role of cytoplasmic Ca2+ in mediating phytohormones activity is discussed

  15. 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.

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

    OpenAIRE

    Leermakers, F.A.M.

    1988-01-01

    The aim of the present study was to unravel the general equilibrium physical properties of lipid bilayer membranes. We consider four major questions:1. What determines the morphology of the association colloids (micelles, membranes, vesicles) in general?2. Do the apolar tails of the lipids in the bilayer organise themselves more like matches in a box or rather like hot spaghetti in a pan?3. How does this membrane organisation depend on temperature?4. How do additives like surfactants or polym...

  17. Vesicle Photonics

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-04-03

    Thin membranes, under appropriate boundary conditions, can self-assemble into vesicles, nanoscale bubbles that encapsulate and hence protect or transport molecular payloads. In this paper, we review the types and applications of light fields interacting with vesicles. By encapsulating light-emitting molecules (e.g. dyes, fluorescent proteins, or quantum dots), vesicles can act as particles and imaging agents. Vesicle imaging can take place also under second harmonic generation from vesicle membrane, as well as employing mass spectrometry. Light fields can also be employed to transport vesicles using optical tweezers (photon momentum) or directly pertrurbe the stability of vesicles and hence trigger the delivery of the encapsulated payload (photon energy).

  18. Using vesicles to study the effect of sterols on the mechanical strength of lipid membranes and the protein-lipid membrane Interaction

    OpenAIRE

    Patty, Philipus Josepus

    2006-01-01

    In this thesis, studies of two biophysical topics will be discussed: the effect of sterols on the mechanical strength of lipid membranes, and the interaction between Cytidine 5'-triphosphate(CTP):phosphocholine cytidylyltransferase (CCT) and lipid membranes. The mechanical strength of lipid membranes was probed by measuring the lysis tension of vesicles, as determined from the minimum pressure required to extrude vesicles through small pores. The vesicles used in these experiments were made f...

  19. Light-induced membrane potential and pH gradient in Halobacterium halobium envelope vesicles.

    Science.gov (United States)

    Renthal, R; Lanyi, J K

    1976-05-18

    Illumination of envelope vesicles prepared from Halobacterium halobium cells causes translocation of protons from inside to outside, due to the light-induced cycling of bacteriorhodopsin. This process results in a pH gradient across the membranes, an electrical potential, and the movements of K+ and Na+. The electrical potential was estimated by following the fluorescence of a cyanine dye, 3,3'-dipentyloxadicarbocyanine. Illumination of H. halobium vesicles resulted in a rapid, reversible decrease of the dye fluorescence, by as much as 35%. This effect was not seen in nonvesicular patches of purple membrane. Observation of maximal fluorescence decreases upon ilumination of vesicles required an optimal dye/membrane protein ratio. The pH optimum for the lightinduced fluorescence decrease was 6.0. The decrease was linear with actinic light intensity up to about 4 X 10(5) ergs cn-2 s-1. Valinomycin, gramicidin, and triphenylmethylphosphonium ion all abolished the fluorescence changes. However, the light-induced pH change was enhanced by these agents. Conversely, buffered vesicles showed no pH change but gave the same or larger fluorescence changes. Thus, we have identified the fluorescence decrease with a light-induced membrane potential, inside negative. By using valinomycin-K+-induced membrane potentials, we calibrated the fluorescence decrease with calculated Nernst diffusion potentials. We found a linear dependence between potential and fluorescence decrease of 3 mV/%, up to 90 mV. When the envelope vesicles were illuminated, the total proton-motive force generated was dependent on the presence of Na+ and K+ and their concentration gradients across the membrane. In general, K+ appeared to be more permeable than Na+ and, thus, permitted development of greater pH gradients and lower electrical potentials. By calculating the total proton-motive force from the sum of the pH and potential terms, we found that the vesicles can produce proton-motive forces near--200 m

  20. Affinity Separation of Lectins Using Porous Membranes Immobilized with Glycopolymer Brushes Containing Mannose or N-Acetyl-D-Glucosamine

    Directory of Open Access Journals (Sweden)

    Yoshiko Miura

    2013-07-01

    Full Text Available Porous membranes with glycopolymer brushes were prepared as biomaterials for affinity separation. Glycopolymer brushes contained acrylic acid and D-mannose or N-acetyl-D-glucosamine, and were formed on substrates by surface-initiated atom transfer radical polymerization. The presence of glycopolymer brush was confirmed by X-ray photoelectron spectroscopy, contact angle, and ellipsometry measurements. The interaction between lectin and the glycopolymer immobilized on glass slides was confirmed using fluorescent-labeled proteins. Glycopolymer-immobilized surfaces exhibited specific adsorption of the corresponding lectin, compared with bovine serum albumin. Lectins were continuously rejected by the glycopolymer-immobilized membranes. When the protein solution was permeated through the glycopolymer-immobilized membrane, bovine serum albumin was not adsorbed on the membrane surface. In contrast, concanavalin A and wheat germ agglutinin were rejected by membranes incorporating D-mannose or N-acetyl-D-glucosamine, respectively. The amounts of adsorbed concanavalin A and wheat germ agglutinin was increased five- and two-fold that of adsorbed bovine serum albumin, respectively.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  2. Numerical observation of non-axisymmetric vesicles in fluid membranes

    CERN Document Server

    Jie, Y; Ji Xing Liu; Zhong Can Ou Yang; Jie, Yan; Quan-Hui, Liu; Ji-Xing, Liu; Zhong-Can, Ou-Yang

    1998-01-01

    By means of Surface Evolver (Exp. Math,1,141 1992), a software package of brute-force energy minimization over a triangulated surface developed by the geometry center of University of Minnesota, we have numerically searched the non-axisymmetric shapes under the Helfrich spontaneous curvature (SC) energy model. We show for the first time there are abundant mechanically stable non-axisymmetric vesicles in SC model, including regular ones with intrinsic geometric symmetry and complex irregular ones. We report in this paper several interesting shapes including a corniculate shape with six corns, a quadri-concave shape, a shape resembling sickle cells, and a shape resembling acanthocytes. As far as we know, these shapes have not been theoretically obtained by any curvature model before. In addition, the role of the spontaneous curvature in the formation of irregular crenated vesicles has been studied. The results shows a positive spontaneous curvature may be a necessary condition to keep an irregular crenated shap...

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

    International Nuclear Information System (INIS)

    A platelet membrane preparation, enriched in plasma membrane markers, took up 45Ca2+ 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 Ca2+ released by IP3 was eliminated by the addition of vanadate to inhibit Ca+-ATPase-mediated DTS Ca2+ sequestration and by the finding that only plasma membrane vesicles exhibit Na+-dependent Ca2+ uptake. Ca2+ released by IP3 was dependent on low extravesicular Ca2+ concentrations. IP3-induced Ca2+ release was additive to that released by Na+ addition while GTP or polyethylene glycol (PEG) had no effect. These results strongly suggest that IP3 facilitates extracellular Ca2+ influx in addition to release from DTS membranes

  4. High flux, positively charged loose nanofiltration membrane by blending with poly (ionic liquid) brushes grafted silica spheres

    International Nuclear Information System (INIS)

    Highlights: • SiO2 spheres were modified by poly (ionic liquid) brushes via RATRP. • Positively charged NF membranes were fabricated by incorporation of SiO2-PIL. • The membranes exhibited higher rejection for dyes and superior penetration for salts. - Abstract: Silica spheres modified by poly (ionic liquid) brushes, a novel positively charged nanomaterial is prepared by atom transfer radical polymerization (ATRP). A high flux positively charged loose nanofiltration membrane is fabricated via “blending-phase inversion” method. The morphology structures, hydrophilicity, thermal and mechanical properties, permeation performance of these membranes are investigated in detail. The results reveal that the hybrid membranes have enhanced surface hydrophilicity, water permeability, thermal stability, and mechanical properties. Characterization of membrane separation properties shows that the hybrid membranes possess higher salt permeability and relatively higher rejection for reactive dyes, which may open opportunities for the recycling of reactive dyes wastewater. Moreover, such hybrid membranes have an outstanding operational stability and salts concentration showed little effect on the separation properties

  5. High flux, positively charged loose nanofiltration membrane by blending with poly (ionic liquid) brushes grafted silica spheres

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Liang [School of Chemical Engineering and Energy, Zhengzhou University, Zhengzhou 450001 (China); Zhang, Yatao, E-mail: zhangyatao@zzu.edu.cn [School of Chemical Engineering and Energy, Zhengzhou University, Zhengzhou 450001 (China); UNESCO Center for Membrane Science and Technology, School of Chemical Engineering, University of New South Wales, Sydney, NSW 2052 (Australia); Wang, Yuanming; Zhang, Haoqin [School of Chemical Engineering and Energy, Zhengzhou University, Zhengzhou 450001 (China); Liu, Jindun, E-mail: liujindun@zzu.edu.cn [School of Chemical Engineering and Energy, Zhengzhou University, Zhengzhou 450001 (China)

    2015-04-28

    Highlights: • SiO{sub 2} spheres were modified by poly (ionic liquid) brushes via RATRP. • Positively charged NF membranes were fabricated by incorporation of SiO{sub 2}-PIL. • The membranes exhibited higher rejection for dyes and superior penetration for salts. - Abstract: Silica spheres modified by poly (ionic liquid) brushes, a novel positively charged nanomaterial is prepared by atom transfer radical polymerization (ATRP). A high flux positively charged loose nanofiltration membrane is fabricated via “blending-phase inversion” method. The morphology structures, hydrophilicity, thermal and mechanical properties, permeation performance of these membranes are investigated in detail. The results reveal that the hybrid membranes have enhanced surface hydrophilicity, water permeability, thermal stability, and mechanical properties. Characterization of membrane separation properties shows that the hybrid membranes possess higher salt permeability and relatively higher rejection for reactive dyes, which may open opportunities for the recycling of reactive dyes wastewater. Moreover, such hybrid membranes have an outstanding operational stability and salts concentration showed little effect on the separation properties.

  6. Cholesterol depletion of enterocytes. Effect on the Golgi complex and apical membrane trafficking

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Niels-Christiansen, L L; Thorsen, Evy; Immerdal, Lissi; Danielsen, E M

    2000-01-01

    %. Morphologically, the Golgi complex/trans-Golgi network was partially transformed into numerous 100-200 nm vesicles. By immunogold electron microscopy, aminopeptidase N was localized in these Golgi-derived vesicles as well as at the basolateral cell surface, indicating a partial missorting. Biochemically, the...... rates of the Golgi-associated complex glycosylation and association with rafts of newly synthesized aminopeptidase N were reduced, and less of the enzyme had reached the brush border membrane after 2 h of labeling. In contrast, the basolateral Na(+)/K(+)-ATPase was neither missorted nor raft......-associated. Our results implicate the Golgi complex/trans-Golgi network in raft formation and suggest a close relationship between this event and apical membrane trafficking....

  7. Multiple Membrane Interactions and Versatile Vesicle Deformations Elicited by Melittin

    Directory of Open Access Journals (Sweden)

    Kingo Takiguchi

    2013-04-01

    Full Text Available Melittin induces various reactions in membranes and has been widely studied as a model for membrane-interacting peptide; however, the mechanism whereby melittin elicits its effects remains unclear. Here, we observed melittin-induced changes in individual giant liposomes using direct real-time imaging by dark-field optical microscopy, and the mechanisms involved were correlated with results obtained using circular dichroism, cosedimentation, fluorescence quenching of tryptophan residues, and electron microscopy. Depending on the concentration of negatively charged phospholipids in the membrane and the molecular ratio between lipid and melittin, melittin induced the “increasing membrane area”, “phased shrinkage”, or “solubilization” of liposomes. In phased shrinkage, liposomes formed small particles on their surface and rapidly decreased in size. Under conditions in which the increasing membrane area, phased shrinkage, or solubilization were mainly observed, the secondary structure of melittin was primarily estimated as an α-helix, β-like, or disordered structure, respectively. When the increasing membrane area or phased shrinkage occurred, almost all melittin was bound to the membranes and reached more hydrophobic regions of the membranes than when solubilization occurred. These results indicate that the various effects of melittin result from its ability to adopt various structures and membrane-binding states depending on the conditions.

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

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

    2016-03-01

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

  10. Multiple Membrane Interactions and Versatile Vesicle Deformations Elicited by Melittin

    OpenAIRE

    Kingo Takiguchi; Michio Homma; Yasunori Yokoyama; Yohko Tanaka-Takiguchi; Tomoyoshi Takahashi; Fumimasa Nomura

    2013-01-01

    Melittin induces various reactions in membranes and has been widely studied as a model for membrane-interacting peptide; however, the mechanism whereby melittin elicits its effects remains unclear. Here, we observed melittin-induced changes in individual giant liposomes using direct real-time imaging by dark-field optical microscopy, and the mechanisms involved were correlated with results obtained using circular dichroism, cosedimentation, fluorescence quenching of tryptophan residues, and e...

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

    Science.gov (United States)

    Yamashita, Akihiro; Fujimoto, Masaru; Katayama, Kenta; Yamaoka, Shohei; Tsutsumi, Nobuhiro; Arimura, Shin-ichi

    2016-01-01

    Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (mitochondrial outer-membrane protrusions; MOPs), while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (mitochondrial-derived vesicles). Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN). The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence. PMID:26752045

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

    Directory of Open Access Journals (Sweden)

    Akihiro Yamashita

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

  13. Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli.

    OpenAIRE

    Chen, L.; Rhoads, D; Tai, P C

    1985-01-01

    We previously described a system for translocating the periplasmic enzyme alkaline phosphatase and the outer membrane protein OmpA into inverted membrane vesicles of Escherichia coli. We have now optimized and substantially improved the translocation system by including polyamines and by reducing the amount of membrane used. Under these conditions, efficient translocation was seen even posttranslationally, i.e., when vesicles were not added until after protein synthesis was stopped. This was ...

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

    DEFF Research Database (Denmark)

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

    1992-01-01

    Microvillar membrane vesicle preparations with varying content of aminopeptidase N were prepared from enterocytes of the pig small intestine. Postembedding immunogold labelling of aminopeptidase N was performed on these glutaraldehyde/paraformaldehyde-fixed, osmium tetroxide-treated and Epon......-embedded microvillar membrane vesicles. The number of gold particles per micrometre microvillar membrane (labelling intensity) was calculated and compared to the corresponding enzymatic activity. A very close relationship was found between labelling intensity and aminopeptidase N activity, demonstrating that...

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

    Science.gov (United States)

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

    2016-07-01

    Their biodegradable nature and ability to target cells make biological vesicles potential nanocarriers for bioactives delivery. In this work, the interaction between proteoliposomes enriched in aquaporins derived from broccoli plants and the glucosinolates was evaluated. The vesicles were stored at different temperatures and their integrity was studied. Determination of glucosinolates, showed that indolic glucosinolates were more sensitive to degradation in aqueous solution than aliphatic glucosinolates. Glucoraphanin was stabilized by leaf and root proteoliposomes at 25°C through their interaction with aquaporins. An extensive hydrogen bond network, including different aquaporin residues, and hydrophobic interactions, as a consequence of the interaction between the linear alkane chain of glucoraphanin and Glu31 and Leu34 protein residues, were established as the main stabilizing elements. Combined our results showed that plasma membrane vesicles from leaf and root tissues of broccoli plants may be considered as suitable carriers for glucosinolate which stabilization can be potentially attributed to aquaporins. PMID:27022872

  16. Tubular lipid membranes pulled from vesicles: Dependence of system equilibrium on lipid bilayer curvature

    Science.gov (United States)

    Golushko, I. Yu.; Rochal, S. B.

    2016-01-01

    Conditions of joint equilibrium and stability are derived for a spherical lipid vesicle and a tubular lipid membrane (TLM) pulled from this vesicle. The obtained equations establish relationships between the geometric and physical characteristics of the system and the external parameters, which have been found to be controllable in recent experiments. In particular, the proposed theory shows that, in addition to the pressure difference between internal and external regions of the system, the variable spontaneous average curvature of the lipid bilayer (forming the TLM) also influences the stability of the lipid tube. The conditions for stability of the cylindrical phase of TLMs after switching off the external force that initially formed the TLM from a vesicle are discussed. The loss of system stability under the action of a small axial force compressing the TLM is considered.

  17. Proton-stimulated Cl-HCO3 antiport by basolateral membrane vesicles of lobster hepatopancreas

    International Nuclear Information System (INIS)

    Purified epithelial basolateral membrane vesicles were prepared from lobster hepatopancreas by sorbitol gradient centrifugation. Na+-K+-adenosinetriphosphatase, alkaline phosphatase, and cytochrome-c oxidase enzyme activities in the final membrane preparation were enriched 9.6-, 1.4-, and 0.4-fold, respectively, compared with their activities in the original tissue homogenate. Vesicle osmotic reactivity was demonstrated using 60-min equilibrium 36Cl uptake experiments at a variety of transmembrane osmotic gradients. 36Cl uptake into vesicles preloaded with HCO3 was significantly greater than into vesicles lacking HCO3. This exchange process was stimulated by a transmembrane proton gradient (internal pH greater than external pH). Proton-gradient-dependent Cl-HCO3 exchange was potential sensitive and stimulated by an electrically negative vesicle interior. 36Cl influx (4-s exposures) into HCO3-loaded vesicles occurred by the combination of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid sensitive, carrier-mediated transfer and apparent diffusion. 36Cl influx was a hyperbolic function of both internal [HCO3] and internal [Cl]. The two internal anions displayed a 100-fold difference in apparent affinity constants with HCO3 being strongly preferred. 36Cl influx was stimulated more by preloaded monovalent than by divalent anions. Na was an inhibitor of proton-dependent anion antiport, whereas K had no effect. A model for HCl-HCO3 antiport is suggested that employs combined transmembrane concentration gradients of Cl and HCO3 to power anion exchange and transfer protons against a concentration gradient

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

    Energy Technology Data Exchange (ETDEWEB)

    Slone, J.H.

    1985-01-01

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

  19. "Nonclassical" secretion of annexin A2 to the lumenal side of the enterocyte brush border membrane

    DEFF Research Database (Denmark)

    Danielsen, E Michael; van Deurs, Bo; Hansen, Gert H

    2003-01-01

    side of the microvilli, showing an apical secretion by a "nonclassical" mechanism. In addition, annexin A2 was associated with surface-connected, deep apical tubules in the apical terminal web region and with an underlying pleiomorphic, tubulo-vesicular compartment (subapical compartment...

  20. Production of plasma membrane vesicles with chloride salts and their utility as a cell membrane mimetic for biophysical characterization of membrane protein interactions

    OpenAIRE

    Del Piccolo, Nuala; Placone, Jesse; He, Lijuan; Agudelo, Sandra Carolina; Hristova, Kalina

    2012-01-01

    Plasma membrane derived vesicles are used as a model system for the biochemical and biophysical investigations of membrane proteins and membrane organization. The most widely used vesiculation procedure relies on formaldehyde and dithiothreitol (DTT), but these active chemicals may introduce artifacts in the experimental results. Here we describe a procedure to vesiculate Chinese hamster ovary (CHO) cells, widely used for the expression of recombinant proteins, using a hypertonic vesiculation...

  1. Permeability of phospholipid membrane for small polar molecules determined from osmotic swelling of giant phospholipid vesicles

    CERN Document Server

    Peterlin, Primoz; Diamant, Haim; Haleva, Emir

    2012-01-01

    A method for determining permeability of phospholipid bilayer based on the osmotic swelling of micrometer-sized giant unilamellar vesicles (GUVs) is presented as an alternative to the two established techniques, dynamic light scattering on liposome suspension, and electrical measurements on planar lipid bilayers. In the described technique, an individual GUV is transferred using a micropipette from a sucrose/glucose solution into an isomolar solution containing the solute under investigation. Throughout the experiment, vesicle cross-section is monitored and recorded using a digital camera mounted on a phase-contrast microscope. Using a least-squares procedure for circle fitting, vesicle radius R is computed from the recorded images of vesicle cross-section. Two methods for determining membrane permeability from the obtained R(t) dependence are described: the first one uses the slope of R(t) for a spherical GUV, and the second one the R(t) dependence around the transition point at which a flaccid vesicle trans...

  2. 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.

  3. Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells.

    Science.gov (United States)

    Ehringer, W D; Niu, W; Chiang, B; Wang, O L; Gordon, L; Chien, S

    2000-07-01

    Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-alpha-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of 14C-FDP by endothelial cells cultured under hypoxia

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

    Science.gov (United States)

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

    2014-01-01

    Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa. PMID:25426629

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    of the two methods; accordingly, it can be assumed that the two methods provide complementary information about the same leakage process. We use the two methods to investigate the membrane-permeabilizing activities of three well-studied cationic membrane-active peptides: mastoparan X, melittin, and...... magainin 2. The FCS results show that leakage induced by magainin 2 is less dependent on the size of the encapsulated fluorescent molecules than leakage induced by mastoparan X and melittin. The confocal imaging results show that all three peptides induce leakage by a heterogeneous process in which one...... portion of the vesicles are completely emptied of their contents but another portion of the vesicles are only partially emptied. These pieces of information regarding leakage induced by mastoparan X, melittin, and magainin 2 could not readily have been obtained by the established assays for studying...

  6. Impact of reducing complement inhibitor binding on the immunogenicity of native neisseria meningitidis outer membrane vesicles

    OpenAIRE

    Daniels-Treffandier, H; Nie, K.; Marsay, L.; Dold, C.; Sadarangani, M.; Reyes-Sandoval, A.; Langford, PR; Wyllie, D; Hill, F; Pollard, AJ; Rollier, CS

    2016-01-01

    Neisseria meningitidis recruits host human complement inhibitors to its surface to down-regulate complement activation and enhance survival in blood. We have investigated whether such complement inhibitor binding occurs after vaccination with native outer membrane vesicles (nOMVs), and limits immunogenicity of such vaccines. To this end, nOMVs reactogenic lipopolysaccharide was detoxified by deletion of the lpxl1 gene (nOMVlpxl1). nOMVs unable to bind human complement factor H (hfH) were gene...

  7. Biogenesis of Salmonella enterica Serovar Typhimurium Membrane Vesicles Provoked by Induction of PagC▿

    OpenAIRE

    Kitagawa, Ryo; Takaya, Akiko; Ohya, Mai; Mizunoe, Yoshimitsu; Takade, Akemi; Yoshida, Shin-ichi; Isogai, Emiko; Yamamoto, Tomoko

    2010-01-01

    Gram-negative bacteria ubiquitously release membrane vesicles (MVs) into the extracellular milieu. Although MVs are the product of growing bacteria, not of cell lysis or death, the regulatory mechanisms underlying MV formation remained unknown. We have found that MV biogenesis is provoked by the induction of PagC, a Salmonella-specific protein whose expression is activated by conditions that mimic acidified macrophage phagosomes. PagC is a major constituent of Salmonella MVs, and increased ex...

  8. The Fusion of Membranes and Vesicles: Pathway and Energy Barriers from Dissipative Particle Dynamics

    OpenAIRE

    Grafmüller, Andrea; Shillcock, Julian; Lipowsky, Reinhard

    2009-01-01

    The fusion of lipid bilayers is studied with dissipative particle dynamics simulations. First, to achieve control over membrane properties, the effects of individual simulation parameters are studied and optimized. Then, a large number of fusion events for a vesicle and a planar bilayer are simulated using the optimized parameter set. In the observed fusion pathway, configurations of individual lipids play an important role. Fusion starts with individual lipids assuming a splayed tail configu...

  9. Intra- and Interspecies Effects of Outer Membrane Vesicles from Stenotrophomonas maltophilia on β-Lactam Resistance.

    Science.gov (United States)

    Devos, Simon; Stremersch, Stephan; Raemdonck, Koen; Braeckmans, Kevin; Devreese, Bart

    2016-04-01

    The treatment ofStenotrophomonas maltophiliainfection with β-lactam antibiotics leads to increased release of outer membrane vesicles (OMVs), which are packed with two chromosomally encoded β-lactamases. Here, we show that these β-lactamase-packed OMVs are capable of establishing extracellular β-lactam degradation. We also show that they dramatically increase the apparent MICs of imipenem and ticarcillin for the cohabituating speciesPseudomonas aeruginosaandBurkholderia cenocepacia. PMID:26787686

  10. K-Cl transport systems in rabbit renal basolateral membrane vesicles

    International Nuclear Information System (INIS)

    The transport pathways for chloride in basolateral membrane vesicles from the rabbit renal cortex were investigated. 36Cl uptake was stimulated by the presence of potassium in the uptake media compared with sodium of N-methyl-D-glucamine. In addition, potassium (86Rb) uptake was stimulated more by chloride than by nitrate or gluconate. Neither of these processes was further stimulated by potassium gradients plus valinomycin, suggesting the presence of an electrically neutral K-Cl cotransport system. A magnesium-induced chloride conductance was also found in the basolateral membrane vesicles. In the absence of magnesium, the chloride conductance was low; valinomycin and an inwardly directed potassium gradient did not stimulated 36Cl uptake, anthracene-9-carboxylic acid did not inhibit 36Cl uptake, and valinomycin did not stimulated chloride-dependent 86Rb uptake. However, in the presence of 1 mM magnesium, opposite results were obtained; valinomycin and an inwardly directed potassium gradient stimulated 36Cl uptake, anthracene-9-carboxylic acid inhibited 36Cl uptake, and valinomycin stimulated chloride-dependent 86Rb uptake. Therefore, an electrically neutral K-Cl cotransport and magnesium-induced chloride conductance were found in renal cortial basolateral membrane vesicles prepared from the rabbit renal cortex

  11. 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...

  12. Mixing Water, Transducing Energy, and Shaping Membranes: Autonomously Self-Regulating Giant Vesicles.

    Science.gov (United States)

    Ho, James C S; Rangamani, Padmini; Liedberg, Bo; Parikh, Atul N

    2016-03-01

    Giant lipid vesicles are topologically closed compartments bounded by semipermeable flexible shells, which isolate femto- to picoliter quantities of the aqueous core from the surrounding bulk. Although water equilibrates readily across vesicular walls (10(-2)-10(-3) cm(3) cm(-2) s(-1)), the passive permeation of solutes is strongly hindered. Furthermore, because of their large volume compressibility (∼10(9)-10(10) N m(-2)) and area expansion (10(2)-10(3) mN m(-1)) moduli, coupled with low bending rigidities (10(-19) N m), vesicular shells bend readily but resist volume compression and tolerate only a limited area expansion (∼5%). Consequently, vesicles experiencing solute concentration gradients dissipate the available chemical energy through the osmotic movement of water, producing dramatic shape transformations driven by surface-area-volume changes and sustained by the incompressibility of water and the flexible membrane interface. Upon immersion in a hypertonic bath, an increased surface-area-volume ratio promotes large-scale morphological remodeling, reducing symmetry and stabilizing unusual shapes determined, at equilibrium, by the minimal bending-energy configurations. By contrast, when subjected to a hypotonic bath, walls of giant vesicles lose their thermal undulation, accumulate mechanical tension, and, beyond a threshold swelling, exhibit remarkable oscillatory swell-burst cycles, with the latter characterized by damped, periodic oscillations in vesicle size, membrane tension, and phase behavior. This cyclical pattern of the osmotic influx of water, pressure, membrane tension, pore formation, and solute efflux suggests quasi-homeostatic self-regulatory behavior allowing vesicular compartments produced from simple molecular components, namely, water, osmolytes, and lipids, to sense and regulate their microenvironment in a negative feedback loop. PMID:26866787

  13. The effect of ultraviolet radiation on wheat root vesicles enriched in plasma membrane

    International Nuclear Information System (INIS)

    The irradiation of plant cells with UV radiation (254 nm) causes various solutes to leak from the cells. Vesicles enriched in plasma membranes were prepared from wheat roots. These were used to determine whether UV radiation alters membrane function by direct action on the membranes and to distinguish between the chemical effects produced by high and low fluences of UV. The plasma membrane-associated K+-stimulated ATPase was very sensitive to UV radiation (100% inhibition with 2). ATPase activity measured in the absence of K+ and K+-stimulated ATPase activity measured in the presence of diethylstilbestrol were much less sensitive. Lipid breakdown, as measured by malondialdehyde production, occurred only at UV fluences greater than 1.8 kJ/m2. (author)

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

    Directory of Open Access Journals (Sweden)

    Mamata Gurung

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

  15. Structural Determinants for Partitioning of Lipids and Proteins Between Coexisting Fluid Phases in Giant Plasma Membrane Vesicles

    OpenAIRE

    Sengupta, Prabuddha; Hammond, Adam; Holowka, David; Baird, Barbara

    2007-01-01

    The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cel...

  16. Intake of silica nanoparticles by giant lipid vesicles: influence of particle size and thermodynamic membrane state

    Directory of Open Access Journals (Sweden)

    Florian G. Strobl

    2014-12-01

    Full Text Available The uptake of nanoparticles into cells often involves their engulfment by the plasma membrane and a fission of the latter. Understanding the physical mechanisms underlying these uptake processes may be achieved by the investigation of simple model systems that can be compared to theoretical models. Here, we present experiments on a massive uptake of silica nanoparticles by giant unilamellar lipid vesicles (GUVs. We find that this uptake process depends on the size of the particles as well as on the thermodynamic state of the lipid membrane. Our findings are discussed in the light of several theoretical models and indicate that these models have to be extended in order to capture the interaction between nanomaterials and biological membranes correctly.

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

    Science.gov (United States)

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

    2016-06-01

    The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate. PMID:26928127

  18. The fusion of membranes and vesicles: pathway and energy barriers from Dissipative Particle Dynamics

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2009-01-01

    The fusion of lipid bilayers is studied with dissipative particle dynamics simulations. First, to achieve control over membrane properties, the effects of individual simulation parameters are studied and optimized. Then, a large number of fusion events for a vesicle and a planar bilayer...... measure the average work for interbilayer flips of a lipid tail, i.e., the average work to displace one lipid tail from one bilayer to the other. This energy barrier is found to depend strongly on a certain dissipative particle dynamics parameter, and, thus, can be adjusted in the simulations. Overall...

  19. Effects of nucleotides on ATP-dependent protein translocation into Escherichia coli membrane vesicles.

    OpenAIRE

    Chen, L.; Tai, P C

    1986-01-01

    We have shown previously that Escherichia coli can translocate the same protein either co- or posttranslationally and that ATP hydrolysis is essential for the posttranslational translocation of the precursors of alkaline phosphatase and OmpA protein into inverted E. coli membrane vesicles. ATP-dependent protein translocation has now been further characterized. In the absence of exogenous Mg2+, dATP, formycin A-5'-triphosphate, ATP-alpha-S, and N1-oxide-ATP could replace ATP, but many other nu...

  20. Chloride permeability of rat brain membrane vesicles correlates with thiamine triphosphate content.

    Science.gov (United States)

    Bettendorff, L; Hennuy, B; De Clerck, A; Wins, P

    1994-07-25

    Incubation of rat brain homogenates with thiamine or thiamine diphosphate (TDP) leads to a synthesis of thiamine triphosphate (TTP). In membrane vesicles subsequently prepared from the homogenates, increased TTP content correlates with increased 36Cl- uptake. A hyperbolic relationship was obtained with a K0.5 of 0.27 nmol TTP/mg protein. In crude mitochondrial fractions from the brains of animals previously treated with thiamine or sulbutiamine, a positive correlation between 36Cl- uptake and TTP content was found. These results, together with other results previously obtained with the patch-clamp technique, suggest that TTP is an activator of chloride channels having a large unit conductance. PMID:7953714

  1. Interaction of miltefosine with intercellular membranes of stratum corneum and biomimetic lipid vesicles.

    Science.gov (United States)

    Alonso, Lais; Mendanha, Sebastião Antônio; Marquezin, Cássia Alessandra; Berardi, Marina; Ito, Amando Siuiti; Acuña, A Ulises; Alonso, Antonio

    2012-09-15

    Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50°C. This effect becomes negligible at temperatures higher that ca. 60°C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis. PMID:22692081

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

    International Nuclear Information System (INIS)

    The fusion of synaptic vesicles (SVs) with the plasma membrane in neurons is a crucial step in the release of neurotransmitters, which are responsible for carrying signals between nerve cells. While many of the molecular players involved in this fusion process have been identified, a precise molecular description of their roles in the process is still lacking. A case in point is the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Although PIP2 is known to be essential for vesicle fusion, its precise role in the process remains unclear. We have re-investigated the role of this lipid in membrane structure and function using the complementary experimental techniques of x-ray reflectivity, both on lipid monolayers at an air-water interface and bilayers on a solid support, and grazing incidence x-ray diffraction on lipid monolayers. These techniques provide unprecedented access to structural information at the molecular level, and detail the profound structural changes that occur in a membrane following PIP2 incorporation. Further, we also confirm and extend previous findings that the association of SVs with membranes is enhanced by PIP2 incorporation, and reveal the structural changes that underpin this phenomenon. Further, the association is further intensified by a physiologically relevant amount of Ca2+ ions in the subphase of the monolayer, as revealed by the increase in interfacial pressure seen with the lipid monolayer system. Finally, a theoretical calculation concerning the products arising from the fusion of these SVs with proteoliposomes is presented, with which we aim to illustrate the potential future uses of this system.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-10-15

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

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Goldsmith, M.H.M.

