Cytosolic calcium homeostasis in fungi: Roles of plasma membrane transport and intracellular sequestration of calcium

International Nuclear Information System (INIS)

Miller, A.J.; Vogg, G.; Sanders, D.

1990-01-01

Cytosolic free calcium ([Ca 2+ ] c ) has been measured in the mycelial fungus Neurospora crassa with Ca 2+ - selective microelectrodes. The mean value of [Ca 2+ ] c is 92 ± 15 nM and it is insensitive to external pH values between 5.8 and 8.4. Simultaneous measurement of membrane potential enables the electrochemical potential difference for Ca 2+ across the plasma membrane to be estimated as about -60 kJmol -1 - a value that cannot be sustained either by a simple Ca 2+ - ATPase, or, in alkaline conditions, by straightforward H + /Ca 2+ exchange with a stoichiometric ratio of + /Ca 2+ . The authors propose that the most likely alternative mechanism of Ca 2+ efflux is ATP-driven H + /Ca 2+ exchange, with a stoichiometric ratio of at least 2 H + /Ca 2+ . The increase in [Ca 2+ ] c in the presence of CN - at pH 8.4 is compared with 45 Ca 2+ influx under the same conditions. The proportion of entering Ca 2+ remaining free in the cytosol is only 8 x 10 -5 , and since the concentration of available chelation sites on Ca 2+ binding proteins is unlikely to exceed 100 μM, a major role for the fungal vacuole in short-term Ca 2+ homeostasis is indicated. This notion is supported by the observation that cytosolic Ca 2+ homeostasis is disrupted by a protonophore, which rapidly abolishes the driving force for Ca 2+ uptake into fungal vacuoles

Calcium influx affects intracellular transport and membrane repair following nanosecond pulsed electric field exposure.

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Thompson, Gary Lee; Roth, Caleb C; Dalzell, Danielle R; Kuipers, Marjorie; Ibey, Bennett L

2014-05-01

The cellular response to subtle membrane damage following exposure to nanosecond pulsed electric fields (nsPEF) is not well understood. Recent work has shown that when cells are exposed to nsPEF, ion permeable nanopores (2  nm) created by longer micro- and millisecond duration pulses. Nanoporation of the plasma membrane by nsPEF has been shown to cause a transient increase in intracellular calcium concentration within milliseconds after exposure. Our research objective is to determine the impact of nsPEF on calcium-dependent structural and repair systems in mammalian cells. Chinese hamster ovary (CHO-K1) cells were exposed in the presence and absence of calcium ions in the outside buffer to either 1 or 20, 600-ns duration electrical pulses at 16.2  kV/cm, and pore size was determined using propidium iodide and calcium green. Membrane organization was observed with morphological changes and increases in FM1-43 fluorescence. Migration of lysosomes, implicated in membrane repair, was followed using confocal microscopy of red fluorescent protein-tagged LAMP1. Microtubule structure was imaged using mEmerald-tubulin. We found that at high 600-ns PEF dosage, calcium-induced membrane restructuring and microtubule depolymerization coincide with interruption of membrane repair via lysosomal exocytosis.

Characterization of transport of calcium by microsomal membranes from roots maize

International Nuclear Information System (INIS)

Vaughan, M.A.

1985-01-01

This study investigates calcium transport by membranes of roots of maize isolated by differential centrifugation. The preparation was determined to be enriched in plasma membrane using market enzyme and electron microscopy. Using the 45 Ca filtration technique and liquid scintillation counting, vesicular calcium uptake was shown to be stimulated by added calmodulin and specific for and dependent on ATP. Conditions for maximal calcium accumulation were found to be 30 min incubation in the presence of 5 mM ATP, 5 mM MgCl 2 , 50 μM CaCl 2 , at 23 0 C, and at pH 6.5. Calcium uptake was inhibited by the ionophores A23187, X-537A, and ionomycin. Sodium fluoride, ruthenium red, and p-chloromercuribenzoate completely inhibited transport: diamide and vanadate produced slight inhibition; caffeine, caffeic acid, oligomycin, and ouabain produced little or no inhibition. Chlorpromazine, W7, trifluoperazine, and R 24 571 inhibit calcium uptake irrespective of added calmodulin, while W5 showed little effect on uptake. Verapamil, nifedipine, cinnarizine, flunarizine, lidoflazine, and diltiazem decreased calcium uptake by 17%-50%. Electron microscopic localization of calcium by pyroantimonate showed vesicles incubated with calmodulin and ATP showed the greatest amount of precipitate. These results suggest that these vesicles accumulate calcium in an ATP-dependent, calmodulin-stimulated manner

Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane

International Nuclear Information System (INIS)

Reid, D.M.; Molday, R.S.; Friedel, U.; Cook, N.J.

1990-01-01

After neuraminidase treatment the Na + /Ca 2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of M r 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na + /Ca 2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na + /Ca 2+ exchange activation by sodium. The authors further investigated the density of the Na + /Ca 2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na + /Ca 2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as they have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na + /Ca 2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

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Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

2017-05-01

The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Non-enzymatic access to the plasma membrane of Medicago root hairs by laser microsurgery

Energy Technology Data Exchange (ETDEWEB)

Kurkdjian, A.; Leitz, G.; Manigault, P.; Harim, A.; Greulich, K. O.

1993-07-01

Using UV laser microsurgery, the cell walls of root hairs from Medicago sativa (alfalfa) were perforated under plasmolysing conditions, giving direct access to the plasma membrane without enzyme treatment. The opening in the cell wall of a few μm in diameter results in immediate movement of the protoplasm and partial or complete extrusion of the cell contents. The movement of the protoplasm is retarded by increases in calcium concentration. The calcium-dependency of the movement of the protoplasm allows us to obtain preferentially the extrusion of protoplasm, or to gain access to a small area of plasma membrane in situ. The complete protoplasm can be expelled, to form a protoplast. Fluorescein diacetate staining indicated esterase activity and membrane integrity of the protoplasts. Microscopic examination revealed organelle movement and the presence of a nucleus. The plasma membrane was free from cell wall fragments, as shown by Tinopal staining. Conditions for obtaining plasmolysis without disturbing the physiology of the root hairs too much were achieved by slow, stepwise and reversible plasmolysis. Cytoplasmic streaming in root hairs was maintained during plasmolysis and laser microperforation. This laser technique should be suitable for the performance of electrophysiological studies using the patch-clamp technique on plasma membrane from non-enzyme-treated cells. (author)

Non-enzymatic access to the plasma membrane of Medicago root hairs by laser microsurgery

International Nuclear Information System (INIS)

Kurkdjian, A.; Leitz, G.; Manigault, P.; Harim, A.; Greulich, K.O.

1993-01-01

Using UV laser microsurgery, the cell walls of root hairs from Medicago sativa (alfalfa) were perforated under plasmolysing conditions, giving direct access to the plasma membrane without enzyme treatment. The opening in the cell wall of a few μm in diameter results in immediate movement of the protoplasm and partial or complete extrusion of the cell contents. The movement of the protoplasm is retarded by increases in calcium concentration. The calcium-dependency of the movement of the protoplasm allows us to obtain preferentially the extrusion of protoplasm, or to gain access to a small area of plasma membrane in situ. The complete protoplasm can be expelled, to form a protoplast. Fluorescein diacetate staining indicated esterase activity and membrane integrity of the protoplasts. Microscopic examination revealed organelle movement and the presence of a nucleus. The plasma membrane was free from cell wall fragments, as shown by Tinopal staining. Conditions for obtaining plasmolysis without disturbing the physiology of the root hairs too much were achieved by slow, stepwise and reversible plasmolysis. Cytoplasmic streaming in root hairs was maintained during plasmolysis and laser microperforation. This laser technique should be suitable for the performance of electrophysiological studies using the patch-clamp technique on plasma membrane from non-enzyme-treated cells. (author)

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

International Nuclear Information System (INIS)

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

1983-01-01

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

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding[OPEN

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Vincent, Thomas R.; Avramova, Marieta; Canham, James; Higgins, Peter; Bilkey, Natasha; Mugford, Sam T.; Pitino, Marco; Toyota, Masatsugu

2017-01-01

A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction. PMID:28559475

Functional implications of plasma membrane condensation for T cell activation.

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Carles Rentero

2008-05-01

Full Text Available The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC, which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.

Involvement of plasma membrane calcium influx in bacterial induction of the K+/H+ and hypersensitive responses in tobacco

International Nuclear Information System (INIS)

Atkinson, M.M.; Keppler, L.D.; Orlandi, E.W.; Baker, C.J.; Mischke, C.F.

1990-01-01

An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K + /H + response characterized by specific plasma membrane K + efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca 2+ influx of 0.02 to 0.06 micromole per gram per hour as determined from 45 Ca 2+ uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K + /H + response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca 2+ influx was prevented by EGTA and calcium channel blockers such as La 3+ , Co 2+ , and Cd 2+ but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K + /H + response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increase plasmalemma Ca 2+ influx is required for the K + /H + and hypersensitive responses in tobacco

A membrane model for cytosolic calcium oscillations. A study using Xenopus oocytes.

OpenAIRE

Jafri, M S; Vajda, S; Pasik, P; Gillo, B

1992-01-01

Cytosolic calcium oscillations occur in a wide variety of cells and are involved in different cellular functions. We describe these calcium oscillations by a mathematical model based on the putative electrophysiological properties of the endoplasmic reticulum (ER) membrane. The salient features of our membrane model are calcium-dependent calcium channels and calcium pumps in the ER membrane, constant entry of calcium into the cytosol, calcium dependent removal from the cytosol, and buffering ...

Calcium fluxes across the plasma membrane of Commelina communis L. assayed in a cell-free system

International Nuclear Information System (INIS)

Siebers, B.; Graef, P.; Weiler, E.W.

1990-01-01

The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous two-phase partitioning was loaded with 45 Ca 2+ through the action of the plasma membrane Ca 2+ -ATPase. Results suggest the presence of a Ca 2+ channel in the plasma membrane of C. communis. The channel is obtained in a Ca 2+ -inactivated state after preparation and Ca 2+ -loading of the vesicles. The inactivation is removed by TFP [trifluoperazine] or W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], presumably due to the Ca 2+ -mobilizing effect of these compounds. The activated Ca 2+ channel is La 3+ sensitive and, in the cell, would allow for passage of Ca 2+ into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed

Visualisation of an nsPEF induced calcium wave using the genetically encoded calcium indicator GCaMP in U87 human glioblastoma cells.

Science.gov (United States)

Carr, Lynn; Bardet, Sylvia M; Arnaud-Cormos, Delia; Leveque, Philippe; O'Connor, Rodney P

2018-02-01

Cytosolic, synthetic chemical calcium indicators are typically used to visualise the rapid increase in intracellular calcium ion concentration that follows nanosecond pulsed electric field (nsPEF) application. This study looks at the application of genetically encoded calcium indicators (GECIs) to investigate the spatiotemporal nature of nsPEF-induced calcium signals using fluorescent live cell imaging. Calcium responses to 44kV/cm, 10ns pulses were observed in U87-MG cells expressing either a plasma membrane targeted GECI (GCaMP5-G), or one cytosolically expressed (GCaMP6-S), and compared to the response of cells loaded with cytosolic or plasma membrane targeted chemical calcium indicators. Application of 100 pulses, to cells containing plasma membrane targeted indicators, revealed a wave of calcium across the cell initiating at the cathode side. A similar spatial wave was not observed with cytosolic indicators with mobile calcium buffering properties. The speed of the wave was related to pulse application frequency and it was not propagated by calcium induced calcium release. Copyright © 2017 Elsevier B.V. All rights reserved.

Plasma membrane ATPases

DEFF Research Database (Denmark)

Palmgren, Michael Broberg; Bækgaard, Lone; Lopez Marques, Rosa Laura

2011-01-01

The plasma membrane separates the cellular contents from the surrounding environment. Nutrients must enter through the plasma membrane in order to reach the cell interior, and toxic metabolites and several ions leave the cell by traveling across the same barrier. Biological pumps in the plasma me...

Plasma concentration of ionized calcium in healthy iguanas.

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Dennis, P M; Bennett, R A; Harr, K E; Lock, B A

2001-08-01

To measure plasma concentration of ionized calcium in healthy green iguanas. Prospective study. 9 juvenile and 21 (10 male, 11 female) adult iguanas. Blood samples were obtained from each iguana, and plasma calcium, glucose, phosphorus, uric acid, total protein, albumin, globulin, potassium, and ionized calcium concentrations, aspartate transaminase (AST) activity, and pH were measured. Heparinized blood was used for measurement of ionized calcium concentration and blood pH. A CBC was also performed to assess the health of the iguanas. Significant differences were not detected among the 3 groups (juveniles, males, and females) with regard to ionized calcium concentration. Mean ionized calcium concentration measured in blood was 1.47 +/- 0.105 mmol/L. Significant differences were detected between juveniles and adults for values of phosphorus, glucose, total protein, albumin, globulin, and AST activity. Ionized calcium concentration provides a clinical measurement of the physiologically active calcium in circulation. Evaluation of physiologically active calcium in animals with suspected calcium imbalance that have total plasma calcium concentrations within reference range or in gravid animals with considerably increased total plasma calcium concentrations is vital for determining a therapeutic plan. Accurate evaluation of calcium status will provide assistance in the diagnosis of renal disease and seizures and allow for better evaluation of the health status of gravid female iguanas.

Physiological implications of seasonal variation in membrane-associated calcium in red spruce mesophyll cells

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D.H. DeHayes; P.G. Schaberg; G.J. Hawley; C.H. Borer; J.R. Cumming; J.R. Strimbeck

1997-01-01

We examined the pattern of seasonal variation in total foliar calcium (Ca) pools and plasma membrane-associated Ca (mCa) in mesophyll cells of current-year and 1-year-old needles of red spruce (Picea rubens Sarg.) and the relationship between mCa and total foliar Ca on an individual plant and seasonal basis. Foliar samples were collected from...

Every day I'm rufflin': Calcium sensing and actin dynamics in the growth factor-independent membrane ruffling of professional phagocytes.

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Schlam, Daniel; Canton, Johnathan

2017-04-03

Professional phagocytes continuously extend dynamic, actin-driven membrane protrusions. These protrusions, often referred to as membrane ruffles, serve a critical role in the essential phagocyte processes of macropinocytosis and phagocytosis. Small GTPases, such as RAC1/2, spatially and temporally regulate membrane ruffle formation. We have recently shown that extracellular calcium regulates the elaboration of membrane ruffles primarily through the synthesis of phosphatidic acid (PtdOH) at the plasma membrane. RAC1/2 guanine nucleotide exchange factors harbouring polybasic stretches are recruited by PtdOH to sites of ruffle formation. Here we discuss our findings and offer perspectives on how the regulation of dynamic actin structures at the plasma membrane by small GTPases is a critical component of phagocyte function.

Liver plasma membranes: an effective method to analyze membrane proteome.

Science.gov (United States)

Cao, Rui; Liang, Songping

2012-01-01

Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

Comparative immunolocalization of the plasma membrane calcium pump and calbindin D28K in chicken retina during embryonic development

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N. Tolosa de Talamoni

2010-05-01

Full Text Available The immunolocalization of the plasma membrane calcium pump (PMCA was studied in 4-week-old chick retina in comparison with calbindin D28K (CaBP immunostaining. We have demonstrated that the monoclonal anti-PMCA antibody 5F10 from human erythrocyte plasma membrane crossreacts with a Ca2+ pump epitope of the cells from the neural retina. The immunolocalization of both proteins was also studied during the embryonic development of the chicken retina. At age 4.5 days, the cells of the retina were faintly immunoreactive to PMCA and CaBP antibodies, but the lack of cellular aggregation and differentiation did not allow discrimination between the two proteins. A clear difference in the localization was seen from the tenth day of development through post-hatching with slight variation. PMCA localized mainly in the outer and inner plexiform layers, in some cells in the ganglion layer, in the nerve fiber layer and slightly in the photoreceptor cells. CaBP was intensely stained in cones, cone pedicles and some amacrine cells. The number of CaBP positive amacrine cells declined after hatching. A few ganglion cells and several nerve fibers were CaBP 333 immunoreactive. The role of these proteins in the early stages of retinal development is unknown, but the results suggest that Ca2+ homeostasis in the retina is well regulated, probably to avoid excessive accumulation of Ca2+, which often leads to neurodegeneration.

Depletion of intracellular calcium stores facilitates the influx of extracellular calcium in platelet derived growth factor stimulated A172 glioblastoma cells.

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Vereb, G; Szöllösi, J; Mátyus, L; Balázs, M; Hyun, W C; Feuerstein, B G

1996-05-01

Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.

Membrane order in the plasma membrane and endocytic recycling compartment.

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Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

Recognition of acidic phospholipase A2 activity in plasma membranes of resident peritoneal macrophages

International Nuclear Information System (INIS)

Shibata, Y.; Abiko, Y.; Ohno, H.; Araki, T.; Takiguchi, H.

1988-01-01

Phospholipase (PLase) activities in the plasma membrane of guinea pig peritoneal macrophages were studied, as these enzymes having such activity may be candidates for the release of arachidonic acid (AA) from phosphatidylcholine (PC). An AA release system operating at acidic pH was identified in the macrophage plasma membrane and characterized. This membrane-bound acidic PLase A 2 had an optimum pH at 4.5, and enzyme activation was observed in Ca ++ -free medium; but the maximum activity was found at 0.5 mM Ca ++ concentration. The Km value for PC of acidic PLase A 2 was 4.2 μM, and a Michaelis-Menten relationship was evident. Calcium might act as a cofactor at some intermediate step during the activation of acidic PLase A 2 in light of the uncompetitive manner of Ca ++ action. Furthermore, the release of [ 3 H]-AA from preradiolabelled macrophage plasma membranes occurred with the addition of Ca ++ at pH 4.5. These data suggest that the acid PLase A 2 is a component of the plasma membrane and is not due to lysosomal contamination since membrane-bound acidic PLase A 2 properties are opposite to those found for lysosomal PLase A 2

Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion

International Nuclear Information System (INIS)

Blowers, D.P.; Boss, W.F.; Trewavas, A.J.

1988-01-01

Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ- 32 P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M r 22,000. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic 32 P also showed a response to the Driselase treatment. An enzymically active driselas preparation was required for the observed responses

C2-domain containing calcium sensors in neuroendocrine secretion

DEFF Research Database (Denmark)

Pinheiro, Paulo S; Houy, Sébastien; Sørensen, Jakob B

2016-01-01

The molecular mechanisms for calcium-triggered membrane fusion have long been sought for, and detailed models now exist that account for at least some of the functions of the many proteins involved in the process. Key players in the fusion reaction are a group of proteins that, upon binding...... to calcium, trigger the merger of cargo-filled vesicles with the plasma membrane. Low-affinity, fast-kinetics calcium sensors of the synaptotagmin family - especially synaptotagmin-1 and synaptotagmin-2 - are the main calcium sensors for fast exocytosis triggering in many cell types. Their functions extend...... beyond fusion triggering itself, having been implicated in the calcium-dependent vesicle recruitment during activity, docking of vesicles to the plasma membrane and priming, and even in post-fusion steps, such as fusion pore expansion and endocytosis. Furthermore, synaptotagmin diversity imparts distinct...

Biogenesis of plasma membrane cholesterol

International Nuclear Information System (INIS)

Lange, Y.

1986-01-01

A striking feature of the molecular organization of eukaryotic cells is the singular enrichment of their plasma membranes in sterols. The authors studies are directed at elucidating the mechanisms underlying this inhomogeneous disposition. Cholesterol oxidase catalyzes the oxidation of plasma membrane cholesterol in intact cells, leaving intracellular cholesterol pools untouched. With this technique, the plasma membrane was shown to contain 95% of the unesterified cholesterol of cultured human fibroblasts. Cholesterol synthesized from [ 3 H] acetate moved to the plasma membrane with a half-time of 1 h at 37 0 C. They used equilibrium gradient centrifugation of homogenates of biosynthetically labeled, cholesterol oxidase treated cells to examine the distribution of newly synthesized sterols among intracellular pools. Surprisingly, lanosterol, a major precursor of cholesterol, and intracellular cholesterol both peaked at much lower buoyant density than did 3-hydroxy-3-methylglutaryl-CoA reductase. This suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. The cholesterol in the buoyant fraction eventually moved to the plasma membrane. Digitonin treatment increased the density of the newly synthesized cholesterol fractions, indicating that nascent cholesterol in transit is associated with cholesterol-rich membranes. The authors are testing the hypothesis that the pathway of cholesterol biosynthesis is spatially organized in various intracellular membranes such that the sequence of biosynthetic steps both concentrates the sterol and conveys it to the plasma membrane

Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

Science.gov (United States)

Raza Shaikh, Saame; Brown, David A

2013-01-01

Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mechanism of store-operated calcium entry

Indian Academy of Sciences (India)

Activation of receptors coupled to the phospholipase C/IP3 signalling pathway results in a rapid release of calcium from its intracellular stores, eventually leading to depletion of these stores. Calcium store depletion triggers an influx of extracellular calcium across the plasma membrane, a mechanism known as the ...