    1986-01-01

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

  6. Controlled Architecture of Dual-Functional Block Copolymer Brushes on Thin-Film Composite Membranes for Integrated "Defending" and "Attacking" Strategies against Biofouling.

    Science.gov (United States)

    Ye, Gang; Lee, Jongho; Perreault, François; Elimelech, Menachem

    2015-10-21

    We report a new macromolecular architecture of dual functional block copolymer brushes on commercial thin-film composite (TFC) membranes for integrated "defending" and "attacking" strategies against biofouling. Mussel-inspired catechol chemistry is used for a convenient immobilization of initiator molecules to the membrane surface with the aid of polydopamine (PDA). Zwitterionic polymer brushes with strong hydration capacity and quaternary ammonium salt (QAS) polymer brushes with bactericidal ability are sequentially grafted on TFC membranes via activators regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP), an environmentally benign and controlled polymerization method. Measurement of membrane intrinsic transport properties in reverse osmosis experiments shows that the modified TFC membrane maintains the same water permeability and salt selectivity as the pristine TFC membrane. Chemical force microscopy and protein/bacterial adhesion studies are carried out for a comprehensive evaluation of the biofouling resistance and antimicrobial ability, demonstrating low biofouling propensity and excellent bacterial inactivation for the modified TFC membrane. We conclude that this polymer architecture, with complementary "defending" and "attacking" capabilities, can effectively prevent the attachment of biofoulants and formation of biofilms and thereby significantly mitigate biofouling on TFC membranes. PMID:26378606

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

    OpenAIRE

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

    2016-01-01

    Background We studied the effect of carbon black (CB) agglomerated nanomaterial on biological membranes as revealed by shapes of human erythrocytes, platelets and giant phospholipid vesicles. Diluted human blood was incubated with CB nanomaterial and observed by different microscopic techniques. Giant unilamellar phospholipid vesicles (GUVs) created by electroformation were incubated with CB nanomaterial and observed by optical microscopy. Populations of erythrocytes and GUVs were analyzed: t...

  8. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms.

    Science.gov (United States)

    Turnbull, Lynne; Toyofuku, Masanori; Hynen, Amelia L; Kurosawa, Masaharu; Pessi, Gabriella; Petty, Nicola K; Osvath, Sarah R; Cárcamo-Oyarce, Gerardo; Gloag, Erin S; Shimoni, Raz; Omasits, Ulrich; Ito, Satoshi; Yap, Xinhui; Monahan, Leigh G; Cavaliere, Rosalia; Ahrens, Christian H; Charles, Ian G; Nomura, Nobuhiko; Eberl, Leo; Whitchurch, Cynthia B

    2016-01-01

    Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs. PMID:27075392

  9. Biological and Immunological Evaluation of Neisseria meningitidis Serogroup A Outer Membrane Vesicle as Vaccine Candidates

    Directory of Open Access Journals (Sweden)

    Seyed Ali Delbaz

    2013-05-01

    Full Text Available Background: Neisseria meningitidis Serogroup A, is a major cause of bacterial meningitidis outbreaks in Africa and the Middle East. While polysaccharide vaccines have been available for many years, these vaccines have many disadvantages including the induction of T-cell independent responses which do not induce memory responses.Objectives: Thus to overcome this problem, in this research outer membrane vesicle (OMV containing PorA was extracted and evaluated by biological and immunological methods.Materials and Methods: OMVs were extracted with deoxycholate and EDTA, and purification was performed by sequential ultracentrifugation. Physicochemical properties of extracted OMVs were analyzed by electron microscopy and SDS-PAGE. The toxicity of LPS content in its was assayed by LAL test. The Presence of PorA as a major component of OMV was confirmed by western blot. To study antibodies synthesis after immunization with OMV, ELISA method was used. Also serum bactericidal assay (SBA was performed to determine the serum bactericidal activity against N.meningitidis serogroup A.Results: The results revealed that the content of protein extracted was 0.1mg/ml. The electron microscopy showed that intactness of the vesicle in these preparation ranged more than 70%. The SDS-PAGE showed that PorA as a major immunological part of outer membrane vesicle was located in 35-40kDa. LAL test showed that the endotoxin activity was around 126EU/ml which is safe for using. The ELISA test revealed that the IgG total titer was elevated after the first injection. SBA indicates that bactericidal antibodies rise after the second dose of booster.Conclusions: The results showed that the extracted OMVs were conformationally stable, and there were no pyrogenic determinants in OMV. Also the results showed that the OMV elicited high level of specific antibodies against N. meningitidis serogroup A. These results indicate that the OMV obtained here, can be used as a meningococcal

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

    International Nuclear Information System (INIS)

    Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using 45Ca2+. Uptake of 45Ca2+ by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of 45Ca2+ uptake to ATP utilization via ΔμH+, no evidence for a secondary H+/Ca2+ antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca2+ and an imposed pH gradient could not drive 45Ca2+ uptake. Optimal uptake of 45Ca2+ occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of 45Ca2+ uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K/sub m/ values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving 45Ca2+ uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell

  11. Calcium transport in sealed vesicles from red beet (Beta vulgaris L. ) storage tissue. II. Characterization of /sup 45/Ca/sup 2 +/ uptake into plasma membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-12-01

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

  12. Low temperature-induced cell surface membrane vesicle shedding is associated with DNA fragmentation

    International Nuclear Information System (INIS)

    Temperature shift conditions of 0 degree to 22 degrees C or 0 degree to 37 degrees C induce the formation and shedding of membrane vesicles (MV) from P815 tumor cell surfaces. When the MV shedding process takes place at 22 degrees C it occurs without changes in cell surface membrane permeability, whereas at 37 degrees C, changes in permeability to 51Cr and trypan blue do occur, thus mimicking the lymphocyte-mediated lytic process of tumor cells. The present studies demonstrate that nuclear DNA fragmentation also occurs in both 0 degree to 22 degrees C and 0 degree to 37 degrees C temperature shifts. However, cell surface membrane permeability to DNA fragments occurs only in the latter condition, i.e., 0 degree to 37 degrees C. The microtubule-stabilizing agent deuterium oxide (D2O) inhibited the MV shedding process, the changes in membrane permeability, and DNA fragmentation. When P815 cells which had been induced to shed MV by the 0 degree to 22 degrees C temperature shift were labeled with 51Cr and used as targets for alloimmune lymphocytes, they were found to be as susceptible to T-cell lysis as control P815 cells. This result indicates that the lytic effect of alloimmune T lymphocytes can be exerted at the target cell surface membrane level independently of nuclear DNA fragmentation

  13. Outer Membrane Vesicle Biosynthesis in Salmonella: Is There More to Gram-Negative Bacteria?

    Science.gov (United States)

    Reidl, Joachim

    2016-01-01

    Recent research has focused on the biological role of outer membrane vesicles (OMVs), which are derived from the outer membranes (OMs) of Gram-negative bacteria, and their potential exploitation as therapeutics. OMVs have been characterized in many ways and functions. Until recently, research focused on hypothetical and empirical models that addressed the molecular mechanisms of OMV biogenesis, such as vesicles bulging from the OM in various ways. The recently reported study by Elhenawy et al. (mBio 7:e00940-16, 2016, http://dx.doi.org/10.1128/mBio.00940-16) provided further insights into OMV biogenesis of Salmonella enterica serovar Typhimurium. That study showed that deacylation of lipopolysaccharides (LPS) influences the level of OMV production and, furthermore, determines a sorting of high versus low acylated LPS in OMs and OMVs, respectively. Interestingly, deacylation may inversely correlate with other LPS modifications, suggesting some synergy toward optimized host resistance via best OM compositions for S Typhimurium. PMID:27531914

  14. Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT from Campylobacter jejuni

    Directory of Open Access Journals (Sweden)

    Uhlin Bernt

    2009-10-01

    Full Text Available Abstract Background Background: Cytolethal distending toxin (CDT is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment. Results Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50% of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form. Conclusion Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of C. jejuni to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC to the surrounding environment, including infected host tissue.

  15. Multiscale modeling of mechanosensing channels on vesicles and cell membranes in 3D constricted flows and shear flows

    Science.gov (United States)

    Peng, Zhangli; Pak, On Shun; Young, Yuan-Nan; Liu, Allen; Stone, Howard

    2015-11-01

    We investigate the gating of mechanosensing channels (Mscls) on vesicles and cell membranes under different flow conditions using a multiscale approach. At the cell level (microns), the membrane tension is calculated using a 3D two-component whole-cell membrane model based on dissipative particle dynamics (DPD), including the cortex cytoskeleton and its interactions with the lipid bilayer. At the Mscl level (nanometers), we predict the relation between channel gating and the membrane tension obtained from a cell-level model using a semi-analytical model based on the bilayer hydrophobic mismatch energy. We systematically study the gating of Mscls of vesicles and cell membranes in constricted channel flows and shear flows, and explore the dependence of the gating on flow rate, cell shape and size. The results provide guidance for future experiments in inducing Mscl opening for various purposes such as drug delivery.

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

    Science.gov (United States)

    Surve, Manalee Vishnu; Anil, Anjali; Kamath, Kshama Ganesh; Bhutda, Smita; Sthanam, Lakshmi Kavitha; Pradhan, Arpan; Srivastava, Rohit; Basu, Bhakti; Dutta, Suryendu; Sen, Shamik; Modi, Deepak; Banerjee, Anirban

    2016-09-01

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

  17. Membrane vesicles: A simplified system for studying auxin transport. Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    Goldsmith, M.H.M.

    1989-12-31

    Indoleacetic acid (IAA), the auxin responsible for regulation of growth, is transported polarly in plants. Several different models have been suggested to account for IAA transport by cells and its accumulation by membrane vesicles. One model sees diffusion of IAA driven by a pH gradient. The anion of a lipophilic weak acid like IAA or butyrate accumulates in an alkaline compartment in accord with the size of the pH gradient The accumulation of IAA may be diminished by the permeability of its lipophilic anion. This anion leak may be blocked by NPA. With anion efflux blocked, a gradient of two pH units would support an IAA accumulation of less than 50-fold at equilibrium (2) Another model sees diffusion of IAA in parallel with a saturable symport (IAA{sup {minus}} + nH{sup +}), driven by both the pH gradient and membrane voltage. Such a symport should be highly accumulative, however, with a lipophilic weak acid such as IAA, net diffusive efflux of IAAH whenever IAAHI{sub i} > IAAH{sub o} would constitute a leak. (3) A third model sees a pH change driven IAA uptake and saturable symport enhanced by internal binding sites. Following pH gradient-driven accumulation of IAA, the anion may bind to an intravesicular site, permitting further uptake of IAA. NPA, by blocking anion efflux, enhances this binding. We have reported that membrane vesicles isolated from actively growing plant tissues are a good system for studying the mechanisms involved in the transport and accumulation of auxin.

  18. Proteomic analysis of outer membrane vesicles from the probiotic strain Escherichia coli Nissle 1917.

    Science.gov (United States)

    Aguilera, Laura; Toloza, Lorena; Giménez, Rosa; Odena, Antonia; Oliveira, Eliandre; Aguilar, Juan; Badia, Josefa; Baldomà, Laura

    2014-02-01

    Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria and have a relevant role in bacteria-host interactions. Using 1D SDS-PAGE and highly sensitive LC-MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain-linked genes and 57 were common to pathogen-derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic-derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 (http://proteomecentral.proteomexchange.org/dataset/PXD000367). PMID:24307187

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

    International Nuclear Information System (INIS)

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

  20. Voltage-dependent calcium channels in skeletal muscle transverse tubules. Measurements of calcium efflux in membrane vesicles

    International Nuclear Information System (INIS)

    Transverse tubule membranes isolated from rabbit skeletal muscle consist mainly of sealed vesicles that are oriented primarily inside out. These membranes contain a high density of binding sites for 1,4-dihydropyridine calcium channel antagonists. The presence of functional voltage-dependent calcium channels in these membranes has been demonstrated by their ability to mediate 45Ca2+ efflux in response to changes in membrane potential. Fluorescence changes of the voltage-sensitive dye, 3,3'-dipropyl-2,2'-thiadicarbocyanine, have shown that transverse tubule vesicles may generate and maintain membrane potentials in response to establishing potassium gradients across the membrane in the presence of valinomycin. A two-step procedure has been developed to measure voltage-dependent calcium fluxes. Vesicles loaded with 45Ca2+ are first diluted into a buffer designed to generate a membrane potential mimicking the resting state of the cell and to reduce the extravesicular Ca2+ to sub-micromolar levels. 45Ca2+ efflux is then measured upon subsequent depolarization. Flux responses are modulated with appropriate pharmacological specificity by 1,4-dihydropyridines and are inhibited by other calcium channel antagonists such as lanthanum and verapamil

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

    DEFF Research Database (Denmark)

    Kristiansen, S; Youn, J; Richter, Erik

    1996-01-01

    vanadate (NaVO3) on glucose transporter (GLUT4) intrinsic activity (V(max) = intrinsic activity x [GLUT4 protein]) was studied in muscle plasma membrane giant vesicles. Giant vesicles (average diameter 7.6 microns) were produced by collagenase treatment of rat skeletal muscle. The vesicles were incubated......) 55% and 60%, respectively, compared with control. The plasma membrane GLUT4 protein content was not changed in response to vanadate. It is concluded that vanadate decreased glucose transport per GLUT4 (intrinsic activity). This finding suggests that regulation of glucose transport in skeletal muscle......Maximally effective concentrations of vanadate (a phosphotyrosine phosphatase inhibitor) increase glucose transport in muscle less than maximal insulin stimulation. This might be due to vanadate-induced decreased intrinsic activity of GLUT4 accompanying GLUT4 translocation. Thus, the effect of...

  2. Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1.

    Science.gov (United States)

    Huang, Ping; Altshuller, Yelena M; Hou, June Chunqiu; Pessin, Jeffrey E; Frohman, Michael A

    2005-06-01

    Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion. PMID:15772157

  3. Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/β

    Institute of Scientific and Technical Information of China (English)

    Quanlong Lu; Zhigang Lu; Qinying Liu; Li Guo; He Ren; Jingyan Fu; Qing Jiang; Paul R Clarke; Chuanmao Zhang

    2012-01-01

    The mechanism for nuclear envelope (NE) assembly is not fully understood.Importin-β and the small GTPase Ran have been implicated in the spatial regulation of NE assembly process.Here we report that chromatin-bound NLS (nuclear localization sequence) proteins provide docking sites for the NE precursor membrane vesicles and nucleoporins via importin-α and -β during NE assembly in Xenopus egg extracts.We show that along with the fast recruitment of the abundant NLS proteins such as nucleoplasmin and histones to the demembranated sperm chromatin in the extracts,importin-α binds the chromatin NLS proteins rapidly.Meanwhile,importin-β binds cytoplasmic NE precursor membrane vesicles and nucleoporins.Through interacting with importin-α on the chromatin NLS proteins,importin-β targets the membrane vesicles and nucleoporins to the chromatin surface.Once encountering RanGTP on the chromatin generated by RCC1,importin-β preferentially binds Ran-GTP and releases the membrane vesicles and nucleoporins for NE assembly.NE assembly is disrupted by blocking the interaction between importin-α and NLS proteins with excess soluble NLS proteins or by depletion of importin-β from the extract.Our findings reveal a novel molecular mechanism for NE assembly in Xenopus egg extracts.

  4. Short-term regulation of NHE3 by EGF and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells and fibroblasts.

    Science.gov (United States)

    Donowitz, M; Janecki, A; Akhter, S; Cavet, M E; Sanchez, F; Lamprecht, G; Zizak, M; Kwon, W L; Khurana, S; Yun, C H; Tse, C M

    2000-01-01

    NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na(+)-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in Vmax of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)i of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number. PMID:11193592

  5. Differential inactivation of two transport systems in E. coli membrane vesicles by gamma radiation

    International Nuclear Information System (INIS)

    Gamma-irradiation of membrane vesicles isolated from E. coli ML 308-225 is shown to decrease the active accumulation of α-methyl glucoside, glutamic acid, and proline. The phosphorylation and transport components of the phosphoenolpyruvate:glucose phosphotransferase system (PEP-PTS) are equally sensitive to radiation. Thus, the decrease in the uptake activity of the PEP-PTS is not due to radiation-induced permeability changes in the membrane, but to inactivation of the components by radiation. When the D-lactate dehydrogenase (D-LDH)-coupled amino-acid transport system was assayed for its permease and metabolic energy components, data showed that the permease system responsible for the active transport of amino acids is more radiosensitive than the electron transport chain, indicating that the radiosensitive component of the permease system is situated in a shunt outside the main flow of electrons in the electron transport chain. The dose-effect response curves for the two transport systems showed that the PEP-PTS is more sensitive to radiation than the amino-acid transport system. Observed changes in the activities of the two transport systems proved due to the action of free radicals. Cysteamine exerts greater radioprotective effect on the PEP-PTS than on the amino-acid transport system. A radiosensitivity gradient appears to exist in the membrane, further supported by spin-label results. The differential radiosensitivities of the two transport systems are due to differences in the radiosensitivities of their respective components and in their accessibility to free radicals in the aqueous medium. The spin-label study also revealed two populations of sulphydryl groups in the membrane which differ with respect to their location in the bilayer, sensitivity to radiation, and extent of protection by free radical scavengers

  6. Biogenesis of Salmonella enterica serovar typhimurium membrane vesicles provoked by induction of PagC.

    Science.gov (United States)

    Kitagawa, Ryo; Takaya, Akiko; Ohya, Mai; Mizunoe, Yoshimitsu; Takade, Akemi; Yoshida, Shin-ichi; Isogai, Emiko; Yamamoto, Tomoko

    2010-11-01

    Gram-negative bacteria ubiquitously release membrane vesicles (MVs) into the extracellular milieu. Although MVs are the product of growing bacteria, not of cell lysis or death, the regulatory mechanisms underlying MV formation remained unknown. We have found that MV biogenesis is provoked by the induction of PagC, a Salmonella-specific protein whose expression is activated by conditions that mimic acidified macrophage phagosomes. PagC is a major constituent of Salmonella MVs, and increased expression accelerates vesiculation. Expression of PagC is regulated at the posttranscriptional and/or posttranslational level in a sigmaS (RpoS)-dependent manner. Serial quantitative analysis has demonstrated that MV formation can accelerate when the quantity of the MV constituents, OmpX and PagC, rises. Overproduction of PagC dramatically impacts the difference in the relative amount of vesiculation, but the corresponding overproduction of OmpX was less pronounced. Quantitative examination of the ratios of PagC and OmpX in the periplasm, outer membrane, and MVs demonstrates that PagC is preferentially enriched in MVs released from Salmonella cells. This suggests that specific protein sorting mechanisms operate when MVs are formed. The possible role(s) of PagC-MV in host cells is discussed. PMID:20802043

  7. Insulin-stimulated Plasma Membrane Fusion of Glut4 Glucose Transporter-containing Vesicles Is Regulated by Phospholipase D1D⃞

    OpenAIRE

    Huang, Ping; Altshuller, Yelena M.; Hou, June Chunqiu; Jeffrey E Pessin; Frohman, Michael A.

    2005-01-01

    Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insu...

  8. Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages.

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N.; Heffron, Fred

    2011-06-01

    We have previously shown that the regulators SpvR, FruR, IHF, PhoP/PhoQ, SsrA/SsrB, SlyA, Hnr, RpoE, SmpB, CsrA, RpoS, Crp, OmpR/EnvZ, and Hfq are essential for Salmonella Typhimurium virulence in mice. Here we use quantitative LC-MS-based proteomics profiling of in-frame deletion mutants of these 14 regulators to identify proteins that are coordinately regulated by these virulence regulators and are thus presumably novel factors contributing to Salmonella pathogenesis. Putative candidate proteins from proteomics analysis were determined, which exhibited similar abundance profiles to those of Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS) proteins. A subset of 5 proteins including STM0082, STM1548, PdgL, STM1633, and STM3595 was selected for further analysis. All 5 proteins were expressed inside macrophage cells and STM0082 (SrfN) was secreted into host cytoplasm. Furthermore, deletion of STM0082 attenuated virulence in mice when administered intraperitoneally as determined by competitive index. srfN transcription was positively regulated by SsrAB, however, secretion was independent of SPI-2 TTSS as well as SPI-1 TTSS and flagella. Proteins including PagK and STM2585A, which are positively regulated by PhoP/PhoQ, have sec signal peptides as predicted for SrfN and were secreted into macrophage cytoplasm regardless of SPI-2 TTSS. Isolation of outer membrane vesicles (OMVs) revealed the presence of SrfN, PagK, and STM2585A inside vesicle compartments. This result is the first case showing delivery of virulence effectors via OMVs in S. Typhimurium. Moreover, Hfq regulation of SrfN translation suggests that small non-coding RNAs may be responsible for regulating effector protein expression.

  9. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.

    Science.gov (United States)

    Zielke, Ryszard A; Wierzbicki, Igor H; Weber, Jacob V; Gafken, Philip R; Sikora, Aleksandra E

    2014-05-01

    Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC

  10. Characterization of calcium transport by basolateral membrane vesicles of human small intestine

    International Nuclear Information System (INIS)

    The present studies investigated the mechanism of Ca2+ transport across basolateral membrane vesicles (BLMVs) prepared from human small intestine. Ca2+ uptake represented transport into the intravesicular space as evident by osmolality study and by the demonstration of Ca2+ efflux from the intravesicular space by Ca2+ ionophore A23187. Ca2+ uptake was stimulated by Mg2+-ATP. Kinetic parameters for ATP-dependent Ca2+ uptake revealed a Michaelis constant (Km) of 0.02±0.01 μM and a maximum rate of uptake (Vmax) of 1.00±0.03 nmol·mg protein-1·min-1. Ca2+ uptake in the presence of Mg2+ was inhibited by 75%. The Km of ATP concentration required for half-maximal Ca2+ uptake was 0.50±0.1 mM. Basolateral membranes depleted of calmodulin by EDTA osmotic shock decreased ATP-dependent Ca2+ uptake by 65%. Trifluoperazine, an anticalmodulin drug, inhibited ATP-dependent Ca2+ uptake by 50%, while no inhibition was noted in calmodulin-depleted membranes. Efflux of Ca2+ in the BLMVs was stimulated by trans-Na+. Na+-dependent Ca2+ uptake was saturable with respect to Ca2+ concentration and exhibited a Km of 0.09±0.03 μM and a Vmax of 1.08±0.01 nmol·mg protein-1·min-1. These results are consistent with the existence of a Na+-Ca2+ exchange system and ATP and Mg2+-dependent, calmodulin-regulated Ca2+, transport mechanism in BLMVs of human enterocytes

  11. Recruitment and SNARE-mediated fusion of vesicles in furrow membrane remodeling during cytokinesis in zebrafish embryos

    International Nuclear Information System (INIS)

    Cytokinesis is the final stage in cell division that serves to partition cytoplasm and daughter nuclei into separate cells. Membrane remodeling at the cleavage plane is a required feature of cytokinesis in many species. In animal cells, however, the precise mechanisms and molecular interactions that mediate this process are not yet fully understood. Using real-time imaging in live, early stage zebrafish embryos, we demonstrate that vesicles labeled with the v-SNARE, VAMP-2, are recruited to the cleavage furrow during deepening in a microtubule-dependent manner. These vesicles then fuse with, and transfer their VAMP-2 fluorescent label to, the plasma membrane during both furrow deepening and subsequent apposition. This observation indicates that new membrane is being inserted during these stages of cytokinesis. Inhibition of SNAP-25 (a cognate t-SNARE of VAMP-2), using a monoclonal antibody, blocked VAMP-2 vesicle fusion and furrow apposition. Transient expression of mutant forms of SNAP-25 also produced defects in furrow apposition. SNAP-25 inhibition by either method, however, did not have any significant effect on furrow deepening. Thus, our data clearly indicate that VAMP-2 and SNAP-25 play an essential role in daughter blastomere apposition, possibly via the delivery of components that promote the cell-to-cell adhesion required for the successful completion of cytokinesis. Our results also support the idea that new membrane addition, which occurs during late stage cytokinesis, is not required for furrow deepening that results from contractile band constriction

  12. Dolichol phosphate induces non-bilayer structures, vesicle fusion and transbilayer movements of lipids in model membranes

    Energy Technology Data Exchange (ETDEWEB)

    de Kruijff, B.; Van Duijn, G.; Valtersson, C.; Chojnacki, T.; Verkleij, A.J.; Dallner, G.

    1987-05-01

    The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichol phosphate on the structure and fluidity of model membranes was studied using different biophysical techniques. These studies suggest that (1) dolichol and dolichol derivatives destabilize unsaturated PE-containing bilayers and promote hexagonal II phase formation; (2) high concentrations of dolichol induce lipid structures characterized by isotropic T P NMR and particulate fracture faces. The effect of dolichol and dolichyl phosphate on fusion between large unilamellar vesicles of DOPC and DOPE was studied using a fluroescence resonance energy transfer assay. The influence of dolichyl phosphate on the transbilary movement of DOPC in multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) composed of DOPC and DOPE (1:2) was investigated by using the PC-specified transfer protein. The results indicate that: (1) both dolichol and dolichyl phosphate enhance vesicle fusion in a comparable and concentration-dependent way; (2) the amount of exchangeable PC from MLVs is increased by dolichyl phosphate probably as a result of fusion processes. It thus appears that these polyprenols are potent destabilizers of bilayer structure and that this process is accompanied by membrane fusion and transbilayer transport of phospholipids.

  13. Dolichol phosphate induces non-bilayer structures, vesicle fusion and transbilayer movements of lipids in model membranes

    International Nuclear Information System (INIS)

    The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichol phosphate on the structure and fluidity of model membranes was studied using different biophysical techniques. These studies suggest that (1) dolichol and dolichol derivatives destabilize unsaturated PE-containing bilayers and promote hexagonal II phase formation; (2) high concentrations of dolichol induce lipid structures characterized by isotropic 31P NMR and particulate fracture faces. The effect of dolichol and dolichyl phosphate on fusion between large unilamellar vesicles of DOPC and DOPE was studied using a fluroescence resonance energy transfer assay. The influence of dolichyl phosphate on the transbilary movement of DOPC in multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) composed of DOPC and DOPE (1:2) was investigated by using the PC-specified transfer protein. The results indicate that: (1) both dolichol and dolichyl phosphate enhance vesicle fusion in a comparable and concentration-dependent way; (2) the amount of exchangeable PC from MLVs is increased by dolichyl phosphate probably as a result of fusion processes. It thus appears that these polyprenols are potent destabilizers of bilayer structure and that this process is accompanied by membrane fusion and transbilayer transport of phospholipids

  14. Engineered Asymmetric Synthetic Vesicles

    Science.gov (United States)

    Lu, Li; Chiarot, Paul

    2013-11-01

    Synthetic vesicles are small, fluid-filled spheres that are enclosed by a bilayer of lipid molecules. They can be used as models for investigating membrane biology and as delivery vehicles for pharmaceuticals. In practice, it is difficult to simultaneously control membrane asymmetry, unilamellarity, vesicle size, vesicle-to-vesicle uniformity, and luminal content. Membrane asymmetry, where each leaflet of the bilayer is composed of different lipids, is of particular importance as it is a feature of most natural membranes. In this study, we leverage microfluidic technology to build asymmetric vesicles at high-throughput. We use the precise flow control offered by microfluidic devices to make highly uniform emulsions, with controlled internal content, that serve as templates to build the synthetic vesicles. Flow focusing, dielectrophoretic steering, and interfacial lipid self-assembly are critical procedures performed on-chip to produce the vesicles. Fluorescent and confocal microscopy are used to evaluate the vesicle characteristics.

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

    OpenAIRE

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

    1980-01-01

    Vesicles have been prepared from 18 : 1c/18 : 1c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose, 31P-NMR and K+ trap measurements. Pools of phosphatidylcholine available for exchange have been investigated using phosphatidylcholine exchange protein from bovine liver. The protein-containing vesicl...

  16. ANALYSIS OF A MIXED FINITE ELEMENT METHOD FOR A PHASE FIELD BENDING ELASTICITY MODEL OF VESICLE MEMBRANE DEFORMATION

    Institute of Scientific and Technical Information of China (English)

    Qiang Du; Liyong Zhu

    2006-01-01

    In this paper, we study numerical approximations of a recently proposed phase field model for the vesicle membrane deformation governed by the variation of the elastic bending energy. To overcome the challenges of high order nonlinear differential systems and the nonlinear constraints associated with the problem, we present the phase field bending elasticity model in a nested saddle point formulation. A mixed finite element method is then employed to compute the equilibrium configuration of a vesicle membrane with prescribed volume and surface area. Coupling the approximation results for a related linearized problem and the general theory of Brezzi-Rappaz-Raviart, optimal order error estimates for the finite element approximations of the phase field model are obtained. Numerical results areprovided to substantiate the derived estimates.

  17. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens.

    Science.gov (United States)

    Cecil, Jessica D; O'Brien-Simpson, Neil M; Lenzo, Jason C; Holden, James A; Chen, Yu-Yen; Singleton, William; Gause, Katelyn T; Yan, Yan; Caruso, Frank; Reynolds, Eric C

    2016-01-01

    Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis. PMID:27035339

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

    Directory of Open Access Journals (Sweden)

    Bruno Haas

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

  19. Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles

    Science.gov (United States)

    Alves, Nathan J.; Turner, Kendrick B.; Medintz, Igor L.; Walper, Scott A.

    2016-01-01

    Bacteria possess innate machinery to transport extracellular cargo between cells as well as package virulence factors to infect host cells by secreting outer membrane vesicles (OMVs) that contain small molecules, proteins, and genetic material. These robust proteoliposomes have evolved naturally to be resistant to degradation and provide a supportive environment to extend the activity of encapsulated cargo. In this study, we sought to exploit bacterial OMV formation to package and maintain the activity of an enzyme, phosphotriesterase (PTE), under challenging storage conditions encountered for real world applications. Here we show that OMV packaged PTE maintains activity over free PTE when subjected to elevated temperatures (>100-fold more activity after 14 days at 37 °C), iterative freeze-thaw cycles (3.4-fold post four-cycles), and lyophilization (43-fold). We also demonstrate how lyophilized OMV packaged PTE can be utilized as a cell free reagent for long term environmental remediation of pesticide/chemical warfare contaminated areas. PMID:27117743

  20. Membrane Vesicles as a Novel Strategy for Shedding Encrusted Cell Surfaces

    Directory of Open Access Journals (Sweden)

    Paul P. Shao

    2014-02-01

    Full Text Available Surface encrustation by minerals, which impedes cellular metabolism, is a potential hazard for microbes. The reduction of U(VI to U(IV by Shewanella oneidensis strain MR-1 leads to the precipitation of the mineral uraninite, as well as a non-crystalline U(IV product. The wild-type (WT strain can produce extracellular polymeric substances (EPS, prompting precipitation of U some distance from the cells and precluding encrustation. Using cryo-transmission electron microscopy and scanning transmission X-ray microscopy we show that, in the biofilm-deficient mutant ∆mxdA, as well as in the WT strain to a lesser extent, we observe the formation of membrane vesicles (MVs as an additional means to lessen encrustation. Additionally, under conditions in which the WT does not produce EPS, formation of MVs was the only observed mechanism to mitigate cell encrustation. Viability studies comparing U-free controls to cells exposed to U showed a decrease in the number of viable cells in conditions where MVs alone are detected, yet no loss of viability when cells produce both EPS and MVs. We conclude that MV formation is a microbial strategy to shed encrusted cell surfaces but is less effective at maintaining cell viability than the precipitation of U on EPS.

  1. Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles. [Pisum sativum. L

    Energy Technology Data Exchange (ETDEWEB)

    Gabathuler, R.; Cleland, R.E.

    1985-12-01

    Pea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K/sub m/ of about 0.23 millimolar for MgATP, is only slightly affected by K/sup +/ or Cl/sup -/ and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO/sub 3//sup -/ > Cl/sup -/) and has a K/sub m/ of about 0.7 millimolar. Auxins decrease the K/sub m/ to about 0.35 millimolar, with no significant effect on the V/sub max/, while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport.