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level

Science.gov (United States)

Belyavskaya, N. A.

The changes of [Ca^2+]_i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station ``Salyut 6'' /1/. These results: 1) indicate that observed Ca^2+-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca^2+ influx through membranes. In model presented, I propose that Ca^2+-activated channels in plasma membrane in response to microgravity allow the movement of Ca^2+ into the root cells, causing a rise in cytoplasmic free Ca^2+ levels. The latter, in its turn, may induce the inhibition of a Ca^2+ efflux by Ca^2+-activated ATPases and through a Ca^2+/H^+ antiport. It is possible that increased cytosolic levels of Ca^2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca^2+]_i. Plant cell can response to such a Ca^2+ rise by an enhancement of membranous Ca^2+-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca^2+-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca^2+ to plant cell.

Plasma oxalic acid and calcium levels in oxalate poisoning

Science.gov (United States)

Zarembski, P. M.; Hodgkinson, A.

1967-01-01

Observations are reported on five cases of suicide or attempted suicide by poisoning with oxalic acid or ethylene glycol. Elevated oxalic acid levels were observed in the plasma, stomach contents, and a number of tissues. Raised oxalic acid levels in plasma were associated with reduced total and ultrafilterable calcium levels. It is suggested that the reduction in plasma total calcium level is due mainly to the deposition of calcium oxalate in the soft tissues, but inhibition of the parathyroid glands may be a contributory factor. Microscopic examination of various tissues indicated that oxalic acid is deposited in the tissues in two forms: (1) crystalline calcium oxalate dihydrate in the kidney and (2) a non-crystalline complex of calcium oxalate and lipid in liver and other tissues. PMID:5602563

Proteomics and the dynamic plasma membrane

DEFF Research Database (Denmark)

Sprenger, Richard R; Jensen, Ole Nørregaard

2010-01-01

plasma membrane is of particular interest, by not only serving as a barrier between the "cell interior" and the external environment, but moreover by organizing and clustering essential components to enable dynamic responses to internal and external stimuli. Defining and characterizing the dynamic plasma...... the challenges in functional proteomic studies of the plasma membrane. We review the recent progress in MS-based plasma membrane proteomics by presenting key examples from eukaryotic systems, including mammals, yeast and plants. We highlight the importance of enrichment and quantification technologies required...... for detailed functional and comparative analysis of the dynamic plasma membrane proteome....

Further characterization of the red beet plasma membrane Ca2+-ATPase using GTP as an alternative substrate

International Nuclear Information System (INIS)

Williams, L.E.; Schueler, S.B.; Briskin, D.P.

1990-01-01

The GTP-driven component of Ca 2+ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca 2+ -translocating ATPase and assess its utility as a probe for this transport system. Uptake of 45 Ca 2+ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca 2+ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca 2+ -ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of 45 Ca 2+ -uptake by exogeneous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven 45 Ca 2+ uptake represents the capacity of the plasma membrane Ca 2+ -translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ- 32 P]GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca 2+ -ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca 2+ -ATPases present in animal cells

KB-R7943, a plasma membrane Na(+)/Ca(2+) exchanger inhibitor, blocks opening of the mitochondrial permeability transition pore.

Science.gov (United States)

Wiczer, Brian M; Marcu, Raluca; Hawkins, Brian J

2014-01-31

The isothiourea derivative, KB-R7943, inhibits the reverse-mode of the plasma membrane sodium/calcium exchanger and protects against ischemia/reperfusion injury. The mechanism through which KB-R7943 confers protection, however, remains controversial. Recently, KB-R7943 has been shown to inhibit mitochondrial calcium uptake and matrix overload, which may contribute to its protective effects. While using KB-R7943 for this purpose, we find here no evidence that KB-R7943 directly blocks mitochondrial calcium uptake. Rather, we find that KB-R7943 inhibits opening of the mitochondrial permeability transition pore in permeabilized cells and isolated liver mitochondria. Furthermore, we find that this observation correlates with protection against calcium ionophore-induced mitochondrial membrane potential depolarization and cell death, without detrimental effects to basal mitochondrial membrane potential or complex I-dependent mitochondrial respiration. Our data reveal another mechanism through which KB-R7943 may protect against calcium-induced injury, as well as a novel means to inhibit the mitochondrial permeability transition pore. Copyright © 2014 Elsevier Inc. All rights reserved.

Glucagon effects on the membrane potential and calcium uptake rate of rat liver mitochondria

International Nuclear Information System (INIS)

Wingrove, D.E.; Amatruda, J.M.; Gunter, T.E.

1984-01-01

It has been widely reported that the in vivo administration of glucagon to rats results in the stimulation of calcium influx in subsequently isolated liver mitochondria. The mechanism of this effect is investigated through simultaneous measurements of calcium uptake rate and mitochondrial membrane potential. This allows the measurement of the calcium uniporter conductance independent of hormonal effects on electron transport or respiration. Two experimental approaches are used. The first involves measuring the uptake of 40-50 nmol of Ca 2+ /mg of mitochondrial protein with the calcium dye antipyrylazo III; the second uses 45 Ca 2+ to follow uptake in the presence of 0.5 to 1.5 μM free calcium, buffered with HEDTA. In both cases a tetraphenyl phosphonium electrode is used to follow membrane potential, and membrane potential is varied using either malonate or butylmalonate in the presence of rotenone. The relative merits of these two approaches are discussed. The conductance of the calcium uniporter is found not to be stimulated by glucagon pretreatment. Also, the relative glucagon stimulation of both calcium influx and membrane potential is found to increase with increasing malonate concentration. These results imply that there is no direct stimulation of calcium uptake into liver mitochondria following glucagon treatment. The results are consistent with a glucagon stimulation of substrate transport, substrate oxidation, or a stimulation of electron transport resulting in an increased membrane potential and secondary stimulation of calcium uptake

Lipid organization of the plasma membrane

NARCIS (Netherlands)

Ingólfsson, Helgi I; Melo, Manuel N; van Eerden, Floris J; Arnarez, Clément; Lopez, Cesar A; Wassenaar, Tsjerk A; Periole, Xavier; de Vries, Alex H; Tieleman, D Peter; Marrink, Siewert J

2014-01-01

The detailed organization of cellular membranes remains rather elusive. Based on large-scale molecular dynamics simulations, we provide a high-resolution view of the lipid organization of a plasma membrane at an unprecedented level of complexity. Our plasma membrane model consists of 63 different

Mg(2+,ATP-dependent plasma membrane calcium pump of smooth muscle cells. ІІ. Regulation of activity

Directory of Open Access Journals (Sweden)

T. О. Veklich

2015-04-01

Full Text Available Plasma membrane Ca2+-pump is one of key proteins, which takes part in Ca2+ exchange in smooth muscle cells. It has a lot of diverse functions from control of basal cytoplasmal Ca2+ concentration to regulation of proteins involved in Ca2+-dependent signal pathway. Ca2+ pump function is often depen­dent on the isoform or even form of alternative splicing. Allowing for a variety of Ca2+-pump functions and properties, which were reviewed in detail in the first part of our review article cycle (Ukr. Biochem. J., 2015; 87(1, the precise control of the mentioned pump activity is very important for cell functioning­. The other part of this article is dedicated to different regulation factors of smooth muscle plasma membrane Ca2+-pump activity: endogenous and exo­genous, biotic and abiotic factors. Special attention is given to literature data and own results about design and the search of selective plasma membrane Ca2+-pump inhibitor which would allow examining its functioning in smooth muscle cells more meticulously.

The plasma membrane as radiosensitive target

International Nuclear Information System (INIS)

Koeteles, Gy.J.

1986-01-01

Components and conditions rendering the plasma membrane susceptible for ionizing radiation are discussed. The list of reviews and articles pointing to various aspects of radiation effects on membranes is analyzed. Radiation induced alterations of plasma membrane and energy deposition in cellular microstructures are overviewed. The possible role of membrane alterations in the fate of irradiated cell is also discussed. (author)

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

Science.gov (United States)

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

2017-11-09

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

Plasma membrane potential depolarization and cytosolic calcium flux are early events involved in tomato (Solanum lycopersicon) plant-to-plant communication.

Science.gov (United States)

Zebelo, Simon A; Matsui, Kenji; Ozawa, Rika; Maffei, Massimo E

2012-11-01

Tomato plants respond to herbivory by emitting volatile organic compounds (VOCs), which are released into the surrounding atmosphere. We analyzed the tomato herbivore-induced VOCs and tested the ability of tomato receiver plants to detect tomato donor volatiles by analyzing early responses, including plasma membrane potential (V(m)) variations and cytosolic calcium ([Ca²⁺](cyt)) fluxes. Receiver tomato plants responded within seconds to herbivore-induced VOCs with a strong V(m) depolarization, which was only partly recovered by fluxing receiver plants with clean air. Among emitted volatiles, we identified by GC-MS some green leaf volatiles (GLVs) such as (E)-2-hexenal, (Z)-3-hexenal, (Z)-3-hexenyl acetate, the monoterpene α-pinene, and the sesquiterpene β-caryophyllene. GLVs were found to exert the stronger V(m) depolarization, when compared to α-pinene and β-caryophyllene. Furthermore, V(m) depolarization was found to increase with increasing GLVs concentration. GLVs were also found to induce a strong [Ca²⁺](cyt) increase, particularly when (Z)-3-hexenyl acetate was tested both in solution and with a gas. On the other hand, α-pinene and β-caryophyllene, which also induced a significant V(m) depolarization with respect to controls, did not exert any significant effect on [Ca²⁺](cyt) homeostasis. Our results show for the first time that plant perception of volatile cues (especially GLVs) from the surrounding environment is mediated by early events, occurring within seconds and involving the alteration of the plasma membrane potential and the [Ca²⁺](cyt) flux. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

[Effects of desulfurization waste on calcium distribution, Ca(2+)-ATPase activity, and antioxidant characteristics of rice leaf under alkali stress].

Science.gov (United States)

Mao, Gui-Lian; Xu, Xing; Zeng, Jin; Yue, Zi-Hui; Yang, Shu-Juan

2012-02-01

To approach the action mechanisms of desulfurization waste on alleviating alkali stress-induced injury of rice, a pot experiment was conducted to study the variations of leaf total calcium content, calcium distribution, plasma membrane Ca(2+)-ATPase activity, and reactive oxygen content of rice seedlings under alkali stress after the application of desulfurization waste. In the control, a few calcium particulates scattered in the cell wall and chloroplasts, while applying desulfurization waste or CaSO4 increased the calcium particulates in the plasma membrane, intercellular space, cell wall, and vacuole significantly. With the increasing application rate of desulfurization waste or CaSO4, the leaf total calcium content increased, Ca(2+)-ATPase activity in plasma membrane and tonoplast presented an increasing trend, plasma membrane relative permeability, MDA content, and O2 production rate decreased, and SOD and POD activities increased. The desulfurization waste could relieve the alkali stress to rice in some extent, and the main reactive compound in the waste could be CaSO4.

Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

Science.gov (United States)

Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

2007-08-17

Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

On reactive suspension plasma spraying of calcium titanate

OpenAIRE

Kotlan, J. (Jiří); Pala, Z. (Zdeněk); Mušálek, R. (Radek); Ctibor, P. (Pavel)

2016-01-01

This study shows possibility of preparation of calcium titanate powder and coatings by reactive suspension plasma spraying. Suspension of mixture of calcium carbonate (CaCO3) and titanium dioxide (TiO2) powders in ethanol was fed into hybrid plasma torch with a DC-arc stabilized by a water–argon mixture (WSP-H 500). Various feeding distances and angles were used in order to optimize suspension feeding conditions. In the next step, the coatings were deposited on stainless steel substrates and ...

Regulation of intracellular calcium in resting and stimulated rat basophilic leukemia cells

International Nuclear Information System (INIS)

Mohr, F.C.

1988-01-01

Intracellular calcium regulation was studied in a cell line of mast cells, the rat basophilic leukemia (RBL) cells with the purpose of determining (1) The properties of the plasma membrane calcium permeability pathway and (2) The role of intracellular calcium stores. The first set of experiments showed that depolarization did not induce calcium entry or secretion in resting cells and did inhibit antigen-stimulated calcium uptake and secretion. In the second set of experiments the ionic basis of antigen-induced depolarization was studied using the fluorescent potential-sensitive probe bis-oxonol. The properties of the calcium entry pathway were more consistent with a calcium channel than a calcium transport mechanism such as Na:Ca exchange. The third set of experiments examined the effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) on RBL cells. CCCP inhibited antigen-stimulated 45 Ca uptake and secretion by depolarizing the plasma membrane

Membrane Targeting of P-type ATPases in Plant Cells

International Nuclear Information System (INIS)

Harper, Jeffrey F.

2004-01-01

How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

Plasma membrane isolation using immobilized concanavalin A magnetic beads.

Science.gov (United States)

Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

2012-01-01

Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells 1

Science.gov (United States)

Arora, Rajeev; Palta, Jiwan P.

1988-01-01

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K+, along with small but significant loss of cellular Ca2+. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca2+ from membrane by extracellular K+ and subsequent perturbation of K+ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca2+ compared to control. Loss of membrane-associated Ca2+ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca2+-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl2. Our results suggest that the loss of membrane-associated Ca2+, in part, plays a role in initiation and progression of freezing injury. Images Fig. 1 Fig. 2 PMID:16666196

Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's β-Amyloid Protein

Directory of Open Access Journals (Sweden)

Masahiro Kawahara

2011-01-01

Full Text Available Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer's β-amyloid protein (AβP play crucial roles in the pathogenesis of Alzheimer's disease (AD. Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”, and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.

Hydrogen superpermeable membrane operation under plasma conditions

International Nuclear Information System (INIS)

Bacal, M.; Bruneteau, A.M.; Livshits, A.I.; Alimov, V.N.; Notkin, M.E.

2003-01-01

The effect of ion bombardment on hydrogen plasma-driven permeation through a superpermeable niobium membrane was investigated. It was found that the increase of membrane temperature and the doping of membrane material with oxygen results in the decrease of ion bombardment effect and in permeability increase. It was demonstrated that membrane decarbonization leads to the formation of a membrane state resistant to sputtering. Possible applications of the membrane resistant to ion bombardment as plasma facing components are considered

Bcl-2 overexpression: effects on transmembrane calcium movement

International Nuclear Information System (INIS)

Rangaswami, Arun A.; Premack, Brett; Walleczek, Jan; Killoran, Pamela; Gardner, Phyllis; Knox, Susan J.

1996-01-01

Purpose/Objective: High levels of expression of the proto-oncogene bcl-2 and its 26 kD protein product Bcl-2 have been correlated with the inhibition of apoptosis and the increased resistance of tumor cells to cytotoxic drugs and ionizing radiation. Unfortunately, the specific mechanism of action of Bcl-2 remains poorly understood. In the studies described here, the role of intracellular calcium fluxes and plasma membrane calcium cycling in the induction of apoptosis, and the effect of Bcl-2 expression on the modulation of transmembrane calcium fluxes following treatment of cells with cytotoxic agents were studied. The relationship between intracellular calcium release, capacitive calcium entry, and the plasma membrane potential were also investigated. Materials and Methods: Human B-cell lymphoma (PW) and human promyelocytic leukemia (HL60) cell lines were transfected with Bcl-2 and a control vector. The Bcl-2 transfectants over expressed the Bcl-2 onco-protein and were more resistant to irradiation than the control cells. Cells were loaded with fluorescent indicators indo-1 and fura-2 AM to quantify the cytosolic calcium concentration and subsequent calcium responses to a variety of cytotoxic stimuli, including the microsomal ATPase inhibitor, thapsigargin, using fluorometric measurements. Comparisons of resting and stimulated cytosolic calcium concentrations were made between the parental, neomycin control, and bcl-2 transfected cells. In order to determine the actual calcium influx rate, cells were loaded with either indo-1 or fura-2 and then exposed to 0.1 mM extracellular manganese, which enters the cells through calcium influx channels and quenches the fluorescent signal in proportion to the calcium influx rate. In order to determine the role of the membrane potential in driving calcium influx, cells were treated with either 0.1 μM Valinomycin or isotonic potassium chloride to either hyper polarize or depolarize the resting membrane potential, and the

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane

OpenAIRE

Doudová, Lenka

2017-01-01

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane Yeast plasma membrane is divided into several different compartments. Membrane compartment of Can1 is specific for its protein and lipid composition, furthermore it creates furrow-like invaginations on the plasma membrane. These invaginations are made by multiprotein complexes called eisosomes, which are located in the cytosolic side of MCCs. It was established that this domain plays an importa...

Caveolae as plasma membrane sensors, protectors and organizers.

Science.gov (United States)

Parton, Robert G; del Pozo, Miguel A

2013-02-01

Caveolae are submicroscopic, plasma membrane pits that are abundant in many mammalian cell types. The past few years have seen a quantum leap in our understanding of the formation, dynamics and functions of these enigmatic structures. Caveolae have now emerged as vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment. Caveolae at the plasma membrane can be removed by endocytosis to regulate their surface density or can be disassembled and their structural components degraded. Coat proteins, called cavins, work together with caveolins to regulate the formation of caveolae but also have the potential to dynamically transmit signals that originate in caveolae to various cellular destinations. The importance of caveolae as protective elements in the plasma membrane, and as membrane organizers and sensors, is highlighted by links between caveolae dysfunction and human diseases, including muscular dystrophies and cancer.

Metabolism of phosphatidylinositol in plasma membranes and synaptosomes of rat cerebral cortex: A comparison between endogenous vs exogenous substrate pools

International Nuclear Information System (INIS)

Navidi, M.; MacQuarrie, R.A.; Sun, G.Y.

1990-01-01

The metabolism of phosphatidylinositols (PI) labeled with [14C]arachidonic acid within plasma membranes or synaptosomes was compared to the metabolism of PI prelabeled with [14C]arachidonic acid and added exogenously to the same membranes. Incubation of membranes containing the endogenously-labeled PI pool in the presence of Ca2+ resulted in the release of labeled arachidonic acid, as well as a small amount of labeled diacylglycerol. Labeled arachidonic acid was effectively reutilized and returned to the membrane phospholipids in the presence of adenosine triphosphate (ATP), CoA, and lysoPI. Although Ca2+ promoted the release of labeled diacylglycerol from prelabeled plasma membranes, this amount was only 17% of the maximal release, i.e., release in the presence of deoxycholate and Ca2+. This latter condition is known to fully activate the PI-phospholipase C, and incubation of prelabeled plasma membranes resulted in a six-fold increase in labeled diacylglycerols. On the other hand, when exogenously labeled PI were incubated with plasma membranes in the presence of Ca2+, the labeled diacylglycerols released were 59% of that compared to the fully activated condition. The phospholipase C action was calcium-dependent, regardless of whether exogenous or endogenous substrates were used in the incubation. In contrast to plasma membranes, intact synaptosomes had limited ability to metabolize exogenous PI even in the presence of Ca2+, although the activity of phospholipase C was similar to that in the plasma membranes when assayed in the presence of deoxycholate and Ca2+. These results suggest that discrete pools of PI are present in plasma membranes, and that the pool associated with the acyltransferase is apparently not readily accessible to hydrolysis by phospholipase C

Autophagosomal membranes assemble at ER-plasma membrane contact sites.

Science.gov (United States)

Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

2017-01-01

The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

Characterization of branchial transepithelial calcium fluxes in freshwater trout, Salmo gairdneri

International Nuclear Information System (INIS)

Perry, S.F.; Flik, G.

1988-01-01

Experiments were performed to determine whether gill transepithelial calcium fluxes in the freshwater trout (Salmo gairdneri) are passive or require active transport and to characterize the mechanism involved. A comparison of the in vivo unidirectional flux ratios with the flux ratios calculated according to the transepithelial electrochemical gradients revealed that calcium uptake from the water requires active transport of Ca 2+ . The inhibition of calcium uptake by external lanthanum, the specific deposition of lanthanum on the apical surface of chloride cells, and the favorable electrochemical gradient for calcium across the apical membrane suggest that the initial step in branchial calcium uptake is the passive entry of calcium into the cytosol of chloride cells through apical channels that are permeable to calcium. The study of gill basolateral plasma membrane vesicles demonstrated the existence of a high-affinity calmodulin-dependent calcium-transporting system. This system actively transports calcium from the cytosol of chloride cells into the plasma against a sizeable electrochemical gradient, thereby completing the transepithelial uptake of calcium. Calcium efflux occurs passively through paracellular pathways between chloride cells and adjacent pavement cells or between neighboring pavement cells

Experimental study of membrane pump for plasma devices

International Nuclear Information System (INIS)

Suzuki, Hajime; Ohyabu, Nobuyoshi; Nakamura, Yukio; Sagara, Akio; Motojima, Osamu; Livshits, A.; Notkin, M.; Busnyuk, A.; Komatsu, Kazuyuki

1998-01-01

Recycling control is a key to improve fusion plasma performance. The membrane pump has potential advantages for hydrogen pumping in fusion devices. However, there are unsolved issues for using membrane pump in LHD (Large Helical Device). The first issue is characteristics of the membrane pump under high incident hydrogen atom flux. The second issue is relationship between the surface condition and the pumping efficiency. Impurities from plasma may change the surface condition of the membrane. In order to solve these issues, a membrane pump system was fabricated and installed in a linear plasma device at NIFS (National Institute for Fusion Science). The membrane pump was successfully operated. (author)

Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

International Nuclear Information System (INIS)

Haylett, T.; Thilo, L.

1986-01-01

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D 1 , was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

Plasma membrane disruption: repair, prevention, adaptation

Science.gov (United States)

McNeil, Paul L.; Steinhardt, Richard A.

2003-01-01

Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

Membrane potential, serum calcium and serum selenium decrease in preeclampsia subjects in Owerri

Directory of Open Access Journals (Sweden)

Johnkennedy Nnodim

2017-08-01

Full Text Available Background Pre-eclampsia is a serious hypertensive condition of pregnancy associated with high maternal and fetal morbidity and mortality. Women who have had pre-eclampsia have a greater risk of developing hypertension, stroke and ischemic heart disease in later life. The etiology of pre-eclampsia remains unclear. Placental insufficiency plays a key role in the progression of this disease. The aim of this study was to determine membrane potential, serum calcium and serum selenium levels in preeclampsia subjects in Owerri.   Methods A case control study involving 200 primigravida (100 preeclamptic and 100 apparently healthy between the ages of 20 and 32 years attending General Hospital Owerri. Fasting venous blood was collected for the determination of serum selenium and serum calcium while membrane potential was calculated using the Nernst equation. The serum calcium was estimated using Randox Kit and serum selenium by atomic absorption spectrophotometry. The Independent Student t test was used for statistical analysis.   Results The results revealed that membrane potential and serum selenium as well as serum calcium were significantly decreased in preeclampsia when compared with the controls, at p<0.05.   Conclusion Our study demonstrated that the decrease in membrane potential, serum calcium and serum selenium levels may play a critical role in the pathogenesis of pre-eclampsia. There may be a need for increasing the dietary intake of these essential trace metals during pregnancy to prevent pre-eclampsia in Owerri.

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

International Nuclear Information System (INIS)

Hu Chuan; Hardee, Deborah; Minnear, Fred

2007-01-01

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins

Quantitative analysis of self-association and mobility of annexin A4 at the plasma membrane.

Science.gov (United States)

Crosby, Kevin C; Postma, Marten; Hink, Mark A; Zeelenberg, Christiaan H C; Adjobo-Hermans, Merel J W; Gadella, Theodorus W J

2013-05-07

Annexins, found in most eukaryotic species, are cytosolic proteins that are able to bind negatively-charged phospholipids in a calcium-dependent manner. Annexin A4 (AnxA4) has been implicated in diverse cellular processes, including the regulation of exocytosis and ion-transport; however, its precise mechanistic role is not fully understood. AnxA4 has been shown to aggregate on lipid layers upon Ca(2+) binding in vitro, a characteristic that may be critical for its function. We have utilized advanced fluorescence microscopy to discern details on the mobility and self-assembly of AnxA4 after Ca(2+) influx at the plasma membrane in living cells. Total internal reflection microscopy in combination with Förster resonance energy transfer reveals that there is a delay between initial plasma membrane binding and the beginning of self-assembly and this process continues after the cytoplasmic pool has completely relocated. Number-and-brightness analysis suggests that the predominant membrane bound mobile form of the protein is trimeric. There also exists a pool of AnxA4 that forms highly immobile aggregates at the membrane. Fluorescence recovery after photobleaching suggests that the relative proportion of these two forms varies and is correlated with membrane morphology. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The plasma membrane proteome of germinating barley embryos

DEFF Research Database (Denmark)

Hynek, Radovan; Svensson, Birte; Jensen, O.N.

2009-01-01

Cereal seed germination involves a complex coordination between different seed tissues. Plasma membranes must play crucial roles in coordination and execution of germination; however, very little is known about seed plasma membrane proteomes due to limited tissue amounts combined...... with amphiphilicity and low abundance of membrane proteins. A fraction enriched in plasma membranes was prepared from embryos dissected from 18 h germinated barley seeds using aqueous two-phase partitioning. Reversed-phase chromatography on C-4 resin performed in micro-spin columns with stepwise elution by 2-propanol...... was used to reduce soluble protein contamination and enrich for hydrophobic proteins. Sixty-one proteins in 14 SDS-PAGE bands were identified by LC-MS/MS and database searches. The identifications provide new insight into the plasma membrane functions in seed germination....

Single cell wound generates electric current circuit and cell membrane potential variations that requires calcium influx.

Science.gov (United States)

Luxardi, Guillaume; Reid, Brian; Maillard, Pauline; Zhao, Min

2014-07-24

Breaching of the cell membrane is one of the earliest and most common causes of cell injury, tissue damage, and disease. If the compromise in cell membrane is not repaired quickly, irreversible cell damage, cell death and defective organ functions will result. It is therefore fundamentally important to efficiently repair damage to the cell membrane. While the molecular aspects of single cell wound healing are starting to be deciphered, its bio-physical counterpart has been poorly investigated. Using Xenopus laevis oocytes as a model for single cell wound healing, we describe the temporal and spatial dynamics of the wound electric current circuitry and the temporal dynamics of cell membrane potential variation. In addition, we show the role of calcium influx in controlling electric current circuitry and cell membrane potential variations. (i) Upon wounding a single cell: an inward electric current appears at the wound center while an outward electric current is observed at its sides, illustrating the wound electric current circuitry; the cell membrane is depolarized; calcium flows into the cell. (ii) During cell membrane re-sealing: the wound center current density is maintained for a few minutes before decreasing; the cell membrane gradually re-polarizes; calcium flow into the cell drops. (iii) In conclusion, calcium influx is required for the formation and maintenance of the wound electric current circuitry, for cell membrane re-polarization and for wound healing.

Fatty acid profiles from the plasma membrane and detergent resistant membranes of two plant species.

Science.gov (United States)

Carmona-Salazar, Laura; El Hafidi, Mohammed; Gutiérrez-Nájera, Nora; Noyola-Martínez, Liliana; González-Solís, Ariadna; Gavilanes-Ruíz, Marina

2015-01-01

It is essential to establish the composition of the plant plasma membrane in order to understand its organization and behavior under continually changing environments. Knowledge of the lipid phase, in particular the fatty acid (FA) complex repertoire, is important since FAs determine many of the physical-chemical membrane properties. FAs are constituents of the membrane glycerolipid and sphingolipid backbones and can also be linked to some sterols. In addition, FAs are components of complex lipids that can constitute membrane micro-domains, and the use of detergent-resistant membranes is a common approach to study their composition. The diversity and cellular allocation of the membrane lipids containing FAs are very diverse and the approaches to analyze them provide only general information. In this work, a detailed FA analysis was performed using highly purified plasma membranes from bean leaves and germinating maize embryos and their respective detergent-resistant membrane preparations. The analyses showed the presence of a significant amount of very long chain FAs (containing 28C, 30C and 32C), in both plasma membrane preparations from bean and maize, that have not been previously reported. Herein is demonstrated that a significant enrichment of very long chain saturated FAs and saturated FAs can occur in detergent-resistant membrane preparations, as compared to the plasma membranes from both plant species. Considering that a thorough analysis of FAs is rarely performed in purified plasma membranes and detergent-resistant membranes, this work provides qualitative and quantitative evidence on the contributions of the length and saturation of FAs to the organization of the plant plasma membrane and detergent-resistant membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs.

Science.gov (United States)

Tetteroo, P A; Bluemink, J G; Dictus, W J; van Zoelen, E J; de Laat, S W

1984-07-01

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.

Introducing Membrane Charge and Membrane Potential to T Cell Signaling

Directory of Open Access Journals (Sweden)

Yuanqing Ma

2017-11-01

Full Text Available While membrane models now include the heterogeneous distribution of lipids, the impact of membrane charges on regulating the association of proteins with the plasma membrane is often overlooked. Charged lipids are asymmetrically distributed between the two leaflets of the plasma membrane, resulting in the inner leaflet being negatively charged and a surface potential that attracts and binds positively charged ions, proteins, and peptide motifs. These interactions not only create a transmembrane potential but they can also facilitate the formation of charged membrane domains. Here, we reference fields outside of immunology in which consequences of membrane charge are better characterized to highlight important mechanisms. We then focus on T cell receptor (TCR signaling, reviewing the evidence that membrane charges and membrane-associated calcium regulate phosphorylation of the TCR–CD3 complex and discuss how the immunological synapse exhibits distinct patterns of membrane charge distribution. We propose that charged lipids, ions in solution, and transient protein interactions form a dynamic equilibrium during T cell activation.

Isolation of plasma membrane-associated membranes from rat liver.

Science.gov (United States)

Suski, Jan M; Lebiedzinska, Magdalena; Wojtala, Aleksandra; Duszynski, Jerzy; Giorgi, Carlotta; Pinton, Paolo; Wieckowski, Mariusz R

2014-02-01

Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.

Binding of [125I]iodipine to parathyroid cell membranes: Evidence of a dihydropyridine-sensitive calcium channel

International Nuclear Information System (INIS)

Jones, J.I.; Fitzpatrick, L.A.

1990-01-01

The parathyroid cell is unusual, in that an increase in extracellular calcium concentrations inhibits PTH release. Calcium channels are glycoproteins that span cell membranes and allow entry of extracellular calcium into cells. We have demonstrated that the calcium channel agonist (+)202-791, which opens calcium channels, inhibits PTH release and that the antagonist (-)202-791, which closes calcium channels, stimulates PTH release. To identify the calcium channels responsible for these effects, we used a radioligand that specifically binds to calcium channels. Bovine parathyroid cell membranes were prepared and incubated under reduced lighting with [125I] iodipine (SA, 2000 Ci/mmol), which recognizes 1,4-dihydropyridine-sensitive calcium channels. Bound ligand was separated from free ligand by rapid filtration through Whatman GF/B filters. Nonspecific binding was measured by the inclusion of nifedipine at 10 microM. Specific binding represented approximately 40% of the total binding. The optimal temperature for [125I] iodipine binding was 4 C, and binding reached equilibrium by 30 min. The equilibrium dissociation constant (Kd) was approximately 550 pM, and the maximum number of binding sites was 780 fmol/mg protein. Both the calcium channel agonist (+)202-791 and antagonist (-)202-791 competitively inhibited [125I] iodipine binding, with 50% inhibition concentrations of 20 and 300 nM, respectively. These data indicate the presence of dihydropyridine-sensitive calcium channels on parathyroid cell membranes

Calcium transport across the membrane of Paramecium caudatum (protozoa)

International Nuclear Information System (INIS)

Martinac, B.

1980-06-01

Calcium transport across the membrane of Paramecium caudatum was studied by measuring calcium uptake and release by means of flow-through-technique, which was developed especially for this purpose. The method allows continuous flow of the cells suspension with radioactive and inactive solution, respectively, combined with simultaneous electrical stimulation of the cells by means of extracellular electrodes. The results obtained were compared to and interpreted according to behavioral patterns of Paramecium, which were registered by the time exposure dark-field macrophotographic technique under the same experimental conditions. (orig.) [de

Plant plasma membrane proteomics for improving cold tolerance

Directory of Open Access Journals (Sweden)

Daisuke eTakahashi

2013-04-01

Full Text Available Plants are always exposed to various stresses. We have focused on freezing stress, which causes serious problems for agricultural management. When plants suffer freeze-induced damage, the plasma membrane is thought to be the primary site of injury because of its central role in regulation of various cellular processes. Cold tolerant species, however, adapt to such freezing conditions by modifying cellular components and functions (cold acclimation. One of the most important adaptation mechanisms to freezing is alteration of plasma membrane compositions and functions. Advanced proteomic technologies have succeeded in identification of many candidates that may play roles in adaptation of the plasma membrane to freezing stress. Proteomics results suggest that adaptations of plasma membrane functions to low temperature are associated with alterations of protein compositions during cold acclimation. Some of proteins identified by proteomic approaches have been verified their functional roles in freezing tolerance mechanisms further. Thus, accumulation of proteomic results in the plasma membrane is of importance for application to molecular breeding efforts to increase cold tolerance in crops.

At the border: the plasma membrane-cell wall continuum.

Science.gov (United States)

Liu, Zengyu; Persson, Staffan; Sánchez-Rodríguez, Clara

2015-03-01

Plant cells rely on their cell walls for directed growth and environmental adaptation. Synthesis and remodelling of the cell walls are membrane-related processes. During cell growth and exposure to external stimuli, there is a constant exchange of lipids, proteins, and other cell wall components between the cytosol and the plasma membrane/apoplast. This exchange of material and the localization of cell wall proteins at certain spots in the plasma membrane seem to rely on a particular membrane composition. In addition, sensors at the plasma membrane detect changes in the cell wall architecture, and activate cytoplasmic signalling schemes and ultimately cell wall remodelling. The apoplastic polysaccharide matrix is, on the other hand, crucial for preventing proteins diffusing uncontrollably in the membrane. Therefore, the cell wall-plasma membrane link is essential for plant development and responses to external stimuli. This review focuses on the relationship between the cell wall and plasma membrane, and its importance for plant tissue organization. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

An Adaptable Spectrin/Ankyrin-Based Mechanism for Long-Range Organization of Plasma Membranes in Vertebrate Tissues.

Science.gov (United States)

Bennett, Vann; Lorenzo, Damaris N

2016-01-01

Ankyrins are membrane-associated proteins that together with their spectrin partners are responsible for micron-scale organization of vertebrate plasma membranes, including those of erythrocytes, excitable membranes of neurons and heart, lateral membrane domains of columnar epithelial cells, and striated muscle. Ankyrins coordinate functionally related membrane transporters and cell adhesion proteins (15 protein families identified so far) within plasma membrane compartments through independently evolved interactions of intrinsically disordered sequences with a highly conserved peptide-binding groove formed by the ANK repeat solenoid. Ankyrins are coupled to spectrins, which are elongated organelle-sized proteins that form mechanically resilient arrays through cross-linking by specialized actin filaments. In addition to protein interactions, cellular targeting and assembly of spectrin/ankyrin domains also critically depend on palmitoylation of ankyrin-G by aspartate-histidine-histidine-cysteine 5/8 palmitoyltransferases, as well as interaction of beta-2 spectrin with phosphoinositide lipids. These lipid-dependent spectrin/ankyrin domains are not static but are locally dynamic and determine membrane identity through opposing endocytosis of bulk lipids as well as specific proteins. A partnership between spectrin, ankyrin, and cell adhesion molecules first emerged in bilaterians over 500 million years ago. Ankyrin and spectrin may have been recruited to plasma membranes from more ancient roles in organelle transport. The basic bilaterian spectrin-ankyrin toolkit markedly expanded in vertebrates through gene duplications combined with variation in unstructured intramolecular regulatory sequences as well as independent evolution of ankyrin-binding activity by ion transporters involved in action potentials and calcium homeostasis. In addition, giant vertebrate ankyrins with specialized roles in axons acquired new coding sequences by exon shuffling. We speculate that

Light-induced modification of plant plasma membrane ion transport.

Science.gov (United States)

Marten, I; Deeken, R; Hedrich, R; Roelfsema, M R G

2010-09-01

Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K(+) channels during membrane potential changes. In most other species, the Ca(2+)-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.

Bile acids modulate signaling by functional perturbation of plasma membrane domains.

Science.gov (United States)

Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

2013-12-13

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Nanoclustering as a dominant feature of plasma membrane organization

NARCIS (Netherlands)

Garcia-Parajo, M.F.; Cambi, A.; Torreno-Pina, J.A.; Thompson, N.; Jacobson, K.

2014-01-01

Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at

Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane

OpenAIRE

1990-01-01

The platelet plasma membrane is lined by a membrane skeleton that appears to contain short actin filaments cross-linked by actin-binding protein. Actin-binding protein is in turn associated with specific plasma membrane glycoproteins. The aim of this study was to determine whether the membrane skeleton regulates properties of the plasma membrane. Platelets were incubated with agents that disrupted the association of the membrane skeleton with membrane glycoproteins. The consequences of this c...

Annexins are instrumental for efficient plasma membrane repair in cancer cells.

Science.gov (United States)

Lauritzen, Stine Prehn; Boye, Theresa Louise; Nylandsted, Jesper

2015-09-01

Plasma membrane stress can cause damage to the plasma membrane, both when imposed by the extracellular environment and by enhanced oxidative stress. Cells cope with these injuries by rapidly activating their plasma membrane repair system, which is triggered by Ca(2+) influx at the wound site. The repair system is highly dynamic, depends on both lipid and protein components, and include cytoskeletal reorganization, membrane replacements, and membrane fusion events. Cancer cells experience enhanced membrane stress when navigating through dense extracellular matrix, which increases the frequency of membrane injuries. In addition, increased motility and oxidative stress further increase the risk of plasma membrane lesions. Cancer cells compensate by overexpressing Annexin proteins including Annexin A2 (ANXA2). Annexin family members can facilitate membrane fusion events and wound healing by binding to negatively charged phospholipids in the plasma membrane. Plasma membrane repair in cancer cells depends on ANXA2 protein, which is recruited to the wound site and forms a complex with the Ca(2+)-binding EF-hand protein S100A11. Here they regulate actin accumulation around the wound perimeter, which is required for wound closure. In this review, we will discuss the requirement for Annexins, S100 proteins and actin cytoskeleton in the plasma membrane repair response of cancer cells, which reveals a novel avenue for targeting metastatic cancers. Copyright © 2015 Elsevier Ltd. All rights reserved.

Plasma-polymerized alkaline anion-exchange membrane: Synthesis and structure characterization

International Nuclear Information System (INIS)

Hu Jue; Meng Yuedong; Zhang Chengxu; Fang Shidong

2011-01-01

After-glow discharge plasma polymerization was developed for alkaline anion-exchange membranes synthesis using vinylbenzyl chloride as monomer. X-ray photoelectron spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy were used to characterize the chemical structure properties of plasma-polymerized membranes. Ion-exchange capacities of quaternized poly(vinylbenzyl chloride) (QPVBC) membranes were measured to evaluate their capability of hydroxyl ion transport. A mechanism of plasma polymerization using VBC as monomer that accounts for the competitive effects of free radicals polymerization and plasma ablation in the plasma polymerization process was proposed. Our results indicate that plasma discharge power influences the contents of functional groups and the structure of the plasma polymer membranes, which attribute to the coactions of polymerization and ablation. The properties of uniform morphology, good adhesion to the substrate, high thermal stability and satisfying anion conduction level suggest the potential application of QPVBC membrane deposited at discharge power of 20 W in alkaline direct methanol fuel cells.

Plasma Calcium and Phosphorus In Weanling Pigs as influenced by ...

African Journals Online (AJOL)

Twenty Yorkshire piglets, weaned at approximately 4 weeks of age, were used in this study to investigate the variations in plasma calcium and phosphorus as influenced by dietary calcium and phosphorus and 3 Cestrum diurnam. In the CCD and TCD pigs, hypercalcemia developed rapidly and persisted following the ...