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

    International Nuclear Information System (INIS)

    Previous studies have shown that gills from M. edulis can accumulate L-alanine from seawater by a saturable process specific for α-neutral amino acids. This uptake occurs against chemical gradients in excess of 106 to 1. To further characterize this uptake, membrane vesicles were prepared from M. edulis gill tissue by differential centrifugation. Enrichments of putative enzyme markers (relative to that in combined initial fractions) were as follows: γ-Glutamyltranspeptidase, 25-30x; Alkaline Phosphatase, 5-6x; K+-dependent para-Nitrophenyl Phosphatase, 3-5x; Succinate Dehydrogenase 0.1-0.2x. These results suggest that the preparation is enriched in plasma membranes, although histochemical studies will be needed to verify this. The time course of 14C-L-alanine uptake in the presence of inwardly-directed Na+ gradient showed a transient overshoot (3-5 fold) at 10 minutes which decreased to equilibrium after six hours. The size of the overshoot and early uptake rates depended on the size of the inwardly-directed Na+ gradient. No overshoot was seen in the presence of inwardly-directed gradients of LiCl or choline-Cl, or with equilibrium concentrations NaCl or mannitol. A reduced overshoot was seen with a gradient of NaSCN. A small overshoot was seen with an inwardly-directed gradient of KCl. Transport of L-alanine included saturable and diffusive components. Uptake of 6 μM L-alanine was inhibited more than 80% by 100 μM α-zwitterionic amino acids (alanine, leucine, glycine); by 30 to 75% by proline, aspartate and lysine; and less than 20% by a β-amino acid, taurine. The results of these experiments agree with those from intact gill studies and support the hypothesis that L-alanine is transported into gill epithelial cells by a secondary active transport process involving Na+

  3. Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris

    Directory of Open Access Journals (Sweden)

    Niehaus Karsten

    2008-06-01

    Full Text Available Abstract Background Outer membrane vesicles (OMVs are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli. Results We have demonstrated that Xanthomonas campestris pv. campestris (Xcc releases OMVs into the culture supernatant during growth. A proteome study identified 31 different proteins that associate with the OMV fraction of which half are virulence-associated. A comparison with the most abundant outer membrane (OM proteins revealed that some proteins are enriched in the OMV fraction. This may be connected to differences in the LPS composition between the OMVs and the OM. Furthermore, a comparison of the OMV proteomes from two different culture media indicated that the culture conditions have an impact on the protein composition. Interestingly, the proteins that are common to both culture conditions are mainly involved in virulence. Conclusion Outer membrane vesicles released from the OM of Xcc contain membrane- and virulence-associated proteins. Future experiments will prove whether these structures can serve as "vehicles" for the transport of virulence factors into the host membrane.

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

    International Nuclear Information System (INIS)

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

  5. Detection of membrane cholesterol by filipin in isolated rat liver coated vesicles is dependent upon removal of the clathrin coat

    OpenAIRE

    1984-01-01

    We investigated the cholesterol content of highly purified populations of coated vesicles from rat liver by biochemical quantitation and by cytochemical electron microscopy using the polyene antibiotic filipin. Failure of this reagent to elicit its typical response for a cholesterol-containing membrane, i.e., a characteristically corrugated or rippled appearance by thin section analysis, had led to the hypothesis (Montesano, R., A. Perrelet, P. Vassalli, and L. Orci, 1979, Proc. Natl. Acad. S...

  6. Influence of O Polysaccharides on Biofilm Development and Outer Membrane Vesicle Biogenesis in Pseudomonas aeruginosa PAO1

    OpenAIRE

    Murphy, Kathleen; Park, Amber J.; Hao, Youai; Brewer, Dyanne; Lam, Joseph S.; Khursigara, Cezar M.

    2014-01-01

    Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to...

  7. Immunization of Mice With Vibrio cholerae Outer-Membrane Vesicles Protects Against Hyperinfectious Challenge and Blocks Transmission

    OpenAIRE

    Bishop, Anne L.; Tarique, Abdullah A.; Patimalla, Bharathi; Calderwood, Stephen B.; Qadri, Firdausi; Camilli, Andrew

    2011-01-01

    Background. Vibrio cholerae excreted by cholera patients is “hyperinfectious” (HI), which can be modeled by passage through infant mice. Immunization of adult female mice with V. cholerae outer-membrane vesicles (OMVs) passively protects suckling mice from challenge. Although V. cholerae is unable to colonize protected pups, the bacteria survive passage and have the potential to be transmitted to susceptible individuals. Here, we investigated the impact of OMV immunization and the HI state on...

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

    Directory of Open Access Journals (Sweden)

    Daniela eBoassa

    2015-02-01

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

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2014-09-16

    Outer membrane vesicles (OMVs) of Gram-negative bacteria have been studied intensively in recent years, primarily in their role in delivering virulence factors and antigens during pathogenesis. However, the near ubiquity of their production suggests that they may play other roles, such as responding to envelope stress or trafficking various cargoes to prevent dilution or degradation by other bacterial species. Here we show that OMVs produced by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, block its interaction with various surfaces such as the walls of xylem vessels in host plants. The release of OMVs was suppressed by the diffusible signal factor-dependent quorum-sensing system, and a X. fastidiosa ΔrpfF mutant in which quorum signaling was disrupted was both much more virulent to plants and less adhesive to glass and plant surfaces than the WT strain. The higher virulence of the ΔrpfF mutant was associated with fivefold higher numbers of OMVs recovered from xylem sap of infected plants. The frequency of attachment of X. fastidiosa to xylem vessels was 20-fold lower in the presence of OMVs than in their absence. OMV production thus is a strategy used by X. fastidiosa cells to adjust attachment to surfaces in its transition from adhesive cells capable of insect transmission to an "exploratory" lifestyle for systemic spread within the plant host which would be hindered by attachment. OMV production may contribute to the movement of other bacteria in porous environments by similarly reducing their contact with environmental constituents. PMID:25197068

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-31

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

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

    Science.gov (United States)

    Jung, Anna Lena; Stoiber, Cornelia; Herkt, Christina E; Schulz, Christine; Bertrams, Wilhelm; Schmeck, Bernd

    2016-04-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host. PMID:27105429

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

    Directory of Open Access Journals (Sweden)

    Jian Wu

    2015-01-01

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

  14. Local membrane length conservation in two-dimensional vesicle simulation using a multicomponent lattice Boltzmann equation method

    Science.gov (United States)

    Halliday, I.; Lishchuk, S. V.; Spencer, T. J.; Pontrelli, G.; Evans, P. C.

    2016-08-01

    We present a method for applying a class of velocity-dependent forces within a multicomponent lattice Boltzmann equation simulation that is designed to recover continuum regime incompressible hydrodynamics. This method is applied to the problem, in two dimensions, of constraining to uniformity the tangential velocity of a vesicle membrane implemented within a recent multicomponent lattice Boltzmann simulation method, which avoids the use of Lagrangian boundary tracers. The constraint of uniform tangential velocity is carried by an additional contribution to an immersed boundary force, which we derive here from physical arguments. The result of this enhanced immersed boundary force is to apply a physically appropriate boundary condition at the interface between separated lattice fluids, defined as that region over which the phase-field varies most rapidly. Data from this enhanced vesicle boundary method are in agreement with other data obtained using related methods [e.g., T. Krüger, S. Frijters, F. Günther, B. Kaoui, and J. Harting, Eur. Phys. J. 222, 177 (2013), 10.1140/epjst/e2013-01834-y] and underscore the importance of a correct vesicle membrane condition.

  15. 13C-labeled 18 : 2n-6 recovered in brush border membrane phospholipids short time after administration

    DEFF Research Database (Denmark)

    Vistisen, Bodil; Høy, Carl-Erik

    2004-01-01

    administered intragastrically a bolus of ML*M, M*LM*, L*L*L* or M*M*M* (* = C-13- labeled fatty acid). Rats were decapitated 2 hours and 6 hours later, and the fatty acid composition and C-13-enrichment of BBM-PC and -PE were determined. C-13-enriched 18:2n-6 was observed in BBM-PC after intragastric...... administration of L*L*L* and ML*M, whereas no C-13-labeled fatty acids were recovered after administration of M*LM* or M*M*M*. Interestingly, no C-13-labeled fatty acids were detected in the BBM-PE fraction. This could be due to a lower turnover of PE than PC and to a different ratio of saturated and unsaturated...... fatty acids in the two phospholipid pools. Minor effects on BBM-PC and BBM-PE fatty acid profiles (mole-%) were observed. The present study demonstrated for the first time incorporation of C-13-labeled 18:2n-6 into BBM-PC 2 hours and 6 hours after intragastric administration of L*L*L* or ML*M. This...

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

    International Nuclear Information System (INIS)

    The wheat roots plasma membrane (PM) vesicles were obtained by sucrose gradient centrifugation. The experiment results shows that the wheat roots of Zhengyin No.1 PM H+-ATPase latent activity was 24%, and PM inside-out vesicle (IOV) accounts for 76%. With -1.0 MPa stress of 24h, PM Ca2+-ATPase activity of both orientation wheat roots were increased. Under normal water condition and PEG stress, 62% and 53% of the enzyme activity was inhibited respectively by EGTA, radioactive calcium-45 transport amount was 22.09 nmol/mg pro and 4.17 nmol/mg pro. respectively with PM-IOV.PEG stress results in a decrease of 45Ca2+ transport amount of wheat roots PM-IOV by 81%

  17. Active auxin uptake by zucchini membrane vesicles: quantitation using ESR volume and delta pH determinations

    Energy Technology Data Exchange (ETDEWEB)

    Lomax, T.L.; Mehlhorn, R.J.; Briggs, W.R.

    1985-10-01

    Closed and pH-tight membrane vesicles prepared from hypocotyls of 5-day-old dark-grown seedlings of Cucurbita pepo accumulate the plant growth hormone indole-3-acetic acid along an imposed proton gradient (pH low outside, high inside). The use of electron paramagnetic spin probes permitted quantitation both of apparent vesicle volume and magnitude of the pH gradient. Under the experimental conditions used, hormone accumulation was at minimum 20-fold, a value 4 times larger than what one would predict if accumulation reflected only diffusional equilibrium at the measured pH gradient. It is concluded that hormone uptake is an active process, with each protonated molecule of hormone accompanied by an additional proton. Experiments with ionophores confirm that it is the pH gradient itself which drives the uptake.

  18. Acellular approaches for regenerative medicine: on the verge of clinical trials with extracellular membrane vesicles?

    OpenAIRE

    Fuster-Matanzo, Almudena; Gessler, Florian; Leonardi, Tommaso; Iraci, Nunzio; Pluchino, Stefano

    2015-01-01

    Extracellular vesicles (EVs) are a heterogeneous population of naturally occurring secreted small vesicles, with distinct biophysical properties and different functions both in physiology and under pathological conditions. In recent years, a number of studies have demonstrated that EVs might hold remarkable potential in regenerative medicine by acting as therapeutically promising nanodrugs. Understanding their final impact on the biology of specific target cells as well as clarification of th...

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

    International Nuclear Information System (INIS)

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

  20. Ureteral retrograde brush biopsy

    Science.gov (United States)

    Biopsy - brush - urinary tract; Retrograde ureteral brush biopsy cytology; Cytology - ureteral retrograde brush biopsy ... to be biopsied is rubbed with the brush. Biopsy forceps may be used instead to collect a ...

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

    DEFF Research Database (Denmark)

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

    1992-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Goldsmith, M.H.

    1985-01-01

    The accumulation of indoleacetic acid (IAA) inside microsomal vesicles depends on the presence of a pH gradient and is reversible when the ..delta..pH is collapsed by ionosphores. Accumulation is stimulated by either napthylphthalamic acid or TIBA. The accumulation of IAA by the vesicles can be saturated. At concentrations of 1 ..mu..M or less, IAA, synthetic auxins, or auxin antagonists do not affect the pH gradient, but decrease the accumulation of /sup 3/H-IAA, and therefore compete specifically for uptake. Concentrations of 10 ..mu..M and above, uptake of either the auxins or weak acids is sufficient to overcome the buffering capacity of the solution within the vesicles. The collapse of the pH gradient by such high concentrations affects uptake of either /sup 3/H-IAA or /sup 14/C-BA to similar extents and thus is nonspecific. 3 refs.

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

    Science.gov (United States)

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

    2015-08-12

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

  4. The effect of acclimation temperature on the fusion kinetics of lipid vesicles derived from endoplasmic reticulum membranes of rainbow trout (Oncorhynchus mykiss) liver.

    Science.gov (United States)

    Miranda, Estuardo J; Hazel, Jeffrey R

    2002-02-01

    Membrane fusion is an obligatory step in many vital cellular processes. The well-established enrichment of bilayer-destabilizing lipids in membranes of poikilotherms subjected to growth at low temperatures leads to the prediction that such membranes will possess a greater propensity to undergo fusion. This hypothesis was explicitly tested in the present study by determining the kinetics of fusion between small unilamellar vesicles (SUVs) prepared from endoplasmic reticulum (ER) membranes of thermally-acclimated (to 5 and 20 degrees C) rainbow trout (Oncorhynchus mykiss) liver and bovine brain phosphatidylserine (BBPS). At temperatures above 10 degrees C, ER vesicles from 5 degrees C-acclimated trout, fused more rapidly and to a greater extent with BBPS vesicles (by average factors of 1.25- and 1.45-fold, respectively) than ER vesicles of 20 degrees C-acclimated trout. At temperatures >35 degrees C, apparent fusion rates declined while the extent of fusion increased in both acclimation groups. Fusion kinetics were found to be well correlated with and limited by the physical properties and phase state of the BBPS vesicles. These results indicate that dynamic attributes of biological membranes, such as the propensity to undergo fusion, are of potential regulatory significance and are partially conserved when growth or environmental temperature changes. PMID:11818217

  5. Bilayer vesicles of amphiphilic cyclodextrines: host membranes that recognize guest molecules

    NARCIS (Netherlands)

    Falvey, P.; Lim, C.W.; Darcy, R.; Revermann, T.; Karst, U.; Marcelis, A.T.M.; Coleman, A.W.; Reinhoudt, D.N.; Ravoo, B.J.

    2005-01-01

    A family of amphiphilic cyclodextrins (6, 7) has been prepared through 6-S-alkylation (alkyl=n-dodecyl and n-hexadecyl) of the primary side and 2-O-PEGylation of the secondary side of alpha-, beta-, and gamma-cyclodextrins (PEG=poly(ethylene glycol)). These cyclodextrins form nonionic bilayer vesicl

  6. Bilayer Vesicles of Amphiphilic Cyclodextrins: Host Membranes That Recognize Guest Molecules

    NARCIS (Netherlands)

    Falvey, Patrick; Lim, Choon Woo; Darcy, Raphael; Revermann, Tobias; Karst, Uwe; Giesbers, Marcel; Marcelis, Antonius T.M.; Lazar, Adina; Coleman, Anthony W.; Reinhoudt, David N.; Ravoo, Bart Jan

    2005-01-01

    A family of amphiphilic cyclodextrins (6, 7) has been prepared through 6-S-alkylation (alkyl=n-dodecyl and n-hexadecyl) of the primary side and 2-O-PEGylation of the secondary side of a-, B-, and Y-cyclodextrins (PEG=poly(ethylene glycol)). These cyclodextrins form nonionic bilayer vesicles in aqueo

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

    NARCIS (Netherlands)

    Hoekstra, D.

    1982-01-01

    The relationship between lipid phase separation and fusion of small unilamellar phosphatidylserine-containing vesicles was investigated. The kinetics of phase separation were monitored by following the increase of self-quenching of the fluorescent phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol

  8. 38,000-DALTON MEMBRANE PROTEIN (P38) PRESENT IN SYNAPTIC VESICLES

    Science.gov (United States)

    A protein with an apparent molecular mass of 38,000 daltons designated p38 was found in synaptic vesicles from rat brain. The subcellular distribution of p38 and some of its properties were determined with the aid of polyclonal and monoclonal antibodies. The subcellular distribut...

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

    DEFF Research Database (Denmark)

    Shillcock, J. C.

    2012-01-01

    parallel dissipative particle dynamics (DPD) to follow spontaneous vesicle self-assembly for up to 445 mu s with near-molecular resolution. The mean mass and radius of gyration of growing amphiphilic clusters obey power laws with exponents of 0.85 +/- 0.03 and 0.41 +/- 0.02, respectively. We show that DPD...

  10. Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles

    International Nuclear Information System (INIS)

    Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions

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

    Directory of Open Access Journals (Sweden)

    Reinhard Rachel

    2002-01-01

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

  12. Development of a cost-effective vaccine candidate with outer membrane vesicles of a tolA-disrupted Shigella boydii strain.

    Science.gov (United States)

    Mitra, Soma; Sinha, Ritam; Mitobe, Jiro; Koley, Hemanta

    2016-04-01

    Our previous studies on outer membrane vesicles based vaccine development against shigellosis, revealed the inability of Shigella to release significant amount of vesicles naturally, during growth. Disruption of tolA, one of the genes of the Tol-Pal system of Gram negative bacterial membrane, has increased the vesicle release rate of a Shigella boydii type 4 strain to approximately 60% higher. We also noticed the vesicles, released from tolA-disrupted strain captured more OmpA protein and lipopolysaccharide, compared to the vesicles released from its wild type prototype. Six to seven weeks old BALB/c mice, immunized with 25μg of three oral doses of the vesicles, released by tolA mutant, conferred 100% protection against lethal homologous challenge through nasal route, compared to only 60% protection after the same dose of wild type immunogen. Mice, immunized with the vesicles from tolA-mutant, manifested significant secretion of mucosal IgG and IgA. A sharp and significant response of pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ) were also observed in the lung lavage of these groups of mice, within 6h post challenge; but at 24h, these inflammatory cytokines showed the sign of subsidence and the system was taken over by the release of anti-inflammatory cytokines (IL-4 and IL-10). Studies with naïve peritoneal macrophages, proved further, the potency of these vesicles to stimulate nitric oxide and TNF-α, IL-12p70, IL-6 and IL-10 productions in-vitro. The ability of these vesicles to trigger polarization of CD4(+) T cells toward Th1 adaptive immune response, had also been observed along with the presence of anti-inflammatory cytokines in the system. Our study demonstrated, the vesicles from tolA-disrupted Shigella were able to suppress Shigella-mediated inflammation in the host and could balance between inflammation and anti-inflammation, promoting better survival and health of the infected mice. Outer membrane vesicles from tolA-mutant, could be a potential

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

    Science.gov (United States)

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

    2016-06-01

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

  14. Structure of the human vitreoretinal border region

    DEFF Research Database (Denmark)

    Heegaard, Steffen

    1994-01-01

    Øjenpatologi, vitreoretinal border region, inner limiting membrane, retina, topographical variation, human......Øjenpatologi, vitreoretinal border region, inner limiting membrane, retina, topographical variation, human...

  15. 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.

  16. Open Syntaxin Docks Synaptic Vesicles

    OpenAIRE

    Marc Hammarlund; Mark T Palfreyman; Shigeki Watanabe; Shawn Olsen; Erik M. Jorgensen

    2007-01-01

    Author Summary Like Olympic swimmers crouched on their starting blocks, synaptic vesicles prepare for fusion with the neuronal plasma membrane long before the starting gun fires. This preparation enables vesicles to fuse rapidly, synchronously, and in the correct place when the signal finally arrives. A well-known but poorly understood part of vesicle preparation is docking, in which vesicles prepare for release by attaching to the plasma membrane at the eventual site of release. Here, we out...

  17. Dipalmitoylphosphatidylcholine/linoleic acid mixed unilamellar vesicles as model membranes for studies on novel free-radical scavengers.

    Science.gov (United States)

    Castelli, F; Trombetta, D; Tomaino, A; Bonina, F; Romeo, G; Uccella, N; Saija, A

    1997-04-01

    Large unilamellar vesicles (LUVs) are generally accepted to be a suitable model for peroxidation studies. In the present report, dipalmitoylphosphatidylcholine (DPPC)/linoleic acid-mixed LUVs were employed as model membranes to verify the inhibitory effect of tocopherol (an efficient representative antioxidant) against 2,2'-azobis(2-amidinopropane)hydrochloride-induced peroxidation (evaluated by monitoring conjugated diene accumulation). In this model, the appropriate experimental conditions (particularly, liposome composition and peroxidation temperature) were selected following characterization of bilayer physical state, and not only by evaluation of peroxidation rate. Thus, the experiments described provide a routine screening procedure that would be appropriate for assessing the activity profile of novel free-radical scavengers. PMID:9253749

  18. LLC-PK1 cells express Na+-lactate cotransport in apical membranes after confluency

    International Nuclear Information System (INIS)

    L-[3H]lactate uptake was characterized in LLC-PK1 cell apical membrane vesicles obtained by intensive culture on microcarrier beads. The apical membrane preparation technique involved MgCl2 precipitation. Na+-dependent L-[3H]lactate uptake was present only after confluency; its appearance paralleled the subcellular localization of aminopeptidase in apical membranes. L-[3H]lactate uptake was Na+-dependent and electrogenic. Only the Na+-dependent component of L[3H]lactate uptake was saturable with one family of independent carriers. The apparent affinity constant was 1.1 ± 0.25 mM and the apparent maximal velocity was 29 ± 3 nmol·mg-1·min-1. The Na+-lactate cotransport stoichiometry was 2 Na+ for 1 lactate. The specificity of the L-lactate transport system was compatible with that of the monocarboxylic acid pathway described previously brush-border membranes of kidney cortex and discrete from the tricarboxylic acid carrier, the D-glucose transporter, and the general pathway for anions. The LLC-PK1 cell line appears to be a useful tool for study of the regulation of L-lactate uptake and biosynthesis of the renal monocarboxylic acid transporter

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    -labeled cholera toxin B subunit which labels ganglioside GM(1) enriched plasma membrane rafts. Cholesterol depletion with methyl-beta-cyclodextrin enhanced DiI vesiculation, GM(1)/DiI domain separation and was accompanied by a detachment of the subcortical cytoskeleton from the plasma membrane. Based on these...

  20. Acellular approaches for regenerative medicine: on the verge of clinical trials with extracellular membrane vesicles?

    Science.gov (United States)

    Fuster-Matanzo, Almudena; Gessler, Florian; Leonardi, Tommaso; Iraci, Nunzio; Pluchino, Stefano

    2015-01-01

    Extracellular vesicles (EVs) are a heterogeneous population of naturally occurring secreted small vesicles, with distinct biophysical properties and different functions both in physiology and under pathological conditions. In recent years, a number of studies have demonstrated that EVs might hold remarkable potential in regenerative medicine by acting as therapeutically promising nanodrugs. Understanding their final impact on the biology of specific target cells as well as clarification of their overall therapeutic impact remains a matter of intense debate. Here we review the key principles of EVs in physiological and pathological conditions with a specific highlight on the most recently described mechanisms regulating some of the EV-mediated effects. First, we describe the current debates and the upcoming research on EVs as potential novel therapeutics in regenerative medicine, either as unmodified agents or as functionalized small carriers for targeted drug delivery. Moreover, we address a number of safety aspects and regulatory limitations related to the novel nature of EV-mediated therapeutic applications. Despite the emerging possibilities of EV treatments, these issues need to be overcome in order to allow their safe and successful application in future explorative clinical studies. PMID:26631254

  1. Size-selective permeation of water-soluble polymers through the bilayer membrane of cyclodextrin vesicles investigated by PFG-NMR.

    Science.gov (United States)

    Himmelein, Sabine; Sporenberg, Nora; Schönhoff, Monika; Ravoo, Bart Jan

    2014-04-15

    Cyclodextrin vesicles (CDVs) consist of a bilayer of amphiphilic cyclodextrins (CDs). CDVs exhibit CD cavities at their surface that are able to recognize and bind hydrophobic guest molecules via size-selective inclusion. In this study, the permeability of α- and β-CDVs is investigated by pulsed field gradient-stimulated echo (PFG-STE) nuclear magnetic resonance. Diffusion experiments with water and two types of water-soluble polymers, polyethylene glycol (PEG) and polypropylene glycol (PPG), revealed three main factors that influence the exchange rate and permeability of CDVs. First, the length of the hydrophobic chain of the CD amphiphile plays a crucial role. Reasonably, vesicles consisting of amphiphiles with a longer aliphatic chain are less permeable since both membrane thickness and melting temperature T(m) increase. Second, the exchange rate through the bilayer membrane depends on the molecular weight of the polymer and decreases with increasing weight of the polymer. Most interestingly, a size-selective distinction of permeation due to the embedded CDs in the bilayer membrane was found. The mechanism of permeation is shown to occur through the CD cavity, such that depending on the size of the cavity, permeation of polymers with different cross-sectional diameters takes place. Whereas PPG permeates through the membrane of β-CD vesicles, it does not permeate α-CD vesicles. PMID:24650278

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

    Science.gov (United States)

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

    2010-10-01

    The present study deals with the morphological modifications of giant dioleoyl phosphatidylcholine vesicles (DOPC GUVs) induced by the nonionic surfactant n-octyl β,D-glucopyranoside at sublytic levels, i.e., in the first steps of the vesicle-to-micelle transition process, when surfactant inserts into the vesicle bilayer without disruption. Experimental conditions were perfected to exactly control the surfactant bilayer composition of the vesicles, in line with former work focused on the mechanical properties of the membrane of magnetic-fluid-loaded DOPC GUVs submitted to a magnetic field. The purpose here was to systematically examine, in the absence of any external mechanical constraint, the dynamics of giant vesicle shape and membrane deformations as a function of surfactant partitioning between the aqueous phase and the lipid membrane, beforehand established by turbidity measurements from small unilamellar vesicles. PMID:20825201

  3. Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes.

    Science.gov (United States)

    Olety, Balaji; Veatch, Sarah L; Ono, Akira

    2016-01-01

    The structural protein of HIV-1, Pr55(Gag) (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a "cell-like" environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy. PMID:27500610

  4. Raft domains of variable properties and compositions in plasma membrane vesicles

    OpenAIRE

    LEVENTAL, Ilya; Grzybek, Michal; Simons, Kai

    2011-01-01

    Biological membranes are compartmentalized for functional diversity by a variety of specific protein–protein, protein–lipid, and lipid–lipid interactions. A subset of these are the preferential interactions between sterols, sphingolipids, and saturated aliphatic lipid tails responsible for liquid–liquid domain coexistence in eukaryotic membranes, which give rise to dynamic, nanoscopic assemblies whose coalescence is regulated by specific biochemical cues. Microscopic phase separation recently...

  5. In vitro approach to the mechanics of lipid membrane area regulation: vesicle absorption and tube formation

    Science.gov (United States)

    Staykova, Margarita; Holmes, Douglas; Read, Clarke; Stone, Howard A.

    2011-03-01

    We have designed an experimental approach that allows us to study the response of supported lipid bilayers to cycles of biaxial expansion and compression. We observed that the bilayer effectively adjusts its area during dilatational or compressive strains in order to reduce its tension. For example, if there is a sufficient lipid reservoir in the form of attached vesicles, then a lipid bilayer may accommodate strains tens of times larger than the critical strain for rupture by expanding its area. Additionally, upon compression the bilayer reduces its area by expelling lipid tubes out of its plane. These observations offer new insights into how cells regulate their surface area in response to various mechanical stimuli, i.e. during physiological volume changes, locomotion, cyclic expansion and compression of the uro- and the alveolar- epithelium, etc.

  6. 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.

  7. Membrane retrieval in the guinea-pig neurohypophysis. Isolation and characterization of secretory vesicles and coated microvesicles after radiolabel incorporation in vivo.

    Science.gov (United States)

    Saermark, T; Jones, P M; Robinson, I C

    1984-03-01

    We have developed small-scale methods for the isolation and biochemical characterization of subcellular fractions from single guinea-pig posterior-pituitary glands. Secretory vesicles and coated microvesicles produced in this way were of similar purity to those isolated from large amounts of tissue by conventional ultracentrifugation. [35S]Cysteine injected into the hypothalamus was found in the soluble contents of secretory vesicles isolated from the neural lobes 24 h later. High-pressure liquid-chromatographic analysis revealed that the radiolabel was incorporated into the expected neurosecretory products (oxytocin, vasopressin and neurophysin) and also into a biosynthetic intermediate in the vasopressin system. The membranes of secretory vesicles were labelled with [3H]choline 24 h after its hypothalamic injection. Little or no [3H]choline could be demonstrated in coated microvesicles at this time, although these structures were labelled 5 days after injection. Stimulating hormone secretion by chronic dehydration produced a significant fall in [3H]choline content of the secretory-vesicle membranes without any transfer of label into coated microvesicles, suggesting that coated microvesicles are not involved in membrane retrieval in the neurohypophysis. PMID:6712633

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

    DEFF Research Database (Denmark)

    Klymchenko, Andrey; Oncul, Sule; Didier, Pascal;

    2009-01-01

    vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization....... Taking into account the high selectivity of F2N12S for the cell plasma membranes and its suitability for both single- and two-photon excitation, applications of this probe to study membrane lateral heterogeneity in biological membranes are foreseen....... of the individual phases from the fluorescence intensity ratio of its two emission bands. By using a linearly polarized excitation light, a strong photoselection was observed for F2N12S in the Lo phase, indicating that its fluorophore is nearly parallel to the lipid chains of the bilayer. In contrast...

  9. The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles

    NARCIS (Netherlands)

    Gerritsen, A.; Verkleij, A.J.; Deenen, L.L.M. van

    1979-01-01

    Triton X-100 (in concentrations which did not cause a significant solubilization of membrane material) caused aggregation of the intramembrane particles of human erythrocyte ghosts. Ghosts from which the extrinsic proteins had been removed by alkali treatment showed a temperature-induced aggregatio

  10. Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue

    OpenAIRE

    1981-01-01

    Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles. Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto t...

  11. Effects of La3+ on ATPase Activities of Plasma Membrane Vesicles Isolated from Casuarina Equisetifolia Seedlings under Acid Rain Stress

    Institute of Scientific and Technical Information of China (English)

    李裕红; 严重玲; 刘景春; 陈英华; 胡俊; 薛博

    2003-01-01

    The effects of La3+ on the growth and the ATPases activities of plasma membrane(PM) vesicles isolated from Casuarina equisetifolia seedlings under artificial acid rain(pH 4.5) stress were studied. The results show that the height, length of roots, fresh weight and PM H+-ATPase activites of Casuarina equisetifolia seedlings increase by the treatments of soaking seeds in LaCl3 solutions with lower concentrations, and those can reach their peak values by treating with 200 mg·L-1 La3+. However, in comparison with the CK, those are inhibited by the higher La3+ concentrations; PM Ca2+-ATPase activity is inhibited with the treatments of La3+. The results also reveal that the H+-ATPase activity and the growth of cell enlarge have a remarkable positive correlation, and La3+ activating H+-ATPase can facilitate plant growth. La3+ also can alleviate cytosolic acidification of plant under acid rain stress and indirectly maintain the stability of intracellular environment. In order to resistant to acid rain and accelerate the growth of Casuarina equisetifolia, the suitable range of La3+ concentrations to soak seeds for 8 h is 50~200 mg*L-1.

  12. The Pseudomonas aeruginosa extracellular secondary metabolite, Paerucumarin, chelates iron and is not localized to extracellular membrane vesicles.

    Science.gov (United States)

    Qaisar, Uzma; Kruczek, Cassandra J; Azeem, Muhammed; Javaid, Nasir; Colmer-Hamood, Jane A; Hamood, Abdul N

    2016-08-01

    Proteins encoded by the Pseudomonas aeruginosa pvcA-D operon synthesize a novel isonitrile functionalized cumarin termed paerucumarin. The pvcA-D operon enhances the expression of the P. aeruginosa fimbrial chaperone/usher pathway (cup) genes and this effect is mediated through paerucumarin. Whether pvcA-D and/or paerucumarin affect the expression of other P. aeruginosa genes is not known. In this study, we examined the effect of a mutation in pvcA-D operon the global transcriptome of the P. aeruginosa strain PAO1-UW. The mutation reduced the expression of several ironcontrolled genes including pvdS, which is essential for the expression of the pyoverdine genes. Additional transcriptional studies showed that the pvcA-D operon is not regulated by iron. Exogenously added paerucumarin enhanced pyoverdine production and pvdS expression in PAO1-UW. Iron-chelation experiments revealed that purified paerucumarin chelates iron. However, exogenously added paerucumarin significantly reduced the growth of a P. aeruginosa mutant defective in pyoverdine and pyochelin production. In contrast to other secondary metabolite, Pseudomonas quinolone signal (PQS), paerucumarin is not localized to the P. aeruginosa membrane vesicles. These results suggest that paerucumarin enhances the expression of iron-controlled genes by chelating iron within the P. aeruginosa extracellular environment. Although paerucumarin chelates iron, it does not function as a siderophore. Unlike PQS, paerucumarin is not associated with the P. aeruginosa cell envelope. PMID:27480638

  13. Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholerae.

    Science.gov (United States)

    Mondal, Ayan; Tapader, Rima; Chatterjee, Nabendu Sekhar; Ghosh, Amit; Sinha, Ritam; Koley, Hemanta; Saha, Dhira Rani; Chakrabarti, Manoj K; Wai, Sun Nyunt; Pal, Amit

    2016-05-01

    Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses. PMID:26930702

  14. Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from Biofilm and Planktonic Phase Associated with Extracellular DNA (eDNA)

    OpenAIRE

    Grande, Rossella; Di Marcantonio, Maria C.; Robuffo, Iole; Pompilio, Arianna; Celia, Christian; Di Marzio, Luisa; Paolino, Donatella; Codagnone, Marilina; Muraro, Raffaella; Stoodley, Paul; Hall-Stoodley, Luanne; Mincione, Gabriella

    2015-01-01

    Helicobacter pylori persistence is associated with its capacity to develop biofilms as a response to changing environmental conditions and stress. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances (EPS) and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the e...

  15. Intranasal Delivery of Group B Meningococcal Native Outer Membrane Vesicle Vaccine Induces Local Mucosal and Serum Bactericidal Antibody Responses in Rabbits

    OpenAIRE

    Shoemaker, David R.; Saunders, Nancy B.; Brandt, Brenda L.; Moran, E. Ellen; LaClair, Andrew D.; Zollinger, Wendell D.

    2005-01-01

    We have previously shown that intranasal immunization of mice with meningococcal native outer membrane vesicles (NOMV) induces both a good local mucosal antibody response and a good systemic bactericidal antibody response. However, in the intranasal mouse model, some of the NOMV entered the lung and caused an acute granulocytic response. We therefore developed an alternate animal model using the rabbit. This model reduces the probability of lung involvement and more closely mimics intranasal ...