Surface modification of nanoporous alumina membranes by plasma polymerization

Energy Technology Data Exchange (ETDEWEB)

Losic, Dusan; Cole, Martin A; Dollmann, Bjoern; Vasilev, Krasimir; Griesser, Hans J [Ian Wark Research Institute, University of South Australia, Mawson Lakes, Adelaide, SA 5095 (Australia)], E-mail: dusan.losic@unisa.edu.au

2008-06-18

The deposition of plasma polymer coatings onto porous alumina (PA) membranes was investigated with the aim of adjusting the surface chemistry and the pore size of the membranes. PA membranes from commercial sources with a range of pore diameters (20, 100 and 200 nm) were used and modified by plasma polymerization using n-heptylamine (HA) monomer, which resulted in a chemically reactive polymer surface with amino groups. Heptylamine plasma polymer (HAPP) layers with a thickness less than the pore diameter do not span the pores but reduce their diameter. Accordingly, by adjusting the deposition time and thus the thickness of the plasma polymer coating, it is feasible to produce any desired pore diameter. The structural and chemical properties of modified membranes were studied by scanning electron microscopy (SEM), atomic force microscopy (AFM) and x-ray electron spectroscopy (XPS). The resultant PA membranes with specific surface chemistry and controlled pore size are applicable for molecular separation, cell culture, bioreactors, biosensing, drug delivery, and engineering complex composite membranes.

Surface modification of nanoporous alumina membranes by plasma polymerization

International Nuclear Information System (INIS)

Losic, Dusan; Cole, Martin A; Dollmann, Bjoern; Vasilev, Krasimir; Griesser, Hans J

2008-01-01

The deposition of plasma polymer coatings onto porous alumina (PA) membranes was investigated with the aim of adjusting the surface chemistry and the pore size of the membranes. PA membranes from commercial sources with a range of pore diameters (20, 100 and 200 nm) were used and modified by plasma polymerization using n-heptylamine (HA) monomer, which resulted in a chemically reactive polymer surface with amino groups. Heptylamine plasma polymer (HAPP) layers with a thickness less than the pore diameter do not span the pores but reduce their diameter. Accordingly, by adjusting the deposition time and thus the thickness of the plasma polymer coating, it is feasible to produce any desired pore diameter. The structural and chemical properties of modified membranes were studied by scanning electron microscopy (SEM), atomic force microscopy (AFM) and x-ray electron spectroscopy (XPS). The resultant PA membranes with specific surface chemistry and controlled pore size are applicable for molecular separation, cell culture, bioreactors, biosensing, drug delivery, and engineering complex composite membranes

Giant plasma membrane vesicles: models for understanding membrane organization.

Science.gov (United States)

Levental, Kandice R; Levental, Ilya

2015-01-01

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

Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

Science.gov (United States)

Michaelis, Mary L; Jiang, Lei; Michaelis, Elias K

2017-01-01

Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved

Characteristics of polyimide-based composite membranes fabricated by low-temperature plasma polymerization

International Nuclear Information System (INIS)

Dung Thi Tran; Mori, Shinsuke; Suzuki, Masaaki

2008-01-01

Composite membranes were prepared by the deposition of plasma-polymerized allylamine films onto a porous polyimide substrate. The relationship between the plasma conditions and the membrane characteristics was described in terms of monomer flow rate, plasma discharge power, plasma polymerization time, and so on. Scanning electron microscope (SEM) images indicate that the thickness of the plasma polymer layer increased and the membrane skin pore size decreased gradually with the increasing of plasma polymerization time. Fourier transform infrared (FTIR) spectra demonstrate the appearance of amine groups in the plasma deposited polymer and the contact angle measurements indicate that the hydrophilicity of the membrane surfaces increased significantly after plasma polymerization. The composite membranes can reject salt from sodium chloride feed solution, and membrane separation performance depends strongly on the plasma conditions applied during the preparation of the plasma deposited polymer films

β-Microseminoprotein Endows Post Coital Seminal Plasma with Potent Candidacidal Activity by a Calcium- and pH-Dependent Mechanism

Science.gov (United States)

Edström Hägerwall, Anneli M. L.; Rydengård, Victoria; Fernlund, Per; Mörgelin, Matthias; Baumgarten, Maria; Cole, Alexander M.; Malmsten, Martin; Kragelund, Birthe B.; Sørensen, Ole E.

2012-01-01

The innate immune factors controlling Candida albicans are mostly unknown. Vulvovaginal candidiasis is common in women and affects approximately 70–75% of all women at least once. Despite the propensity of Candida to colonize the vagina, transmission of Candida albicans following sexual intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as β-microseminoprotein. At neutral pH, the fungicidal activity of β-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E71 was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of β-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. A homologous fragment from porcine β-microseminoprotein demonstrated calcium-dependent fungicidal activity in a CFU assay, suggesting this may be a common feature for members of the β-microseminoprotein family. By electron microscopy, β-microseminoprotein was found to cause lysis of Candida. Liposome experiments demonstrated that β-microseminoprotein was active towards ergosterol-containing liposomes that mimic fungal membranes, offering an explanation for the selectivity against fungi. These data identify β-microseminoprotein as an important innate immune factor active against C. albicans and may help explain the low sexual transmission rate of Candida

Study on surface adhesion of Plasma modified Polytetrafluoroethylene hollow fiber membrane

Science.gov (United States)

Chen, Jiangrong; Zhang, Huifeng; Liu, Guochang; Guo, Chungang; Lv, Jinglie; Zhangb, Yushan

2018-01-01

Polytetrafluoroethylene (PTFE) is popular membrane material because of its excellent thermal stability, chemical stability and mechanical stability. However, the low surface energy and non-sticky property of PTFE present challenges for modification. In the present study, plasma treatment was performed to improve the surface adhesion of PTFE hollow fiber membrane. The effect of discharge voltage, treatment time on the adhesion of PTFE hollow fiber membrane was symmetrically evaluated. Results showed that the plasma treatment method contributed to improve the surface activity and roughness of PTFE hollow fiber membrane, and the adhesion strength depend significantly on discharge voltage, which was beneficial to seepage pressure of PTFE hollow fiber membrane module. The adhesion strength of PTFE membrane by plasma treated at 220V for 3min reached as high as 86.2 N, far surpassing the adhesion strength 12.7 N of pristine membrane. Furthermore, improvement of content of free radical and composition analysis changes of the plasma modified PTFE membrane were investigated. The seepage pressure of PTFE membrane by plasma treated at 220V for 3min was 0.375 MPa, which means that the plasma treatment is an effective technique to improve the adhesion strength of membrane.

Calcium movements and the cellular basis of gravitropism

Science.gov (United States)

Roux, S. J.; Biro, R. L.; Hale, C. C.

An early gravity-transduction event in oat coleoptiles which precedes any noticeable bending is the accumulation of calcium on their prospective slower-growing side. Sub-cellular calcium localization studies indicate that the gravity-stimulated redistribution of calcium results in an increased concentration of calcium in the walls of responding cells. Since calcium can inhibit the extension growth of plant cell walls, this selective accumulation of calcium in walls may play a role in inducing the asymmetry of growth which characterizes gravitropism. The active transport of calcium from cells into walls is performed by a calcium-dependent ATPase localized in the plasma membrane. Evidence is presented in support of the hypothesis that this calcium pump is regulated by a feed-back mechanism which includes the participation of calmodulin.

Effect of plasma membrane fluidity on serotonin transport by endothelial cells

International Nuclear Information System (INIS)

Block, E.R.; Edwards, D.

1987-01-01

To evaluate the effect of plasma membrane fluidity of lung endothelial cells on serotonin transport, porcine pulmonary artery endothelial cells were incubated for 3 h with either 0.1 mM cholesterol hemisuccinate, 0.1 mM cis-vaccenic acid, or vehicle (control), after which plasma membrane fluidity and serotinin transport were measured. Fluorescence spectroscopy was used to measure fluidity in the plasma membrane. Serotonin uptake was calculated from the disappearance of [ 14 C]-serotonin from the culture medium. Cholesterol decreased fluidity in the subpolar head group and central and midacyl side-chain regions of the plasma membrane and decreased serotonin transport, whereas cis-vaccenic acid increased fluidity in the central and midacyl side-chain regions of the plasma membrane and also increased serotonin transport. Cis-vaccenic acid had no effect of fluidity in the subpolar head group region of the plasma membrane. These results provide evidence that the physical state of the central and midacyl chains within the pulmonary artery endothelial cell plasma membrane lipid bilayer modulates transmembrane transport of serotonin by these cells

An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes.

Science.gov (United States)

Bezrukov, Ludmila; Blank, Paul S; Polozov, Ivan V; Zimmerberg, Joshua

2009-11-15

A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-beta-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-beta-cyclodextrin plasma membrane extraction properties.

Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.

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Saarikangas, Juha; Zhao, Hongxia; Lappalainen, Pekka

2010-01-01

The plasma membrane and the underlying cortical actin cytoskeleton undergo continuous dynamic interplay that is responsible for many essential aspects of cell physiology. Polymerization of actin filaments against cellular membranes provides the force for a number of cellular processes such as migration, morphogenesis, and endocytosis. Plasma membrane phosphoinositides (especially phosphatidylinositol bis- and trisphosphates) play a central role in regulating the organization and dynamics of the actin cytoskeleton by acting as platforms for protein recruitment, by triggering signaling cascades, and by directly regulating the activities of actin-binding proteins. Furthermore, a number of actin-associated proteins, such as BAR domain proteins, are capable of directly deforming phosphoinositide-rich membranes to induce plasma membrane protrusions or invaginations. Recent studies have also provided evidence that the actin cytoskeleton-plasma membrane interactions are misregulated in a number of pathological conditions such as cancer and during pathogen invasion. Here, we summarize the wealth of knowledge on how the cortical actin cytoskeleton is regulated by phosphoinositides during various cell biological processes. We also discuss the mechanisms by which interplay between actin dynamics and certain membrane deforming proteins regulate the morphology of the plasma membrane.

Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography

Directory of Open Access Journals (Sweden)

Geisler Markus

1998-01-01

Full Text Available In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993 et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 &mgr;M and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

Calcium, membranes and accumulation of alkaloids in plants

International Nuclear Information System (INIS)

Lovkova, M.Ya.; Buzuk, G.N.; Grinkevich, N.I.

1983-01-01

Ca 2+ effect upon metabolism of aporphines and protopines has been studied in Glaucium flavun, which alkaloids are of an essential interest for the medicine practice. It has been shown that calcium produces the inhibiting effect both on catabolitic splitting and metabolism of glaucine and protopine. It has been anticipated that calcuium introduced into an expert plant stabilizes membranes of intracellular structures and prevents 14 C alkaloid entering from an environment to metabolically active cell compartments, which contain ferments realizing transformations of the above compounds. The level of membrane permeability is probably the main mechanism, through which a control of metabolism processes occurs, and hence, a control of alkaloid accumulation processes under in vivo conditions

A plasma membrane H + ATPase gene is germinationinduced in ...

African Journals Online (AJOL)

A plasma membrane H + ATPase gene is germinationinduced in wheat embryos. ... African Journal of Biotechnology ... of a germination specific plasma membrane H+-ATPase was analyzed by RTPCR and in situ RNA hybridization methods.

Calcium waves.

Science.gov (United States)

Jaffe, Lionel F

2008-04-12

Waves through living systems are best characterized by their speeds at 20 degrees C. These speeds vary from those of calcium action potentials to those of ultraslow ones which move at 1-10 and/or 10-20 nm s(-1). All such waves are known or inferred to be calcium waves. The two classes of calcium waves which include ones with important morphogenetic effects are slow waves that move at 0.2-2 microm s(-1) and ultraslow ones. Both may be propagated by cycles in which the entry of calcium through the plasma membrane induces subsurface contraction. This contraction opens nearby stretch-sensitive calcium channels. Calcium entry through these channels propagates the calcium wave. Many slow waves are seen as waves of indentation. Some are considered to act via cellular peristalsis; for example, those which seem to drive the germ plasm to the vegetal pole of the Xenopus egg. Other good examples of morphogenetic slow waves are ones through fertilizing maize eggs, through developing barnacle eggs and through axolotl embryos during neural induction. Good examples of ultraslow morphogenetic waves are ones during inversion in developing Volvox embryos and across developing Drosophila eye discs. Morphogenetic waves may be best pursued by imaging their calcium with aequorins.

Composite plasma polymerized sulfonated polystyrene membrane for PEMFC

Energy Technology Data Exchange (ETDEWEB)

Nath, Bhabesh Kumar; Khan, Aziz; Chutia, Joyanti, E-mail: jchutiaiasst@gmail.com

2015-10-15

Highlights: • Methyl methane sulfonate (MMS) is used as the sulfonating agent. • The proton conductivity of the membrane is found to be 0.141 S cm{sup −1}. • Power density of fuel cell with styrene/MMS membrane is 0.5 W cm{sup −2}. • The membrane exhibits thermal stability up to 140 °C. - Abstract: This work presents the introduction of an organic compound methyl methane sulfonate (MMS) for the first time in fabrication of polystyrene based proton exchange membrane (PEM) by plasma polymerization process. The membrane is fabricated by co-polymerizing styrene and MMS in capacitively coupled continuous RF plasma. The chemical composition of the plasma polymerized polymer membrane is investigated using Fourier Transform Infrared Spectroscopy which reveals the formation of composite structure of styrene and MMS. The surface morphology studied using AFM and SEM depicts the effect of higher partial pressure of MMS on surface topography of the membrane. The proton transport property of the membrane studied using electrochemical impedance spectroscopy shows the achievement of maximum proton conductivity of 0.141 S cm{sup −1} which is comparable to Nafion 117 membrane. Fuel cell performance test of the synthesized membrane shows a maximum power density of 500 mW cm{sup −2} and current density of 0.62 A cm{sup −2} at 0.6 V.

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It.

Science.gov (United States)

Kraft, Mary L

2016-01-01

Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with

Changes in plasma membrane structure upon irradiation on thymocytes

International Nuclear Information System (INIS)

Dreval', V.I.

1993-01-01

Thymocytes were irradiated with doses of 4 to 10 4 Gy. The binding of 1-anilinonaphtalene-8-sulphonate and Ca 2+ to plasma membranes; viscosity and lipid peroxidation; Stern-Folmer constant; and the number of Sh-groups of membrane proteins were determined. The structural changes in plasma membranes after irradiation of thymocytes were found to be cooperative

Protein diffusion in plant cell plasma membranes: the cell-wall corral.

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Martinière, Alexandre; Runions, John

2013-01-01

Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

Protein diffusion in plant cell plasma membranes: The cell-wall corral

Directory of Open Access Journals (Sweden)

Alexandre eMartinière

2013-12-01

Full Text Available Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

Plasma membrane and salinity tolerance of barley plants

International Nuclear Information System (INIS)

Al-Rahmani, F. H.; Al-Mashhadani, M. S.; Al-Delemee, N. H.

1997-01-01

Barley cultivar, California Mario ut, was grown in a nutrient solution containing increasing Nacl concentrations up to 250 mm. The effect of Nacl on growth, mineral compost ion ant integrity of the plasma membrane was studied. Growth of the shoot'and root was stimulated or little affected by 10 and 20 ml Nacl. Further increase in Nacl concentrations depressed the growth. The depression was conspicuous between 100 and 250 mm Nacl. Increasing Nacl concentration decreased potassium content in the shoots and roots and led to steep increase in sodium accumulation. The integrity of the plasma membrane was measured in term of potassium leakage from the root tips. Rapid leakage of potassium was obtained at Nacl concentrations ranging from 100 to 250 mm. At the same concentrations of Nacl, adenosine triphosphatase activity in the root tips was increased. Results indicate that the plasma membrane of root cells was damaged by the increased levels of salinity. It was concluded that the plasma membrane of root cells is the primary site of salinity toxicity. (authors). 40 refs., 5 tabs. 3 figs

Quantitative changes in adipocyte plasma membrane in response to nutritional manipulations

International Nuclear Information System (INIS)

Lewis, D.S.; Masoro, E.J.; Yu, B.P.

1981-01-01

The effects of changes in adipocyte size and the effects of nutritional manipulations on the quantity of plasma membrane per adipocyte were investigated. A method for estimating the quantity of plasma membrane was developed based on the specific labeling of adipocyte plasma membrane protein with the nonpermeable labeling agent 125I-labeled diazotized diiodosulfanilic acid. By studying rats (ranging in age from 50 to 125 days) fed a standard laboratory chow or a low fat diet or a high fat diet, a wide range of mean fat cell sizes was obtained. It was found that as the volume of the fat cell increased, the amount of plasma membrane increased in a linear fashion and that this linear relationship had the same slope whether the size of the adipocyte increased slowly with age or rapidly in response to a high fat diet. In contrast, fasting for up to 3 days caused a marked decrease in the mean volume of the adipocytes, but either no change or much less change in the amount of plasma membrane per cell than would have been predicted from the linear relationship between adipocytes, but either no change or much less change in the amount of plasma membrane per cell than would have been predicted form the linear relationship between adipocyte volume and amount of plasma membrane per cell obtained with fed rats, i.e., adipocytes from fasted rats contain more plasma membrane per cell than do fat cells of the same size from fed rats. Neither feeding a high fat diet nor fasting caused detectable changes in the protein and lipid composition of the adipocyte plasma membrane

Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

Science.gov (United States)

Sun, Bingyun; Hood, Leroy

2014-06-06

The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

Conformational changes and translocation of tissue-transglutaminase to the plasma membranes: role in cancer cell migration

International Nuclear Information System (INIS)

Kumar, Ambrish; Hu, Jianjun; LaVoie, Holly A; Walsh, Kenneth B; DiPette, Donald J; Singh, Ugra S

2014-01-01

membrane fractions and sensitive to intracellular Ca 2+ concentration suggesting a calcium requirement in TG2-regulated cell migration. Taken together, we conclude that resveratrol induces conformational changes in TG2, and that Ca 2+ -mediated TG2 association with the plasma membrane is responsible for the inhibitory effects of resveratrol on cell migration

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris.

Science.gov (United States)

Grillitsch, Karlheinz; Tarazona, Pablo; Klug, Lisa; Wriessnegger, Tamara; Zellnig, Günther; Leitner, Erich; Feussner, Ivo; Daum, Günther

2014-07-01

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production. Copyright © 2014 Elsevier B.V. All rights reserved.

Isolation and characterization of plasma membranes from guinea pig ileum

International Nuclear Information System (INIS)

Anon.

1986-01-01

A plasma membrane fraction from guinea pig ileum has been isolated by extraction of a crude microsomal fraction with a low ionic strength buffer containing ATP and Ca 2+ . The extracted microsomes were subjected to sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membranes were substantially free from contamination with contractile proteins, mitochondria and sarco-plasmic reticulum. The plasma membrane vesicles were enriched 30-to-40-fold in Na + -K + -ATPase and 5'-nucleotidase activities. The plasma membrane vesicles accumulated Ca 2+ in the presence of ATP. The addition of Ca 2+ ionophore A23187 to vesicles loaded with Ca 2+ in the presence of ATP removed Ca 2+ completely from the vesicles in one minute. The Km values for the Ca 2+ -dependent phosphorylated intermediates of Ca 2+ -Mg 2+ -ATPase and Ca 2+ uptake were approximately 0.8 μM indicating that the phosphorylated intermediates represent phosphorylation of Ca 2+ pump ATPase. The 3 H-nitrendipine binding to plasma membranes was characterized by high affinity with Kd of 185 pM and B/sub max/ 1280 fmol/mg protein. The plasma membrane vesicles prepared by these procedures can prove useful for the study of ion transport

Cholesterol asymmetry in synaptic plasma membranes.

Science.gov (United States)

Wood, W Gibson; Igbavboa, Urule; Müller, Walter E; Eckert, Gunter P

2011-03-01

Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: (i) chronic ethanol consumption; (ii) statins; (iii) aging; and (iv) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, P-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Mechanisms underlying anomalous diffusion in the plasma membrane.

Science.gov (United States)

Krapf, Diego

2015-01-01

The plasma membrane is a complex fluid where lipids and proteins undergo diffusive motion critical to biochemical reactions. Through quantitative imaging analyses such as single-particle tracking, it is observed that diffusion in the cell membrane is usually anomalous in the sense that the mean squared displacement is not linear with time. This chapter describes the different models that are employed to describe anomalous diffusion, paying special attention to the experimental evidence that supports these models in the plasma membrane. We review models based on anticorrelated displacements, such as fractional Brownian motion and obstructed diffusion, and nonstationary models such as continuous time random walks. We also emphasize evidence for the formation of distinct compartments that transiently form on the cell surface. Finally, we overview heterogeneous diffusion processes in the plasma membrane, which have recently attracted considerable interest. Copyright © 2015. Published by Elsevier Inc.

Properties of Plasma Membrane from Pea Root Seedlings under Altered Gravity

Science.gov (United States)

Klymchuk, D.; Baranenko, V.; Vorobyova, T. V.; Kurylenko, I.; Chyzhykova, O.; Dubovoy, V.

In this study, the properties of pea (Pisum sativum L.) plasma membrane were examined to determine how the membrane structure and functions are regulated in response to clinorotation (2 rev/min) conditions. Membrane preparations enriched by plasma membrane vesicles were obtained by aqueous two-phase partitioning from 6-day seedling roots. The specific characteristics of H^+-ATPase, lípid composition and peroxidation intensity as well as fluidity of lipid bilayer were analysed. ATP hydrolytic activity was inhibited by ortovanadate and was insensitive to aside and nitrate in sealed plasma membrane vesicles isolated from both clinorotated and control seedlings. Plasma membrane vesicles from clinorotated seedlings in comparison to controls were characterised by increase in the total lipid/protein ratio, ATP hydrolytic activity and intensifying of lipid peroxidation. Sitosterol and campesterol were the predominant free sterol species. Clinorotated seedlings contained a slightly higher level of unsaturated fatty acid than controls. Plasma membrane vesicles were labelled with pyrene and fluorescence originating from monomeric (I_M) molecules and excimeric (I_E) aggregates were measured. The calculated I_E/I_M values were higher in clinorotated seedlings compared with controls reflecting the reduction in membrane microviscosity. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H^+-ATPase activity in response of pea seedlings to altered gravity is discussed.

Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs ofLymnaea stagnalis andBithynia tentaculata (Mollusca).

Science.gov (United States)

Zivkovic, Danica; Créton, Robbert; Zwaan, Gideon; de Bruijn, Willem C; Dohmen, M René

1990-11-01

During extrusion of the first polar body in eggs ofLymnaea stagnalis andBithynia tentaculata a localized Ca 2+ /Mg 2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs ofLymnaea it was found only in the vegetal hemisphere, whereas inBithynia eggs it was localized both in the vegetal hemisphere and at the animal pole. This pattern of enzyme activity corresponds to the polar pattern of transcellular ionic currents measured with the vibrating probe, which we showed to be partially carried or regulated by calcium [Zivkovic and Dohmen (1989) Biol Bull (Woods Hole) 176 (Suppl):103-109]. The characteristics of the ATPase were studied using a variety of approaches such as ion and substrate depletions and substitutions, addition of specific inhibitors of ATPase activity, treatment with EDTA/EGTA and electron energy-loss spectrometry. The results indicate that, inLymnaea, there are at least two enzymatic entities. The first one is a Ca 2+ /Mg 2+ ATPase localized along the membrane and in the cortex of the vegetal hemisphere. The second one is a Ca 2+ -stimulated ATPase (calcium pump of the plasma membrane) localized in a small region of the membrane at the vegetal pole. We speculate that in the eggs ofLymnaea andBithynia a functional relationship exists between the plasma-membrane-associated ATPase activity and the transcellular ionic currents measured in the same region.

Plasma deposited fluorinated films on porous membranes

Energy Technology Data Exchange (ETDEWEB)

Gancarz, Irena [Department of Polymer and Carbon Materials, Wrocław University of Technology, 50-370 Wrocław (Poland); Bryjak, Marek, E-mail: marek.bryjak@pwr.edu.pl [Department of Polymer and Carbon Materials, Wrocław University of Technology, 50-370 Wrocław (Poland); Kujawski, Jan; Wolska, Joanna [Department of Polymer and Carbon Materials, Wrocław University of Technology, 50-370 Wrocław (Poland); Kujawa, Joanna; Kujawski, Wojciech [Nicolaus Copernicus University, Faculty of Chemistry, 7 Gagarina St., 87-100 Torun (Poland)

2015-02-01

75 KHz plasma was used to modify track etched poly(ethylene terephthalate) membranes and deposit on them flouropolymers. Two fluorine bearing monomers were used: perflourohexane and hexafluorobenzene. The modified surfaces were analyzed by means of attenuated total reflection infra-red spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy and wettability. It was detected that hexaflourobenxene deposited to the larger extent than perflourohaxane did. The roughness of surfaces decreased when more fluoropolymer was deposited. The hydrophobic character of surface slightly disappeared during 20-days storage of hexaflourobenzene modified membrane. Perfluorohexane modified membrane did not change its character within 120 days after modification. It was expected that this phenomenon resulted from post-reactions of oxygen with radicals in polymer deposits. The obtained membranes could be used for membrane distillation of juices. - Highlights: • Plasma deposited hydrophobic layer of flouropolymers. • Deposition degree affects the surface properties. • Hydrohilization of surface due to reaction of oxygen with entrapped radicals. • Possibility to use modified porous membrane for water distillation and apple juice concentration.

Plants and fungi in the era of heterogeneous plasma membranes.

Science.gov (United States)

Opekarová, M; Malinsky, J; Tanner, W

2010-09-01

Examples from yeast and plant cells are described that show that their plasma membrane is laterally compartmented. Distinct lateral domains encompassing both specific lipids and integral proteins coexist within the plane of the plasma membrane. The compartments are either spatially stable and include distinct sets of proteins, or they are transiently formed to accomplish diverse functions. They are not related to lipid rafts or their clusters, as defined for mammalian cells. This review summarises only well-documented compartments of plasma membranes from plants and fungi, which have been recognised using microscopic approaches. In several cases, physiological functions of the membrane compartmentation are revealed.

Neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

International Nuclear Information System (INIS)

Stevenson, K.B.; Nauseef, W.M.; Clark, R.A.

1987-01-01

The glucoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3 H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody. Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove adsorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

Science.gov (United States)

Stone, Matthew B; Shelby, Sarah A; Veatch, Sarah L

2017-06-14

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

The dynamics of plant plasma membrane proteins: PINs and beyond.

Science.gov (United States)

Luschnig, Christian; Vert, Grégory

2014-08-01

Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment. © 2014. Published by The Company of Biologists Ltd.

An atmospheric-pressure cold plasma leads to apoptosis in Saccharomyces cerevisiae by accumulating intracellular reactive oxygen species and calcium

International Nuclear Information System (INIS)

Ma, R N; Zhang, Q; Tian, Y; Su, B; Zhang, J; Fang, J; Feng, H Q; Liang, Y D

2013-01-01

A non-thermal plasma is known to induce apoptosis of various cells but the mechanism is not yet clear. A eukaryotic model organism Saccharomyces cerevisiaewas used to investigate the cellular and biochemical regulations of cell apoptosis and cell cycle after an atmospheric-pressure cold plasma treatment. More importantly, intracellular calcium (Ca 2+ ) was first involved in monitoring the process of plasma-induced apoptosis in this study. We analysed the cell apoptosis and cell cycle by flow cytometry and observed the changes in intracellular reactive oxygen species (ROS) and Ca 2+ concentration, cell mitochondrial membrane potential (Δψ m ) as well as nuclear DNA morphology via fluorescence staining assay. All experimental results indicated that plasma-generated ROS leads to the accumulation of intracellular ROS and Ca 2+ that ultimately contribute to apoptosis associated with cell cycle arrest at G1 phase through depolarization of Δψ m and fragmenting nuclear DNA. This work provides a novel insight into the physical and biological mechanism of apoptosis induced by a plasma which could benefit for promoting the development of plasmas applied to cancer therapy. (paper)

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

International Nuclear Information System (INIS)

Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

1988-01-01

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn 2+ , while Fe 2+ and Mn 2+ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca 2+ , phorbol ester, or antigen

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

International Nuclear Information System (INIS)

Thatte, H.S.; Mickelson, J.R.; Addis, P.B.; Louis, C.F.

1987-01-01

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45 Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration

Atmospheric-pressure plasma activation and surface characterization on polyethylene membrane separator

Science.gov (United States)

Tseng, Yu-Chien; Li, Hsiao-Ling; Huang, Chun

2017-01-01

The surface hydrophilic activation of a polyethylene membrane separator was achieved using an atmospheric-pressure plasma jet. The surface of the atmospheric-pressure-plasma-treated membrane separator was found to be highly hydrophilic realized by adjusting the plasma power input. The variations in membrane separator chemical structure were confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Chemical analysis showed newly formed carbonyl-containing groups and high surface concentrations of oxygen-containing species on the atmospheric-pressure-plasma-treated polymeric separator surface. It also showed that surface hydrophilicity primarily increased from the polar component after atmospheric-pressure plasma treatment. The surface and pore structures of the polyethylene membrane separator were examined by scanning electron microscopy, revealing a slight alteration in the pore structure. As a result of the incorporation of polar functionalities by atmospheric-pressure plasma activation, the electrolyte uptake and electrochemical impedance of the atmospheric-pressure-plasma-treated membrane separator improved. The investigational results show that the separator surface can be controlled by atmospheric-pressure plasma surface treatment to tailor the hydrophilicity and enhance the electrochemical performance of lithium ion batteries.

Influence of ionizing radiation on the plasma membrane proteins

International Nuclear Information System (INIS)

Dreval', V.I.

1992-01-01

The effect of ionizing radiation on the meat cattle thymocytes plasma membranes was studied. Using fluorescence quenching technique the effect of irradiation of proteins conformation was investigated. The influence of ionizing radiation on the plasma membranes was shown to be followed by changes of the protein structure-dynamic organization

Interaction between La(III) and proteins on the plasma membrane of horseradish

Science.gov (United States)

Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

2012-06-01

Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

Solubilization of rat kidney plasma membrane proteins associated with 3H-aldosterone

International Nuclear Information System (INIS)

Ozegovic, B.; Dobrovic-Jenik, D.; Milkovic, S.

1988-01-01

The treatment of rat kidney plasma membranes with sodium dodecyl sulphate (SDS) did not essentially affect the ability of the membranes for 3 H-aldosterone binding as compared with the intact plasma membranes (Ozegovic et al., 1977). A gel filtration of 3 H-aldosterone - kidney plasma membranes complex on Sepharose 6B yielded 2 protein and 2 3 H-aldosterone peaks. The proteins which were eluted in the first peak were associated with the first 3 H-aldosterone peak while the second 3 H-aldosterone peak was eluted with Ve corresponding to Ve of free 3 H-aldosterone. Spironolactone, a competitive antagonist of aldosterone, prevented the binding of 3 H-aldosterone to the membrane proteins. The results demonstrated a high affinity of the kidney plasma membranes solubilized with SDS and a specificity of aldosterone binding to the plasma membrane proteins of higher molecular mass. (author)

RNAi-mediated downregulation of poplar plasma membrane intrinsic proteins (PIPs) changes plasma membrane proteome composition and affects leaf physiology.

Science.gov (United States)

Bi, Zhen; Merl-Pham, Juliane; Uehlein, Norbert; Zimmer, Ina; Mühlhans, Stefanie; Aichler, Michaela; Walch, Axel Karl; Kaldenhoff, Ralf; Palme, Klaus; Schnitzler, Jörg-Peter; Block, Katja

2015-10-14

Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement. Copyright © 2015. Published by Elsevier B.V.

Purification of plant plasma membranes by two-phase partitioning and measurement of H+ pumping.

Science.gov (United States)

Lund, Anette; Fuglsang, Anja Thoe

2012-01-01

Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.

Experimental study of fouling and cleaning of sintered stainless steel membrane in electro-microfiltration of calcium salt particles.

Science.gov (United States)

Qin, Frank G F; Mawson, John; Zeng, Xin An

2011-05-30

Sintered stainless steel (SSS) microfiltration membranes, which served as electrode directly, were used for the experiment of separating Alamin, a calcium salt and protein containing particles, found in dairy processing. Fouling and cleaning of the SSS membranes under the application of an external electric field were studied. The imposed electric field was found, diverging the pH of permeate and retentate. This in turn altered the solubility of the calcium salt and impacted the performance of electro microfiltration membrane. Using electric field as an enhanced cleaning-in-place (CIP) method in back flushing SSS membrane was also studied.

Experimental Study of Fouling and Cleaning of Sintered Stainless Steel Membrane in Electro-Microfiltration of Calcium Salt Particles

Directory of Open Access Journals (Sweden)

Frank G. F. Qin

2011-05-01

Full Text Available Sintered stainless steel (SSS microfiltration membranes, which served as electrode directly, were used for the experiment of separating Alamin, a calcium salt and protein containing particles, found in dairy processing. Fouling and cleaning of the SSS membranes under the application of an external electric field were studied. The imposed electric field was found, diverging the pH of permeate and retentate. This in turn altered the solubility of the calcium salt and impacted the performance of electro microfiltration membrane. Using electric field as an enhanced cleaning-in-place (CIP method in back flushing SSS membrane was also studied.

Layered plasma polymer composite membranes

Science.gov (United States)

Babcock, Walter C.

1994-01-01

Layered plasma polymer composite fluid separation membranes are disclosed, which comprise alternating selective and permeable layers for a total of at least 2n layers, where n is .gtoreq.2 and is the number of selective layers.

Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy.

Science.gov (United States)

Jia, Hao-Ran; Wang, Hong-Yin; Yu, Zhi-Wu; Chen, Zhan; Wu, Fu-Gen

2016-03-16

Long-time stable plasma membrane imaging is difficult due to the fast cellular internalization of fluorescent dyes and the quick detachment of the dyes from the membrane. In this study, we developed a two-step synergistic cell surface modification and labeling strategy to realize long-time plasma membrane imaging. Initially, a multisite plasma membrane anchoring reagent, glycol chitosan-10% PEG2000 cholesterol-10% biotin (abbreviated as "GC-Chol-Biotin"), was incubated with cells to modify the plasma membranes with biotin groups with the assistance of the membrane anchoring ability of cholesterol moieties. Fluorescein isothiocyanate (FITC)-conjugated avidin was then introduced to achieve the fluorescence-labeled plasma membranes based on the supramolecular recognition between biotin and avidin. This strategy achieved stable plasma membrane imaging for up to 8 h without substantial internalization of the dyes, and avoided the quick fluorescence loss caused by the detachment of dyes from plasma membranes. We have also demonstrated that the imaging performance of our staining strategy far surpassed that of current commercial plasma membrane imaging reagents such as DiD and CellMask. Furthermore, the photodynamic damage of plasma membranes caused by a photosensitizer, Chlorin e6 (Ce6), was tracked in real time for 5 h during continuous laser irradiation. Plasma membrane behaviors including cell shrinkage, membrane blebbing, and plasma membrane vesiculation could be dynamically recorded. Therefore, the imaging strategy developed in this work may provide a novel platform to investigate plasma membrane behaviors over a relatively long time period.

Glucose rapidly decreases plasma membrane GLUT4 content in rat skeletal muscle.

Science.gov (United States)

Marette, A; Dimitrakoudis, D; Shi, Q; Rodgers, C D; Klip, A; Vranic, M

1999-02-01

We have previously demonstrated that chronic hyperglycemia per se decreases GLUT4 glucose transporter expression and plasma membrane content in mildly streptozotocin- (STZ) diabetic rats (Biochem. J. 284, 341-348, 1992). In the present study, we investigated the effect of an acute rise in glycemia on muscle GLUT4 and GLUT1 protein contents in the plasma membrane, in the absence of insulin elevation. Four experimental groups of rats were analyzed in the postabsorptive state: 1. Control rats. 2. Hyperglycemic STZ-diabetic rats with moderately reduced fasting insulin levels. 3. STZ-diabetic rats made normoglycemic with phlorizin treatment. 4. Phlorizin-treated (normoglycemic) STZ-diabetic rats infused with glucose for 40 min. The uniqueness of the latter model is that glycemia can be rapidly raised without any concomitant increase in plasma insulin levels. Plasma membranes were isolated from hindlimb muscle and GLUT1 and GLUT4 proteins amounts determined by Western blot analysis. As predicted, STZ-diabetes caused a significant decrease in the abundance of GLUT4 in the isolated plasma membranes. Normalization of glycemia for 3 d with phlorizin treatment restored plasma membrane GLUT4 content in muscle of STZ-diabetic rats. A sudden rise in glycemia over a period of 40 min caused the GLUT4 levels in the plasma membrane fraction to decrease to those of nontreated STZ-diabetic rats. In contrast to the GLUT4 transporter, plasma membrane GLUT1 abundance was not changed by the acute glucose challenge. It is concluded that glucose can have regulatory effect by acutely reducing plasma membrane GLUT4 protein contents in rat skeletal muscle. We hypothesize that this glucose-induced downregulation of plasma membrane GLUT4 could represent a protective mechanism against excessive glucose uptake under hyperglycemic conditions accompanied by insulin resistance.

Insights into plant plasma membrane aquaporin trafficking.

Science.gov (United States)

Hachez, Charles; Besserer, Arnaud; Chevalier, Adrien S; Chaumont, François

2013-06-01

Plasma membrane intrinsic proteins (PIPs) are plant aquaporins that facilitate the diffusion of water and small uncharged solutes through the cell membrane. Deciphering the network of interacting proteins that modulate PIP trafficking to and activity in the plasma membrane is essential to improve our knowledge about PIP regulation and function. This review highlights the most recent advances related to PIP subcellular routing and dynamic redistribution, identifies some key molecular interacting proteins, and indicates exciting directions for future research in this field. A better understanding of the mechanisms by which plants optimize water movement might help in identifying new molecular players of agronomical relevance involved in the control of cellular water uptake and drought tolerance. Copyright © 2012 Elsevier Ltd. All rights reserved.

Calcium phosphate nanoparticles as versatile carrier for small and large molecules across cell membranes

Energy Technology Data Exchange (ETDEWEB)

Sokolova, Viktoriya; Rotan, Olga; Klesing, Jan [University of Duisburg-Essen, Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE) (Germany); Nalbant, Perihan [University of Duisburg-Essen, Faculty of Biology, Institute of Molecular Cell Biology (Germany); Buer, Jan; Knuschke, Torben; Westendorf, Astrid M. [University Hospital Essen, University of Duisburg-Essen, Institute of Medical Microbiology (Germany); Epple, Matthias, E-mail: matthias.epple@uni-due.de [University of Duisburg-Essen, Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE) (Germany)

2012-06-15

The successful transport of molecules across the cell membrane is a key point in biology and medicine. In most cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100-250 nm, depending on the functionalization) were loaded with fluorescent oligonucleotides, peptide, proteins, antibodies, polymers or porphyrins and characterized by dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. Any excess of molecules was removed by ultracentrifugation, and the dissolved molecules at the same concentration were used as control. The uptake of such fluorescence-labeled nanoparticles into HeLa cells was monitored by fluorescence microscopy and confocal laser scanning microscopy. Calcium phosphate nanoparticles were able to transport all molecules across the cell membrane, whereas the dissolved molecules alone were taken up only to a very small extent or even not at all.

Calcium phosphate nanoparticles as versatile carrier for small and large molecules across cell membranes

Science.gov (United States)

Sokolova, Viktoriya; Rotan, Olga; Klesing, Jan; Nalbant, Perihan; Buer, Jan; Knuschke, Torben; Westendorf, Astrid M.; Epple, Matthias

2012-06-01

The successful transport of molecules across the cell membrane is a key point in biology and medicine. In most cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100-250 nm, depending on the functionalization) were loaded with fluorescent oligonucleotides, peptide, proteins, antibodies, polymers or porphyrins and characterized by dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. Any excess of molecules was removed by ultracentrifugation, and the dissolved molecules at the same concentration were used as control. The uptake of such fluorescence-labeled nanoparticles into HeLa cells was monitored by fluorescence microscopy and confocal laser scanning microscopy. Calcium phosphate nanoparticles were able to transport all molecules across the cell membrane, whereas the dissolved molecules alone were taken up only to a very small extent or even not at all.