  16. Cholesterol induces surface localization of polyphenols in model membranes thus enhancing vesicle stability against lysozyme, but reduces protection of distant double bonds from reactive-oxygen species.

    Science.gov (United States)

    de Athayde Moncorvo Collado, Alejandro; Dupuy, Fernando G; Morero, Roberto D; Minahk, Carlos

    2016-07-01

    The main scope of the present study was to analyze the membrane interaction of members of different classes of polyphenols, i.e. resveratrol, naringenin, epigallocatechin gallate and enterodiol, in model systems of different compositions and phase states. In addition, the possible association between membrane affinity and membrane protection against both lipid oxidation and bilayer-disruptive compounds was studied. Gibbs monolayer experiments indicated that even though polyphenols showed poor surface activity, it readily interacted with lipid films. Actually, a preferential interaction with expanded monolayers was observed, while condensed and cholesterol-containing monolayers decreased the affinity of these phenolic compounds. On the other hand, fluorescence anisotropy studies showed that polyphenols were able to modulate membrane order degree, but again this effect was dependent on the cholesterol concentration and membrane phase state. In fact, cholesterol induced a surface rather than deep into the hydrophobic core localization of phenolic compounds in the membranes. In general, the polyphenolic molecules tested had a better antioxidant activity when they were allowed to get inserted into the bilayers, i.e. in cholesterol-free membranes. On the other hand, a membrane-protective effect against bilayer permeabilizing activity of lysozyme, particularly in the presence of cholesterol, could be assessed. It can be hypothesized that phenolic compounds may protect membrane integrity by loosely covering the surface of lipid vesicles, once cholesterol push them off from the membrane hydrophobic core. However, this cholesterol-driven distribution may lead to a reduced antioxidant activity of linoleic acid double bonds. PMID:27063609

  17. Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Gaigg, B; Neergaard, T B; Schneiter, R; Hansen, J.K.; Faergeman, N J; Jensen, N A; Andersen, J R; Friis, J; Sandhoff, R; Schrøder, H D; Knudsen, J

    2001-01-01

    -70%. The reduced incorporation of [(3)H]myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport. The plasma membrane of the...... Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids. Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures. The present results...

  18. Penetration of a Single Domain of Bacillus thuringiensis Cry1Ie-Domain I to a Lipid Membrane In vitro

    Institute of Scientific and Technical Information of China (English)

    GUO Shu-yuan; LI Jie; CHEN Zhen; HE Kang-lai

    2014-01-01

    Domain I of the activated Crystal protein from Bacillus thuringiensis has a sevenα-helix bundle structure, which is responsible for membrane channel formation in its insecticidal mechanism. Cry1Ie is toxic to Asian corn borer, Ostrinia furnacalis (Guenée), and plays important roles in insect biological control. The domain I from Cry1Ie has been expressed and puriifed in its normal conformation, as embedded in the full length homologous toxin structure. The membrane insertion ability of this single domain was compared with the full length homologous toxin using a monolayer insertion experiment. The results indicated that the Cry1Ie-domain I had the ability to insert into the lipid monolayer, and this ability is greater than that of the IE648 toxin. However, the state of insertion is not stable and remains for only a short period of time. The Cry1Ie-domain I plays no role in receptor binding as it had a nonspeciifc binding with the brush border membrane vesicles of the Asian corn borer.

  19. Phase-Field Modeling of Lipid Vesicles With Pores

    Science.gov (United States)

    Seifi, Saman; Salac, David

    2013-11-01

    The formation and annihilation of pores in a lipid vesicle membrane is critical to a number of biotechnologies, such as drug delivery. Previous models of vesicle behavior have ignored the influence of topological changes in the vesicle membrane. Here the entire Helfrich model of a vesicle membrane is considered. Topological changes in the vesicle membrane, such as the formation of a pore, are captured through the use of an embedded phase-field model. The numerical method and sample results will be presented.

  20. Brushing your infant's teeth

    Science.gov (United States)

    ... gov/ency/patientinstructions/000769.htm Brushing Your Child’s Teeth To use the sharing features on this page, ... how to brush their teeth on their own. Tooth and Gum Care for Young Children You should ...

  1. Cellular Immune Responses in Humans Induced by Two Serogroup B Meningococcal Outer Membrane Vesicle Vaccines Given Separately and in Combination.

    Science.gov (United States)

    Oftung, Fredrik; Korsvold, Gro Ellen; Aase, Audun; Næss, Lisbeth M

    2016-04-01

    MenBvac and MeNZB are safe and efficacious outer membrane vesicle (OMV) vaccines against serogroup B meningococcal disease. Antibody responses have previously been investigated in a clinical trial with these two OMV vaccines given separately (25 μg/dose) or in combination (12.5 and 12.5 μg/dose) in three doses administered at 6-week intervals. Here, we report the results from analyzing cellular immune responses against MenBvac and MeNZB OMVs in terms of antigen-specific CD4(+)T cell proliferation and secretion of cytokines. The proliferative CD4(+)T cell responses to the combined vaccine were of the same magnitude as the homologous responses observed for each individual vaccine. The results also showed cross-reactivity in the sense that both vaccine groups receiving separate vaccines responded to both homologous and heterologous OMV antigen when assayed for antigen-specific cellular proliferation. In addition, a multiplex bead array assay was used to analyze the presence of Th1 and Th2 cytokines in cell culture supernatants. The results showed that gamma interferon, interleukin-4 (IL-4), and IL-10 responses could be detected as a result of vaccination with both the MenBvac and the MeNZB vaccines given separately, as well as when given in combination. With respect to cross-reactivity, the cytokine results paralleled the observations made for proliferation. In conclusion, the results demonstrate that cross-reactive cellular immune responses involving both Th1 and Th2 cytokines can be induced to the same extent by different tailor-made OMV vaccines given either separately or in combination with half the dose of each vaccine. PMID:26865595

  2. Outer membrane vesicles from Brucella abortus promote bacterial internalization by human monocytes and modulate their innate immune response.

    Directory of Open Access Journals (Sweden)

    Cora N Pollak

    Full Text Available Outer membrane vesicles (OMVs released by some gram-negative bacteria have been shown to exert immunomodulatory effects that favor the establishment of the infection. The aim of the present study was to assess the interaction of OMVs from Brucella abortus with human epithelial cells (HeLa and monocytes (THP-1, and the potential immunomodulatory effects they may exert. Using confocal microscopy and flow cytometry, FITC-labeled OMVs were shown to be internalized by both cell types. Internalization was shown to be partially mediated by clathrin-mediated endocytosis. Pretreatment of THP-1 cells with Brucella OMVs inhibited some cytokine responses (TNF-α and IL-8 to E. coli LPS, Pam3Cys or flagellin (TLR4, TLR2 and TLR5 agonists, respectively. Similarly, pretreatment with Brucella OMVs inhibited the cytokine response of THP-1 cells to B. abortus infection. Treatment of THP-1 cells with OMVs during IFN-γ stimulation reduced significantly the inducing effect of this cytokine on MHC-II expression. OMVs induced a dose-dependent increase of ICAM-1 expression on THP-1 cells and an increased adhesion of these cells to human endothelial cells. The addition of OMVs to THP-1 cultures before the incubation with live B. abortus resulted in increased numbers of adhered and internalized bacteria as compared to cells not treated with OMVs. Overall, these results suggest that OMVs from B. abortus exert cellular effects that promote the internalization of these bacteria by human monocytes, but also downregulate the innate immune response of these cells to Brucella infection. These effects may favor the persistence of Brucella within host cells.

  3. Pseudomonas aeruginosa outer membrane vesicles triggered by human mucosal fluid and lysozyme can prime host tissue surfaces for bacterial adhesion

    Directory of Open Access Journals (Sweden)

    Matteo Maria Emiliano Metruccio

    2016-06-01

    Full Text Available Pseudomonas aeruginosa is a leading cause of human morbidity and mortality that often targets epithelial surfaces. Host immunocompromise, or the presence of indwelling medical devices, including contact lenses, can predispose to infection. While medical devices are known to accumulate bacterial biofilms, it is not well understood why resistant epithelial surfaces become susceptible to P. aeruginosa. Many bacteria, including P. aeruginosa, release Outer Membrane Vesicles (OMVs in response to stress that can fuse with host cells to alter their function. Here, we tested the hypothesis that mucosal fluid can trigger OMV release to compromise an epithelial barrier. This was tested using tear fluid and corneal epithelial cells in vitro and in vivo. After 1 h both human tear fluid, and the tear component lysozyme, greatly enhanced OMV release from P. aeruginosa strain PAO1 compared to PBS controls (~100 fold. TEM and SDS-PAGE showed tear fluid and lysozyme-induced OMVs were similar in size and protein composition, but differed from biofilm-harvested OMVs, the latter smaller with fewer proteins. Lysozyme-induced OMVs were cytotoxic to human corneal epithelial cells in vitro and murine corneal epithelium in vivo. OMV exposure in vivo enhanced Ly6G/C expression at the corneal surface, suggesting myeloid cell recruitment, and primed the cornea for bacterial adhesion (~4-fold, P < 0.01. Sonication disrupted OMVs retained cytotoxic activity, but did not promote adhesion, suggesting the latter required OMV-mediated events beyond cell killing. These data suggest that mucosal fluid induced P. aeruginosa OMVs could contribute to loss of epithelial barrier function during medical device-related infections.

  4. Friction between Polymer Brushes

    OpenAIRE

    Sokoloff, J. B.

    2006-01-01

    By solving the equilibrium equations for a polymer in a neutral polymer brush, the degree of interpenetration of two polymer brushes in contact and near contact is calculated. These results are used to calculate values of the force of static friction in agreement with recent friction measurements for polymer brush lubricated surfaces. It is shown that at sufficiently light loads polymer brush coated surfaces can slide, with the load supported entirely by osmotic pressure, at a sufficiently la...

  5. The membrane composition of coated pits, microvilli, endosomes, and lysosomes is distinctive in the rat kidney proximal tubule cell

    OpenAIRE

    1986-01-01

    The distribution of a number of membrane proteins on plasmalemmal microdomains (microvilli, coated pits) and in endosomes and lysosomes of the proximal tubule epithelial cell was determined in normal rat kidneys by immunofluorescence and immunoelectron microscopy. Two major brush border proteins, 130 and 94 kD, and gamma-glutamyl transpeptidase were detected on the membranes of the microvilli but were not found on membranes of coated pits. Gp330, the Heymann nephritis antigen, and clathrin we...

  6. Radiation inactivation probe of membrane-bound enzymes: gamma-glutamyltranspeptidase, aminopeptidase N, and sucrase

    International Nuclear Information System (INIS)

    gamma-Glutamyltranspeptidase (GGT), aminopeptidase N (AP-N), and sucrase in purified rabbit intestinal brush border membrane vesicles were irradiated in situ at -135 degrees C using high energy electrons. Surviving activities of the enzymes were measured as a function of radiation dose, and the functional unit target sizes (corresponding to carbohydrate-free polypeptides) were determined using target analysis. The in situ functional unit sizes were GGT 59 kDa, AP-N 59 kDa, and sucrase 63 kDa. Together with biochemical data determined previously, it is concluded that the noncovalently attached large (approximately 40 kDa) and small (approximately 25 kDa) subunits of GGT are both required for catalytic activity. Furthermore, these data suggest that (i) the membrane-bound form of AP-N consists of one or more noncovalently attached subunits of 59 kDa, each of which is enzymatically active; and (ii) in situ sucrase activity is associated with a subunit of 63 kDa which is noncovalently attached within the sucrase-isomaltase complex

  7. Synaptic Vesicle Exocytosis

    OpenAIRE

    Südhof, Thomas C; Rizo, Josep

    2011-01-01

    Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap aro...

  8. Translocation of ProOmpA possessing an intramolecular disulfide bridge into membrane vesicles of Escherichia coli. Effect of membrane energization

    International Nuclear Information System (INIS)

    In the absence of delta mu H+, the in vitro translocation of proOmpA resulted in the stable accumulation of a possible translocation intermediate in addition to a transiently accumulating one. The stable intermediate was detected on a polyacrylamide gel as two proteinase K-resistant bands corresponding to a molecular weight of about 28,000. The appearance of the bands was appreciably enhanced when proOmpA was oxidized with ferricyanide. No mature OmpA appeared. When proOmpA reduced with dithiothreitol was used, on the other hand, the bands did not appear at all. Upon the replacement of Cys302 of OmpA with Gly, the intermediate accumulation was abolished. The proOmpA treated with dithiothreitol was labeled with N-[3H]-ethylmaleimide, whereas that treated with ferricyanide was not. The ferricyanide-treated proOmpA was translocated into membrane vesicles in the presence of delta mu H+. The mature OmpA thus translocated and processed was not labeled with N-[3H]ethylmaleimide. It is concluded that proOmpA possessing the Cys290-Cys302 disulfide bridge can be translocated without cleavage of the bridge, when delta mu H+ is imposed. The accumulation of the disulfide bridge-containing intermediate was ATP-dependent, whereas its conversion to the translocated mature form was not blocked in the presence of adenosine 5'-(beta, gamma-imino)triphosphate. It is concluded that the early and late stages of the translocation reaction require ATP and delta mu H+ differently

  9. Na(+)-I- symport activity is present in membrane vesicles from thyrotropin-deprived non-I(-)-transporting cultured thyroid cells.

    OpenAIRE

    Kaminsky, S. M.; Levy, O.; Salvador, C.; Dai, G.; Carrasco, N.

    1994-01-01

    The active accumulation of I- in the thyroid gland is mediated by the Na(+)-I- symporter and driven by the Na+ gradient generated by the Na+/K(+)-ATPase. Thyrotropin (TSH) stimulates thyroidal I- accumulation. Rat thyroid-derived FRTL-5 cells require TSH to accumulate I-. TSH withdrawal for over 7 days results in complete loss of Na(+)-I-symport activity in these cells [Weiss, S. J., Philp, N. J. and Grollman, E. F. (1984) Endocrinology 114, 1090-1098]. Surprisingly, membrane vesicles prepare...

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

    Science.gov (United States)

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

    2013-11-01

    Computed tomography (CT) is the primary non-invasive imaging technique used for most patients with suspected liver disease. In order to improve liver-specific imaging properties and prevent toxic effects in patients with compromised renal function, we investigated the encapsulation of iodine within ethosomal vesicles. As a first step in the development of novel contrast agents using ethosomes for CT imaging applications, iodine was entrapped within ethosomes and iodine-containing ethosomes of the desired size were obtained by extrusion using a polycarbonate membrane with a defined pore size. Ethosomes containing iodine showed a relatively high CT density, which decreased when they were extruded, due to the rupture and re-formation of the lipid bilayer of the ethosome. However, when a solution with a high iodine concentration was used as a dispersion media during the extrusion process, the decrease in CT density could be prevented. In addition, ethosomes containing iodine were taken up efficiently by macrophages, which are abundant in the liver, and these ethosomes exhibited no cellular toxicity. These results demonstrate that iodine could be entrapped within ethosomal vesicles, giving the ethosomes a relatively high CT density, and that the extrusion technique used in this study could conveniently and reproducibly produce ethosomal vesicles with a desired size. Therefore, ethosomes containing iodine, as prepared in this study, have potential as contrast agents with applications in CT imaging. PMID:23857905

  11. Morphometric image analysis of giant vesicles: a new tool for quantitative thermodynamics studies of phase-separation in lipid membranes

    DEFF Research Database (Denmark)

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

    2012-01-01

    We have developed a strategy to determine lengths and orientations of tie lines in the coexistence region of liquid-ordered and liquid-disordered phases of cholesterol containing ternary lipid mixtures. The method combines confocal-fluorescence-microscopy image stacks of giant unilamellar vesicle...

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

    International Nuclear Information System (INIS)

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

  13. Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens.

    OpenAIRE

    MacIntosh, S C; Stone, T B; Jokerst, R S; Fuchs, R L

    1991-01-01

    A laboratory-selected colony of Heliothis virescens displaying a 20- to 70-fold level of resistance to Bacillus thuringiensis proteins was evaluated to identify mechanism(s) of resistance. Brush-border membrane vesicles were isolated from larval midgut epithelium from the susceptible and resistant strains of H. virescens. Two B. thuringiensis proteins, CryIA(b) and CryIA(c), were iodinated and shown to specifically bind to brush-border membrane vesicles of both insect strains. Multiple change...

  14. Structure of the animal vitreoretinal border region

    DEFF Research Database (Denmark)

    Heegaard, Steffen

    1994-01-01

    Øjenpatologi, vitreoretinal border region, inner limiting membrane of the retina, animals, ultrastructure, comparative anatomy......Øjenpatologi, vitreoretinal border region, inner limiting membrane of the retina, animals, ultrastructure, comparative anatomy...

  15. Rotating Brush Seal

    Science.gov (United States)

    Lattime, S. B.; Braun, M. J.; Choy. F. K.; Hendricks, R. C.; Steinetz, B. M.

    2006-01-01

    The proven technology of brush seals has been extended to the mitigation of problems arising from friction and wear at the bristle-rotor interface at high surface speeds. In prototype testing, the brush is mounted on, and free to rotate with the shaft, thus providing a complaint primary seal. A face seal positioned between the backing plate of the brush seal and the housing provides a secondary seal. The purpose of this paper is to demonstrate the interaction between the brush bristles and the shaft at high surface speeds as well as introduce a numerical model to simulate the bristle behavior. A test facility was constructed to study the effects of centrifugal forces on bristle deflection in a single rotating brush seal. The bristle-rotor interface was observed through a video camera, which utilized a high magnification borescope and a high frequency strobe light source. Rotational speeds of the rotor and the brush seal were measured by a magnetic and optical speed sensor, respectively. Preliminary results with speeds up to 11,000 rpm show no speed differential between the brush seal and rotor, or any instability problems associated with the brush seal. Bristle liftoff from the rotor is successfully captured on video.

  16. Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle.

    Science.gov (United States)

    Bertipaglia, Chiara; Schneider, Sarah; Jakobi, Arjen J; Tarafder, Abul K; Bykov, Yury S; Picco, Andrea; Kukulski, Wanda; Kosinski, Jan; Hagen, Wim Jh; Ravichandran, Arvind C; Wilmanns, Matthias; Kaksonen, Marko; Briggs, John Ag; Sachse, Carsten

    2016-07-01

    Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1. PMID:27266708

  17. Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from Biofilm and Planktonic Phase Associated with Extracellular DNA (eDNA).

    Science.gov (United States)

    Grande, Rossella; Di Marcantonio, Maria C; Robuffo, Iole; Pompilio, Arianna; Celia, Christian; Di Marzio, Luisa; Paolino, Donatella; Codagnone, Marilina; Muraro, Raffaella; Stoodley, Paul; Hall-Stoodley, Luanne; Mincione, Gabriella

    2015-01-01

    Helicobacter pylori persistence is associated with its capacity to develop biofilms as a response to changing environmental conditions and stress. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances (EPS) and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori ATCC 43629/NCTC 11639 biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of the OMVs. The presence of eDNA in bOMVs and pOMVs was initially carried out using DNase I-gold complex labeling and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterization carried out using dynamic light scattering (DLS) analysis. eDNA associated with OMVs was detected and quantified using a PicoGreen spectrophotometer assay, while its extraction was performed with a DNA Kit. TEM images showed that eDNA was mainly associated with the OMV membrane surfaces; while PicoGreen staining showed a four-fold increase of dsDNA in bOMVs compared with pOMVs. The eDNA extracted from OMVs was visualized using gel electrophoresis. DLS analysis indicated that both planktonic and biofilm H. pylori phenotypes generated vesicles, with a broad distribution of sizes on the nanometer scale. The DLS aggregation assay suggested that eDNA may play a role in the aggregation of OMVs, in the biofilm phenotype. Moreover, the eDNA associated with vesicle membrane may impede DNase I activity on H. pylori biofilms. These results suggest that OMVs derived from the H. pylori biofilm phenotype may play a structural role by preventing eDNA degradation by nucleases, providing a bridging function between eDNA strands on OMV surfaces and promoting aggregation. PMID:26733944

  18. Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles.

    OpenAIRE

    Rhoads, D B; Tai, P C; Davis, B D

    1984-01-01

    In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA). Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energ...

  19. The 4p16.3 Parkinson Disease Risk Locus Is Associated with GAK Expression and Genes Involved with the Synaptic Vesicle Membrane

    Science.gov (United States)

    Nagle, Michael W.; Latourelle, Jeanne C.; Labadorf, Adam; Dumitriu, Alexandra; Hadzi, Tiffany C.; Beach, Thomas G.; Myers, Richard H.

    2016-01-01

    Genome-wide association studies (GWAS) have identified the GAK/DGKQ/IDUA region on 4p16.3 among the top three risk loci for Parkinson’s disease (PD), but the specific gene and risk mechanism are unclear. Here, we report transcripts containing the 3’ clathrin-binding domain of GAK identified by RNA deep-sequencing in post-mortem human brain tissue as having increased expression in PD. Furthermore, carriers of 4p16.3 PD GWAS risk SNPs show decreased expression of one of these transcripts, GAK25 (Gencode Transcript 009), which correlates with the expression of genes functioning in the synaptic vesicle membrane. Together, these findings provide strong evidence for GAK clathrin-binding- and J-domain transcripts’ influence on PD pathogenicity, and for a role for GAK in regulating synaptic function in PD. PMID:27508417

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

    Directory of Open Access Journals (Sweden)

    Jung Hwa Lee

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

  1. Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area.

    Science.gov (United States)

    Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

    2016-04-01

    To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs. PMID:26825252

  2. Effects of salinity on activities of H+-ATPase, H+-PPase and membrane lipid composition in plasma membrane and tonoplast vesicles isolated from soybean (Glycine max L.) seedlings

    Institute of Scientific and Technical Information of China (English)

    YU Bing-jun; LAM Hon-ming; SHAO Gui-hua; LIU You-ling

    2005-01-01

    The effects of NaCl stress on the H+ -ATPase, H+ -PPase activity and lipid composition of plasma membrane(PM) and tonoplast(TP) vesicles isolated from roots and leaves of two soybean cultivars( Glycine max L. ) differing in salt tolerance(Wenfeng7,salt-tolerant; Union, salt-sensitive) were investigated. When Wenfeng7 was treated with 0.3% (W/V) NaCl for 3 d, the H+ -ATPase activities in PM and TP from roots and leaves exhibited a reduction and an enhancement, respectively. The H+ -PPase activity in TP from roots also increased. Similar effects were not observed in roots of Union. In addition, the increases of phospholipid content and ratios ofphospholipid to galactolipid in PM and TP from roots and leaves of Wenfeng7 may also change membrane permeability and hence affect salt tolerance.

  3. Fouling-resistant polymer brush coatings

    KAUST Repository

    Thérien-Aubin, Héloïse

    2011-11-01

    A major problem to be addressed with thin composite films used in processes such as coatings or water purification is the biofouling of the surface. To address this problem in a model system, functionalized polyaramide membranes containing an atom transfer radical polymerization (ATRP) initiator were synthesized as a versatile approach to easily modify the surface properties of the polyaramide. Poly(methacrylic acid) brushes were grown using surface initiated ATRP followed by the functionalization of the poly(methacrylic acid) brushes with different side-chains chosen to reduce adhesion between the membrane and foulant. The relation between membrane fouling and the physicochemical properties of the surface was investigated in detail. © 2011 Elsevier Ltd. All rights reserved.

  4. Surface grafted polymer brushes: potential applications in dengue biosensors

    International Nuclear Information System (INIS)

    A polymer brush membrane-based ultrasensitive biosensor for dengue diagnosis was constructed using poly(hydroxyethyl methacrylate) (PHEMA) brushes immobilized onto low density polyethylene (LDPE) films. LDPE surface films were initially modified by Ar+ ion irradiation to activate the polymer surface. Subsequently, graft polymerization of 2-hydroxyethyl methacrylate onto the activated LDPE surface was carried out under aqueous conditions to create patterned polymer brushes of PHEMA. The grafted PHEMA brushes were characterized by Fourier transform-infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and contact angle analysis. The SEM observations showed that selective surface activation with Ar+ implantation and graft polymerization on the selectively activated surface had occurred. The PHEMA brushes were electrically characterized in the presence of concentrations of human immunoglobulin (IgG). The proposed amperometric biosensor was successfully used for determination of IgG in physiologic samples with excellent responses. (author)

  5. Surface grafted polymer brushes: potential applications in dengue biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Baratela, Fernando Jose Costa; Higa, Olga Zazuco, E-mail: ozahiga@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Faria, Henrique Antonio Mendonca de; Queiroz, Alvaro Antonio Alencar de, E-mail: alencar@unifei.edu.br [Universidade Federal de Itajuba (UNIFEI), Itajuba, MG (Brazil). Instituto de Fisica e Quimica

    2013-07-01

    A polymer brush membrane-based ultrasensitive biosensor for dengue diagnosis was constructed using poly(hydroxyethyl methacrylate) (PHEMA) brushes immobilized onto low density polyethylene (LDPE) films. LDPE surface films were initially modified by Ar{sup +} ion irradiation to activate the polymer surface. Subsequently, graft polymerization of 2-hydroxyethyl methacrylate onto the activated LDPE surface was carried out under aqueous conditions to create patterned polymer brushes of PHEMA. The grafted PHEMA brushes were characterized by Fourier transform-infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and contact angle analysis. The SEM observations showed that selective surface activation with Ar+ implantation and graft polymerization on the selectively activated surface had occurred. The PHEMA brushes were electrically characterized in the presence of concentrations of human immunoglobulin (IgG). The proposed amperometric biosensor was successfully used for determination of IgG in physiologic samples with excellent responses. (author)

  6. Formation of Oligovesicular Vesicles by Micromanipulation

    Directory of Open Access Journals (Sweden)

    Yukihisa Okumura

    2011-09-01

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

  7. The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

    Directory of Open Access Journals (Sweden)

    Ofir Bahar

    2014-01-01

    Full Text Available Pattern recognition receptors (PRRs play an important role in detecting invading pathogens and mounting a robust defense response to restrict infection. In rice, one of the best characterized PRRs is XA21, a leucine rich repeat receptor-like kinase that confers broad-spectrum resistance to multiple strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo. In 2009 we reported that an Xoo protein, called Ax21, is secreted by a type I-secretion system and that it serves to activate XA21-mediated immunity. This report has recently been retracted. Here we present data that corrects our previous model. We first show that Ax21 secretion does not depend on the predicted type I secretion system and that it is processed by the general secretion (Sec system. We further show that Ax21 is an outer membrane protein, secreted in association with outer membrane vesicles. Finally, we provide data showing that ax21 knockout strains do not overcome XA21-mediated immunity.

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

    Science.gov (United States)

    Kilpinen, Lotta; Impola, Ulla; Sankkila, Lotta; Ritamo, Ilja; Aatonen, Maria; Kilpinen, Sami; Tuimala, Jarno; Valmu, Leena; Levijoki, Jouko; Finckenberg, Piet; Siljander, Pia; Kankuri, Esko; Mervaala, Eero; Laitinen, Saara

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Lotta Kilpinen

    2013-12-01

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  11. Extracellular Membrane Vesicles as Vehicles for Brain Cell-to-Cell Interactions in Physiological as well as Pathological Conditions

    Directory of Open Access Journals (Sweden)

    Gabriella Schiera

    2015-01-01

    Full Text Available Extracellular vesicles are involved in a great variety of physiological events occurring in the nervous system, such as cross talk among neurons and glial cells in synapse development and function, integrated neuronal plasticity, neuronal-glial metabolic exchanges, and synthesis and dynamic renewal of myelin. Many of these EV-mediated processes depend on the exchange of proteins, mRNAs, and noncoding RNAs, including miRNAs, which occurs among glial and neuronal cells. In addition, production and exchange of EVs can be modified under pathological conditions, such as brain cancer and neurodegeneration. Like other cancer cells, brain tumours can use EVs to secrete factors, which allow escaping from immune surveillance, and to transfer molecules into the surrounding cells, thus transforming their phenotype. Moreover, EVs can function as a way to discard material dangerous to cancer cells, such as differentiation-inducing proteins, and even drugs. Intriguingly, EVs seem to be also involved in spreading through the brain of aggregated proteins, such as prions and aggregated tau protein. Finally, EVs can carry useful biomarkers for the early diagnosis of diseases. Herein we summarize possible roles of EVs in brain physiological functions and discuss their involvement in the horizontal spreading, from cell to cell, of both cancer and neurodegenerative pathologies.

  12. Lubrication by glycoprotein brushes.

    Science.gov (United States)

    Zappone, Bruno; Ruths, Marina; Greene, George W.; Israelachvili, Jacob

    2006-03-01

    Grafted polyelectrolyte brushes show excellent lubricating properties under water and have been proposed as a model to study boundary lubrication in biological system. Lubricin, a glycoprotein of the synovial fluid, is considered the major boundary lubricant of articular joints. Using the Surface Force Apparatus, we have measured normal and friction forces between model surfaces (negatively charged mica, positively charged poly-lysine and aminothiol, hydrophobic alkanethiol) bearing adsorbed layers of lubricin. Lubricin layers acts like a versatile anti-adhesive, adsorbing on all the surfaces considered and creating a repulsion similar to the force between end-grafted polymer brushes. Analogies with polymer brushes also appear from bridging experiment, where proteins molecules are end-adsorbed on two opposing surfaces at the same time. Lubricin `brushes' show good lubricating ability at low applied pressures (P<0.5MPa), especially on negatively charged surfaces like mica. At higher load, the adsorbed layers wears and fails lubricating the surfaces, while still protecting the underlying substrate from wearing. Lubricin might thus be a first example of biological polyelectrolytes providing `brush-like' lubrication and wear-protection.

  13. Resident CAPS on dense-core vesicles docks and primes vesicles for fusion.

    Science.gov (United States)

    Kabachinski, Greg; Kielar-Grevstad, D Michelle; Zhang, Xingmin; James, Declan J; Martin, Thomas F J

    2016-02-15

    The Ca(2+)-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro-scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2-dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly. PMID:26700319

  14. Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles

    OpenAIRE

    1992-01-01

    A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fuel...

  15. Deciphering Cell-to-Cell Communication in Acquisition of Cancer Traits: Extracellular Membrane Vesicles Are Regulators of Tissue Biomechanics.

    Science.gov (United States)

    Pokharel, Deep; Wijesinghe, Philip; Oenarto, Vici; Lu, Jamie F; Sampson, David D; Kennedy, Brendan F; Wallace, Vincent P; Bebawy, Mary

    2016-08-01

    Deciphering the role of cell-to-cell communication in acquisition of cancer traits such as metastasis is one of the key challenges of integrative biology and clinical oncology. In this context, extracellular vesicles (EVs) are important vectors in cell-to-cell communication and serve as conduits in the transfer of cellular constituents required for cell function and for the establishment of cellular phenotypes. In the case of malignancy, they have been shown to support the acquisition of common traits defined as constituting the hallmarks of cancer. Cellular biophysics has contributed to our understanding of some of these central traits with changes in tissue biomechanics reflective of cell state. Indeed, much is known about stiffness of the tissue scaffold in the context of cell invasion and migration. This article advances this knowledge frontier by showing for the first time that EVs are mediators of tissue biomechanical properties and, importantly, demonstrates a link between the acquisition of cancer multidrug resistance and increased tissue stiffness of the malignant mass. The methodology used in the study employed optical coherence elastography and atomic force microscopy on breast cancer cell monolayers and tumor spheroids. Specifically, we show here that the acquired changes in tissue stiffness can be attributed to the intracellular transfer of a protein complex comprising ezrin, radixin, moesin, CD44, and P-glycoprotein. This has important implications in facilitating mechano-transduced signaling cascades that regulate the acquisition of cancer traits, such as invasion and metastasis. Finally, this study also introduces novel targets and strategies for diagnostic and therapeutic innovation in oncology, with a view to prevention of metastatic spread and personalized medicine in cancer treatment. PMID:27501296

  16. Friction between Ring Polymer Brushes

    OpenAIRE

    2015-01-01

    Friction between ring-polymer brushes at melt densities sliding past each other are studied using extensive course-grained molecular dynamics simulations and scaling arguments, and the results are compared to the friction between linear-polymer brushes. We show that for a velocity range spanning over three decades, the frictional forces measured for ring-polymer brushes are half the corresponding friction in case of linear brushes. In the linear-force regime, the weak inter-digitation of two ...

  17. Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

    OpenAIRE

    Gratzl, Manfred; Krieger-Brauer, H.; Ekerdt, R

    1981-01-01

    A new procedure is described for the preparation of highly purified and stable secretory vesicles from adrenal medulla. Two forms of acetylcholinesterase, a membrane bound form as well as a soluble form, were found within these vesicles. The secretory vesicles, isolated by differential centrifugation, were further purified on a continuous isotonic Percoll™ gradient. In this way, secretory vesicles were separated from mitochondrial, microsomal and cell membrane contamination. The secretory ves...

  18. Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor.