Calcium phosphate nanoparticles as versatile carrier for small and large molecules across cell membranes

International Nuclear Information System (INIS)

Sokolova, Viktoriya; Rotan, Olga; Klesing, Jan; Nalbant, Perihan; Buer, Jan; Knuschke, Torben; Westendorf, Astrid M.; Epple, Matthias

2012-01-01

The successful transport of molecules across the cell membrane is a key point in biology and medicine. In most cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100–250 nm, depending on the functionalization) were loaded with fluorescent oligonucleotides, peptide, proteins, antibodies, polymers or porphyrins and characterized by dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. Any excess of molecules was removed by ultracentrifugation, and the dissolved molecules at the same concentration were used as control. The uptake of such fluorescence-labeled nanoparticles into HeLa cells was monitored by fluorescence microscopy and confocal laser scanning microscopy. Calcium phosphate nanoparticles were able to transport all molecules across the cell membrane, whereas the dissolved molecules alone were taken up only to a very small extent or even not at all.

A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

International Nuclear Information System (INIS)

Rosiere, T.K.; Marrs, J.A.; Bouck, G.B.

1990-01-01

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39

Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

Science.gov (United States)

Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

2016-08-01

Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

International Nuclear Information System (INIS)

Tidow, Henning; Hein, Kim L.; Baekgaard, Lone; Palmgren, Michael G.; Nissen, Poul

2010-01-01

Plant plasma-membrane Ca 2+ -ATPase is regulated via binding of calmodulin to its autoinhibitory N-terminal domain. In this study, the expression, purification, crystallization and preliminary X-ray diffraction analysis of this protein complex from A. thaliana are reported. Plasma-membrane Ca 2+ -ATPases (PMCAs) are calcium pumps that expel Ca 2+ from eukaryotic cells to maintain overall Ca 2+ homoeostasis and to provide local control of intracellular Ca 2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for presynaptic and postsynaptic Ca 2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium-bound calmodulin (Ca 2+ -CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca 2+ -ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 Å, β = 113.2°. A complete data set was collected to 3.0 Å resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin

Antifouling enhancement of polysulfone/TiO2 nanocomposite separation membrane by plasma etching

Science.gov (United States)

Chen, Z.; Yin, C.; Wang, S.; Ito, K.; Fu, Q. M.; Deng, Q. R.; Fu, P.; Lin, Z. D.; Zhang, Y.

2017-01-01

A polysulfone/TiO2 nanocomposite membrane was prepared via casting method, followed by the plasma etching of the membrane surface. Doppler broadened energy spectra vs. positron incident energy were employed to elucidate depth profiles of the nanostructure for the as-prepared and treated membranes. The results confirmed that the near-surface of the membrane was modified by the plasma treatment. The antifouling characteristics for the membranes, evaluated using the degradation of Rhodamin B, indicated that the plasma treatment enhances the photo catalytic ability of the membrane, suggesting that more TiO2 nanoparticles are exposed at the membrane surface after the plasma treatment as supported by the positron result.

Antifouling enhancement of polysulfone/TiO2 nanocomposite separation membrane by plasma etching

International Nuclear Information System (INIS)

Chen, Z; Yin, C; Wang, S; Fu, Q M; Deng, Q R; Fu, P; Lin, Z D; Zhang, Y; Ito, K

2017-01-01

A polysulfone/TiO 2 nanocomposite membrane was prepared via casting method, followed by the plasma etching of the membrane surface. Doppler broadened energy spectra vs. positron incident energy were employed to elucidate depth profiles of the nanostructure for the as-prepared and treated membranes. The results confirmed that the near-surface of the membrane was modified by the plasma treatment. The antifouling characteristics for the membranes, evaluated using the degradation of Rhodamin B, indicated that the plasma treatment enhances the photo catalytic ability of the membrane, suggesting that more TiO 2 nanoparticles are exposed at the membrane surface after the plasma treatment as supported by the positron result. (paper)

Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury 1

Science.gov (United States)

Iswari, S.; Palta, Jiwan P.

1989-01-01

Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a Km around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its Km was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in Km, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress. Images Figure 1 Figure 2 PMID:16666856

Research on Permeability of Poly(ethylene) Terephthalate Track Membranes Modified in Plasma

CERN Document Server

Dmitriev, S N; Sleptsov, V V; Elinson, V M; Potrjasaj, V V

2001-01-01

The properties of poly(ethylene) terephthalate track membranes subjected to the plasma RF-discharge treatment in air have been investigated. The effect of the treatment conditions in plasma on the structure and the properties of the membranes formed in the gas-discharge etching has been studied. It has been figured out that the influence of the air plasma on the membranes under study leads to a formation of asymmetric membranes with a higher flow rate, the structure and chemical composition of their superficial layer are changed. It is shown that the presence of the modified layer on the surface of the membranes causes changing their hydrodynamic characteristics - water permeability of the membranes treated in plasma in a greater degree depends upon {pH} of the filtered solution.

Effects of intravenous administration of two volumes of calcium solution on plasma ionized calcium concentration and recovery from naturally occurring hypocalcemia in lactating dairy cows.

NARCIS (Netherlands)

Doze, J.G.; Donders, R.; Kolk, J.H. van der

2008-01-01

OBJECTIVE: To compare the effects of administration of 2 volumes of a calcium solution (calcium oxide and calcium gluconate) on plasma ionized calcium concentration (PICaC) and clinical recovery from naturally occurring hypocalcemia (NOHC; milk fever) in lactating dairy cows. ANIMALS: 123 cows with

Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

Science.gov (United States)

Sardo, Luca; Hatch, Steven C; Chen, Jianbo; Nikolaitchik, Olga; Burdick, Ryan C; Chen, De; Westlake, Christopher J; Lockett, Stephen; Pathak, Vinay K; Hu, Wei-Shau

2015-11-01

To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas

Regulation of the Plasma Membrane H+-ATPase

DEFF Research Database (Denmark)

Falhof, Janus

The plasma membrane (PM) H+-ATPase is responsible for generating the electrochemical gradientthat drives the secondary transport of nutrients across the cellular membrane. It belongs to a familyof cation and lipid transporters that are vital to many organisms. PM H+-ATPases are Type P3AATPases...

Hunting for low abundant redox proteins in plant plasma membranes.

Science.gov (United States)

Lüthje, Sabine; Hopff, David; Schmitt, Anna; Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana

2009-04-13

Nowadays electron transport (redox) systems in plasma membranes appear well established. Members of the flavocytochrome b family have been identified by their nucleotide acid sequences and characterized on the transcriptional level. For their gene products functions have been demonstrated in iron uptake and oxidative stress including biotic interactions, abiotic stress factors and plant development. In addition, NAD(P)H-dependent oxidoreductases and b-type cytochromes have been purified and characterized from plasma membranes. Several of these proteins seem to belong to the group of hypothetical or unknown proteins. Low abundance and the lack of amino acid sequence data for these proteins still hamper their functional analysis. Consequently, little is known about the physiological function and regulation of these enzymes. In recent years evidence has been presented for the existence of microdomains (so-called lipid rafts) in plasma membranes and their interaction with specific membrane proteins. The identification of redox systems in detergent insoluble membranes supports the idea that redox systems may have important functions in signal transduction, stress responses, cell wall metabolism, and transport processes. This review summarizes our present knowledge on plasma membrane redox proteins and discusses alternative strategies to investigate the function and regulation of these enzymes.

Gravity Responsive NADH Oxidase of the Plasma Membrane

Science.gov (United States)

Morre, D. James (Inventor)

2002-01-01

A method and apparatus for sensing gravity using an NADH oxidase of the plasma membrane which has been found to respond to unit gravity and low centrifugal g forces. The oxidation rate of NADH supplied to the NADH oxidase is measured and translated to represent the relative gravitational force exerted on the protein. The NADH oxidase of the plasma membrane may be obtained from plant or animal sources or may be produced recombinantly.

Preparation of poly(2-chloroaniline) membrane and plasma surface modification

International Nuclear Information System (INIS)

Kir, E.; Oksuz, L.; Helhel, S.

2006-01-01

P2ClAn membranes were obtained from chemically synthesized poly(2-chloroaniline) (P2ClAn) by casting method. These membranes were cast from dimethyl formamide (DMF) and were in the undoped state. P2ClAn membranes were characterized by Fourier infrared spectroscopy and scanning electron microscopy. Measurements of water content capacity, membrane thickness and ion-exchange capacity of the cast membranes were carried out. P2ClAn membranes were treated by electron cylotron resonance (ECR) plasma for surface modification. Plasma treatment has been successfully utilized for improving the surface properties of P2ClAn membranes such as increasing pore diameters and number of pores for better anion or molecule transportation

Contactless Stimulation and Control of Biomimetic Nanotubes by Calcium Ion Gradients.

Science.gov (United States)

Kirejev, Vladimir; Ali Doosti, Baharan; Shaali, Mehrnaz; Jeffries, Gavin D M; Lobovkina, Tatsiana

2018-04-17

Membrane tubular structures are important communication pathways between cells and cellular compartments. Studying these structures in their native environment is challenging, due to the complexity of membranes and varying chemical conditions within and outside of the cells. This work demonstrates that a calcium ion gradient, applied to a synthetic lipid nanotube, triggers lipid flow directed toward the application site, resulting in the formation of a bulge aggregate. This bulge can be translated in a contactless manner by moving a calcium ion source along the lipid nanotube. Furthermore, entrapment of polystyrene nanobeads within the bulge does not tamper the bulge movement and allows transporting of the nanoparticle cargo along the lipid nanotube. In addition to the synthetic lipid nanotubes, the response of cell plasma membrane tethers to local calcium ion stimulation is investigated. The directed membrane transport in these tethers is observed, but with slower kinetics in comparison to the synthetic lipid nanotubes. The findings of this work demonstrate a novel and contactless mode of transport in lipid nanotubes, guided by local exposure to calcium ions. The observed lipid nanotube behavior can advance the current understanding of the cell membrane tubular structures, which are constantly reshaped during dynamic cellular processes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The stoichiometry of the TMEM16A ion channel determined in intact plasma membranes of COS-7 cells using liquid-phase electron microscopy.

Science.gov (United States)

Peckys, Diana B; Stoerger, Christof; Latta, Lorenz; Wissenbach, Ulrich; Flockerzi, Veit; de Jonge, Niels

2017-08-01

TMEM16A is a membrane protein forming a calcium-activated chloride channel. A homodimeric stoichiometry of the TMEM16 family of proteins has been reported but an important question is whether the protein resides always in a dimeric configuration in the plasma membrane or whether monomers of the protein are also present in its native state within in the intact plasma membrane. We have determined the stoichiometry of the human (h)TMEM16A within whole COS-7 cells in liquid. For the purpose of detecting TMEM16A subunits, single proteins were tagged by the streptavidin-binding peptide within extracellular loops accessible by streptavidin coated quantum dot (QD) nanoparticles. The labeled proteins were then imaged using correlative light microscopy and environmental scanning electron microscopy (ESEM) using scanning transmission electron microscopy (STEM) detection. The locations of 19,583 individual proteins were determined of which a statistical analysis using the pair correlation function revealed the presence of a dimeric conformation of the protein. The amounts of detected label pairs and single labels were compared between experiments in which the TMEM16A SBP-tag position was varied, and experiments in which tagged and non-tagged TMEM16A proteins were present. It followed that hTMEM16A resides in the plasma membrane as dimer only and is not present as monomer. This strategy may help to elucidate the stoichiometry of other membrane protein species within the context of the intact plasma membrane in future. Copyright © 2017 Elsevier Inc. All rights reserved.

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

1988-05-15

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn/sup 2 +/, while Fe/sup 2 +/ and Mn/sup 2 +/ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca/sup 2 +/, phorbol ester, or antigen.

Insulin stimulation of phospholipid methylation in isolated rat adipocyte plasma membranes.

OpenAIRE

Kelly, K L; Kiechle, F L; Jarett, L

1984-01-01

Partially purified plasma membranes prepared from rat adipocytes contain N-methyltransferase(s) that utilize(s) S-adenosyl-L-methionine to synthesize phosphatidylcholine from phosphatidylethanolamine. The incorporation of [3H]methyl from S-adenosyl-L-[methyl-3H]methionine into plasma membrane phospholipids was linear with incubation time and plasma membrane protein concentration and was inhibited in a dose-dependent manner by both S-adenosyl-L-homocysteine and 3-deazadenosine. The addition of...

Membrane oscillations in the channel of a stationary plasma motor

International Nuclear Information System (INIS)

Bugrova, A.I.; Lipatov, A.S.; Morozov, A.I.; Kharchevnikov, V.K.

1999-01-01

Results of measuring the ion flux density in the channel of the stationary plasma drive are presented. Two plane easters move both along and transverse to the plasma flux. During the experiment, the strong low-frequency oscillations (∼ 35 kHz) are observed in the channel of the stationary plasma drive. It is found that membrane oscillations are accompanied by oscillations of the electron temperature. These membrane oscillations affect the divergence of the output plasma jet and the erosion of the output part of the channel of the stationary plasma drive [ru

Membrane junctions in xenopus eggs: their distribution suggests a role in calcium regulation

OpenAIRE

Gardiner, DM; Grey, RD

1983-01-01

We have observed the presence of membrane junctions formed between the plasma membrane and cortical endoplasmic reticulum of mature, unactivated eggs of xenopus laevis. The parallel, paired membranes of the junction are separated by a 10-mn gap within which electron-dense material is present. This material occurs in patches with an average center-to-center distance of approximately 30 nm. These junctions are rare in immature (but fully grown) oocytes (approximately 2 percent of the plasma mem...

Research on permeability of poly(ethylene) terephthalate track membranes modified in plasma

International Nuclear Information System (INIS)

Dmitriev, S.N.; Kravets, L.I.; Sleptsov, V.V.; Elinson, V.M.; Potryasaj, V.V.

2001-01-01

The properties of poly(ethylene) terephthalate track membranes subjected to the plasma RF-discharge treatment in air have been investigated. The effect of the treatment conditions in plasma on the structure and the properties of the membranes formed in the gas-discharge etching has been studied. It has been figured out that the influence of the air plasma on the membranes under study leads to a formation of asymmetric membranes with a higher flow rate, the structure and chemical composition of their superficial layer are changed. It is shown that the presence of the modified layer on the surface of the membranes causes changing their hydrodynamic characteristics - water permeability of the membranes treated in plasma in a greater degree depends upon pH of the filtered solution. (author)

Calcium alpha-ketoglutarate administration to malnourished hemodialysis patients improves plasma arginine concentrations.

Science.gov (United States)

Riedel, E; Hampl, H; Steudle, V; Nündel, M

1996-01-01

Calcium alpha-ketoglutarate administration to 24 malnourished hemodialysis patients for 1 year leads to a significant increase in plasma concentrations of L-arginine from 53.6 +/- 18.3 (compared to a healthy control group: 87.5 +/- 27.3) to 71.1 +/- 15.9 mumol/l (p calcium alpha-ketoglutarate administration.

Novel determinants of H-Ras plasma membrane localization and transformation

DEFF Research Database (Denmark)

Willumsen, B M; Cox, A D; Solski, P A

1996-01-01

cysteine did not abolish palmitoylation. However, despite continued lipid modification the mutant proteins failed to bind to plasma membranes and instead accumulated on internal membranes and, importantly, were not transforming. Addition of an N-terminal myristoylation signal to these defective mutants......, or to proteins entirely lacking the C-terminal 25 residues restored both plasma membrane association and transforming activity. Thus, H-Ras does not absolutely require prenylation or palmitoylation nor indeed its hypervariable domain in order to interact with effectors that ultimately cause transformation....... However, in this native state, the C-terminus appears to provide a combination of lipids and a previously unrecognized signal for specific plasma membrane targeting that are essential for the correct localization and biological function of H-Ras....

Na+/H+ exchange activity in the plasma membrane of Arabidopsis.

Science.gov (United States)

Qiu, Quan-Sheng; Barkla, Bronwyn J; Vera-Estrella, Rosario; Zhu, Jian-Kang; Schumaker, Karen S

2003-06-01

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt.

Reverse-osmosis membranes by plasma polymerization

Science.gov (United States)

Hollahan, J. R.; Wydeven, T.

1972-01-01

Thin allyl amine polymer films were developed using plasma polymerization. Resulting dry composite membranes effectively reject sodium chloride during reverse osmosis. Films are 98% sodium chloride rejective, and 46% urea rejective.

Kinetic identification of an intracellular calcium compartment sensitive to phosphate and dinitrophenol in intact isolated rabbit aorta

International Nuclear Information System (INIS)

Hai, C.M.; Phair, R.D.

1986-01-01

Previous work from this laboratory revealed the presence of at least three distinct intracellular calcium compartments in intact segments of rabbit aorta. In this study one of these intracellular compartments is shown to be sensitive to dinitrophenol and to increased extracellular phosphate. Intact aortic segments were loaded with 45 Ca in bicarbonate-buffered physiologic salt solution for 1 hour, and then transferred to a flow-through chamber perfused with physiologic salt solution. Effluent from the chamber was collected for 8 hours, and 45 Ca efflux curves were analyzed using compartmental analysis. When aortic segments were loaded and washed out in dinitrophenol, the slowest component of the efflux curve was less prominent; in high phosphate it was more prominent. The rate constant changes required to account for these data were primarily in the exchange between the cytosolic and slowest intracellular calcium compartment, suggesting that the slowest calcium compartment resolved during the 8-hour washout was mitochondrial. This compartment contained 5.4 +/- 3.2 nmol calcium/g wet wt. tissue. The calcium flux across its membranes was 0.32 +/- 0.04 nmol min-1g-1. Because this flux is much smaller than the plasma-membrane calcium flux, we suggest that, in normal physiological circumstances, plasma-membrane extrusion is more important for the removal of Ca from the smooth muscle cytosol than is uptake into this slow intracellular compartment

Effect of free calcium concentration and ionic strength on alginate fouling in cross-flow membrane filtration

NARCIS (Netherlands)

Brink, van den P.; Zwijnenburg, A.; Smith, G.; Temmink, B.G.; Loosdrecht, van M.C.

2009-01-01

Extracellular polymeric substances (EPS) are generally negatively charged polymers. Membrane fouling in membrane bioreactors (MBRs) by EPS is therefore influenced by the water chemistry of the mixed liquor (calcium concentration, foulant concentration and ionic strength). We used alginate as a model

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

DEFF Research Database (Denmark)

Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne

2003-01-01

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting...... in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal...... of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx...

Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts

Energy Technology Data Exchange (ETDEWEB)

Steponkus, P.L.

1991-01-01

This project focuses on lesions in the plasma membrane of protoplasts that occur during freezing to temperatures below {minus}5{degrees} which result in changes in the semipermeablity of the plasma membrane. This injury, referred to as loss of osmotic responsiveness, is associated with the formation of large, aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal{sub II} phase transitions in the plasma membrane and subtending lamellar. The goals of this project are to provide a mechanistic understanding of the mechanism by which freeze-induced dehydration effects the formation of aparticulate domains and lamellar-to-hexagonal{sub II} phase transitions and to determine the mechanisms by which cold acclimation and cryoprotectants preclude or diminish these ultrastructural changes. Our working hypothesis is the formation of aparticulate domains and lamellar-to-hexagon{sub II} phase transitions in the plasma membrane and subtending lamellae are manifestations of hydration-dependent bilayer-bilayer interactions.

Quantitative Microscopic Analysis of Plasma Membrane Receptor Dynamics in Living Plant Cells.

Science.gov (United States)

Luo, Yu; Russinova, Eugenia

2017-01-01

Plasma membrane-localized receptors are essential for cellular communication and signal transduction. In Arabidopsis thaliana, BRASSINOSTEROID INSENSITIVE1 (BRI1) is one of the receptors that is activated by binding to its ligand, the brassinosteroid (BR) hormone, at the cell surface to regulate diverse plant developmental processes. The availability of BRI1 in the plasma membrane is related to its signaling output and is known to be controlled by the dynamic endomembrane trafficking. Advances in fluorescence labeling and confocal microscopy techniques enabled us to gain a better understanding of plasma membrane receptor dynamics in living cells. Here we describe different quantitative microscopy methods to monitor the relative steady-state levels of the BRI1 protein in the plasma membrane of root epidermal cells and its relative exocytosis and recycling rates. The methods can be applied also to analyze similar dynamics of other plasma membrane-localized receptors.

In vivo effect of 3,5,3'-triiodothyronine on calcium uptake in several tissues in the rat: Evidence for a physiological role for calcium as the first messenger for the prompt action of thyroid hormone at the level of the plasma membrane

International Nuclear Information System (INIS)

Segal, J.