    Science.gov (United States)

    Pérez, Claudia; Fernandez, Luisa E; Sun, Jianguang; Folch, Jorge Luis; Gill, Sarjeet S; Soberón, Mario; Bravo, Alejandra

    2005-12-20

    Bacillus thuringiensis subsp. israelensis produces crystal proteins, Cry (4Aa, 4Ba, 10Aa, and 11Aa) and Cyt (1Aa and 2Ba) proteins, toxic to mosquito vectors of human diseases. Cyt1Aa overcomes insect resistance to Cry11Aa and Cry4 toxins and synergizes the toxicity of these toxins. However, the molecular mechanism of synergism remains unsolved. Here, we provide evidence that Cyt1Aa functions as a receptor of Cry11Aa. Sequential-binding analysis of Cyt1Aa and Cry11Aa revealed that Cyt1Aa binding to Aedes aegypti brush border membrane vesicles enhanced the binding of biotinylated-Cry11Aa. The Cyt1Aa- and Cry11Aa-binding epitopes were mapped by means of the yeast two-hybrid system, peptide arrays, and heterologous competition assays with synthetic peptides. Two exposed regions in Cyt1Aa, loop beta6-alphaE and part of beta7, bind Cry11Aa. On the other side, Cry11Aa binds Cyt1Aa proteins by means of domain II-loop alpha8 and beta-4, which are also involved in midgut receptor interaction. Characterization of single-point mutations in Cry11Aa and Cyt1Aa revealed key Cry11Aa (S259 and E266) and Cyt1Aa (K198, E204 and K225) residues involved in the interaction of both proteins and in synergism. Additionally, a Cyt1Aa loop beta6-alphaE mutant (K198A) with enhanced synergism to Cry11Aa was isolated. Data provided here strongly indicates that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium because it produces a toxin and also its functional receptor, promoting toxin binding to the target membrane and causing toxicity. PMID:16339907

  19. Mechanistic insight into the TH1-biased immune response to recombinant subunit vaccines delivered by probiotic bacteria-derived outer membrane vesicles.

    Science.gov (United States)

    Rosenthal, Joseph A; Huang, Chung-Jr; Doody, Anne M; Leung, Tiffany; Mineta, Kaho; Feng, Danielle D; Wayne, Elizabeth C; Nishimura, Nozomi; Leifer, Cynthia; DeLisa, Matthew P; Mendez, Susana; Putnam, David

    2014-01-01

    Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV's ability to induce self-adjuvanted robust and protective humoral and T(H)1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered "pathogen-like particles" derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion. PMID:25426709

  20. Mechanistic insight into the TH1-biased immune response to recombinant subunit vaccines delivered by probiotic bacteria-derived outer membrane vesicles.

    Directory of Open Access Journals (Sweden)

    Joseph A Rosenthal

    Full Text Available Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs. Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV's ability to induce self-adjuvanted robust and protective humoral and T(H1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered "pathogen-like particles" derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion.

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

    LENUS (Irish Health Repository)

    Tudor, E L

    2010-05-19

    Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

  2. Helicobacter pylori Outer Membrane Vesicle Proteins Induce Human Eosinophil Degranulation via a β2 Integrin CD11/CD18- and ICAM-1-Dependent Mechanism

    Directory of Open Access Journals (Sweden)

    Su Hyuk Ko

    2015-01-01

    Full Text Available Eosinophil cationic protein (ECP, a cytotoxic protein contained in eosinophils granules, can contribute to various inflammatory responses. Although Helicobacter pylori infection increases infiltration of eosinophils, the mechanisms of eosinophil degranulation by H. pylori infection are largely unknown. The goal of this study was to investigate the role of H. pylori outer membrane vesicles (OMVs in modulating eosinophil degranulation. We found that eosinophils treated with H. pylori OMVs released significantly more ECP compared with untreated controls. In addition, eosinophils cocultured with OMV-preexposed primary gastric epithelial cells exhibited significantly increased ECP release. Similarly, eosinophils cocultured with culture supernatant (CM from primary gastric epithelial cells exposed to OMVs (OMV-CM released significantly higher amounts of ECP compared with eosinophils cocultured with CM from unexposed control cells. Furthermore, OMVs and OMV-CM both induced the upregulation of ICAM-1 on gastric epithelial cells and β2 integrin CD11b on eosinophils. In addition, both transduction of ICAM-1 shRNA into gastric epithelial cells and treatment with neutralizing mAbs to CD18 significantly decreased OMV-mediated or OMV-CM-mediated release of ECP. These results suggest that the eosinophil degranulation response to H. pylori OMVs occurs via a mechanism that is dependent on both β2 integrin CD11/CD18 and ICAM-1.

  3. Campylobacter jejuni outer membrane vesicle-associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E-cadherin and occludin.

    Science.gov (United States)

    Elmi, Abdi; Nasher, Fauzy; Jagatia, Heena; Gundogdu, Ozan; Bajaj-Elliott, Mona; Wren, Brendan; Dorrell, Nick

    2016-04-01

    Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram-negative bacteria. Campylobacter jejuni produces OMVs that trigger IL-8, IL-6, hBD-3 and TNF-α responses from T84 intestinal epithelial cells and are cytotoxic to Caco-2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E-cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co-incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E-cadherin and occludin. The addition of 11168H OMVs to the co-culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time-dependent and dose-dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells. PMID:26451973

  4. Loss of the transcriptional repressor PAG-3/Gfi-1 results in enhanced neurosecretion that is dependent on the dense-core vesicle membrane protein IDA-1/IA-2.

    Directory of Open Access Journals (Sweden)

    Tao Cai

    2009-04-01

    Full Text Available It is generally accepted that neuroendocrine cells regulate dense core vesicle (DCV biogenesis and cargo packaging in response to secretory demands, although the molecular mechanisms of this process are poorly understood. One factor that has previously been implicated in DCV regulation is IA-2, a catalytically inactive protein phosphatase present in DCV membranes. Our ability to directly visualize a functional, GFP-tagged version of an IA-2 homolog in live Caenorhabditis elegans animals has allowed us to capitalize on the genetics of the system to screen for mutations that disrupt DCV regulation. We found that loss of activity in the transcription factor PAG-3/Gfi-1, which functions as a repressor in many systems, results in a dramatic up-regulation of IDA-1/IA-2 and other DCV proteins. The up-regulation of DCV components was accompanied by an increase in presynaptic DCV numbers and resulted in phenotypes consistent with increased neuroendocrine secretion. Double mutant combinations revealed that these PAG-3 mutant phenotypes were dependent on wild type IDA-1 function. Our results support a model in which IDA-1/IA-2 is a critical element in DCV regulation and reveal a novel genetic link to PAG-3-mediated transcriptional regulation. To our knowledge, this is the first mutation identified that results in increased neurosecretion, a phenotype that has clinical implications for DCV-mediated secretory disorders.

  5. Porphyromonas gingivalis Outer Membrane Vesicles Induce Selective Tumor Necrosis Factor Tolerance in a Toll-Like Receptor 4- and mTOR-Dependent Manner.

    Science.gov (United States)

    Waller, Tobias; Kesper, Laura; Hirschfeld, Josefine; Dommisch, Henrik; Kölpin, Johanna; Oldenburg, Johannes; Uebele, Julia; Hoerauf, Achim; Deschner, James; Jepsen, Sören; Bekeredjian-Ding, Isabelle

    2016-04-01

    Porphyromonas gingivalisis an important member of the anaerobic oral flora. Its presence fosters growth of periodontal biofilm and development of periodontitis. In this study, we demonstrated that lipophilic outer membrane vesicles (OMV) shed fromP. gingivalispromote monocyte unresponsiveness to liveP. gingivalisbut retain reactivity to stimulation with bacterial DNA isolated fromP. gingivalisor AIM2 ligand poly(dA·dT). OMV-mediated tolerance ofP. gingivalisis characterized by selective abrogation of tumor necrosis factor (TNF). Neutralization of interleukin-10 (IL-10) during OMV challenge partially restores monocyte responsiveness toP. gingivalis; full reactivity toP. gingivaliscan be restored by inhibition of mTOR signaling, which we previously identified as the major signaling pathway promoting Toll-like receptor 2 and Toll-like receptor 4 (TLR2/4)-mediated tolerance in monocytes. However, despite previous reports emphasizing a central role of TLR2 in innate immune recognition ofP. gingivalis, our current findings highlight a selective role of TLR4 in the promotion of OMV-mediated TNF tolerance: only blockade of TLR4-and not of TLR2-restores responsiveness toP. gingivalis Of further note, OMV-mediated tolerance is preserved in the presence of cytochalasin B and chloroquine, indicating that triggering of surface TLR4 is sufficient for this effect. Taking the results together, we propose thatP. gingivalisOMV contribute to local immune evasion ofP. gingivalisby hampering the host response. PMID:26857578

  6. Endobiliary brush biopsy

    DEFF Research Database (Denmark)

    Adamsen, Sven; Olsen, Marianne; Jendresen, Marianne Bille;

    2006-01-01

    OBJECTIVE: Obtaining cytological specimens by wire-guided endobiliary brushing at the time of endoscopic retrograde cholangiopancreatography (ERCP) is a convenient way to reach a diagnosis. Sensitivity for malignant disease is generally around 50% and specificity around 100%. The present study wa...

  7. Smart photonic carbon brush

    Science.gov (United States)

    Morozov, Oleg G.; Kuznetsov, Artem A.; Morozov, Gennady A.; Nureev, Ilnur I.; Sakhabutdinov, Airat Z.; Faskhutdinov, Lenar M.; Artemev, Vadim I.

    2016-03-01

    Aspects of the paper relate to a wear monitoring system for smart photonic carbon brush. There are many applications in which regular inspection is not feasible because of a number of factors including, for example, time, labor, cost and disruptions due to down time. Thus, there is a need for a system that can monitor the wear of a component while the component is in operation or without having to remove the component from its operational position. We propose a new smart photonic method for characterization of carbon brush wear. It is based on the usage of advantages of the multiplicative response of FBG and LPFG sensors and its double-frequency probing. Additional measuring parameters are the wear rate, the brush temperature, the engine rotation speed, the hangs control, and rotor speed. Sensor is embedded in brush. Firstly the change of sensor length is used to measure wear value and its central wavelength shift for temperature ones. The results of modeling and experiments are presented.

  8. Phase Transition Induced Fission in Lipid Vesicles

    OpenAIRE

    Leirer, C.; Wunderlich, B.; Myles, V.M.; Schneider, M F

    2009-01-01

    Abstract In this work we demonstrate how the first order phase transition in giant unilamellar vesicles (GUVs) can function as a trigger for membrane fission. When driven through their gel-fluid phase transition GUVs exhibit budding or pearl formation. These buds remain connected to the mother vesicle presumably by a small neck. Cooling these vesicles from the fluid phase (T>Tm) through the phase transition into the gel state (T

  9. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors.

    Science.gov (United States)

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels H H; Borregaard, Niels

    2013-10-01

    Neutrophils are indispensable in the innate immune defense against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in-gel-digested with trypsin. The resulting peptides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes. PMID:23650620

  10. Friction between ring polymer brushes.

    Science.gov (United States)

    Erbaş, Aykut; Paturej, Jarosław

    2015-04-28

    Friction between ring polymer brush bilayers sliding past each other at melt densities is studied using extensive coarse-grained molecular dynamics simulations and scaling arguments, and the results are compared to the friction between bilayers of linear polymer brushes. We show that for a velocity range spanning over three decades, the frictional forces measured for ring polymer brushes are half of the corresponding friction in the case of linear brushes. In the linear-force regime, the weak inter-digitation between ring brush layers as compared to linear brushes leads also to a lower number of binary collisions between the monomers from opposing brushes. At high velocities, where the thickness of the inter-digitation between bilayers is on the order of monomer size regardless of brush topology, stretched segments of ring polymers adopt the double-stranded conformation. As a result, monomers of the double-stranded segments collide on average less with the monomers of the opposing ring brush even though a similar number of monomers occupies the inter-digitation layer for ring and linear brush bilayers. The numerical data obtained from our simulations are consistent with the proposed scaling analysis. Conformation-dependent friction reduction observed in ring brushes can have important consequences in non-equilibrium bulk systems. PMID:25747253

  11. Pericellular Brush and Mechanics of Guinea Pig Fibroblast Cells Studied with AFM.

    Science.gov (United States)

    Dokukin, Maxim; Ablaeva, Yulija; Kalaparthi, Vivekanand; Seluanov, Andrei; Gorbunova, Vera; Sokolov, Igor

    2016-07-12

    The atomic force microscopy (AFM) indentation method combined with the brush model can be used to separate the mechanical response of the cell body from deformation of the pericellular layer surrounding biological cells. Although self-consistency of the brush model to derive the elastic modulus of the cell body has been demonstrated, the model ability to characterize the pericellular layer has not been explicitly verified. Here we demonstrate it by using enzymatic removal of hyaluronic content of the pericellular brush for guinea pig fibroblast cells. The effect of this removal is clearly seen in the AFM force-separation curves associated with the pericellular brush layer. We further extend the brush model for brushes larger than the height of the AFM probe, which seems to be the case for fibroblast cells. In addition, we demonstrate that an extension of the brush model (i.e., double-brush model) is capable of detecting the hierarchical structure of the pericellular brush, which, for example, may consist of the pericellular coat and the membrane corrugation (microridges and microvilli). It allows us to quantitatively segregate the large soft polysaccharide pericellular coat from a relatively rigid and dense membrane corrugation layer. This was verified by comparison of the parameters of the membrane corrugation layer derived from the force curves collected on untreated cells (when this corrugation membrane part is hidden inside the pericellular brush layer) and on treated cells after the enzymatic removal of the pericellular coat part (when the corrugations are exposed to the AFM probe). We conclude that the brush model is capable of not only measuring the mechanics of the cell body but also the parameters of the pericellular brush layer, including quantitative characterization of the pericellular layer structure. PMID:27410750

  12. Border markets

    DEFF Research Database (Denmark)

    Walther, Olivier

    2014-01-01

    their unequal economic development: the presence of a trade community, the combination of trading and productive activities, and the relative porosity of borders. In a second part, I examine how border markets on the U.S.-Mexico border must simultaneously guarantee the security of the state while......The objective of this issue of Articulo – Journal of Urban Research is to examine the characteristics of border markets in a comparative perspective. In this introductory paper, I first discuss what makes African border markets different from other markets, and examine several factors that explain...... favoring regional trade. The last part of the paper argues that more policy attention should be paid to border markets which, despite being at the margin of states, are a vital component of their economy. Fifty years after most West African states became independent and just as NAFTA turns 20, it is high...

  13. Thermodynamics and dynamics of the formation of spherical lipidic vesicles

    CERN Document Server

    Zapata, E Hernandez; Santamaría-Holek, I

    2009-01-01

    We propose a free energy expression accounting for the formation of spherical vesicles from planar lipidic membranes and derive a Fokker-Planck equation for the probability distribution describing the dynamics of vesicle formation. We found that formation may occur as an activated process for small membranes and as a transport process for sufficiently large membranes. We give explicit expressions for the transition rates and the characteristic time of vesicle formation in terms of the relevant physical parameters.

  14. Interaction of insulin with SDS/CTAB catanionic Vesicles

    International Nuclear Information System (INIS)

    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

  15. 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...

  16. Activation of Immune and Defense Responses in the Intestinal Mucosa by Outer Membrane Vesicles of Commensal and Probiotic Escherichia coli Strains.

    Science.gov (United States)

    José Fábrega, María; Aguilera, Laura; Giménez, Rosa; Varela, Encarna; Alexandra Cañas, María; Antolín, María; Badía, Josefa; Baldomà, Laura

    2016-01-01

    The influence of microbiota in human health is well-known. Imbalances in microbiome structure have been linked to several diseases. Modulation of microbiota composition through probiotic therapy is an attempt to harness the beneficial effects of commensal microbiota. Although, there is wide knowledge of the responses induced by gut microbiota, the microbial factors that mediate these effects are not well-known. Gram-negative bacteria release outer membrane vesicles (OMVs) as a secretion mechanism of microbial factors, which have an important role in intercellular communication. Here, we investigated whether OMVs from the probiotic Escherichia coli strain Nissle 1917 (EcN) or the commensal E. coli strain ECOR12 trigger immune responses in various cellular models: (i) peripheral blood mononuclear cells (PBMCs) as a model of intestinal barrier disruption, (ii) apical stimulation of Caco-2/PMBCs co-culture as a model of intact intestinal mucosa, and (iii) colonic mucosa explants as an ex vivo model. Stimulations with bacterial lysates were also performed. Whereas, both OMVs and lysates activated expression and secretion of several cytokines and chemokines in PBMCs, only OMVs induced basolateral secretion and mRNA upregulation of these mediators in the co-culture model. We provide evidence that OMVs are internalized in polarized Caco-2 cells. The activated epithelial cells elicit a response in the underlying immunocompetent cells. The OMVs effects were corroborated in the ex vivo model. This experimental study shows that OMVs are an effective strategy used by beneficial gut bacteria to communicate with and modulate host responses, activating signaling events through the intestinal epithelial barrier. PMID:27242727

  17. Activation of Immune and Defense Responses in the Intestinal Mucosa by Outer Membrane Vesicles of Commensal and Probiotic Escherichia coli Strains

    Science.gov (United States)

    José Fábrega, María; Aguilera, Laura; Giménez, Rosa; Varela, Encarna; Alexandra Cañas, María; Antolín, María; Badía, Josefa

    2016-01-01

    The influence of microbiota in human health is well-known. Imbalances in microbiome structure have been linked to several diseases. Modulation of microbiota composition through probiotic therapy is an attempt to harness the beneficial effects of commensal microbiota. Although, there is wide knowledge of the responses induced by gut microbiota, the microbial factors that mediate these effects are not well-known. Gram-negative bacteria release outer membrane vesicles (OMVs) as a secretion mechanism of microbial factors, which have an important role in intercellular communication. Here, we investigated whether OMVs from the probiotic Escherichia coli strain Nissle 1917 (EcN) or the commensal E. coli strain ECOR12 trigger immune responses in various cellular models: (i) peripheral blood mononuclear cells (PBMCs) as a model of intestinal barrier disruption, (ii) apical stimulation of Caco-2/PMBCs co-culture as a model of intact intestinal mucosa, and (iii) colonic mucosa explants as an ex vivo model. Stimulations with bacterial lysates were also performed. Whereas, both OMVs and lysates activated expression and secretion of several cytokines and chemokines in PBMCs, only OMVs induced basolateral secretion and mRNA upregulation of these mediators in the co-culture model. We provide evidence that OMVs are internalized in polarized Caco-2 cells. The activated epithelial cells elicit a response in the underlying immunocompetent cells. The OMVs effects were corroborated in the ex vivo model. This experimental study shows that OMVs are an effective strategy used by beneficial gut bacteria to communicate with and modulate host responses, activating signaling events through the intestinal epithelial barrier. PMID:27242727

  18. Water Soluble Responsive Polymer Brushes

    Directory of Open Access Journals (Sweden)

    Andrew J. Parnell

    2011-12-01

    Full Text Available Responsive polymer brushes possess many interesting properties that enable them to control a range of important interfacial behaviours, including adhesion, wettability, surface adsorption, friction, flow and motility. The ability to design a macromolecular response to a wide variety of external stimuli makes polymer brushes an exciting class of functional materials, and has been made possible by advances in modern controlled polymerization techniques. In this review we discuss the physics of polymer brush response along with a summary of the techniques used in their synthesis. We then review the various stimuli that can be used to switch brush conformation; temperature, solvent quality, pH and ionic strength as well as the relatively new area of electric field actuation We discuss examples of devices that utilise brush conformational change, before highlighting other potential applications of responsive brushes in real world devices.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    This paper describes a method to create giant protein vesicles (GPVs) of ≥10 μm by solvent‐driven fusion of large vesicles (0.1–0.2 μm) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein‐reconstituted large unilamellar vesicles (LUVs...

  20. Trafficking of astrocytic vesicles in hippocampal slices

    International Nuclear Information System (INIS)

    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.

  1. 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.

  2. Phase Transition Induced Fission in Lipid Vesicles

    CERN Document Server

    Leirer, C; Myles, V M; Schneider, M F

    2010-01-01

    In this work we demonstrate how the first order phase transition in giant unilamellar vesicles (GUVs) can function as a trigger for membrane fission. When driven through their gel-fluid phase transition GUVs exhibit budding or pearl formation. These buds remain connected to the mother vesicle presumably by a small neck. Cooling these vesicles from the fluid phase (T>Tm) through the phase transition into the gel state (Tvesicle remains intact. Pearling tubes which formed upon heating break-up and decay into multiple individual vesicles which then diffuse freely. Finally we demonstrate that mimicking the intracellular bulk viscosity by increasing the bulk viscosity to 40cP does not affect the overall fission process, but leads to a significant decrease in size of the released vesicles.

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

    Directory of Open Access Journals (Sweden)

    FredericAMeunier

    2013-10-01

    Here we discuss the functions of the cortical actin network, myosins and their effectors in controlling the processes that lead to tethering, directed transport, docking, and fusion of exocytotic vesicles in regulated exocytosis.

  4. Bottle-Brush Brushes: Cylindrical Molecular Brushes of Poly(2-oxazoline) on Glassy Carbon

    KAUST Repository

    Zhang, Ning

    2009-07-28

    We report on the synthesis of brushes of bottle-brushes of poly(2-oxazoline)s on polished glassy carbon (GC) substrates. First, homogeneous and stable poly(2-isopropenyl-2-oxazoline) (PIPOx) brush layers with thicknesses up to 160 nm were created directly onto GC by the self-initiated photografting and photopolymerization (SIPGP) of 2-isopropenyl-2-oxazoline (IPOx). Kinetic studies reveal a linear increase in thickness with the polymerization time. In a second reaction, the pendant 2-oxazoline ring of the PIPOx brushes were used for the living cationic ring-opening polymerization (LCROP) with different substituted 2-oxazoline monomers to form the side chains. Also for the second surface-initiatedLCROPfrom the surface-bound macroinitiator brushes, the thickness increase with the polymerization time was found to be linear and reproducible. Characterization of the resulting bottle-brush brushes by FTIR spectroscopy,contact angle, and AFM indicates a high side chain grafting density and quantitative reactions. Finally, we have demonstrated the possibility of functionalizing the bottle-brush brushes side chain end groups with sterically demanding molecules. © 2009 American Chemical Society.

  5. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

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

  6. Continuous microfluidic fabrication of synthetic asymmetric vesicles.

    Science.gov (United States)

    Lu, Li; Schertzer, Jeffrey W; Chiarot, Paul R

    2015-09-01

    We report on a novel microfluidic strategy for the continuous fabrication of monodisperse asymmetric vesicles with customized membrane composition, size, and luminal content. The microfluidic device encompasses a triangular post region and two flow-focusing regions. The major steps involved in the vesicle fabrication process include: (1) forming highly uniform water emulsions in an oil/inner-leaflet-lipid solution, (2) replacing the inner-leaflet-lipid solution with an outer-leaflet-lipid solution inside the microchannel network, (3) forming water-in-oil-in-water double emulsions, and (4) extracting excess oil/outer-leaflet-lipid solution from the double emulsions. Bilayer membrane asymmetry and unilamellarity are evaluated using a fluorescence quenching assay and a transmembrane protein insertion assay, respectively. Our approach addresses many of the deficiencies found in existing technologies for building vesicles, and yields strong membrane asymmetry. The ability to create and sustain membrane asymmetry is an important feature, as it is a characteristic of nearly all natural membranes. Over 80% of the vesicles remain stable for at least 6 weeks and the membrane asymmetry is maintained for over 30 hours. The asymmetric vesicles built using this strategy are collected off-chip and hold the potential to be used as model systems in membrane biology or as vehicles for drug delivery. PMID:26220822

  7. Vesicle Size Regulates Nanotube Formation in the Cell

    OpenAIRE

    Qian Peter Su; Wanqing Du; Qinghua Ji; Boxin Xue; Dong Jiang; Yueyao Zhu; Jizhong Lou; Li Yu; Yujie Sun

    2016-01-01

    Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro st...

  8. Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

    Science.gov (United States)

    Solberg, L B; Stang, E; Brorson, S-H; Andersson, G; Reinholt, F P

    2015-02-01

    Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis. PMID:25201349

  9. The puzzle of chloroplast vesicle transport – involvement of GTPases

    OpenAIRE

    Karim, Sazzad; Aronsson, Henrik

    2014-01-01

    In the cytosol of plant cells vesicle transport occurs via secretory pathways among the endoplasmic reticulum network, Golgi bodies, secretory granules, endosome, and plasma membrane. Three systems transfer lipids, proteins and other important molecules through aqueous spaces to membrane-enclosed compartments, via vesicles that bud from donor membranes, being coated and uncoated before tethered and fused with acceptor membranes. In addition, molecular, biochemical and ultrastructural evidence...

  10. A transient solution for vesicle electrodeformation and relaxation

    CERN Document Server

    Zhang, Jia; Tan, Wenchang; Lin, Hao

    2012-01-01

    A transient analysis for vesicle deformation under DC electric fields is developed. The theory extends from a droplet model, with the additional consideration of a lipid membrane separating two fluids of arbitrary properties. For the latter, both a membrane-charging and a membrane-mechanical model are supplied. The vesicle is assumed to remain spheroidal in shape for all times. The main result is an ODE governing the evolution of the vesicle aspect ratio. The effects of initial membrane tension and pulse length are examined. The model prediction is extensively compared with experimental data, and is shown to accurately capture the system behavior in the regime of no or weak electroporation. More importantly, the comparison reveals that vesicle relaxation obeys a universal behavior regardless of the means of deformation. The process is governed by a single timescale that is a function of the vesicle initial radius, the fluid viscosity, and the initial membrane tension. This universal scaling law can be used to...

  11. 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.

  12. 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. PMID:27053351

  13. Forces between asymmetric polymer brushes

    OpenAIRE

    Shim, D.F.K.; Cates, M. E.

    1990-01-01

    We study the equilibrium compression of asymmetric polymer brushes grafted on flat plates, under athermal and theta solvent conditions, using a lattice self-consistent field (SCF) approach. We find that the separation d between two plates coated asymmetrically with brushes of type 1 and 2, as a function of the force F, obeys the "bisection rule", d(F) = (d1(F) + d 2(F)) /2 where d1(F)and d 2(F) are the corresponding separations for the symmetric brushes of type 1 and 2 respectively.The bisect...

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

    International Nuclear Information System (INIS)

    The effect of osmotic pressure on the structure of large uni-lamellar vesicle (LUV) of the lipid mixtures of monosialoganglioside (GM1)-cholesterol-dioleoyl-phosphatidylcholine (DOPC) was studies by using wide-angle X-ray scattering (WAXS) method. The molar ratios of the mixtures were 0.1/0.1/1, 0/0.1/1, and 0/0/1. The ternary lipid mixture is a model of lipid rafts. The value of osmotic pressure was varied from 0 to 4.16x105 N/m2 by adding the polyvinylpyrrolidone (PVP) in the range from 0 to 25 % w/v. In the case of the mixtures without GM1, the rise of the osmotic pressure just enhances the multi-lamellar stacking with deceasing the inter-lamellar spacing. On the other hand, the mixture containing GM1 shows the structural transition from a uni-lamellar vesicle to a double-layered vesicle (a liposome including a smaller one inside) by the rise of osmotic pressure. In this morphology transition the total surface area of the double-layered vesicle is mostly as same as that of the LUV at the initial state. The polar head region of GM1 is bulky and highly hydrophilic due to the oligosaccharide chain containing a sialic acid residue. Then, the present results suggest that the existence of GM1 in the outer-leaflet of the LUV is essentially important for such a double-layered vesicle formation. Alternatively, a phenomenon similar to an endo- and/or exocytosis in cells can be caused simply by a variation of osmotic pressure.

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

    Energy Technology Data Exchange (ETDEWEB)

    Onai, Teruaki; Hirai, Mitsuhiro, E-mail: mhirai@fs.aramaki.gunma-u.ac.j [Department of Physics, Gunma University, Maebashi 371-8510 (Japan)

    2010-10-01

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

  16. Measuring the Energetics of Membrane Protein Dimerization in Mammalian Membranes

    OpenAIRE

    Chen, Lirong; Novicky, Lawrence; Merzlyakov, Mikhail; Hristov, Tihomir; Hristova, Kalina

    2010-01-01

    Thus far, methods that give quantitative information about lateral interactions in membranes have been restricted peptides or simplified protein constructs studied in detergents, lipid vesicles or bacterial membranes. None of the available methods have been extended to complex or full length membrane proteins. Here we show how free energies of membrane protein dimerization can be measured in mammalian plasma membrane-derived vesicles. The measurements, performed in single vesicles, utilize th...

  17. Cell mediated calcification and matrix vesicles

    International Nuclear Information System (INIS)

    This publication on calcification and the sequence of events directed by the cell to facilitate this process contains the following topics: New Ultrastructural Techniques for Study of Calcification; Mechanisms of Matrix Vesicle Calcification; Role of Mitochondria, Matrix Proteins and Cytokines in Calcification; Role of Phospholipids and Membranes in Calcification; Biogenesis of Matrix Vesicles in Vivo and in Vitro; Calcification and Ossification in Vitro; Calcific Diseases and Abnormal Bone Mineralization. (Auth.)

  18. Protein tyrosine phosphorylation in synaptic vesicles.

    OpenAIRE

    Pang, D T; Wang, J K; Valtorta, F; Benfenati, F; Greengard, P.

    1988-01-01

    Protein tyrosine phosphorylation in purified synaptic vesicles from rat forebrain has been studied in the presence of Mn2+ and orthovanadate. High levels of endogenous protein tyrosine phosphorylation were observed. Four major phosphoproteins, with apparent molecular masses of 105, 94, 38, and 30 kDa, were shown to contain phosphotyrosine. The 38-kDa phosphoprotein was identified as synaptophysin (p38), a well-characterized integral membrane protein of synaptic vesicles. The three other phosp...

  19. Compartmentalization and Transport in Synthetic Vesicles

    OpenAIRE

    Schmitt, Christine; Lippert, Anna H.; Bonakdar, Navid; Sandoghdar, Vahid; Voll, Lars M

    2016-01-01

    Nanoscale 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, such as permeability, stability, or chemical reactivity. In this review, we focus on the application of simple and nested artificial vesicles in...

  20. Water Soluble Responsive Polymer Brushes

    OpenAIRE

    Parnell, Andrew J.; Weir, Michael P

    2011-01-01

    Responsive polymer brushes possess many interesting properties that enable them to control a range of important interfacial behaviours, including adhesion, wettability, surface adsorption, friction, flow and motility. The ability to design a macromolecular response to a wide variety of external stimuli makes polymer brushes an exciting class of functional materials, and has been made possible by advances in modern controlled polymerization techniques. In this review we discuss the physics of ...

  1. Shedding of the Matrix Metalloproteinases MMP-2, MMP-9, and MT1-MMP as Membrane Vesicle-Associated Components by Endothelial Cells

    OpenAIRE

    Taraboletti, Giulia; D’Ascenzo, Sandra; Borsotti, Patrizia; Giavazzi, Raffaella; Pavan, Antonio; Dolo, Vincenza

    2002-01-01

    Production of matrix-degrading proteases, particularly matrix metalloproteinases (MMPs), by endothelial cells is a critical event during angiogenesis, the process of vessel neoformation that occurs in normal and pathological conditions. MMPs are known to be highly regulated at the level of synthesis and activation, however, little is known about the regulation of MMP secretion by endothelial cells. We found that cultured human umbilical vein endothelial cells shed vesicles (300 to 600 nm) ori...

  2. Lectin binding studies on a glycopolymer brush flow-through biosensor by localized surface plasmon resonance.

    Science.gov (United States)

    Rosencrantz, Ruben R; Nguyen, Vu Hoa; Park, Hyunji; Schulte, Christine; Böker, Alexander; Schnakenberg, Uwe; Elling, Lothar

    2016-08-01

    A localized surface plasmon resonance biosensor in a flow-through configuration was applied for investigating kinetics of lectin binding to surface-grafted glycopolymer brushes. Polycarbonate filter membranes with pore sizes of 400 nm were coated with a 114-nm thick gold layer and used as substrate for surface-initiated atom-transfer radical polymerization of a glycomonomer. These grafted from glycopolymer brushes were further modified with two subsequent enzymatic reactions on the surface to yield an immobilized trisaccharide presenting brush. Specific binding of lectins including Clostridium difficile toxin A receptor domain to the glycopolymer brush surface could be investigated in a microfluidic setup with flow-through of the analytes and transmission surface plasmon resonance spectroscopy. Graphical abstract Glycopolymer brushes serve as high affinity ligands for lectin and toxin interactions in a sensitive, disposable flow-through LSPR biosensor. PMID:27277814

  3. Polarized exocyst-mediated vesicle fusion directs intracellular lumenogenesis within the C. elegans excretory cell

    OpenAIRE

    Armenti, Stephen T.; Chan, Emily; Nance, Jeremy

    2014-01-01

    Lumenogenesis of small seamless tubes occurs through intracellular membrane growth and directed vesicle fusion events. Within the C. elegans excretory cell, which forms seamless intracellular tubes (canals) that mediate osmoregulation, lumens grow in length and diameter when vesicles fuse with the expanding lumenal surface. Here, we show that lumenal vesicle fusion depends on the small GTPase RAL-1, which localizes to vesicles and acts through the exocyst vesicle-tethering complex. Loss of ei...