1990-01-01

Calcium has been shown in vitro to serve as the first messenger for the rapid effect of thyroid hormone at the level of the plasma membrane. In the present study the physiological relevance of this mechanism is examined in the whole animal. To this end, the effect of T3 on 45calcium uptake and sugar 2-deoxyglucose (2-DG) uptake, an effect that requires extracellular calcium, and the influence of calcium blockers thereon were measured in ventricles, atria, diaphragm, fat, and liver in the rat. In the first three tissues, T3 produced comparable changes in 45Ca uptake and 2-DG uptake (T3 increased 2-DG uptake in fat, where 45Ca uptake was undetected, and had no effect in liver); this activity was blocked by the calcium channel blocker cadmium. The effect of T3 on 45Ca uptake, like its effect on the in vivo uptake of 2-DG described previously, was biphasic and time related; at physiological doses of 0.01 and 0.1 micrograms/100 g BW, T3 increased 45Ca uptake, whereas at greater (pharmacological) doses of 1 and 100 micrograms/100 g BW, T3 was without effect or inhibited 45Ca uptake. In ventricles and atria, the stimulatory effect of T3 on 45Ca uptake was very rapid within 2 min, at which time it was at or near maximum (50-90% above control) and then declined gradually and was not seen after 10-20 min. Of the several calcium blockers employed, verapamil (organic) and cadmium (inorganic) were found to be the most effective. Verapamil and cadmium produced a rapid, transient, and dose-related inhibition of 45Ca uptake in the tissues examined (except fat tissue where, under the experimental conditions employed, 45Ca uptake was undetected). Verapamil, given iv (200 micrograms/100 g BW) or ip (1 mg/100 g BW), reduced tissue 45Ca uptake by 50-90% within 2 or 10 min, respectively, and then its inhibitory effect diminished rapidly and was not seen after 20-30 min

Plasma membrane proteomics and its application in clinical cancer biomarker discovery

DEFF Research Database (Denmark)

Leth-Larsen, Rikke; Lund, Rikke; Ditzel, Henrik J

2010-01-01

Plasma membrane proteins that are exposed on the cell surface have important biological functions, such as signaling into and out of the cells, ion transport, and cell-cell and cell-matrix interactions. The expression level of many of the plasma membrane proteins involved in these key functions...... targeted by protein drugs, such as human antibodies, that have enhanced survival of several groups of cancer patients. The combination of novel analytical approaches and subcellular fractionation procedures has made it possible to study the plasma membrane proteome in more detail, which will elucidate...... cancer biology, particularly metastasis, and guide future development of novel drug targets. The technical advances in plasma membrane proteomics and the consequent biological revelations will be discussed herein. Many of the advances have been made using cancer cell lines, but because the main goal...

Deletion of the Intestinal Plasma Membrane Calcium Pump, Isoform 1, Atp2b1, in Mice is Associated with Decreased Bone Mineral Density and Impaired Responsiveness to 1, 25-Dihydroxyvitamin D3

Science.gov (United States)

Ryan, Zachary C.; Craig, Theodore A.; Filoteo, Adelaida G.; Westendorf, Jennifer J.; Cartwright, Elizabeth J.; Neyses, Ludwig; Strehler, Emanuel E.; Kumar, Rajiv

2016-01-01

The physiological importance of the intestinal plasma membrane calcium pump, isoform 1, (Pmca1, Atp2b1), in calcium absorption and homeostasis has not been previously demonstrated in vivo. Since global germ-line deletion of the Pmca1 in mice is associated with embryonic lethality, we selectively deleted the Pmca1 in intestinal absorptive cells. Mice with loxP sites flanking exon 2 of the Pmca1 gene (Pmca1fl/fl) were crossed with mice expressing Cre recombinase in the intestine under control of the villin promoter to give mice in which the Pmca1 had been deleted in the intestine (Pmca1EKO mice). Pmca1EKO mice were born at a reduced frequency and were small at the time of birth when compared to wild-type (Wt) litter mates. At two months of age, Pmca1EKO mice fed a 0.81% calcium, 0.34% phosphorus, normal vitamin D diet had reduced whole body bone mineral density (P <0.037), and reduced femoral bone mineral density (P <0.015). There was a trend towards lower serum calcium and higher serum parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) concentrations in Pmca1EKO mice compared to Wt mice but the changes were not statistically significant. The urinary phosphorus/creatinine ratio was increased in Pmca1EKO mice (P <0.004). Following the administration of 200 ng of 1α,25(OH)2D3 intraperitoneally to Wt mice, active intestinal calcium transport increased ∼2-fold, whereas Pmca1EKO mice administered an equal amount of 1α,25(OH)2D3 failed to show an increase in active calcium transport. Deletion of the Pmca1 in the intestine is associated with reduced growth and bone mineralization, and a failure to up-regulate calcium absorption in response to 1α,25(OH)2D3. PMID:26392310

Plasma membrane aquaporins mediates vesicle stability in broccoli.

Directory of Open Access Journals (Sweden)

Maria Del Carmen Martínez-Ballesta

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

Assessment of membrane protection by 31P-NMR effects of lidocaine on calcium-paradox in myocardium

International Nuclear Information System (INIS)

Sakai, Hirosumi; Yoshiyama, Minoru; Teragaki, Masakazu; Takeuchi, Kazuhide; Takeda, Takeda; Ikata, Mari; Ishikawa, Makoto; Miura, Iwao

1989-01-01

In studying calcium paradox, perfused rat hearts were used to investigate the myocardial protective effects of lidocaine. Intracellular contents of phosphates were measured using the 31 P-NMR method. In hearts reexposed to calcium, following 3 minute calcium-free perfusion, a rapid contracture occurred, followed by rapid and complete disappearance of intracellular phosphates with no resumption of cardiac function. In hearts where lidocaine was administered from the onset of the calcium-free perfusion until 2 minutes following the onset of reexposure to calcium, both intracellular phosphates and cardiac contractility were maintained. Therefore, it can be said that cell membranes were protected by lidocaine

Identification and characterization of polyphosphoinositides in the tonoplast membrane

International Nuclear Information System (INIS)

Cho, Myeon; Memon, A.R.; Boss, W.F.

1989-01-01

In vitro data indicate that the tonoplast membrane is the major target site for IP 3 action and Ca +2 is released from this compartment. If this is true, then several questions arise: (1) Is the plasma membrane or tonoplast the source of IP 3 ? (2) If the plasma membrane is the source of IP 3 , how does IP 3 get to the vacuole? In this connection, the results from in vivo labeling of carrot cells with [ 3 H] inositol show that the major phospholipid components in the tonoplast are PI and lysoPIP. In vitro phosphorylation studies using [γ- 32 P]ATP indicate that PI kinase is present in the tonoplast membrane even though PIP was not detected with inositol labeling. If PIP and PIP 2 are not present in the tonoplast, then IP 3 must diffuse or be transported from the plasma membrane. Since IP 3 is rapidly metabolized by cytosolic phosphatases there may be an IP 3 -sensitive calcium store other than the vacuole closely associated with the plasma membrane

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

Science.gov (United States)

Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

2015-09-01

Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American

Plasma surface modification of polypropylene track-etched membrane to improve its performance properties

Science.gov (United States)

Kravets, L. I.; Elinson, V. M.; Ibragimov, R. G.; Mitu, B.; Dinescu, G.

2018-02-01

The surface and electrochemical properties of polypropylene track-etched membrane treated by plasma of nitrogen, air and oxygen are studied. The effect of the plasma-forming gas composition on the surface morphology is considered. It has been found that the micro-relief of the membrane surface formed under the gas-discharge etching, changes. Moreover, the effect of the non-polymerizing gas plasma leads to formation of oxygen-containing functional groups, mostly carbonyl and carboxyl. It is shown that due to the formation of polar groups on the surface and its higher roughness, the wettability of the plasma-modified membranes improves. In addition, the presence of polar groups on the membrane surface layer modifies its electrochemical properties so that conductivity of plasma-treated membranes increase.

Routine detection of calcium-binding proteins following their adsorption to nitrocellulose membrane filters

International Nuclear Information System (INIS)

Hincke, M.T.

1988-01-01

A routine semiquantitative procedure which permits soluble calcium-binding proteins to be detected following their adsorption to nitrocellulose membrane filters by liquid scintillation counting of specifically bound 45 Ca is described. Proteins with high affinity for calcium such as calmodulin and troponin can be detected with a detection threshold of about 2 μg per 400 μl. Modifications to decrease this limit are feasible and are discussed. This technique should allow calcium-binding proteins of unknown function to be assayed during their purification. It was necessary to treat solutions containing 45 Ca with chelex-100 in order to prevent loss of calcium binding which occurred as the decay product (SC 3+ ) accumulated, suggesting that all studies utilizing 45 Ca as a tracer should evaluate possible interference by this ion

Effect of Plasma Membrane Semipermeability in Making the Membrane Electric Double Layer Capacitances Significant.

Science.gov (United States)

Sinha, Shayandev; Sachar, Harnoor Singh; Das, Siddhartha

2018-01-30

Electric double layers (or EDLs) formed at the membrane-electrolyte interface (MEI) and membrane-cytosol interface (MCI) of a charged lipid bilayer plasma membrane develop finitely large capacitances. However, these EDL capacitances are often much larger than the intrinsic capacitance of the membrane, and all of these capacitances are in series. Consequently, the effect of these EDL capacitances in dictating the overall membrane-EDL effective capacitance C eff becomes negligible. In this paper, we challenge this conventional notion pertaining to the membrane-EDL capacitances. We demonstrate that, on the basis of the system parameters, the EDL capacitance for both the permeable and semipermeable membranes can be small enough to influence C eff . For the semipermeable membranes, however, this lowering of the EDL capacitance can be much larger, ensuring a reduction of C eff by more than 20-25%. Furthermore, for the semipermeable membranes, the reduction in C eff is witnessed over a much larger range of system parameters. We attribute such an occurrence to the highly nonintuitive electrostatic potential distribution associated with the recently discovered phenomena of charge-inversion-like electrostatics and the attainment of a positive zeta potential at the MCI for charged semipermeable membranes. We anticipate that our findings will impact the quantification and the identification of a large number of biophysical phenomena that are probed by measuring the plasma membrane capacitance.

MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

Science.gov (United States)

Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

2017-03-01

Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins. Copyright © 2016 Elsevier GmbH. All rights reserved.

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

DEFF Research Database (Denmark)

Lee, Yu-Chen; Block, Gregory; Chen, Huiwen

2008-01-01

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane...... proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment...... of plasma membrane marker 5'-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5'-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1...

Plasma treatment of polyethersulfone membrane for benzene removal from water by air gap membrane distillation.

Science.gov (United States)

Pedram, Sara; Mortaheb, Hamid Reza; Arefi-Khonsari, Farzaneh

2018-01-01

In order to obtain a durable cost-effective membrane for membrane distillation (MD) process, flat sheet polyethersulfone (PES) membranes were modified by an atmospheric pressure nonequilibrium plasma generated using a dielectric barrier discharge in a mixture of argon and hexamethyldisiloxane as the organosilicon precursor. The surface properties of the plasma-modified membranes were characterized by water contact angle (CA), liquid entry pressure, X-ray photoelectron spectroscopy, scanning electron microscopy, and atomic force microscopy. The water CA of the membrane was increased from 64° to 104° by depositing a Si(CH 3 )-rich thin layer. While the pristine PES membrane was not applicable in the MD process, the modified PES membrane could be applied for the first time in an air gap membrane distillation setup for the removal of benzene as a volatile organic compound from water. The experimental design using central composite design and response surface methodology was applied to study the effects of feed temperature, concentration, and flow rate as well as their binary interactions on the overall permeate flux and separation factor. The separation factor and permeation flux of the modified PES membrane at optimum conditions were comparable with those of commercial polytetrafluoroethylene membrane.

Isolation of plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover

International Nuclear Information System (INIS)

Cook, H.W.; Palmer, F.B.; Byers, D.M.; Spence, M.W.

1988-01-01

A rapid and reliable method for the isolation of plasma membranes and microsomes of high purity and yield from cultured glioma cells is described. The procedure involves disruption by N2 cavitation, preliminary separation by centrifugation in Tricine buffer, and final separation on a gradient formed from 40% Percoll at pH 9.3. Enzyme and chemical markers indicated greater than 60% yield with six- to eightfold enrichment for plasma membranes and greater than 25% yield with three- to fourfold enrichment for a microsomal fraction consisting mainly of endoplasmic reticulum. The final fractions were obtained with high reproducibility in less than 1 h from the time of cell harvesting. Application of this procedure to human fibroblasts in culture is assessed. The isolation procedure was applied to investigations of synthesis and turnover of sphingomyelin and phosphatidylcholine in plasma membranes of glioma cells following incubation for 4-24 h with [methyl- 3 H]choline. These studies indicated that radioactivity from phosphatidylcholine synthesized in microsomes from exogenous choline may serve as a precursor of the head-group of sphingomyelin accumulating in the plasma membrane

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

Science.gov (United States)

Csermely, P; Szamel, M; Resch, K; Somogyi, J

1988-05-15

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

Research on water permeability of poly(ethylene) terephthalate track membranes modified with plasma

International Nuclear Information System (INIS)

Kravets, L.I.; Dmitriev, S.N.; Sleptsov, V.V.; Elinson, V.M.; Potryasay, V.V.

2001-01-01

The properties of poly(ethylene) terephthalate track membranes subjected to effect of plasma of the RF-discharge in air have been investigated. The influence conditions of a plasma treatment on the surface properties and hydrodynamic characteristics of the membranes has been studied. It has been found that the effect of the air plasma on the researched membranes results in a formation of asymmetric track membranes with a higher flow rate, the structure and chemical composition of their superficial layer are changed. It was shown that the availability of the modified layer on the membrane surface caused changing in their hydrodynamic characteristics - the water permeability of the membranes, processed in plasma, in a greater degree depends upon pH of a filtered solution. (author)

Therapeutic plasma exchange performed in tandem with hemodialysis without supplemental calcium in the apheresis circuit.

Science.gov (United States)

Zhao, Yong; Ibrahim, Hiba; Bailey, Jeffrey A; Linden, Jeanne; Hickson, Elda; Haynes, Stefanie; Greene, Mindy; Vauthrin, Michelle; Weinstein, Robert

2017-06-01

Therapeutic plasma exchange (TPE) and hemopoietic progenitor cell (HPC) collection are apheresis procedures that can safely be performed in tandem with hemodialysis. Despite the return of citrate-anticoagulated blood to the patient during HPC collection, it is not necessary to administer supplemental calcium during these procedures because the ionized calcium concentration is restored as the returning blood passes through the dialyzer. It is not known whether this applies to TPE, in which a mixture of blood and pharmaceutical albumin, an avid binder of plasma ionized calcium, is returned to the patient through the dialyzer. We report on three dialysis-dependent patients who required TPE and underwent tandem treatments without supplemental calcium in the apheresis circuit. Overall, ionized calcium fell 4-12% (P = 0.0.024) and patients reported no symptoms of hypocalcemic toxicity. Tandem hemodialysis/TPE can be performed without supplemental calcium in the apheresis circuit. J. Clin. Apheresis 32:154-157, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

OpenAIRE

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

2010-01-01

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

G-protein activity in Percoll-purified plasma membranes, bulk plasma membranes, and low-density plasma membranes isolated from rat cerebral cortex

Czech Academy of Sciences Publication Activity Database

Bouřová, Lenka; Stöhr, Jiří; Lisý, Václav; Rudajev, Vladimír; Novotný, Jiří; Svoboda, Petr

2009-01-01

Roč. 15, č. 4 (2009), BR111-BR122 ISSN 1234-1010 R&D Projects: GA MŠk(CZ) LC554; GA MŠk(CZ) LC06063; GA ČR(CZ) GA309/06/0121; GA AV ČR(CZ) IAA500110606 Institutional research plan: CEZ:AV0Z50110509 Keywords : rat cerebral cortex * plasma membrane * G-protein activity Subject RIV: CE - Biochemistry Impact factor: 1.543, year: 2009

Towards Enhanced Performance Thin-film Composite Membranes via Surface Plasma Modification

Science.gov (United States)

Reis, Rackel; Dumée, Ludovic F.; Tardy, Blaise L.; Dagastine, Raymond; Orbell, John D.; Schutz, Jürg A.; Duke, Mikel C.

2016-01-01

Advancing the design of thin-film composite membrane surfaces is one of the most promising pathways to deal with treating varying water qualities and increase their long-term stability and permeability. Although plasma technologies have been explored for surface modification of bulk micro and ultrafiltration membrane materials, the modification of thin film composite membranes is yet to be systematically investigated. Here, the performance of commercial thin-film composite desalination membranes has been significantly enhanced by rapid and facile, low pressure, argon plasma activation. Pressure driven water desalination tests showed that at low power density, flux was improved by 22% without compromising salt rejection. Various plasma durations and excitation powers have been systematically evaluated to assess the impact of plasma glow reactions on the physico-chemical properties of these materials associated with permeability. With increasing power density, plasma treatment enhanced the hydrophilicity of the surfaces, where water contact angles decreasing by 70% were strongly correlated with increased negative charge and smooth uniform surface morphology. These results highlight a versatile chemical modification technique for post-treatment of commercial membrane products that provides uniform morphology and chemically altered surface properties. PMID:27363670

Gel chromatography of immunoextracted plasma calcitonin in response to the calcium clamp in healthy males

International Nuclear Information System (INIS)

Bucht, E.; Toerring, O.; Sjoeberg, H.E.

1985-01-01

A radioimmunoassay (RIA) was developed for immunoreactive calcitonin (iCT) in human plasma. Antibodies against synthetic human calcitonin (hCT) coupled to bovine serum albumin (BSA) were raised in rabbits and were directed against the carboxy terminal part of CT. The detection limit of the assay was 8 pg/ml. In 7 males the iCT response to a calcium-clamp was studied. Blood was collected at 0,30 and 60 min after the start of the calcium infusion. iCT was measured directly in plasma and in extracts obtained after purification of plasma iCT by means of immobilized CT antiboides. There was a good correlation between iCT in plasma samples and extracts, r = 0.993, n = 14 (P< 0.001). Dilution curves of extracts and plasma were parallel with the hCT standard curves. Gel chromatography of the extracts on Sephadex G-50 and G-75 disclosed heterogeneity of iCT in normal plasma during basal conditions as well as during calcium stimulation. Thirty min after the start of the calcium clamp all molecular forms, most likely constituting monomeric and dimeric CT and larger forms, were increased, while after 60 min iCT seemed to constitute predominantly forms larger than monomeric TCY. Basal levels of unextracted iCT in healthy males (n = 44, 37 +/- 10 years) were 15 +/- 9 pg-equivalents/ml (mean +/- SD), which was higher than in females (n = 40, 32 +/- 9 years) 11 +/- 4 pg-equivalents/ml (P < 0.05). (author)

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

Science.gov (United States)

Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B; Zhang, Jinzhong; Catz, Sergio D

2016-02-12

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Plasma Membranes Modified by Plasma Treatment or Deposition as Solid Electrolytes for Potential Application in Solid Alkaline Fuel Cells

Science.gov (United States)

Reinholdt, Marc; Ilie, Alina; Roualdès, Stéphanie; Frugier, Jérémy; Schieda, Mauricio; Coutanceau, Christophe; Martemianov, Serguei; Flaud, Valérie; Beche, Eric; Durand, Jean

2012-01-01

In the highly competitive market of fuel cells, solid alkaline fuel cells using liquid fuel (such as cheap, non-toxic and non-valorized glycerol) and not requiring noble metal as catalyst seem quite promising. One of the main hurdles for emergence of such a technology is the development of a hydroxide-conducting membrane characterized by both high conductivity and low fuel permeability. Plasma treatments can enable to positively tune the main fuel cell membrane requirements. In this work, commercial ADP-Morgane® fluorinated polymer membranes and a new brand of cross-linked poly(aryl-ether) polymer membranes, named AMELI-32®, both containing quaternary ammonium functionalities, have been modified by argon plasma treatment or triallylamine-based plasma deposit. Under the concomitant etching/cross-linking/oxidation effects inherent to the plasma modification, transport properties (ionic exchange capacity, water uptake, ionic conductivity and fuel retention) of membranes have been improved. Consequently, using plasma modified ADP-Morgane® membrane as electrolyte in a solid alkaline fuel cell operating with glycerol as fuel has allowed increasing the maximum power density by a factor 3 when compared to the untreated membrane. PMID:24958295

Plasma membranes modified by plasma treatment or deposition as solid electrolytes for potential application in solid alkaline fuel cells.