  4. Border Battle.

    Science.gov (United States)

    Berlin, Joey

    2015-11-01

    The Texas Medical Association, the Texas Alliance for Patient Access, and other organizations ask for reversal of a court ruling that allows a Texas negligence case to proceed in New Mexico. The case is sounding alarms all over Texas' medical community, conjuring fears that if the Supreme Court upholds the application of New Mexico law, it could have a significant impact on Texas doctors who operate near the state's vast borders and treat out-of-state patients. PMID:26536518

  5. The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins.

    OpenAIRE

    C. Du; Nickerson, K W

    1996-01-01

    Brush border membrane vesicles from larvae of the tobacco hornworm, Manduca sexta, contain protein bands of 85 and 120 kDa which react directly with streptavidin conjugated to alkaline phosphatase. The binding could be prevented either by including 10 microM biotin in the reaction mixture or by prior incubation of the brush border membrane vesicles with an activated 60- to 65-kDa toxin from Bacillus thuringiensis HD-73. The ability of B. thuringiensis toxins to recognize biotin-containing pro...

  6. Three-dimensional visualization of coated vesicle formation in fibroblasts

    OpenAIRE

    1980-01-01

    Fibroblasts apparently ingest low density lipoproteins (LDL) by a selective mechanism of receptor-mediated endocytosis involving the formation of coated vesicles from the plasma membrane. However, it is not known exactly how coated vesicles collect LDL receptors and pinch off from the plasma membrane. In this report, the quick-freeze, deep- etch, rotary-replication method has been applied to fibroblasts; it displays with unusual clarity the coats that appear under the plasma membrane at the s...

  7. 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.

  8. Concentration-Dependent Staining of Lactotroph Vesicles by FM 4-64

    OpenAIRE

    Stenovec, Matjaž; Poberaj, Igor; Kreft, Marko; Zorec, Robert

    2005-01-01

    Hormones are released from neuroendocrine cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. An elegant way to study this process at the single-vesicle level is to use styryl dyes, which stain not only the membrane, but also the matrix of individual vesicles in some neuroendocrine cells. However, the mechanism by which the vesicle matrix is stained is not completely clear. One possibility is that molecules of the styryl dye in the bath solution di...

  9. POLYELEOSTEARIC ACID VESICLES

    Institute of Scientific and Technical Information of China (English)

    LI Zichen; XIE Ximng; FAN Qinghua; FANG Yifei

    1992-01-01

    α-Eleostearic acid and β-eleostearic acid formed vesicles in aqueous medium when an ethanol solutionofeleostearic acid was injected rapidly into a vigorously vortexed aqueous phase. Formation of the vesicles was demonstrated by electron microscopic observation and bromothymol blue encapsulation experiments. Polymerizations of the eleostearic acids in the formed vesicles carried out by UV irradiation produced poly-α-eleostearic acid and poly-β-eleostearic acid vesicles.

  10. Slow sedimentation and deformability of charged lipid vesicles.

    Directory of Open Access Journals (Sweden)

    Iván Rey Suárez

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

  11. ETHOSOMES AS ELASTIC VESICLES IN TRANSDERMAL DRUG DELIVERY: AN OVERVIEW

    OpenAIRE

    N. B. Gupta et al.

    2012-01-01

    Ethosomes are as novel vesicles in transdermal drug delivery show significant effects of drug penetration through the biological membrane with slight modification of well established drug carrier liposomes. Ethosomes are soft, malleable vesicles composed mainly of phospholipids, ethanol and water. The size of ethosome vesicles can be modulated from tens of nanometer to microns. The ethosomes can be prepared by Hot as well as Cold method. The evaluation parameters of ethosomes include visualiz...

  12. From Vesicles to Protocells: The Roles of Amphiphilic Molecules

    OpenAIRE

    Yuka Sakuma; Masayuki Imai

    2015-01-01

    It is very challenging to construct protocells from molecular assemblies. An important step in this challenge is the achievement of vesicle dynamics that are relevant to cellular functions, such as membrane trafficking and self-reproduction, using amphiphilic molecules. Soft matter physics will play an important role in the development of vesicles that have these functions. Here, we show that simple binary phospholipid vesicles have the potential to reproduce the relevant functions of adhes...

  13. Outer Membrane Vesicles from the Probiotic Escherichia coli Nissle 1917 and the Commensal ECOR12 Enter Intestinal Epithelial Cells via Clathrin-Dependent Endocytosis and Elicit Differential Effects on DNA Damage

    Science.gov (United States)

    Cañas, María-Alexandra; Giménez, Rosa; Fábrega, María-José; Toloza, Lorena; Badia, Josefa

    2016-01-01

    Interactions between intestinal microbiota and the human host are complex. The gut mucosal surface is covered by a mucin layer that prevents bacteria from accessing the epithelial cells. Thus, the crosstalk between microbiota and the host mainly rely on secreted factors that can go through the mucus layer and reach the epithelium. In this context, vesicles released by commensal strains are seen as key players in signaling processes in the intestinal mucosa. Studies with Gram-negative pathogens showed that outer membrane vesicles (OMVs) are internalized into the host cell by endocytosis, but the entry mechanism for microbiota-derived vesicles is unknown. Escherichia coli strains are found as part of normal human gut microbiota. In this work, we elucidate the pathway that mediate internalization of OMVs from the probiotic E.coli Nissle 1917 (EcN) and the commensal ECOR12 strains in several human intestinal epithelial cell lines. Time course measurement of fluorescence and microscopy analysis performed with rhodamine B-R18-labeled OMVs in the presence of endocytosis inhibitors showed that OMVs from these strains enter epithelial cells via clathrin-mediated endocytosis. Vesicles use the same endocytosis pathway in polarized epithelial monolayers. Internalized OMVs are sorted to lysosomal compartments as shown by their colocalization with clathrin and specific markers of endosomes and lysosomes. OMVs from both strains did not affect cell viability, but reduce proliferation of HT-29 cells. Labeling of 8-oxo-dG adducts in DNA revealed that neither OMVs from EcN nor from ECOR12 promoted oxidative DNA damage. In contrast, flow cytometry analysis of phosphorylated γH2AX evidenced that OMVs from the probiotic EcN significantly produced more double strand breaks in DNA than ECOR12 OMVs. The EcN genotoxic effects have been attributed to the synthesis of colibactin. However, it is not known how colibactin is exported and delivered into host cells. Whether colibactin is secreted

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

    Energy Technology Data Exchange (ETDEWEB)

    Mohd, Hur Munawar Kabir; Ahmad, Ainee Fatimah; Radiman, Shahidan; Mohamed, Faizal; Rosli, Nur Ratasha Alia Md; Ayob, Muhammad Taqiyuddin Mawardi; Rahman, Irman Abdul [School of Applied Physics, Faculty of Science and Technology, University Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)

    2014-09-03

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

  15. Dynamics of a compound vesicle: numerical simulations

    Science.gov (United States)

    Veerapaneni, Shravan; Young, Yuan-Nan; Vlahovska, Petia; Blawzdziewicz, Jerzy

    2010-11-01

    Vesicles (self-enclosing lipid membranes) in simple linear flows are known to exhibit rich dynamics such as tank-treading, tumbling, trembling (swinging), and vacillating breathing. Recently, vesicles have been used as a multi-functional platform for drug-delivery. In this work, the dynamics of simplified models for such compound vesicles is investigated numerically using a state-of-the-art boundary-integral code that has been validated with high accuracy and efficiency. Results show that for a vesicle enclosing a rigid particle in a simple shear flow, transition from tank-treading to tumbling is possible even in the absence of viscosity mismatch in the interior and exterior fluids. We will discuss the shape transformations, multiple particle interactions and the flow properties. Comparison with results from analytical modeling gives insights to the underlying physics for such novel dynamics.

  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. Charge transferred in brush discharges

    Science.gov (United States)

    Talarek, M.; Kacprzyk, R.

    2015-10-01

    Electrostatic discharges from surfaces of plastic materials can be a source of ignition, when appear in explosive atmospheres. Incendivity of electrostatic discharges can be estimated using the transferred charge test. In the case of brush discharges not all the energy stored at the tested sample is released and the effective surface charge density (or surface potential) crater is observed after the discharge. Simplified model, enabling calculation of a charge transferred during electrostatic brush discharge, was presented. Comparison of the results obtained from the simplified model and from direct measurements of transferred charge are presented in the paper.

  18. SGLT2 inhibitors act from the extracellular surface of the cell membrane

    OpenAIRE

    Ghezzi, Chiara; Hirayama, Bruce A.; Gorraitz, Edurne; Loo, Donald D. F.; Liang, Yin; Wright, Ernest M.

    2014-01-01

    Abstract SGLT2 inhibitors are a new class of drugs that have been recently developed to treat type II diabetes. They lower glucose levels by inhibiting the renal Na+/glucose cotransporter SGLT2, thereby increasing the amount of glucose excreted in the urine. Pharmacodynamics studies have raised questions about how these inhibitors reach SGLT2 in the brush border membrane of the S1 and S2 segments of the renal proximal tubule: are these drugs filtered by the glomerulus and act extracellularly,...

  19. Macroion Interaction at Polyelectrolyte Brush Interfaces

    Science.gov (United States)

    Qu, Chen

    2015-03-01

    The effect of macroions, including synthetic polyelectrolytes, DNA and proteins, on the structure and surface properties of charged polymer thin films remains inadequately understood partially due to the complexity involving the hydrophobic effect and the conformational change of polymeric macroions. In this work, we explore a group of inorganic nanocluster based macroions, hydrophilic polyoxometalates (POMs) of robust nanocluster structure and carrying high surface charges (~ 2-42 negative charges) to investigate their interaction with surface tethered poly-2-vinylpyridine (P2VP) brush-like thin films immersed in aqueous solution. We observe the collapse of swollen P2VP chains by adding POM macroions of increased concentration by AFM, QCM and contact goniometer measurements, in sharp contrast to the increased chain stretching by adding monovalent salts. A careful comparison is made between distinct POMs based on their charge, size and chemical nature. These findings serve as a good reference for theoretical model modification and design of new mesoporous composite membranes.

  20. Putative interaction of brush cells with bicarbonate secreting cells in the proximal corpus mucosa

    Directory of Open Access Journals (Sweden)

    Julia Anna-Maria Eberle

    2013-07-01

    Full Text Available The gastric epithelium is protected from the highly acidic luminal content by alkaline mucus which is secreted from specialized epithelial cells. In the stomach of mice strong secretion of alkaline fluid was observed at the gastric groove, the border between corpus and fundus mucosa. Since this region is characterized by numerous brush cells it was proposed that these cells might secrete alkaline solution as suggested for brush cells in the bile duct. In fact, it was found that in this region multiple cells express elements which are relevant for the secretion of bicarbonate, including carbonic anhydrase (CAII, the cystic fibrosis transmembrane conductance regulator (CFTR and the Na+/H+ exchanger (NHE1. However, this cell population was distinct from brush cells which express the TRP-channel TRPM5 and are considered as putative sensory cells. The location of both cell populations in close proximity implies the possibility for a paracrine interaction. This view was substantiated by the finding that brush cells express prostaglandin synthase-1 (COX-1 and the neighbouring cells a specific receptor type for prostaglandins. The notion that brush cells may be able to sense a local acidification was supported by the observation that they express the channel PKD1L3 which contributes to the acid responsiveness of gustatory sensory cells. The results support the concept that brush cells may sense the luminal content and influence via prostaglandins the secretion of alkaline solution.

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

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2008-01-01

    The dynamics of tension-induced fusion of two vesicles is studied using dissipative particle dynamics (DPD) simulations. The vesicle membranes use an improved DPD parameter set that results in their sustaining only a 10–30% relative area stretch before rupturing on the microsecond timescale...

  2. Lipids and NADPH-dependent superoxide production in plasma membrane vesicles from roots of wheat grown under copper deficiency or excess.

    Science.gov (United States)

    Quartacci, M F; Cosi, E; Navari-Izzo, F

    2001-01-01

    The effects of in vivo copper on the lipid composition of root plasma membrane and the activities of membrane-bound enzymes, such as NADPH-dependent oxidases and lipoxygenase, were studied. Plants were grown in hydroponic culture for 11 d without Cu supply or in the presence of 50 microM Cu. Control plants were supplied with 0.3 microM Cu. Growth of roots was severely affected in the 50 microM Cu-grown plants, whereas roots grown in Cu-deficient solution did not show any difference in comparison with the control. The 50 microM Cu concentration caused an increase in the leakage of K(+) ions as well. Excess metal supply resulted in a decrease in the total lipid content of plasma membrane, a higher phospholipid amount and a reduction of steryl lipids (free sterols, steryl glycosides and acylated steryl glycosides). Cu depletion in the growth solution had only a slight effect on the plasma membrane lipid composition. In comparison with the control, only the excess of Cu caused a decrease in the lipid to protein ratio as well as a change in the phospholipid composition, with a lower phosphatidylcholine to phosphatidylethanolamine ratio. The degree of unsaturation of root plasma membranes decreased following the 0 Cu treatment and even more after the 50 microM Cu supply. Plasma membranes of wheat grown under metal deficiency and excess showed increased NADPH-dependent superoxide-producing oxidase activities, whereas membrane-bound lipoxygenase was not increased or activated due to Cu treatments. The consequences of changes in plasma membrane lipid composition and activated oxygen production as a result of Cu treatments are discussed. PMID:11181715

  3. From vesicles to protocells: the roles of amphiphilic molecules.

    Science.gov (United States)

    Sakuma, Yuka; Imai, Masayuki

    2015-01-01

    It is very challenging to construct protocells from molecular assemblies. An important step in this challenge is the achievement of vesicle dynamics that are relevant to cellular functions, such as membrane trafficking and self-reproduction, using amphiphilic molecules. Soft matter physics will play an important role in the development of vesicles that have these functions. Here, we show that simple binary phospholipid vesicles have the potential to reproduce the relevant functions of adhesion, pore formation and self-reproduction of vesicles, by coupling the lipid geometries (spontaneous curvatures) and the phase separation. This achievement will elucidate the pathway from molecular assembly to cellular life. PMID:25738256

  4. From Vesicles to Protocells: The Roles of Amphiphilic Molecules

    Directory of Open Access Journals (Sweden)

    Yuka Sakuma

    2015-03-01

    Full Text Available It is very challenging to construct protocells from molecular assemblies. An important step in this challenge is the achievement of vesicle dynamics that are relevant to cellular functions, such as membrane trafficking and self-reproduction, using amphiphilic molecules. Soft matter physics will play an important role in the development of vesicles that have these functions. Here, we show that simple binary phospholipid vesicles have the potential to reproduce the relevant functions of adhesion, pore formation and self-reproduction of vesicles, by coupling the lipid geometries (spontaneous curvatures and the phase separation. This achievement will elucidate the pathway from molecular assembly to cellular life.

  5. Compartmentalization and Transport in Synthetic Vesicles.

    Science.gov (United States)

    Schmitt, Christine; Lippert, Anna H; Bonakdar, Navid; Sandoghdar, Vahid; Voll, Lars M

    2016-01-01

    Nanoscale 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, such as 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 multicompartmented vesosomes as compartmentalized nanoscale bioreactors. In the bottom-up development of protocells from vesicular nanoreactors, 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. PMID:26973834

  6. 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.

  7. Effect of the achondroplasia mutation on FGFR3 dimerization and FGFR3 structural response to fgf1 and fgf2: A quantitative FRET study in osmotically derived plasma membrane vesicles.

    Science.gov (United States)

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-07-01

    The G380R mutation in the transmembrane domain of FGFR3 is a germline mutation responsible for most cases of Achondroplasia, a common form of human dwarfism. Here we use quantitative Fӧster Resonance Energy Transfer (FRET) and osmotically derived plasma membrane vesicles to study the effect of the achondroplasia mutation on the early stages of FGFR3 signaling in response to the ligands fgf1 and fgf2. Using a methodology that allows us to capture structural changes on the cytoplasmic side of the membrane in response to ligand binding to the extracellular domain of FGFR3, we observe no measurable effects of the G380R mutation on FGFR3 ligand-bound dimer configurations. Instead, the most notable effect of the achondroplasia mutation is increased propensity for FGFR3 dimerization in the absence of ligand. This work reveals new information about the molecular events that underlie the achondroplasia phenotype, and highlights differences in FGFR3 activation due to different single amino-acid pathogenic mutations. PMID:27040652

  8. Exosomes : Nano-vesicles in immune regulation

    OpenAIRE

    Johansson, Sara M

    2008-01-01

    Nano-vesicles (30-100 nm) with an endosome-derived limiting membrane are called exosomes. These are released from the cell when the endosome fuses with the outer cell membrane. Exosomes from antigen presenting cells (APC) carry MHC class I and class II as well as integrins, tetraspanins and co-stimulatory molecules. They can either stimulate T cell responses or induce tolerance. Exosomes are presently being evaluated as therapeutic tools but still little is known about their...

  9. Lipid trafficking sans Vesicles: where, why, how?

    OpenAIRE

    Prinz, William A.

    2010-01-01

    Eukaryotic cells possess a remarkable diversity of lipids, which distribute among cellular membranes by well-characterized vesicle trafficking pathways. However, transport of lipids by alternate or “nonvesicular” routes is also critical for lipid synthesis, metabolism, and proper membrane partitioning. In the past few years, considerable progress has been made in characterizing the mechanisms of nonvesicular lipid transport and how it may go awry in particular diseases, but many fundamental q...

  10. Loop polymer brushes from polymer single crystals

    Science.gov (United States)

    Zhou, Tian; Li, Christopher

    2014-03-01

    Loop polymer brushes represent a category of polymer brushes with both chain ends being tethered to a surface or interface with sufficiently high density. Due to this morphological difference, loop brushes exhibit distinct properties compared with traditional polymer brushes with single chain end being tethered. In our study, α, ω-functionalized polycaprolactone (PCL) single crystals were prepared as templates for polymer brush synthesis. By carefully controlling crystallization condition and immobilization, looped polymer brushes were successfully prepared. Comprehensive studies on the morphology and physical properties of these polymer brushes were carried out using Atomic Force Microscopy and FTIR. Advantages of using this method include exclusive loop morphology, high grafting density, controlled tethering sites and tunable loop size.

  11. 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.

  12. 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.

  13. Brush seals and common issues in brush seal applications

    OpenAIRE

    Akşit, Mahmut Faruk; Aksit, Mahmut Faruk

    2012-01-01

    Brush seals are receiving wider acceptance with growing number of successful turbomachinery sealing applications over the past couple of decades. Due to their complaint nature these seals can recover from large transient rotor interference occurrences without any appreciable sustained damage or permanent performance loss. They are formed by a multitude of flexible fine bristles tightly clamped between two metal plates. When subjected to operating pressures and rotor interference, the friction...

  14. Osmotic and curvature stress affect PEG-induced fusion of lipid vesicles but not mixing of their lipids.

    OpenAIRE

    Malinin, Vladimir S.; Frederik, Peter; Lentz, Barry R

    2002-01-01

    Poly (ethylene glycol) (PEG) in the external environment of membrane vesicles creates osmotic imbalance that leads to mechanical stress in membranes and may induce local membrane curvature. To determine the relative importance of membrane stress and curvature in promoting fusion, we monitored contents mixing (CM) and lipid mixing (LM) between different sized vesicles under a variety of osmotic conditions. CM between highly curved vesicles (SUV, 26 nm diameter) was up to 10 times greater than ...

  15. Extracellular Vesicles as Novel Delivery Tools for Cancer Treatment.

    Science.gov (United States)

    Erkan, Erdogan Pekcan; Saydam, Okay

    2016-01-01

    Extracellular vesicles (EVs) are different types of membrane-derived vesicles that originate from the endosomal pathway or the plasma membrane. These vesicles are used as "carriers" in intercellular communication, and are responsible for the transfer of biological cargo (lipids, proteins, RNA species, and DNA) between different cells. Despite the shortcomings in our knowledge of EV biology, attempts to employ EVs as natural delivery tools for therapeutic purposes have been partly successful in different settings. In this review, we highlight this unique potential of EVs, and discuss previous examples and future scenarios. PMID:26412464

  16. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  17. Extracellular Vesicle (EV) Array

    DEFF Research Database (Denmark)

    Jørgensen, Malene; Bæk, Rikke; Pedersen, Shona;

    2013-01-01

    Exosomes are one of the several types of cell-derived vesicles with a diameter of 30-100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define...

  18. Fusion of Nonionic Vesicles

    DEFF Research Database (Denmark)

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

    2010-01-01

    We present an experimental study of vesicle fusion using light and neutron scattering to monitor fusion events. Vesicles are reproducibly formed with an extrusion procedure using an single amphiphile triethylene glycol mono-n-decyl ether in water. They show long-term stability for temperatures...... around 20 C, but at temperatures above 26 C we observe an increase in the scattered intensity due to fusion. The system is unusually well suited for the study of basic mechanisms of vesicle fusion. The vesicles are flexible with a bending rigidity of only a few k(H)T. The monolayer spontaneous curvature......, Ho, depends strongly on temperature in a known way and is thus tunable. For temperatures where H-0 > 0 vesicles tyre long-term stable, while in the range H-0 fusion rate increases the more negative the Spontaneous curvature Through a quantitative;analysis of the fusion rate we arrive tit...

  19. Identification of minor components of coated vesicles by use of permeation chromatography

    OpenAIRE

    1981-01-01

    Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurification we have made use of the relatively uniform diameter of coated vesicles (50-150 nm) to fractionate conventionally purified coated vesicles according to size in glass bead columns of 200-nm po...

  20. Nanotribology of charged polymer brushes

    Science.gov (United States)

    Klein, Jacob

    Polymers at surfaces, whose modern understanding may be traced back to early work by Sam Edwards1, have become a paradigm for modification of surface properties, both as steric stabilizers and as remarkable boundary lubricants2. Charged polymer brushes are of particular interest, with both technological implications and especially biological relevance where most macromolecules are charged. In the context of biolubrication, relevant in areas from dry eye syndrome to osteoarthritis, charged polymer surface phases and their complexes with other macromolecules may play a central role. The hydration lubrication paradigm, where tenaciously-held yet fluid hydration shells surrounding ions or zwitterions serve as highly-efficient friction-reducing elements, has been invoked to understand the excellent lubrication provided both by ionized3 and by zwitterionic4 brushes. In this talk we describe recent advances in our understanding of the nanotribology of such charged brush systems. We consider interactions between charged end-grafted polymers, and how one may disentangle the steric from the electrostatic surface forces5. We examine the limits of lubrication by ionized brushes, both synthetic and of biological origins, and how highly-hydrated zwitterionic chains may provide extremely effective boundary lubrication6. Finally we describe how the lubrication of articular cartilage in the major joints, a tribosystem presenting some of the greatest challenges and opportunities, may be understood in terms of a supramolecular synergy between charged surface-attached polymers and zwitterionic groups7. Work supported by European Research Council (HydrationLube), Israel Science Foundation (ISF), Petroleum Research Fund of the American Chemical Society, ISF-NSF China Joint Program.

  1. 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.

  2. Lipid-Targeting Peptide Probes for Extracellular Vesicles.

    Science.gov (United States)

    Flynn, Aaron D; Yin, Hang

    2016-11-01

    Extracellular vesicles released from cells are under intense investigation for their roles in cell-cell communication and cancer progression. However, individual vesicles have been difficult to probe as their small size renders them invisible by conventional light microscopy. However, as a consequence of their small size these vesicles possess highly curved lipid membranes that offer an unconventional target for curvature-sensing probes. In this article, we present a strategy for using peptide-based biosensors to detect highly curved membranes and the negatively charged membrane lipid phosphatidylserine, we delineate several assays used to validate curvature- and lipid-targeting mechanisms, and we explore potential applications in probing extracellular vesicles released from sources such as apoptotic cells, cancer cells, or activated platelets. J. Cell. Physiol. 231: 2327-2332, 2016. © 2016 Wiley Periodicals, Inc. PMID:26909741

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

    International Nuclear Information System (INIS)

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

  4. Comparing the efficiency of Denture brush and Ordinary brush in complete Denture cleaning

    OpenAIRE

    Amir Fayaz; Mohaddeseh Shakerian; Ghassem Ansari

    2013-01-01

    Please cite this article as: Fayaz A, Shakerian M, Ansari GH. Comparing the Efficiency of Denture brush and Ordinary brush in complete Denture cleaning. Novel Biomed 2013;1(2):62-65.Background & objective: Denture cleansing is a key element in retaining mucosa free of any inflammation. However, many denture users usually ignore this important factor. This investigation was designed to compare the efficacy of a denture brush and an ordinary brush in cleansing process of complete dentures.M...

  5. Nanopatterned polymer brushes: conformation, fabrication and applications

    Science.gov (United States)

    Yu, Qian; Ista, Linnea K.; Gu, Renpeng; Zauscher, Stefan; López, Gabriel P.

    2015-12-01

    Surfaces with end-grafted, nanopatterned polymer brushes that exhibit well-defined feature dimensions and controlled chemical and physical properties provide versatile platforms not only for investigation of nanoscale phenomena at biointerfaces, but also for the development of advanced devices relevant to biotechnology and electronics applications. In this review, we first give a brief introduction of scaling behavior of nanopatterned polymer brushes and then summarize recent progress in fabrication and application of nanopatterned polymer brushes. Specifically, we highlight applications of nanopatterned stimuli-responsive polymer brushes in the areas of biomedicine and biotechnology.

  6. Budding and Fission of a multiphase vesicle

    CERN Document Server

    Allain, J M; Allain, Jean-Marc; Amar, Martine Ben

    2005-01-01

    We present a model of bi-phasic vesicle in the limit of large surface tension. In this regime, the vesicle is completely stretched and well described by two spherical caps with a fold which concentrates the membrane stress. The conservation laws and geometric constraints restrict the space of possible shapes to a pair of solutions labeled by a parameter $\\tau$ given by {\\it line tension/pressure}. For a given $\\tau$ value, the two solutions differ by the length of the interface between domains. For a critical value $\\tau\\_c$, the two vesicle shapes become identical and no solution exists above this critical value. This model sheds new light on two proposed mechanisms (osmotic shocks and molecule absorption) to explain the budding and the fission in recent experiments.

  7. Comparison of Intranasal Outer Membrane Vesicles with Cholera Toxin and Injected MF59C.1 as Adjuvants for Malaria Transmission Blocking Antigens AnAPN1 and Pfs48/45

    Science.gov (United States)

    Pritsch, Michael; Ben-Khaled, Najib; Chaloupka, Michael; Kobold, Sebastian; Berens-Riha, Nicole; Peter, Annabell; Liegl, Gabriele; Schubert, Sören; Hoelscher, Michael; Löscher, Thomas; Wieser, Andreas

    2016-01-01

    Purified protein vaccines often require adjuvants for efficient stimulation of immune responses. There is no licensed mucosal adjuvant on the market to adequately boost the immune response to purified antigens for intranasal applications in humans. Bacterial outer membrane vesicles (OMV) are attractive candidates potentially combining antigenic and adjuvant properties in one substance. To more precisely characterize the potential of Escherichia coli OMV for intranasal vaccination with heterologous antigens, immune responses for AnAPN1 and Pfs48/45 as well as ovalbumin as a reference antigen were assessed in mice. The intranasal adjuvant cholera toxin (CT) and parenteral adjuvant MF59C.1 were used in comparison. Vaccinations were administered intranasally or subcutaneously. Antibodies (total IgG and IgM as well as subclasses IgG1, IgG2a, IgG2b, and IgG3) were measured by ELISA. T cell responses (cytotoxic T cells, Th1, Th17, and regulatory T cells) were determined by flow cytometry. When OMV were used as adjuvant for intranasal immunization, antibody and cellular responses against all three antigens could be induced, comparable to cholera toxin and MF59C.1. Antigen-specific IgG titres above 1 : 105 could be detected in all groups. This study provides the rationale for further development of OMV as a vaccination strategy in malaria and other diseases.

  8. MAA-1, a novel acyl-CoA-binding protein involved in endosomal vesicle transport in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Larsen, Morten K; Tuck, Simon; Færgeman, Nils J.;

    2006-01-01

    The budding and fission of vesicles during membrane trafficking requires many proteins, including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted by the hydro......The budding and fission of vesicles during membrane trafficking requires many proteins, including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted......-CoA in vitro and that this ligand-binding ability is important for its function in vivo. Our results are consistent with a role for MAA-1 in an acyl-CoA-dependent process during vesicle formation....

  9. Activation of calcineurin by phosphotidylserine containing vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Politino, M.; King, M.M.

    1986-05-01

    Calcineurin (CaN) is a Ca/sup 2 +/- and calmodulin-regulated phosphatase. Recent findings suggested an association of CaN with biological membranes and prompted the present investigation into the interactions of the phosphatase with phospholipids in vitro. In the absence of calmodulin, sonicated preparations of phosphatidylserine (PS) provided a five-fold activation of the Ni- and Mn-supported activities of CaN towards (/sup 32/P) histone Hl; activation in the presence of calmodulin was much less pronounced. Half-maximal activation in the absence of calmodulin required approximately 0.1 mg/ml of PS. Activation of CaN was also observed with mixed vesicles of phosphatidylcholine (PC) containing 20% PS but not with PC alone, or with phosphatidylethanolamine (PE). Molecular sieve chromatography on Ultrogel AcA 34 provided further evidence that CaN associates with phospholipid vesicles composed of PS, or PC containing 20% PS, but not with vesicles of PC or PE. Complete association with medium sized vesicles of PS and PC/PS required Ca/sup 2 +/ ions; in the absence of the metal ion at least 60% of the enzyme failed to interact with the lipids while the remainder preferentially migrated with larger vesicles. These results suggest a role for Ca/sup 2 +/ in regulating CaN's interaction with phospholipids.

  10. Bacillus thuringiensis Cry34Ab1/Cry35Ab1 interactions with western corn rootworm midgut membrane binding sites.

    Directory of Open Access Journals (Sweden)

    Huarong Li

    Full Text Available BACKGROUND: Bacillus thuringiensis (Bt Cry34Ab1/Cry35Ab1 are binary insecticidal proteins that are co-expressed in transgenic corn hybrids for control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Bt crystal (Cry proteins with limited potential for field-relevant cross-resistance are used in combination, along with non-transgenic corn refuges, as a strategy to delay development of resistant rootworm populations. Differences in insect midgut membrane binding site interactions are one line of evidence that Bt protein mechanisms of action differ and that the probability of receptor-mediated cross-resistance is low. METHODOLOGY/PRINCIPAL FINDINGS: Binding site interactions were investigated between Cry34Ab1/Cry35Ab1 and coleopteran active insecticidal proteins Cry3Aa, Cry6Aa, and Cry8Ba on western corn rootworm midgut brush border membrane vesicles (BBMV. Competitive binding of radio-labeled proteins to western corn rootworm BBMV was used as a measure of shared binding sites. Our work shows that (125I-Cry35Ab1 binds to rootworm BBMV, Cry34Ab1 enhances (125I-Cry35Ab1 specific binding, and that (125I-Cry35Ab1 with or without unlabeled Cry34Ab1 does not share binding sites with Cry3Aa, Cry6Aa, or Cry8Ba. Two primary lines of evidence presented here support the lack of shared binding sites between Cry34Ab1/Cry35Ab1 and the aforementioned proteins: 1 No competitive binding to rootworm BBMV was observed for competitor proteins when used in excess with (125I-Cry35Ab1 alone or combined with unlabeled Cry34Ab1, and 2 No competitive binding to rootworm BBMV was observed for unlabeled Cry34Ab1 and Cry35Ab1, or a combination of the two, when used in excess with (125I-Cry3Aa, or (125I-Cry8Ba. CONCLUSIONS/SIGNIFICANCE: Combining two or more insecticidal proteins active against the same target pest is one tactic to delay the onset of resistance to either protein. We conclude that Cry34Ab1/Cry35Ab1 are compatible with Cry3Aa, Cry6Aa, or Cry8Ba

  11. 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.

  12. The Border Multiple

    DEFF Research Database (Denmark)

    of European borders by looking at border practices in the light of the mobility turn, and thus as dynamic, multiple, diverse and best expressed in everyday experiences of people living at and with borders, rather than focusing on static territorial divisions between states and regions at geopolitical...... level. It provides border scholars and researchers as well as policymakers with new empirical and theoretical evidence on the de- and re-bordering processes going on in diverse border regions in Europe, both within and outside of the EU....

  13. Imaging Exocytosis of Single Synaptic Vesicles at a Fast CNS Presynaptic Terminal.

    Science.gov (United States)

    Midorikawa, Mitsuharu; Sakaba, Takeshi

    2015-11-01

    Synaptic vesicles are tethered to the active zone where they are docked/primed so that they can fuse rapidly upon Ca(2+) influx. To directly study these steps at a CNS presynaptic terminal, we used total internal reflection fluorescence (TIRF) microscopy at the live isolated calyx of Held terminal and measured the movements of single synaptic vesicle just beneath the plasma membrane. Only a subset of vesicles within the TIRF field underwent exocytosis. Following exocytosis, new vesicles (newcomers) approached the membrane and refilled the release sites slowly with a time constant of several seconds. Uniform elevation of the intracellular Ca(2+) using flash photolysis elicited an exocytotic burst followed by the sustained component, representing release of the readily releasable vesicles and vesicle replenishment, respectively. Surprisingly, newcomers were not released within a second of high Ca(2+). Instead, already-tethered vesicles became release-ready and mediated the replenishment. Our results reveal an important feature of conventional synapses. PMID:26539890

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

    OpenAIRE

    Lagow, Robert D; Hong Bao; Cohen, Evan N; Daniels, Richard W.; Aleksej Zuzek; Williams, Wade H; Gregory T Macleod; R. Bryan Sutton; Bing Zhang

    2007-01-01

    Author Summary Most living cells constantly renew their membrane compositions and frequently communicate with neighboring cells by delivering cargo molecules from small vesicles. A key step in cargo delivery requires the fusion of the vesicle membrane with the target membrane mediated by SNARE proteins. In most cellular compartments, fusion occurs constitutively, requiring little participation of other molecules. In other cellular compartments, such as synapses in the nervous system, vesicle ...

  15. Astrocytic vesicles and gliotransmitters: Slowness of vesicular release and synaptobrevin2-laden vesicle nanoarchitecture.

    Science.gov (United States)

    Zorec, R; Verkhratsky, A; Rodríguez, J J; Parpura, V

    2016-05-26

    Neurotransmitters released at synapses activate neighboring astrocytes, which in turn, modulate neuronal activity by the release of diverse neuroactive substances that include classical neurotransmitters such as glutamate, GABA or ATP. Neuroactive substances are released from astrocytes through several distinct molecular mechanisms, for example, by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release regulated by a stimulus-mediated increase in cytosolic calcium involves soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE)-dependent merger of the vesicle membrane with the plasmalemma. Up to 25 molecules of synaptobrevin 2 (Sb2), a SNARE complex protein, reside at a single astroglial vesicle; an individual neuronal, i.e. synaptic, vesicle contains ∼70 Sb2 molecules. It is proposed that this paucity of Sb2 molecules in astrocytic vesicles may determine the slow secretion. In the present essay we shall overview multiple aspects of vesicular architecture and types of vesicles based on their cargo and dynamics in astroglial cells. PMID:25727638

  16. Dual wound dc brush motor gearhead

    Science.gov (United States)

    Henson, Barrie W.

    1986-01-01

    The design requirements, the design, development tests and problems, the qualification and life test and the findings of the strip examination of a dual wound DC brushed motor gearhead are described. It is the only space qualified dual wound dc brushed motor gearhead in Europe.

  17. Interfacial properties of bottle-brush polyelectrolytes

    DEFF Research Database (Denmark)

    Claesson, P. M.; Naderi, A.; Iruthayaraj, J.; Pettersson, T.; Vareikis, A.; Makuska, R.; Iruthayaraj, Joseph

    This article is focused on interfacial properties of bottle brush polyelectrolytes, where side-chains are attached along a polymer backbone. This class of polymer has been much less studied than block copolymers, which is particularly true for bottle brush polyelectrolytes with a high graft density...

  18. Salt-induced contraction of polyelectrolyte brushes

    International Nuclear Information System (INIS)

    We present an experimental study dedicated to understanding the behaviour of polyelectrolyte chains when salt goes into a polyelectrolyte brush. We use the ability of asymmetric neutral-charged diblock copolymers to anchor to a selective interface or to self-assemble in water, to examine polyelectrolyte brushes both in planar and in spherical geometries. Using neutron reflectivity, the monomer profile of planar brushes has been found to switch from a Gaussian profile to a parabolic profile, showing that at large spatial scale a salted planar brush behaves like a neutral one in good solvent. Using small angle neutron scattering, it is found that spherical brushes exhibit the same behaviour at a large spatial scale although polyelectrolyte chains remain rod-like at a small spatial scale whatever the salinity is. The charged chains inside a polyelectrolyte brush could be viewed as 'surveyor's chains', which fold their rod-like segments of persistence length lp. In the last part, spherical polyelectrolyte brushes in contact have been examined. Their behaviour is discussed in terms of interdigitation or contraction of the brushes

  19. BSA adsorption on bimodal PEO brushes

    NARCIS (Netherlands)

    Bosker, W.T.E.; Iakovlev, P.A.; Norde, W.; Cohen Stuart, M.A.

    2005-01-01

    BSA adsorption onto bimodal PEO brushes at a solid surface was measured using optical reflectometry. Bimodal brushes consist of long (N=770) and short (N=48) PEO chains and were prepared on PS surfaces, applying mixtures of PS 29-PEO48 and PS37-PEO770 block copolymers and using the Langmuir-Blodgett

  20. BSA adsorption on bimodal PEO brushes

    NARCIS (Netherlands)

    Bosker, WTE; Iakovlev, PA; Norde, W; Stuart, MAC

    2005-01-01

    BSA adsorption onto bimodal PEO brushes at a solid surface was measured using optical reflectometry. Bimodal brushes consist of long (N = 770) and short (N = 48) PEO chains and were prepared on PS surfaces, applying mixtures of PS29-PEO48 and PS37-PEO770 block copolymers and using the Langmuir-Blodg

  1. Brush-Wheel Samplers for Planetary Exploration

    Science.gov (United States)

    Rivellini, Tommaso

    2003-01-01

    A report proposes brush-wheel mechanisms for acquiring samples of soils from remote planets. In simplest terms, such a mechanism would contain brush wheels that would be counter-rotated at relatively high speed. The mechanism would be lowered to the ground from a spacecraft or other exploratory vehicle. Upon contact with the ground, the counter-rotating brush wheels would kick soil up into a collection chamber. Thus, in form and function, the mechanism would partly resemble traditional street and carpet sweepers. The main advantage of using of brush wheels (in contradistinction to cutting wheels or other, more complex mechanisms) is that upon encountering soil harder than expected, the brushes could simply deflect and the motor(s) could continue to turn. That is, sufficiently flexible brushes would afford resistance to jamming and to overloading of the motors used to rotate the brushes, and so the motors could be made correspondingly lighter and less power hungry. Of course, one could select the brush stiffnesses and motor torques and speeds for greatest effectiveness in sampling soil of a specific anticipated degree of hardness.

  2. ETHOSOMES AS ELASTIC VESICLES IN TRANSDERMAL DRUG DELIVERY: AN OVERVIEW

    Directory of Open Access Journals (Sweden)

    N. B. Gupta et al.

    2012-03-01

    Full Text Available Ethosomes are as novel vesicles in transdermal drug delivery show significant effects of drug penetration through the biological membrane with slight modification of well established drug carrier liposomes. Ethosomes are soft, malleable vesicles composed mainly of phospholipids, ethanol and water. The size of ethosome vesicles can be modulated from tens of nanometer to microns. The ethosomes can be prepared by Hot as well as Cold method. The evaluation parameters of ethosomes include visualization, vesicle size and zeta potential, transition temperature, surface tension activity measurement, vesicle stability, drug content, penetration and permeation studies. Ethosomes have been found to be much more efficient at delivering drug to the skin than either liposomes or hydroalcoholic solution. Thus, it can be a logical conclusion that ethosomal formulation possesses promising future in effective dermal/transdermal delivery of bioactive agents.

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

    DEFF Research Database (Denmark)

    Surma, Michal A; Klose, Christian; Klemm, Robin W; Ejsing, Christer S.; Simons, Kai

    2011-01-01

    Previous work has showed that ergosterol and sphingolipids become sorted to secretory vesicles immunoisolated using a chimeric, artificial raft membrane protein as bait. In this study, we have extended this analysis to three populations of secretory vesicles isolated using natural yeast plasma...... membrane (PM) proteins: Pma1p, Mid2p and Gap1*p as baits. We compared the lipidomes of the immunoisolated vesicles with each other and with the lipidomes of the donor compartment, the trans-Golgi network, and the acceptor compartment, the PM, using a quantitative mass spectrometry approach that provided a...... complete lipid overview of the yeast late secretory pathway. We could show that vesicles captured with different baits carry the same cargo and have almost identical lipid compositions; being highly enriched in ergosterol and sphingolipids. This finding indicates that lipid raft sorting is a generic...

  4. RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border.

    Science.gov (United States)

    Marcos-Ramiro, Beatriz; García-Weber, Diego; Barroso, Susana; Feito, Jorge; Ortega, María C; Cernuda-Morollón, Eva; Reglero-Real, Natalia; Fernández-Martín, Laura; Durán, Maria C; Alonso, Miguel A; Correas, Isabel; Cox, Susan; Ridley, Anne J; Millán, Jaime

    2016-05-01

    Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation. PMID:27138256

  5. Focus on Extracellular Vesicles: Development of Extracellular Vesicle-Based Therapeutic Systems

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Ohno

    2016-02-01

    Full Text Available Many types of cells release phospholipid membrane vesicles thought to play key roles in cell-cell communication, antigen presentation, and the spread of infectious agents. Extracellular vesicles (EVs carry various proteins, messenger RNAs (mRNAs, and microRNAs (miRNAs, like a “message in a bottle” to cells in remote locations. The encapsulated molecules are protected from multiple types of degradative enzymes in body fluids, making EVs ideal for delivering drugs. This review presents an overview of the potential roles of EVs as natural drugs and novel drug-delivery systems.

  6. Semiflexible polymer brushes and the brush-mushroom crossover.

    Science.gov (United States)

    Egorov, Sergei A; Hsu, Hsiao-Ping; Milchev, Andrey; Binder, Kurt

    2015-04-01

    Semiflexible polymers end-grafted to a repulsive planar substrate under good solvent conditions are studied by scaling arguments, computer simulations, and self-consistent field theory. Varying the chain length N, persistence length lp, and grafting density σg, the chain linear dimensions and distribution functions of all monomers and of the free chain ends are studied. Particular attention is paid to the limit of very small σg, where the grafted chains behave as "mushrooms" no longer interacting with each other. Unlike a flexible mushroom, which has a self-similar structure from the size (a) of an effective monomer up to the mushroom height (h/a ∝ N(v), ν ≈ 3/5), a semiflexible mushroom (like a free semiflexible chain) exhibits three different scaling regimes, h/a ∝ N for contour length L = Na volume, h/a ∝ (lp/a)(1/5)N(ν). The semiflexible brush is predicted to scale as h/a ∝ (lpaσg)(1/3)N in the excluded volume regime, and h/a ∝ (lpa(3)σ(2))(1/4)N in the Gaussian regime. Since in the volume taken by a semiflexible mushroom excluded-volume interactions are much weaker in comparison to a flexible mushroom, there occurs an additional regime where semiflexible mushrooms overlap without significant chain stretching. Moreover, since the size of a semiflexible mushroom is much larger than the size of a flexible mushroom with the same N, the crossover from mushroom to brush behavior is predicted to take place at much smaller densities than for fully flexible chains. The numerical results, however, confirm the scaling predictions only qualitatively; for chain lengths that are relevant for experiments, often intermediate effective exponents are observed due to extended crossovers. PMID:25687784

  7. Rheological properties of sheared vesicle and cell suspensions

    OpenAIRE

    Lamura, A.; Gompper, G.

    2014-01-01

    Numerical simulations of vesicle suspensions are performed in two dimensions to study their dynamical and rheological properties. An hybrid method is adopted, which combines a mesoscopic approach for the solvent with a curvature-elasticity model for the membrane. Shear flow is induced by two counter-sliding parallel walls, which generate a linear flow profile. The flow behavior is studied for various vesicle concentrations and viscosity ratios between the internal and the external fluid. Both...

  8. 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.

  9. Vesicle Size Regulates Nanotube Formation in the Cell

    Science.gov (United States)

    Su, Qian Peter; Du, Wanqing; Ji, Qinghua; Xue, Boxin; Jiang, Dong; Zhu, Yueyao; Lou, Jizhong; Yu, Li; Sun, Yujie

    2016-01-01

    Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance. Here, we use lysosomes and autolysosomes, whose sizes range between 100 nm and 1 μm, as model systems to study the size effects on nanotube formation both in vivo and in vitro. Single molecule observations indicate that driven by kinesin motors, small vesicles (100–200 nm) are mainly transported along the tracks while a remarkable portion of large vesicles (500–1000 nm) form nanotubes. This size effect is further confirmed by in vitro reconstitution assays on liposomes and purified lysosomes and autolysosomes. We also apply Atomic Force Microscopy (AFM) to measure the initiation force for nanotube formation. These results suggest that the size-dependence may be one of the mechanisms for cells to regulate cellular processes involving membrane-deformation, such as the timing of tubulation-mediated vesicle recycling. PMID:27052881

  10. Interfacial Friction and Adhesion of Polymer Brushes

    KAUST Repository

    Landherr, Lucas J. T.

    2011-08-02

    A bead-probe lateral force microscopy (LFM) technique is used to characterize the interfacial friction and adhesion properties of polymer brushes. Our measurements attempt to relate the physical structure and chemical characteristics of the brush to their properties as thin-film, tethered lubricants. Brushes are synthesized at several chain lengths and surface coverages from polymer chains of polydimethylsiloxane (PDMS), polystyrene (PS), and a poly(propylene glycol)-poly(ethylene glycol) block copolymer (PPG/PEG). At high surface coverage, PDMS brushes manifest friction coefficients (COFs) that are among the lowest recorded for a dry lubricant film (μ ≈ 0.0024) and close to 1 order of magnitude lower than the COF of a bare silicon surface. Brushes synthesized from higher molar mass chains exhibit higher friction forces than those created using lower molar mass polymers. Increased grafting density of chains in the brush significantly reduces the COF by creating a uniform surface of stretched chains with a decreased surface viscosity. Brushes with lower surface tension and interfacial shear stresses manifest the lowest COF. In particular, PDMS chains exhibit COFs lower than PS by a factor of 3.7 and lower than PPG/PEG by a factor of 4.7. A scaling analysis conducted on the surface coverage (δ) in relation to the fraction (ε) of the friction force developing from adhesion predicts a universal relation ε ∼ δ4/3, which is supported by our experimental data. © 2011 American Chemical Society.

  11. Polymer Release out of a Spherical Vesicle through a Pore

    OpenAIRE

    Park, Pyeong Jun; Sung, Wokyung

    1998-01-01

    Translocation of a polymer out of curved surface or membrane is studied via mean first passage time approach. Membrane curvature gives rise to a constraint on polymer conformation, which effectively drives the polymer to the outside of membrane where the available volume of polymer conformational fluctuation is larger. Considering a polymer release out of spherical vesicle, polymer translocation time $\\tau$ is changed to the scaling behavior $\\tau\\sim L^2$ for $R

  12. Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes

    OpenAIRE

    Solberg, L. B.; Stang, E.; Brorson, S.-H.; Andersson, G; Reinholt, F.P.

    2014-01-01

    Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in o...

  13. Effectiveness of oscillatory gutter brushes in removing street sweeping waste.

    Science.gov (United States)

    Vanegas-Useche, Libardo V; Abdel-Wahab, Magd M; Parker, Graham A

    2015-09-01

    In this paper, the novel concept of oscillatory gutter brushes of road sweepers is studied experimentally. Their effectiveness in removing different debris types is studied by means of a brushing test rig. The debris types dealt with are medium-size gravel, small and fine particles, wet thin debris, and compacted debris. The performance of two types of brushes, cutting and F128, under diverse operating conditions is investigated. The purpose of the tests is to ascertain whether brush oscillations superimposed onto brush rotation improve sweeping effectiveness. According to the results, brush oscillations seem to be useful for increasing brushing effectiveness in the case of bonded particles and wet thin debris, especially for bonded debris, but they seem to be of no value for other loosed debris. Also, appropriate values of brush penetration, sweeper speed, brush angle of attack, rotational speed, and frequency of brush speed oscillations, for the debris types studied are provided. PMID:26026947

  14. Evidence that phospholipase D mediates ADP ribosylation factor- dependent formation of Golgi coated vesicles

    OpenAIRE

    1996-01-01

    Formation of coatomer-coated vesicles from Golgi-enriched membranes requires the activation of a small GTP-binding protein, ADP ribosylation factor (ARF). ARF is also an efficacious activator of phospholipase D (PLD), an activity that is relatively abundant on Golgi- enriched membranes. It has been proposed that ARF, which is recruited onto membranes from cytosolic pools, acts directly to promote coatomer binding and is in a 3:1 stoichiometry with coatomer on coated vesicles. We present evide...

  15. A novel class of clathrin-coated vesicles budding from endosomes

    OpenAIRE

    1996-01-01

    Clathrin-coated vesicles transport selective integral membrane proteins from the plasma membrane to endosomes and from the TGN to endosomes. Recycling of proteins from endosomes to the plasma membrane occurs via unidentified vesicles. To study this pathway, we used a novel technique that allows for the immunoelectron microscopic examination of transferrin receptor-containing endosomes in nonsectioned cells. Endosomes were identified as separate discontinuous tubular-vesicular entities. Each e...

  16. Comparing the efficiency of Denture brush and Ordinary brush in complete Denture cleaning

    Directory of Open Access Journals (Sweden)

    Amir Fayaz

    2013-11-01

    Full Text Available Please cite this article as: Fayaz A, Shakerian M, Ansari GH. Comparing the Efficiency of Denture brush and Ordinary brush in complete Denture cleaning. Novel Biomed 2013;1(2:62-65.Background & objective: Denture cleansing is a key element in retaining mucosa free of any inflammation. However, many denture users usually ignore this important factor. This investigation was designed to compare the efficacy of a denture brush and an ordinary brush in cleansing process of complete dentures.Materials & Methods: A group of 31 individuals aged 44-76 years were included in this study. Each patient was then instructed to use the denture brush for a period of 4 weeks while an ordinary brush was to be used for the following 4 weeks. Dentures were photographed and evaluated at every two week intervals using a computer photographic software assessment method. Pictures were compared using the image tool for plaque remaining on the denture surfaces. Student t-test was used to analyse data collected.Results: Comparison of the brush type efficacy at 2 and 4 weeks did not show any significant difference ( P>0.05 , however , clinical evaluation indicated that denture brush leaves much less plaque bio-film compare to the ordinary one, with mean plaque traced at 6.88 to 9.24 in 4 weeks.Conclusion: There were no significant differences found between the two brushes’ efficacy, with clinical evaluation significantly in favor of denture brush.

  17. Wolbachia bacteria reside in host Golgi-related vesicles whose position is regulated by polarity proteins.

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Cho

    Full Text Available Wolbachia pipientis are intracellular symbiotic bacteria extremely common in various organisms including Drosophila melanogaster, and are known for their ability to induce changes in host reproduction. These bacteria are present in astral microtubule-associated vesicular structures in host cytoplasm, but little is known about the identity of these vesicles. We report here that Wolbachia are restricted only to a group of Golgi-related vesicles concentrated near the site of membrane biogenesis and minus-ends of microtubules. The Wolbachia vesicles were significantly mislocalized in mutant embryos defective in cell/planar polarity genes suggesting that cell/tissue polarity genes are required for apical localization of these Golgi-related vesicles. Furthermore, two of the polarity proteins, Van Gogh/Strabismus and Scribble, appeared to be present in these Golgi-related vesicles. Thus, establishment of polarity may be closely linked to the precise insertion of Golgi vesicles into the new membrane addition site.

  18. Shape transitions of high-genus fluid vesicles

    Science.gov (United States)

    Noguchi, Hiroshi

    2015-12-01

    The morphologies of genus-2 to -8 fluid vesicles are studied by using dynamically triangulated membrane simulations with area-difference elasticity. It is revealed that the alignments of the membrane pores alter the vesicle shapes and the types of shape transitions for the genus g ≥ 3 . At a high reduced volume, a stomatocyte with a circular alignment of g + 1 pores continuously transforms into a discocyte with a line of g pores with increasing intrinsic area difference. In contrast, at a low volume, a stomatocyte transforms into a (g+1) -hedral shape and subsequently exhibits a discrete phase transition to a discocyte.

  19. 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

  20. Cycling of dense core vesicles involved in somatic exocytosis of serotonin by leech neurons

    Directory of Open Access Journals (Sweden)

    Citlali eTrueta

    2012-06-01

    Full Text Available We studied the cycling of dense core vesicles producing somatic exocytosis of serotonin. Our experiments were made using electron microscopy and vesicle staining with fluorescent dye FM1-43 in Retzius neurons of the leech, which secrete serotonin from clusters of dense core vesicles in a frequency-dependent manner. Electron micrographs of neurons at rest or after 1 Hz stimulation showed two pools of dense core vesicles. A perinuclear pool near Golgi apparatuses, from which vesicles apparently form, and a peripheral pool with vesicle clusters at a distance from the plasma membrane. By contrast, after 20 Hz electrical stimulation 47% of the vesicle clusters were apposed to the plasma membrane, with some omega exocytosis structures. Dense core and small clear vesicles apparently originating from endocytosis were incorporated in multivesicular bodies. In another series of experiments, neurons were stimulated at 20 Hz while bathed in a solution containing peroxidase. Electron micrographs of these neurons contained gold particles coupled to anti-peroxidase antibodies in dense core vesicles and multivesicular bodies located near the plasma membrane. Cultured neurons depolarized with high potassium in the presence of FM1-43 displayed superficial fluorescent spots, each reflecting a vesicle cluster. A partial bleaching of the spots followed by another depolarization in the presence of FM1-43 produced restaining of some spots, other spots disappeared, some remained without restaining and new spots were formed. Several hours after electrical stimulation the FM1-43 spots accumulated at the center of the somata. This correlated with electron micrographs of multivesicular bodies releasing their contents near Golgi apparatuses. Our results suggest that dense core vesicle cycling related to somatic serotonin release involves two steps: the production of clear vesicles and multivesicular bodies after exocytosis, and the formation of new dense core vesicles in

  1. 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

  2. How pure are your vesicles?

    Directory of Open Access Journals (Sweden)

    Jason Webber

    2013-01-01

    Full Text Available 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.

  3. Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals

    OpenAIRE

    1994-01-01

    The fluorophore FM1-43 appears to stain membranes of recycled synaptic vesicles. We used FM1-43 to study mechanisms of synaptic vesicle clustering and mobilization in living frog motor nerve terminals. FM1- 43 staining of these terminals produces a linear series of fluorescent spots, each spot marking the cluster of several hundred synaptic vesicles at an active zone. Most agents we tested did not affect staining, but the phosphatase inhibitor okadaic acid (OA) disrupted the fluorescent spots...

  4. Vesicle recycling at ribbon synapses in the finely branched axon terminals of mouse retinal bipolar neurons

    OpenAIRE

    LoGiudice, Lisamarie; Sterling, Peter; Matthews, Gary

    2009-01-01

    In retinal bipolar neurons, synaptic ribbons mark the presence of exocytotic active zones in the synaptic terminal. It is unknown, however, where compensatory vesicle retrieval is localized in this cell type and by what mechanism(s) excess membrane is recaptured. To determine whether endocytosis is localized or diffuse in mouse bipolar neurons, we imaged FM4-64 to track vesicles in cells whose synaptic ribbons were tagged with a fluorescent peptide. In synaptic terminals, vesicle retrieval oc...

  5. Identification of three coated vesicle components as alpha- and beta- tubulin linked to a phosphorylated 50,000-dalton polypeptide

    OpenAIRE

    1983-01-01

    Coated vesicles are involved in the intracellular transport of membrane proteins between a variety of membrane compartments. The coats of bovine brain coated vesicles contain at least six polypeptides in addition to an 180,000-dalton polypeptide called clathrin. In this report we show that the 54,000- and 56,000-dalton coated vesicle polypeptides are alpha- and beta-tubulin, determined by immunoblotting and two-dimensional gel electrophoresis. An affinity-purified tubulin antiserum can precip...

  6. Multiple Modes of Endophilin-mediated Conversion of Lipid Vesicles into Coated Tubes: IMPLICATIONS FOR SYNAPTIC ENDOCYTOSIS*

    OpenAIRE

    Mizuno, Naoko; Jao, Christine C; Langen, Ralf; Steven, Alasdair C

    2010-01-01

    Endophilin A1 is a BAR (Bin/amphiphysin/Rvs) protein abundant in neural synapses that senses and induces membrane curvature, contributing to neck formation in presynaptic endocytic vesicles. To investigate its role in membrane remodeling, we used cryoelectron microscopy to characterize structural changes induced in lipid vesicles by exposure to endophilin. The vesicles convert rapidly to coated tubules whose morphology reflects the local concentration of endophilin. Their diameters and curvat...

  7. Pulsatile lipid vesicles under osmotic stress

    CERN Document Server

    Chabanon, Morgan; Liedberg, Bo; Parikh, Atul N; Rangamani, Padmini

    2016-01-01

    The response of lipid bilayers to osmotic stress is an important part of cellular function. Previously, in [Oglecka et al. 2014], we reported that cell-sized giant unilamellar vesicles (GUVs) exposed to hypotonic media, respond to the osmotic assault by undergoing a cyclical sequence of swelling and bursting events, coupled to the membrane's compositional degrees of freedom. Here, we seek to deepen our quantitative understanding of the essential pulsatile behavior of GUVs under hypotonic conditions, by advancing a comprehensive theoretical model for vesicle dynamics. The model quantitatively captures our experimentally measured swell-burst parameters for single-component GUVs, and reveals that thermal fluctuations enable rate dependent pore nucleation, driving the dynamics of the swell-burst cycles. We further identify new scaling relationships between the pulsatile dynamics and GUV properties. Our findings provide a fundamental framework that has the potential to guide future investigations on the non-equili...

  8. Application of polymer brush prepared by RIGP to enzyme immobilization

    International Nuclear Information System (INIS)

    A diethylamino-group-containing polymer brush was grafted onto a porous hollow-fiber membrane by radiation-induced graft polymerization (RIGP) of an epoxy group-containing vinyl monomer, followed by a reaction of the epoxy group with diethylamine. Anion-exchange porous hollow-fiber membranes with a thickness of about 1.2 mm and a pore size of about 0.3 μm were used as a supporting matrix to immobilize cycloisomaltooligosaccharide glucanotransferase (CITase). CTTase was immobilized to the membrane via anion-exchange adsorption and by subsequent enzymatic cross-linking with transglutaminase, the amount of which ranged from 3 to 110 mg per g of the membrane. The degree of enzyme multilayer binding was equivalent to 0.3 to 9.8. Dextran, as the substrate, was converted into seven- to nine-glucose-membered cycloisomaltooligosaccharides (CI-7, -8 and -9) at a maximum yield of 28% in weight at a space velocity of 10 per hour during the permeation of 2.0 (w/w)% dextran solution across the CITase-immobilized porous hollow-fiber membrane. The yield of CIs increased with increasing degree of CITase multilayering. (author)

  9. DNA-mediated self-assembly of artificial vesicles.

    Directory of Open Access Journals (Sweden)

    Maik Hadorn

    Full Text Available BACKGROUND: Although multicompartment systems made of single unilamellar vesicles offer the potential to outperform single compartment systems widely used in analytic, synthetic, and medical applications, their use has remained marginal to date. On the one hand, this can be attributed to the binary character of the majority of the current tethering protocols that impedes the implementation of real multicomponent or multifunctional systems. On the other hand, the few tethering protocols theoretically providing multicompartment systems composed of several distinct vesicle populations suffer from the readjustment of the vesicle formation procedure as well as from the loss of specificity of the linking mechanism over time. METHODOLOGY/PRINCIPAL FINDINGS: In previous studies, we presented implementations of multicompartment systems and resolved the readjustment of the vesicle formation procedure as well as the loss of specificity by using linkers consisting of biotinylated DNA single strands that were anchored to phospholipid-grafted biotinylated PEG tethers via streptavidin as a connector. The systematic analysis presented herein provides evidences for the incorporation of phospholipid-grafted biotinylated PEG tethers to the vesicle membrane during vesicle formation, providing specific anchoring sites for the streptavidin loading of the vesicle membrane. Furthermore, DNA-mediated vesicle-vesicle self-assembly was found to be sequence-dependent and to depend on the presence of monovalent salts. CONCLUSIONS/SIGNIFICANCE: This study provides a solid basis for the implementation of multi-vesicle assemblies that may affect at least three distinct domains. (i Analysis. Starting with a minimal system, the complexity of a bottom-up system is increased gradually facilitating the understanding of the components and their interaction. (ii Synthesis. Consecutive reactions may be implemented in networks of vesicles that outperform current single compartment

  10. Straddling the border

    DEFF Research Database (Denmark)

    Eilenberg, Michael

    2011-01-01

    border between the Indonesian province of West Kalimantan and the Malaysian state of Sarawak on the island of Borneo. Based on local narratives, the aim of this paper is to unravel the little known history of how the Iban segment of the border population in West Kalimantan became entangled in the highly...

  11. The Border Pedagogy Revisited

    Science.gov (United States)

    Kazanjian, Christopher John

    2011-01-01

    Border pedagogy is a multicultural educational approach utilized in multicultural settings to help students understand their histories and experiences and how it affects their identities and cultures. The approach seeks to produce intellectuals that transcend physical and metaphysical boundaries. The goal of border pedagogy is to remove cultural…

  12. Release of canine parvovirus from endocytic vesicles

    International Nuclear Information System (INIS)

    Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A2 like domain in N-terminus of VP1. In this study we characterized the role of PLA2 activity on CPV entry process. PLA2 activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA2 inhibitors inhibited the viral proliferation suggesting that PLA2 activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA2 activity might have a role during endocytic entry. The presence of drugs modifying endocytosis (amiloride, bafilomycin A1, brefeldin A, and monensin) caused viral proteins to remain in endosomal/lysosomal vesicles, even though the drugs were not able to inhibit the exposure of VP1 N-terminal end. These results indicate that the exposure of N-terminus of VP1 alone is not sufficient to allow CPV to proliferate. Some other pH-dependent changes are needed for productive infection. In addition to blocking endocytic entry, amiloride was able to block some postendocytic steps. The ability of CPV to permeabilize endosomal membranes was demonstrated by feeding cells with differently sized rhodamine-conjugated dextrans together with the CPV in the presence or in the absence of amiloride, bafilomycin A1, brefeldin A, or monensin. Dextran with a molecular weight of 3000 was released from vesicles after 8 h of infection, while dextran with a molecular weight of 10,000 was mainly retained in vesicles. The results suggest that CPV infection does not cause disruption of endosomal vesicles. However, the permeability of endosomal membranes apparently changes during CPV infection, probably due to the PLA2 activity of the virus. These results suggest that parvoviral PLA2 activity is essential for productive infection and presumably

  13. Business, brokers and borders

    DEFF Research Database (Denmark)

    Walther, Olivier

    developed with foreign partners from a different origin, religion or culture. In the second part, we study the spatial structure of trade networks and the influence of national borders on the development of social ties. The paper shows that the spatial form of trade networks is constrained by the historical......Using social network analysis, this paper studies the structure of trade networks that developed across West African borders. The first part aims to understand the centralization of cross-border trade networks. In a business environment where transaction costs are extremely high, we find that...... origin of the traders engaged in cross-border activities. In those markets where trade is recent and where most of the traders are not native of the region, national borders are likely to exert a greater influence than in those regions where trade has pre-colonial roots....

  14. Layer-by-layer deposition of vesicles mediated by supramolecular interactions.

    Science.gov (United States)

    Roling, Oliver; Wendeln, Christian; Kauscher, Ulrike; Seelheim, Patrick; Galla, Hans-Joachim; Ravoo, Bart Jan

    2013-08-13

    Vesicles are dynamic supramolecular structures with a bilayer membrane consisting of lipids or synthetic amphiphiles enclosing an aqueous compartment. Lipid vesicles have often been considered as mimics for biological cells. In this paper, we present a novel strategy for the preparation of three-dimensional multilayered structures in which vesicles containing amphiphilic β-cyclodextrin are interconnected by proteins using cyclodextrin guests as bifunctional linker molecules. We compared two pairs of adhesion molecules for the immobilization of vesicles: mannose-concanavalin A and biotin-streptavidin. Microcontact printing and thiol-ene click chemistry were used to prepare suitable substrates for the vesicles. Successful immobilization of intact vesicles through the mannose-concanavalin A and biotin-streptavidin motifs was verified by fluorescence microscopy imaging and dynamic light scattering, while the vesicle adlayer was characterized by quartz crystal microbalance with dissipation monitoring. In the case of the biotin-streptavidin motif, up to six layers of intact vesicles could be immobilized in a layer-by-layer fashion using supramolecular interactions. The construction of vesicle multilayers guided by noncovalent vesicle-vesicle junctions can be taken as a minimal model for artificial biological tissue. PMID:23898918

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

    International Nuclear Information System (INIS)

    Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca2+-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca2+ level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca2+-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles

  16. Polish Eastern Border as an External European Union Border

    OpenAIRE

    Mazurek, Tomasz; Barwiński, Marek

    2009-01-01

    1. Apart from Finnish-Russian border, Polish eastern border is one of the longest external European Union and Schengen Agreement land borders under the control of a single country. 2. For the last few years, Polish Government has significantly improved the infrastructure of Border Guard at the eastern border in order to prevent “flooding” of Europe by illegal immigrants and smuggled goods. 3. Despite this fact, Polish eastern border does not have sufficient infrastructure, especially wh...

  17. Microfluidic Fabrication of Pluronic Vesicles with Controlled Permeability.

    Science.gov (United States)

    do Nascimento, Débora F; Arriaga, Laura R; Eggersdorfer, Max; Ziblat, Roy; Marques, Maria de Fátima V; Reynaud, Franceline; Koehler, Stephan A; Weitz, David A

    2016-05-31

    Block copolymers with a low hydrophilic-to-lipophilic balance form membranes that are highly permeable to hydrophilic molecules. Polymersomes with this type of membrane enable the controllable release of molecules without membrane rupture. However, these polymersomes are difficult to assemble because of their low hydrophobicity. Here, we report a microfluidic approach to the production of these polymersomes using double-emulsion drops with ultrathin shells as templates. The small thickness of the middle oil phase enables the attraction of the hydrophobic blocks of the polymers adsorbed at each of the oil/water interfaces of the double emulsions; this results in the dewetting of the oil from the surface of the innermost water drops of the double emulsions and the ultimate formation of the polymersome. This approach to polymersome fabrication enables control of the vesicle size and results in the efficient encapsulation of hydrophilic ingredients that can be released through the polymer membrane without membrane rupture. We apply our approach to the fabrication of Pluronic L121 vesicles and characterize the permeability of their membranes. Furthermore, we show that membrane permeability can be tuned by blending different Pluronic polymers. Our work thus describes a route to producing Pluronic vesicles that are useful for the controlled release of hydrophilic ingredients. PMID:27192611

  18. Motor Brush Testing for Mars and Vacuum

    Science.gov (United States)

    Noon, Don E.

    1999-01-01

    Brush motors have been qualified and flown successfully on Mars missions, but upcoming missions require longer life and higher power. A test program was therefore undertaken to identify the best brush material for operation in the Mars atmosphere. Six different brush materials were used in 18 identical motors and operated under various load conditions for a period of four weeks in low-pressure CO2. All motors performed acceptably, with accumulated motor revolutions between 98 and 144 million revolutions, depending on load. A proprietary silver-graphite material from Superior Carbon (SG54-27) appears to be the best choice for long life, but even the stock copper-graphite brushes performed reliably with acceptable wear. The motors from the CO2 test were then cleaned and run in vacuum for 2 weeks. The difference in results was dramatic, with 5 motors failing catastrophically and wear rates increasing by orders of magnitude for the SG54-27 material. Three brush materials survived the test with no failures: SG54-27 with a proprietary Ball Aerospace impregnation, a silver-graphite-molybdenum disulfide material from Superior Carbon (SG59), and a copper sulfide-graphite material also from Superior Carbon (BG91).

  19. Molecular and Functional Characterisation of a new Mammalian Family of Proton-coupled Amino Acid Transporters

    OpenAIRE

    Foltz, Martin

    2006-01-01

    Proton-coupled amino acid transport (PAT) systems in the apical membrane of epithelial cells of the small intestine and the renal tubule have been demonstrated by use of the human intestinal cell line Caco-2 and renal brush border membrane vesicles. The present thesis summarises the revelation of the molecular entity of the PAT system and provides deeper insights into this new mammalian family of proton/amino acid cotransporters. The identified family comprises four structural similar murine ...

  20. Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance.

    Directory of Open Access Journals (Sweden)

    Vicky Goler-Baron

    Full Text Available Multidrug resistance (MDR is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and

  1. Thermal undulations of quasi-spherical vesicles stabilized by gravity

    DEFF Research Database (Denmark)

    Henriksen, Jonas Rosager; Ipsen, John Hjorth

    2002-01-01

    The classical treatment of quasi-spherical vesicle undulations has, in the present work, been reviewed and extended to systems, which are affected by a gravitational field caused by a density difference across the membrane. The effects have been studied by the use of perturbation theory leading to...

  2. Spontaneous Vesicles Modulated by Polymers

    OpenAIRE

    Francisco Ortega; M. Mercedes Velázquez; Margarita Valero

    2011-01-01

    Vesicles are widely used in technological applications including cosmetic products, in microencapsulation for drug delivery, as anticancer agents and in the technology of adhesives, paints and inks. The vesicle size and the surface charge are very important properties from a technological point of view. Thus, the challenge in formulation is to find inexpensive stable vesicles with well-defined sizes and to modulate the surface charge of these aggregates. In this work we analyze the effect of ...

  3. Two-stage eutectic metal brushes

    Science.gov (United States)

    Hsu, John S [Oak Ridge, TN

    2009-07-14

    A two-stage eutectic metal brush assembly having a slip ring rigidly coupled to a shaft, the slip ring being electrically coupled to first voltage polarity. At least one brush is rigidly coupled to a second ring and slidingly engaged to the slip ring. Eutectic metal at least partially fills an annulus between the second ring and a stationary ring. At least one conductor is rigidly coupled to the stationary ring and electrically coupled to a second voltage polarity. Electrical continuity is maintained between the first voltage polarity and the second voltage polarity. Periodic rotational motion is present between the stationary ring and the second ring. Periodic rotational motion is also present between the brush and the slip ring.

  4. Charge Transport Properties in Polymer Brushes

    Science.gov (United States)

    Moog, Mark; Tsui, Frank; Vonwald, Ian; You, Wei

    Electrical transport properties in poly(3-methyl)thiophene (P3MT) brushes have been studied. The P3MT brushes correspond to a new type of surface-tethered, vertically oriented conjugated molecular wires, sandwiched between two metallic electrodes to form the electrode-molecule-electrode (EME) devices. P3MT is a highly conjugated polymer, a ''workhorse'' material for organic electronics and photonics. The P3MT brushes were grown on ITO surfaces with controlled length (between 2 and 100 nm). The top electrodes were transfer-printed Au films with lateral dimensions between 200 nm and 50 μm. I-V and differential conductance measurements were performed using conductive AFM and 4-terminal techniques. Tunneling and field-emission measurements in EME devices with molecular lengths mobility and the interplay between intra- and intermolecular transport have been investigated.

  5. New mechanisms of vesicles migration.

    Science.gov (United States)

    Aursulesei, Viviana; Vasincu, Decebal; Timofte, Daniel; Vrajitoriu, Lucia; Gatu, Irina; Iacob, Dan D; Ghizdovat, Vlad; Buzea, Calin; Agop, Maricel

    2016-07-01

    In multicellular organisms, both health and disease are defined by means of communication patterns involving the component cells. Despite the intricate networks of soluble mediators, cells are also programed to exchange complex messages pre-assembled as multimolecular cargo of membranous structures known as extracellular vesicles (EVs). Several biogenetic pathways produce EVs with different properties able to orchestrate neighboring cell reactions or to establish an environment ripe for spreading tumor cells. Such an effect is in fact an extension of similar physiological roles played by exosomes in guiding cell migration under nontumoral tissue remodeling and organogenesis. We start with a biological thought experiment equivalent to Bénard's experiment, involving a fluid layer of EVs adherent to an extracellular matrix, in a haptotactic gradient, then, we build and present the first Lorenz model for EVs migration. Using Galerkin's method of reducing a system of partial differential equations to a system of ordinary differential equations, a biological Lorenz system is developed. Such a physical frame distributing individual molecular or exosomal type cell-guiding cues in the extracellular matrix space could serve as a guide for tissue neoformation of the budding pattern in nontumoral or tumoral instances. PMID:27045674

  6. Vesicles in a Poiseuille flow

    CERN Document Server

    Danker, Gerrit; Misbah, Chaouqi

    2008-01-01

    Vesicle dynamics in unbounded Poiseuille flow is analyzed using a small-deformation theory. Our analytical results quantitatively describe vesicle migration and provide new physical insights. At low ratio between the inner and outer viscosity $\\lambda$ (i.e. in the tank-treading regime), the vesicle always migrates towards the flow centerline, unlike other soft particles such as drops. Above a critical $\\lambda$, vesicle tumbles and cross-stream migration vanishes. A novel feature is predicted, namely the coexistence of two types of nonequilibrium configurations at the centreline, a bullet-like and a parachute-like shapes.

  7. Multiple N-ethylmaleimide-sensitive components are required for endosomal vesicle fusion.

    OpenAIRE

    Rodriguez, L; Stirling, C J; Woodman, P. G.

    1994-01-01

    This report examines the inhibition of endosomal vesicle fusion by the alkylating agent N-ethylmaleimide (NEM). The concentration of NEM required to inhibit vesicle fusion depended upon whether membrane and cytosolic fractions were treated separately or together, enabling the resolution of at least two components to the inhibition. The first component is inactivated at low levels of NEM when cytosolic and membrane fractions are treated together. On the contrary, inhibition of the second compo...

  8. A method for analysis of lipid vesicle domain structure from confocal image data

    DEFF Research Database (Denmark)

    Husen, Peter Rasmussen; Fidorra, Matthias; Hartel, Steffen; Bagatolli, Luis; Ipsen, John Hjort

    2012-01-01

    Quantitative characterization of the lateral structure of curved membranes based on fluorescence microscopy requires knowledge of the fluorophore distribution on the surface. We present an image analysis approach for extraction of the fluorophore distribution on a spherical lipid vesicle from...... confocal imaging stacks. The technique involves projection of volumetric image data onto a triangulated surface mesh representation of the membrane, correction of photoselection effects and global motion of the vesicle during image acquisition and segmentation of the surface into domains using histograms...

  9. Oligomerization of a Cargo Receptor Directs Protein Sorting into COPII-coated Transport Vesicles

    OpenAIRE

    Sato, Ken; Nakano, Akihiko

    2003-01-01

    Secretory proteins are transported from the endoplasmic reticulum (ER) to the Golgi complex in vesicles coated with coat protein complex II (COPII). The incorporation of certain transport molecules (cargo) into the COPII vesicles is thought to be mediated by cargo receptors. Here we show that Emp47p, a type-I membrane protein, is specifically required for the transport of an integral membrane protein, Emp46p, from the ER. Exit of Emp46p from the ER was saturable and de...

  10. Dissipative particle dynamics simulation study of the bilayer-vesicle transition

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A bilayer structure is an important immediate for the vesicle formation. However,the mechanism for the bilayer-vesicle transition remains unclear. In this work,a dissipative particle dynamics(DPD) simulation method was employed to study the mechanism of the bilayer-vesicle transition. A coarse-grained model was built based on a lipid molecule termed dimyristoylphosphatidylcholine(DMPC). Simulations were performed from two different initial configurations:a random dispersed solution and a tensionless bilayer. It was found that the bilayer-vesicle transition was driven by the minimization of the water-tail hydrophobic interaction energy,and was accompanied with the increase of the position entropy due to the redistribution of water molecules. The bulk pressure was reduced during the bilayer-vesicle transition,suggesting the evolved vesicle morphology was at the relatively low free energy state. The membrane in the product vesicle was a two-dimensional fluid. It can be concluded that the membrane of a vesicle is not interdigitated and most of the bonds in lipid chains are inclined to orient along the radical axis of the vesicle.

  11. Dissipative particle dynamics simulation study of the bilayer-vesicle transition

    Institute of Scientific and Technical Information of China (English)

    WU ShaoGui; GUO HongXia

    2008-01-01

    A bilayer structure is an important immediate for the vesicle formation. However, the mechanism for the bilayer-vesicle transition remains unclear. In this work, a dissipative particle dynamics (DPD) simula-tion method was employed to study the mechanism of the bilayer-vesicle transition. A coarse-grained model was built based on a lipid molecule termed dimyristoylphosphatidylcholine (DMPC). Simulations were performed from two different initial configurations: a random dispersed solution and a ten-sionless bilayer. It was found that the bilayer-vesicle transition was driven by the minimization of the water-tail hydrophobic interaction energy, and was accompanied with the increase of the position en-tropy due to the redistribution of water molecules. The bulk pressure was reduced during the bi-layer-vesicle transition, suggesting the evolved vesicle morphology was at the relatively low free en-ergy state. The membrane in the product vesicle was a two-dimensional fluid. It can be concluded that the membrane of a vesicle is not interdigitated and most of the bonds in lipid chains are inclined to orient along the radical axis of the vesicle.

  12. A new approach to follow a single extracellular vesicle-cell interaction using optical tweezers.

    Science.gov (United States)

    Prada, Ilaria; Amin, Ladan; Furlan, Roberto; Legname, Giuseppe; Verderio, Claudia; Cojoc, Dan

    2016-01-01

    Extracellular vesicles (EVs) are spherical membrane structures released by most cells. These highly conserved mediators of intercellular communication carry proteins, lipids, and nucleic acids, and transfer these cellular components between cells by different mechanisms, such as endocytosis, macropinocytosis, or fusion. However, the temporal and spatial dynamics of vesicle-cell interactions still remain largely unexplored. Here we used optical tweezers to drive single EVs produced by microglial cells onto the surface of astrocytes or microglia in primary culture. By visualizing single EV-cell contacts, we observed that microglial vesicles displayed different motilities on the surface of astrocytes compared with microglia. After contact, EVs positioned on astrocytes displayed some minor oscillatory motion around the point of adhesion, while vesicles dragged to microglia displayed quite regular directional movement on the plasma membrane. Both the adhesion and motion of vesicles on glial cells were strongly reduced by cloaking phosphatidylserine (PS) residues, which are externalized on the vesicle membrane and act as determinants for vesicle recognition by target cells. These data identify optical manipulation as a powerful tool to monitor in vitro vesicle-cell dynamics with high temporal and spatial resolution and to determine in a quantitative manner the contribution of surface receptors/extracellular protein ligands to the contact. PMID:26757810

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

    CERN Document Server

    Peterlin, Primoz

    2010-01-01

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

  14. Calmodulin stimulation of calcium transport in carrot microsomal vesicles

    International Nuclear Information System (INIS)

    ATP-dependent 45Ca2+ uptake into microsomal vesicles isolated from cultured carrot cells (Daucus carota Danvers) was stimulated 2-3 fold by 5 ug/ml calmodulin (CaM). Microsomal vesicles separated with a linear sucrose gradient showed two peaks with CaM-stimulated Ca2+ uptake activities. One peak (at 1.12 g/cc) comigrated with the activity of the antimycin A-insensitive NADH-dependent cytochrome c reductase. This transport activity was enhanced 10-20 fold by 10 mM oxalate and appeared to be associates with vesicles derived primarily from the ER. The other peak of CaM-stimulated Ca2+ uptake (at 1.17 g/cc) was not affected by oxalate. These vesicles are probably derived from the plasma membrane. Preliminary experiments with the low-density vesicles (ER) vesicles, indicate that inositol-1,4,5-trisphosphate caused a transient reduction in intravesicular Ca2+. These results are consistent with the ER being an important site of intracellular Ca2+ regulation

  15. Bubble-induced microstreaming: guiding and destroying lipid vesicles

    Science.gov (United States)

    Marmottant, Philippe; Hilgenfeldt, Sascha

    2002-11-01

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

  16. The Arabidopsis P4-ATPase ALA3 requires a ß-subunit to function in phospholipid translocation and secretory vesicle formation

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura

    shoots. The root growth defect is accompanied by the failure of the root to release border cells of the root cap. Electron micrograph data suggest that functioning and shedding of border cells are dependent on ALA3, as ala3 mutants are devoid of the characteristic proliferation of slime vesicles at the...

  17. Toric Border Basis

    OpenAIRE

    Mourrain, Bernard; Trebuchet, Philippe

    2014-01-01

    International audience We extend the theory and the algorithms of Border Bases to systems of Laurent polynomial equations, defining ''toric'' roots. Instead of introducing new variables and new relations to saturate by the variable inverses, we propose a more efficient approach which works directly with the variables and their inverse. We show that the commutation relations and the inversion relations characterize toric border bases. We explicitly describe the first syzygy module associate...

  18. A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins.

    Science.gov (United States)

    Diao, Jiajie; Ishitsuka, Yuji; Lee, Hanki; Joo, Chirlmin; Su, Zengliu; Syed, Salman; Shin, Yeon-Kyun; Yoon, Tae-Young; Ha, Taekjip

    2012-05-01

    SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d. PMID:22582418

  19. Biological reactivity of nanoparticles: mosaics from optical microscopy videos of giant lipid vesicles

    Science.gov (United States)

    Zupanc, Jernej; Dobnikar, Andrej; Drobne, Damjana; Valant, Janez; Erdogmus, Deniz; Bas, Erhan

    2011-02-01

    Emerging fields such as nanomedicine and nanotoxicology, demand new information on the effects of nanoparticles on biological membranes and lipid vesicles are suitable as an experimental model for bio-nano interaction studies. This paper describes image processing algorithms which stitch video sequences into mosaics and recording the shapes of thousands of lipid vesicles, which were used to assess the effect of CoFe2O4 nanoparticles on the population of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipid vesicles. The applicability of this methodology for assessing the potential of engineered nanoparticles to affect morphological properties of lipid membranes is discussed.

  20. 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

  1. Molecular characterization of exosome-like vesicles from breast cancer cells

    International Nuclear Information System (INIS)

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

  2. Vesicle Priming in a SNAP

    OpenAIRE

    Müller, Martin; Davis, Graeme W.

    2010-01-01

    In this issue of Neuron, Burgalossi et al. (2010) investigate synaptic vesicle priming using presynaptic Ca2+ uncaging at a small, glutamatergic, central synapse. Combining this technique with mouse genetics, the authors demonstrate that vesicle priming during ongoing neural activity can be limited by the recycling of recently used SNARE complexes.

  3. Phototriggered functionalization of hierarchically structured polymer brushes.

    Science.gov (United States)

    de los Santos Pereira, Andres; Kostina, Nina Yu; Bruns, Michael; Rodriguez-Emmenegger, Cesar; Barner-Kowollik, Christopher

    2015-06-01

    The precise design of bioactive surfaces, essential for the advancement of many biomedical applications, depends on achieving control of the surface architecture as well as on the ability to attach bioreceptors to antifouling surfaces. Herein, we report a facile avenue toward hierarchically structured antifouling polymer brushes of oligo(ethylene glycol) methacrylates via surface-initiated atom transfer radical polymerization (SI-ATRP) presenting photoactive tetrazole moieties, which permitted their functionalization via nitrile imine-mediated tetrazole-ene cyclocloaddition (NITEC). A maleimide-functional ATRP initiator was photoclicked to the side chains of a brush enabling a subsequent polymerization of carboxybetaine acrylamide to generate a micropatterned graft-on-graft polymer architecture as evidenced by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Furthermore, the spatially resolved biofunctionalization of the tetrazole-presenting brushes was accessed by the photoligation of biotin-maleimide and subsequent binding of streptavidin. The functionalized brushes bearing streptavidin were able to resist the fouling from blood plasma (90% reduction with respect to bare gold). Moreover, they were employed to demonstrate a model biosensor by immobilization of a biotinylated antibody and subsequent capture of an antigen as monitored in real time by surface plasmon resonance. PMID:25961109

  4. Curvature elasticity of a grafted polyelectrolyte brush

    Science.gov (United States)

    Lei, Zhen; Miao, Bing; Yang, Shuang; Chen, Er-Qiang

    2015-06-01

    The curvature elasticity of a polyelectrolyte brush monolayer attached to curved surface is investigated theoretically. An analytical method based on the strong-stretching theory for a Gaussian chain is developed to calculate the elastic modulus induced by a polyelectrolyte brush. In particular, the scaling relations for the bending or Gaussian modulus with respect to system parameters related to the electrostatic interaction (degree of ionization and salt concentration) are derived. Using the numerical self-consistent-field theory, the inner structural, free-energy, and elastic moduli are computed for the polyelectrolyte brush with excluded-volume interactions. Compared to the analytical result, the curvature elasticity has a weaker dependence on the system parameters, which is attributed to the linearization for the Poisson-Boltzmann equation in the analytical treatment. Furthermore, our results are compared to the curvature elasticity of a bare charged surface, wherefrom the unique polyelectrolyte brush effect on the surface elasticity is clarified clearly. The scaling relations derived in our paper can serve as a guide to experimental studies on the related systems.

  5. Spherical Polyelectrolyte Brushes and Their Films

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Nano-sized spherical polyelectrolyte brushes (SPB), which consist of a solid polystyrene core and linear poly(acrylic acid) (PAA) chains covalently attached on the core surface by one end with different lengths and grafting densities, were prepared by photo-emulsion polymerization and thermo- controlled emulsion polymerization.

  6. Chinese Brush Calligraphy Character Retrieval and Learning

    Science.gov (United States)

    Zhuang, Yueting; Zhang, Xiafen; Lu, Weiming; Wu, Fei

    2007-01-01

    Chinese brush calligraphy is a valuable civilization legacy and a high art of scholarship. It is still popular in Chinese banners, newspaper mastheads, university names, and celebration gifts. There are Web sites that try to help people enjoy and learn Chinese calligraphy. However, there lacks advanced services such as content-based retrieval or…

  7. Conjugated Polymer Brushes (Poly(3-hexylthiophene) brushes): new electro- and photo-active molecular architectures

    OpenAIRE

    Khanduyeva, Natalya

    2009-01-01

    The aim of the present work was to screen the main methods for the synthesis of conjugated polymers for their suitability in the preparation of conductive polymer brushes. The main focus was put on the grafting of intrinsically soluble substituted regioregular polyalkylthiophenes because of their excellent optoelectronic properties. The resulting polymer films were characterized and their optoelectrical properties studied. For the first time, a synthesis of conductive polymer brushes on solid...

  8. 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

  9. Synthesis and characterization of metal oxide nanorod brushes

    Indian Academy of Sciences (India)

    Kalyan Raidongia; M Eswaramoorthy

    2008-02-01

    Nanorod brushes of -Al2O3, MoO3 and ZnO have been synthesized using amorphous carbon nanotube (-CNT) brushes as the starting material. The brushes of -Al2O3 and MoO3 are made up of single crystalline nanorods. In the case of ZnO brushes, the nanorod bristles are made by the fusion of 15–25 nm size nanoparticles and are porous in nature. Metal oxide nanorod brushes thus obtained have been characterized by XRD, FESEM, TEM and Raman spectroscopy. Single crystalline ruby nanorods were obtained by introducing chromium ions during the synthesis of alumina rods.

  10. Coated vesicles contain a phosphatidylinositol kinase

    International Nuclear Information System (INIS)

    When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. The authors have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of the CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism

  11. Vesicles and vesicle fusion: coarse-grained simulations

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2010-01-01

    compounds inside vesicles delays their clearance from the blood stream. In this chapter, we survey the biological role and physico-chemical properties of phospholipids, and describe progress in coarse-grained simulations of vesicles and vesicle fusion. Because coarse-grained simulations retain only those...... molecular details that are thought to influence the large-scale processes of interest, they act as a model embodying our current understanding. Comparing the predictions of these models with experiments reveals the importance of the retained microscopic details and also the deficiencies that can suggest...

  12. The aminosterol antibiotic squalamine permeabilizes large unilamellar phospholipid vesicles.

    Science.gov (United States)

    Selinsky, B S; Zhou, Z; Fojtik, K G; Jones, S R; Dollahon, N R; Shinnar, A E

    1998-03-13

    The ability of the shark antimicrobial aminosterol squalamine to induce the leakage of polar fluorescent dyes from large unilamellar phospholipid vesicles (LUVs) has been measured. Micromolar squalamine causes leakage of carboxyfluorescein (CF) from vesicles prepared from the anionic phospholipids phosphatidylglycerol (PG), phosphatidylserine (PS), and cardiolipin. Binding analyses based on the leakage data show that squalamine has its highest affinity to phosphatidylglycerol membranes, followed by phosphatidylserine and cardiolipin membranes. Squalamine will also induce the leakage of CF from phosphatidylcholine (PC) LUVs at low phospholipid concentrations. At high phospholipid concentrations, the leakage of CF from PC LUVs deviates from a simple dose-response relationship, and it appears that some of the squalamine can no longer cause leakage. Fluorescent dye leakage generated by squalamine is graded, suggesting the formation of a discrete membrane pore rather than a generalized disruption of vesicular membranes. By using fluorescently labeled dextrans of different molecular weight, material with molecular weight squalamine, but material with molecular weight >/=10,000 is retained. Negative stain electron microscopy of squalamine-treated LUVs shows that squalamine decreases the average vesicular size in a concentration-dependent manner. Squalamine decreases the size of vesicles containing anionic phospholipid at a lower squalamine/lipid molar ratio than pure PC LUVs. In a centrifugation assay, squalamine solubilizes phospholipid, but only at significantly higher squalamine/phospholipid ratios than required for either dye leakage or vesicle size reduction. Squalamine solubilizes PC at lower squalamine/phospholipid ratios than PG. We suggest that squalamine complexes with phospholipid to form a discrete structure within the bilayers of LUVs, resulting in the transient leakage of small encapsulated molecules. At higher squalamine/phospholipid ratios, these

  13. Receptors for short-chain fatty acids in brush cells at the gastric groove

    Directory of Open Access Journals (Sweden)

    Julia Anna-Maria Eberle

    2014-04-01

    Full Text Available In the stomach of rodents clusters of brush cells are arranged at the gastric groove, immediately at the transition zone from the non-glandular reservoir compartment to the glandular digestive compartment. Based on their taste cell-like molecular phenotype it has been speculated that the cells may be capable to sense constituents of the ingested food, however, searches for nutrient receptors have not been successful. In this study, it was hypothesized that the cells may express receptors for short-chain fatty acids, metabolites generated by microorganisms during the storage of ingested food in the murine forestomach, which lacks the acidic milieu of more posterior regions of the stomach and is colonized with numerous microbiota. Experimental approaches, including RT-PCR analysis and immunohistochemical studies, revealed that the majority of these brush cells express the G-protein coupled receptor types GPR41 (FFAR3 and GPR43 (FFAR2, which are activated by short-chain fatty acids. Both, the GPR41 receptor proteins as well as an appropriate G-protein, α-gustducin, were found to be segregated at the apical brush border of the cells, indicating a direct contact with the luminal content of this gastric region. The exposure of microvillar processes with appropriate receptors and signaling elements to the gastric lumen suggests that the brush cells may in fact be capable to sense the short-chain fatty acids which originate from fermentation processes during the retention of ingested food in the anterior part of the stomach.

  14. Localization of phosphorylated TrkA in carrier vesicles involved in its nuclear translocation in U251 cell line

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A number of transmembrane receptors are targeted to the nucleus and convincingly localized therein. However, what remains a conundrum is how these cell-surface receptors end up in the nucleus. In this study, we reported that the transmembrane receptor phosphorylated TrkA was located in a series of carrier vesicles, including ring-like vesicles near the plasma membrane, large core vesicles and small dense core vesicles around the nuclei, as well as in the nucleus in human glioma cell line U251 using immunocytochemistry and immunofluorescence staining. Meanwhile, we also showed that small dense core vesicles budded from large core vesicles, and interacted with the nuclear envelope. Accordingly, our results suggested that such a series of membrane compartments might be involved in the pathway of nuclear translocation of the transmembrane receptor TrkA.

  15. Localization of phosphorylated TrkA in carrier vesicles involved in its nuclear translocation in U251 cell line

    Institute of Scientific and Technical Information of China (English)

    GONG AiHua; ZHANG ZhiJian; XIAO DeSheng; YANG Yong; WANG YongZhong; CHEN YongChang

    2007-01-01

    A number of transmembrane receptors are targeted to the nucleus and convincingly localized therein.However, what remains a conundrum is how these cell-surface receptors end up in the nucleus. In this study, we reported that the transmembrane receptor phosphorylated TrkA was located in a series of carrier vesicles, including ring-like vesicles near the plasma membrane, large core vesicles and small dense core vesicles around the nuclei, as well as in the nucleus in human glioma cell line U251 using immunocytochemistry and immunofluorescence staining. Meanwhile, we also showed that small dense core vesicles budded from large core vesicles, and interacted with the nuclear envelope. Accordingly,our results suggested that such a series of membrane compartments might be involved in the pathway of nuclear translocation of the transmembrane receptor TrkA.

  16. Toward Infection-Resistant Surfaces: Achieving High Antimicrobial Peptide Potency by Modulating the Functionality of Polymer Brush and Peptide.

    Science.gov (United States)

    Yu, Kai; Lo, Joey C Y; Mei, Yan; Haney, Evan F; Siren, Erika; Kalathottukaren, Manu Thomas; Hancock, Robert E W; Lange, Dirk; Kizhakkedathu, Jayachandran N

    2015-12-30

    Bacterial infection associated with indwelling medical devices and implants is a major clinical issue, and the prevention or treatment of such infections is challenging. Antimicrobial coatings offer a significant step toward addressing this important clinical problem. Antimicrobial coatings based on tethered antimicrobial peptides (AMPs) on hydrophilic polymer brushes have been shown to be one of the most promising strategies to avoid bacterial colonization and have demonstrated broad spectrum activity. Optimal combinations of the functionality of the polymer-brush-tethered AMPs are essential to maintaining long-term AMP activity on the surface. However, there is limited knowledge currently available on this topic. Here we report the development of potent antimicrobial coatings on implant surfaces by elucidating the roles of polymer brush chemistry and peptide structure on the overall antimicrobial activity of the coatings. We screened several combinations of polymer brush coatings and AMPs constructed on nanoparticles, titanium surfaces, and quartz slides on their antimicrobial activity and bacterial adhesion against Gram-positive and Gram-negative bacteria. Highly efficient killing of planktonic bacteria by the antimicrobial coatings on nanoparticle surfaces, as well as potent killing of adhered bacteria in the case of coatings on titanium surfaces, was observed. Remarkably, the antimicrobial activity of AMP-conjugated brush coatings demonstrated a clear dependence on the polymer brush chemistry and peptide structure, and optimization of these parameters is critical to achieving infection-resistant surfaces. By analyzing the interaction of polymer-brush-tethered AMPs with model lipid membranes using circular dichroism spectroscopy, we determined that the polymer brush chemistry has an influence on the extent of secondary structure change of tethered peptides before and after interaction with biomembranes. The peptide structure also has an influence on the density

  17. Acetaminophen Changes Intestinal Epithelial Cell Membrane Properties, Subsequently Affecting Absorption Processes

    Directory of Open Access Journals (Sweden)

    Christine Schäfer

    2013-08-01

    Full Text Available Background/Aims: Acetaminophen (APAP effects on intestinal barrier properties are less investigated. APAP may lead to a changed bioavailability of a subsequently administered drug or diet in the body. We investigated the influence of APAP on enterocytic cell membrane properties that are able to modify the net intestinal absorption of administered substances across the Caco-2 barrier model. Methods: The effect of APAP on cytotoxicity was measured by LDH assay, TER value and cell capacitance label-free using impedance monitoring, membrane permeability by FITC-dextrans, and efflux transporter MDR1 activity by Rh123. APAP levels were determined by HPLC analysis. Cell membrane topography and microvilli were investigated using SEM and intestinal alkaline phosphatase (Alpi and tight junction protein 1 (TJP1 expression by western blot analysis. Results: APAP changed the apical cell surface, reduced the number of microvilli and protein expression of Alpi as a brush border marker and TJP1, increased the membrane integrity and concurrently decreased cell capacitance over time. In addition, APAP decreased the permeability to small molecules and increased the efflux transporter activity, MDR1. Conclusion: APAP alters the Caco-2 cell membrane properties by different mechanisms and reduces the permeability to administered substances. These findings may help to optimize therapeutic implications.

  18. Business, brokers and borders

    DEFF Research Database (Denmark)

    Walther, Olivier

    2015-01-01

    The objective of this paper is to show how a formal approach to networks can make a significant contribution to the study of cross-border trade in West Africa. Building on the formal tools and theories developed by Social Network Analysis, we examine the network organization of 136 large traders in...... and cooperation shared among local traders, and on the distant ties developed with foreign partners from a different origin, religion or culture. Studying the spatial structure of trade networks, we find that in those markets where trade is recent and where most of the traders are not native of the...... region, national borders are likely to exert a greater influence than in those regions where trade has pre-colonial roots. Combining formal network analysis and ethnographic studies, we argue, can make a significant contribution to the current revival of interest in cross-border trade in the policy field....

  19. Effect of polymer brushes on the Self Assembly of 3D Poly(Styrene-Methylmethacrylate) thin films

    Science.gov (United States)

    Lwoya, Baraka; Albert, Julie

    2015-03-01

    It would be instrumental to understand the self-assembly capabilities of polymers especially given their industrial capabilities of templating and membrane application .The ability of block copolymers to self assemble into different morphologies is determined by several factor including type of polymer blocks, volume fraction, substrate preference to a polymer and chain architecture . In this paper Poly(Styrene-Methylmethacrylate) (PS-PMMA) was chosen was chosen to further understand the effect polymer brushes on the substrate had on the self assembly of 3D structured PS-PMMA spin coated thin films (30-150 nm). The polymer brushes were grown using surface initiated atomic transfer radical polymerization (SI-ATRP) with the optimal chain length being confirmed by gel permeation chromatography. By using ellipsometer and contact angle measurement the uniformity of the polymer brushes are characterized, while the morphology of the spin coated thin films after thermal annealing would be characterized using atomic force microscopy (AFM).

  20. THREE INTELLIGENCE METHODOLOGIES FOR BORDER DEFENCE AND BORDER SECURITY

    Directory of Open Access Journals (Sweden)

    Glen Segell

    2011-08-01

    Full Text Available The main security problem that any state faces today is protecting itscitizens in countering organised crime and terrorism. Wars between states are lessfrequent than in previous eras. Border defence and border security are distinctmissions requiring different forces with different training and different equipment.Border defence is predominately against the armed forces of other states requiringtanks, aircraft and ships. Traditionally, border security includes the mission roles ofimmigration, crime, agriculture, finance, disease control and terrorism. Intelligencegathering and analysis using three methodologies - trends and patterns, frequency,and probability – provides a solution to the large and expensive armed forces forterritorial border defence and defines the ability to succeed in border security.