Science.gov (United States)

Reinholdt, Marc; Ilie, Alina; Roualdès, Stéphanie; Frugier, Jérémy; Schieda, Mauricio; Coutanceau, Christophe; Martemianov, Serguei; Flaud, Valérie; Beche, Eric; Durand, Jean

2012-07-30

In the highly competitive market of fuel cells, solid alkaline fuel cells using liquid fuel (such as cheap, non-toxic and non-valorized glycerol) and not requiring noble metal as catalyst seem quite promising. One of the main hurdles for emergence of such a technology is the development of a hydroxide-conducting membrane characterized by both high conductivity and low fuel permeability. Plasma treatments can enable to positively tune the main fuel cell membrane requirements. In this work, commercial ADP-Morgane® fluorinated polymer membranes and a new brand of cross-linked poly(aryl-ether) polymer membranes, named AMELI-32®, both containing quaternary ammonium functionalities, have been modified by argon plasma treatment or triallylamine-based plasma deposit. Under the concomitant etching/cross-linking/oxidation effects inherent to the plasma modification, transport properties (ionic exchange capacity, water uptake, ionic conductivity and fuel retention) of membranes have been improved. Consequently, using plasma modified ADP-Morgane® membrane as electrolyte in a solid alkaline fuel cell operating with glycerol as fuel has allowed increasing the maximum power density by a factor 3 when compared to the untreated membrane.

Plasma membrane of a marine T cell lymphoma: surface labelling, membrane isolation, separation of membrane proteins and distribution of surface label amongst these proteins

International Nuclear Information System (INIS)

Crumpton, M.J.; Marchalonis, J.J.; Haustein, D.; Atwell, J.L.; Harris, A.W.

1976-01-01

Two established techniques for analysis of plasma membranes, namely, lactoperoxidase catalyzed surface radioiodination of intact cells and bulk membrane isolation following disruption of cells by shear forces, were applied in studies of membrane proteins of continuously cultured cells of the monoclonal T lymphoma line WEHI-22. It was found that macromolecular 125 I-iodide incorporated into plasma membrane proteins of intact cells was at least as good a marker for the plasma as was the commonly used enzyme 5'-nucleotidase, T lymphoma plasma membrane proteins were complex when analysed by polyacrylamide gel electrophoresis in sodium dodecylsulphate-containing buffers and more than thirty distinct components were resolved. More than fifteen of the components observed on a mass basis were also labelled with 125 I-iodide. Certain bands, however, exhibited a degree of label disproportionate to their staining properties with Coomassie Blue. This was interpreted in terms of their accessibility to the solvent in the intact cells. (author)

There Is No Simple Model of the Plasma Membrane Organization

Science.gov (United States)

Bernardino de la Serna, Jorge; Schütz, Gerhard J.; Eggeling, Christian; Cebecauer, Marek

2016-01-01

Ever since technologies enabled the characterization of eukaryotic plasma membranes, heterogeneities in the distributions of its constituents were observed. Over the years this led to the proposal of various models describing the plasma membrane organization such as lipid shells, picket-and-fences, lipid rafts, or protein islands, as addressed in numerous publications and reviews. Instead of emphasizing on one model we in this review give a brief overview over current models and highlight how current experimental work in one or the other way do not support the existence of a single overarching model. Instead, we highlight the vast variety of membrane properties and components, their influences and impacts. We believe that highlighting such controversial discoveries will stimulate unbiased research on plasma membrane organization and functionality, leading to a better understanding of this essential cellular structure. PMID:27747212

Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

Science.gov (United States)

Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

2014-01-01

Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

Specificity of Plasma Membrane Targeting by the Rous Sarcoma Virus Gag Protein

OpenAIRE

Scheifele, Lisa Z.; Rhoads, Jonathan D.; Parent, Leslie J.

2003-01-01

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affin...

Plasma-modified polyethylene membrane as a separator for lithium-ion polymer battery

International Nuclear Information System (INIS)

Kim, Jun Young; Lee, Yongbeom; Lim, Dae Young

2009-01-01

The surface of polyethylene (PE) membranes as a separator for lithium-ion polymer battery was modified with acrylonitrile (AN) using the plasma technology. The plasma-induced acrylonitrile coated PE (PiAN-PE) membrane was characterized by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and contact angle measurement. The electrochemical performance of the lithium-ion polymer cell fabricated with the PE and the PiAN-PE membranes were also analyzed. The surface characterization demonstrates that the enhanced adhesion of the PiAN-PE membrane resulted from the increased polar component of surface energy for the PiAN-PE membrane. The presence of the PiAN induced onto the surface of the membrane via the plasma modification plays a critical role in improving the wettability and electrolyte retention, the interfacial adhesion between the electrodes and the separator, the cycle performance of the resulting lithium-ion polymer cell assembly. The PiAN-PE membrane modified by the plasma treatment holds a great potential to be used as a high-performance and cost-effective separator for lithium-ion polymer battery.

Lipid self-assembly and lectin-induced reorganization of the plasma membrane.

Science.gov (United States)

Sych, Taras; Mély, Yves; Römer, Winfried

2018-05-26

The plasma membrane represents an outstanding example of self-organization in biology. It plays a vital role in protecting the integrity of the cell interior and regulates meticulously the import and export of diverse substances. Its major building blocks are proteins and lipids, which self-assemble to a fluid lipid bilayer driven mainly by hydrophobic forces. Even if the plasma membrane appears-globally speaking-homogeneous at physiological temperatures, the existence of specialized nano- to micrometre-sized domains of raft-type character within cellular and synthetic membrane systems has been reported. It is hypothesized that these domains are the origin of a plethora of cellular processes, such as signalling or vesicular trafficking. This review intends to highlight the driving forces of lipid self-assembly into a bilayer membrane and the formation of small, transient domains within the plasma membrane. The mechanisms of self-assembly depend on several factors, such as the lipid composition of the membrane and the geometry of lipids. Moreover, the dynamics and organization of glycosphingolipids into nanometre-sized clusters will be discussed, also in the context of multivalent lectins, which cluster several glycosphingolipid receptor molecules and thus create an asymmetric stress between the two membrane leaflets, leading to tubular plasma membrane invaginations.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

Plasma membrane organization and dynamics is probe and cell line dependent.

Science.gov (United States)

Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

2017-09-01

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment.

Science.gov (United States)

Yu, Yuanjie; Böing, Anita N; Hau, Chi M; Hajji, Najat; Ruf, Wolfram; Sturk, Auguste; Nieuwland, Rienk

2018-06-01

 Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored.  This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains.  Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined.  Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density ( ρ ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI.  Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity. Schattauer GmbH Stuttgart.

Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes.

Science.gov (United States)

Heyno, Eiri; Mary, Véronique; Schopfer, Peter; Krieger-Liszkay, Anja

2011-07-01

Production of reactive oxygen species (hydroxyl radicals, superoxide radicals and hydrogen peroxide) was studied using EPR spin-trapping techniques and specific dyes in isolated plasma membranes from the growing and the non-growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of superoxide in all plasma membrane samples. Hydroxyl radicals were only produced by the membranes of the hypocotyl growing zone when a Fenton catalyst (FeEDTA) was present. By contrast, in membranes from other parts of the seedlings a low rate of spontaneous hydroxyl radical formation was observed due to the presence of small amounts of tightly bound peroxidase. It is concluded that apoplastic hydroxyl radical generation depends fully, or for the most part, on peroxidase localized in the cell wall. In soybean plasma membranes from the growing zone of the hypocotyl pharmacological tests showed that the superoxide production could potentially be attributed to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a quinone reductase.

Mg(2+),ATP-dependent plasma membrane calcium pump of smooth muscle cells. ІІ. Regulation of activity

OpenAIRE

T. О. Veklich; Iu. Iu. Mazur; S. О. Kosterin

2015-01-01

Plasma membrane Ca2+-pump is one of key proteins, which takes part in Ca2+ exchange in smooth muscle cells. It has a lot of diverse functions from control of basal cytoplasmal Ca2+ concentration to regulation of proteins involved in Ca2+-dependent signal pathway. Ca2+ pump function is often depen­dent on the isoform or even form of alternative splicing. Allowing for a variety of Ca2+-pump functions and properties, which were reviewed in detail in the first part of our review article cycle (U...

Photostable bipolar fluorescent probe for video tracking plasma membranes related cellular processes.

Science.gov (United States)

Zhang, Xinfu; Wang, Chao; Jin, Liji; Han, Zhuo; Xiao, Yi

2014-08-13

Plasma membranes can sense the stimulations and transmit the signals from extracellular environment and then make further responses through changes in locations, shapes or morphologies. Common fluorescent membrane markers are not well suited for long time tracking due to their shorter retention time inside plasma membranes and/or their lower photostability. To this end, we develop a new bipolar marker, Mem-SQAC, which can stably insert into plasma membranes of different cells and exhibits a long retention time over 30 min. Mem-SQAC also inherits excellent photostability from the BODIPY dye family. Large two-photon absorption cross sections and long wavelength fluorescence emissions further enhance the competitiveness of Mem-SQAC as a membrane marker. By using Mem-SQAC, significant morphological changes of plasma membranes have been monitored during heavy metal poisoning and drug induced apoptosis of MCF-7 cells; the change tendencies are so distinctly different from each other that they can be used as indicators to distinguish different cell injuries. Further on, the complete processes of endocytosis toward Staphylococcus aureus and Escherichia coli by RAW 264.7 cells have been dynamically tracked. It is discovered that plasma membranes take quite different actions in response to the two bacteria, information unavailable in previous research reports.

ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

Science.gov (United States)

Isono, Erika; Kalinowska, Kamila

2017-12-01

To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fluidity of pea root plasma membranes under altered gravity

Science.gov (United States)

Klymchuk, D. O.; Baranenko, V. V.; Vorobyova, T. V.; Dubovoy, V. D.

This investigation aims to determine whether clinorotation 2 rev min of pea Pisum sativum L seedlings induces the alterations in the physical-chemical properties of cellular membranes including the plasma membrane fluidity The last is an important regulator of functional activity of membrane enzymes The plasma membranes were isolated by aqueous two-phase partitioning from roots of 6-day old pea seedlings The membrane fluidity was examined by fluorescence spectroscopy using pyrene probe The plasma membrane vesicles with known protein concentration were added to the incubation buffer to a final concentration of 50 mu g of protein per ml A small amount by 1 mu l of pyrene solution in 2-propanol was added to the incubation mixture to a final probe concentration 5 mu M at constant mixing Fluorescence spectra were measured using a Perkin-Elmer LS-50 spectrofluorometer Perkin-Elmer England Pyrene was excited at 337 nm and fluorescence intensity of monomers I M and excimers I E were measured at 393 and 470 nm respectively The I E I M ratios were 0 081 pm 0 003 and 0 072 pm 0 004 in preparations obtained from clinorotated and the control seedlings respectively This fact indicates that rotation on the clinostat increases the membrane fluidity Compared with controls clinorotated seedlings have also showed a reduced growth and a higher level of total unsaturated fatty acids determined by gas chromatography The factors that influence on the fluidity of membrane lipids in bilayer appear to be the

Role of phosphatidylinositol 4,5-bisphosphate in regulating EHD2 plasma membrane localization.

Directory of Open Access Journals (Sweden)

Laura C Simone

Full Text Available The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4 play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2's association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy, and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein.

Surface modification of polyacrylonitrile co-polymer membranes using pulsed direct current nitrogen plasma

Energy Technology Data Exchange (ETDEWEB)

Pal, Dipankar; Neogi, Sudarsan; De, Sirshendu, E-mail: sde@che.iitkgp.ernet.in

2015-12-31

Low temperature plasma treatment using pulsed direct current discharge of nitrogen gas was employed to enhance hydrophilicity of the polyacrylonitrile co-polymer membranes. The membranes were characterized in terms of morphology, structure, hydrophilicity, and membrane performance. Properties and functional groups on the surface of polyacrylonitrile co-polymer membranes were investigated by contact angle, scanning electron microscopy, Fourier transform infrared and X-ray photoelectron spectroscopy. Effects of plasma conditions, namely, pulsed voltage, duty cycle and treatment time on increase in membrane hydrophilicity were studied. Permeability of treated membrane was increased by 47% and it was retained up to 70 days. Surface etching due to plasma treatment was confirmed by weight loss of the treated membranes. Due to surface etching, average pore size increased and rejection of 200 kDa polyethylene glycol decreased to about 70% for the treated membrane. Oxygen and nitrogen functional groups were responsible for surface hydrophilicity. - Highlights: • Surface modification of polyacrylonitrile co-polymer membranes by pulsed direct current nitrogen plasma • Hydrophilic functional groups incorporated on the membrane surface • Significant enhancement of the permeability and wettability of the membranes • Water contact angle increased with storage time and finally stabilized.

Surface modification of polyacrylonitrile co-polymer membranes using pulsed direct current nitrogen plasma

International Nuclear Information System (INIS)

Pal, Dipankar; Neogi, Sudarsan; De, Sirshendu

2015-01-01

Low temperature plasma treatment using pulsed direct current discharge of nitrogen gas was employed to enhance hydrophilicity of the polyacrylonitrile co-polymer membranes. The membranes were characterized in terms of morphology, structure, hydrophilicity, and membrane performance. Properties and functional groups on the surface of polyacrylonitrile co-polymer membranes were investigated by contact angle, scanning electron microscopy, Fourier transform infrared and X-ray photoelectron spectroscopy. Effects of plasma conditions, namely, pulsed voltage, duty cycle and treatment time on increase in membrane hydrophilicity were studied. Permeability of treated membrane was increased by 47% and it was retained up to 70 days. Surface etching due to plasma treatment was confirmed by weight loss of the treated membranes. Due to surface etching, average pore size increased and rejection of 200 kDa polyethylene glycol decreased to about 70% for the treated membrane. Oxygen and nitrogen functional groups were responsible for surface hydrophilicity. - Highlights: • Surface modification of polyacrylonitrile co-polymer membranes by pulsed direct current nitrogen plasma • Hydrophilic functional groups incorporated on the membrane surface • Significant enhancement of the permeability and wettability of the membranes • Water contact angle increased with storage time and finally stabilized.

Function of plasma membrane microdomain-associated proteins during legume nodulation.

Science.gov (United States)

Qiao, Zhenzhen; Libault, Marc

2017-10-03

Plasma membrane microdomains are plasma membrane sub-compartments enriched in sphingolipids and sterols, and composed by a specific set of proteins. They are involved in recognizing signal molecules, transducing these signals, and controlling endocytosis and exocytosis processes. In a recent study, applying biochemical and microscopic methods, we characterized the soybean GmFWL1 protein, a major regulator of soybean nodulation, as a new membrane microdomain-associated protein. Interestingly, upon rhizobia inoculation of the soybean root system, GmFWL1 and one of its interacting partners, GmFLOT2/4, both translocate to the root hair cell tip, the primary site of interaction and infection between soybean and Rhizobium. The role of GmFWL1 as a plasma membrane microdomain-associated protein is also supported by immunoprecipitation assays performed on soybean nodules, which revealed 178 GmFWL1 protein partners including a large number of microdomain-associated proteins such as GmFLOT2/4. In this addendum, we provide additional information about the identity of the soybean proteins repetitively identified as GmFWL1 protein partners. Their function is discussed especially in regard to plant-microbe interactions and microbial symbiosis. This addendum will provide new insights in the role of plasma membrane microdomains in regulating legume nodulation.

Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

Science.gov (United States)

Huda, Kazi Md Kamrul; Banu, Mst Sufara Akhter; Pathi, Krishna Mohan; Tuteja, Narendra

2013-01-01

Plasma membrane Ca(2+)ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+)) from the cell, hence regulating Ca(2+) level within cells. Though plant Ca(2+)ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. The 1478 bp promoter sequence of rice plasma membrane Ca(2+)ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+)ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. The rice plasma membrane Ca(2+)ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this

Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

Directory of Open Access Journals (Sweden)

Kazi Md Kamrul Huda

Full Text Available Plasma membrane Ca(2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+ from the cell, hence regulating Ca(2+ level within cells. Though plant Ca(2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.The 1478 bp promoter sequence of rice plasma membrane Ca(2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.The rice plasma membrane Ca(2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible

Perforin rapidly induces plasma membrane phospholipid flip-flop.

Directory of Open Access Journals (Sweden)

Sunil S Metkar

Full Text Available The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells.

PLASMA-MEMBRANE LIPID ALTERATIONS INDUCED BY NACL IN WINTER-WHEAT ROOTS

NARCIS (Netherlands)

MANSOUR, MMF; VANHASSELT, PR; KUIPER, PJC

A highly enriched plasma membrane fraction was isolated by two phase partitioning from wheat roots (Triticum aestivum L. cv. Vivant) grown with and without 100 mM NaCl. The lipids of the plasma membrane fraction were extracted and characterized. Phosphatidylcholine and phosphatidylethanolamine were

Zymosterol is located in the plasma membrane of cultured human fibroblasts

International Nuclear Information System (INIS)

Echevarria, F.; Norton, R.A.; Nes, W.D.; Lange, Y.

1990-01-01

Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. (1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. (2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. (3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool

Na+/H+ Exchange Activity in the Plasma Membrane of Arabidopsis1

Science.gov (United States)

Qiu, Quan-Sheng; Barkla, Bronwyn J.; Vera-Estrella, Rosario; Zhu, Jian-Kang; Schumaker, Karen S.

2003-01-01

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt. PMID:12805632

Palmitoylation of POTE family proteins for plasma membrane targeting

International Nuclear Information System (INIS)

Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

2007-01-01

The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane

Mechanism and evolution of calcium transport across the plant plasma membrane

Science.gov (United States)

Calcium is an essential plant nutrient, thus the influx of Ca(2+) into plant cells is a critical process. In addition, the efflux of Ca(2+) out of a cell is important to prevent toxicity resulting from Ca(2+) excess, and to modulate levels of cytosolic Ca(2+) required for signaling functions. Bioc...

Analysis of the effects of cerium on calcium ion in the protoplasts of ...

African Journals Online (AJOL)

Administrator

2011-09-12

Sep 12, 2011 ... African Journal of Biotechnology Vol. 10(52), pp. ... Paper. Analysis of the effects of cerium on calcium ion in the .... plasma membrane (Zeng et al., 2003). Recently .... cerium on vegetable growth of wheat (Triticum aestivum L.).

Thymocyte plasma membrane of the rainbow trout, Salmo gairdneri: Associated immunoglobulin and heteroantigens

Science.gov (United States)

Warr, G.W.; DeLuca, D.; Anderson, D.P.

1983-01-01

1. Thymic lymphocytes of the rainbow trout, S. gairdneri were disrupted and a plasma membrane containing fraction isolated by differential and buoyant density centrifugation.2. Radioiodine introduced into the membrane by the lactoperoxidase catalyzed reaction and immunoglobulin (identified by radioimmunoassay with monoclonal antibody) both copurified in the plasma membrane fraction.3. Rabbit antibody raised to the plasma membrane fraction showed a strong reaction with trout lymphocytes in immunofluorescence, was mitogenic for trout lymphocytes, and recognized lymphocyte membrane heteroantigens of molecular weight > 70,000 in the thymus and 45,000–95,000 in the head kidney.

TiO2-Based Phosphoproteomic Analysis of the Plasma Membrane and the Effects of Phosphatase Inhibitor Treatment

DEFF Research Database (Denmark)

Thingholm, Tine; Larsen, Martin Røssel; Ingrell, Christian

2008-01-01

Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies...... of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO 2-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing....... We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10 (7) cells), achieving more than 70% yield of membrane proteins...

Surface physical chemistry properties in coated bacterial cellulose membranes with calcium phosphate.

Science.gov (United States)

de Olyveira, Gabriel Molina; Basmaji, Pierre; Costa, Ligia Maria Manzine; Dos Santos, Márcio Luiz; Dos Santos Riccardi, Carla; Guastaldi, Fernando Pozzi Semeghini; Scarel-Caminaga, Raquel Mantuaneli; de Oliveira Capote, Ticiana Sidorenko; Pizoni, Elisabeth; Guastaldi, Antônio Carlos

2017-06-01

Bacterial cellulose has become established as a new biomaterial, and it can be used for medical applications. In addition, it has called attention due to the increasing interest in tissue engineering materials for wound care. In this work, the bacterial cellulose fermentation process was modified by the addition of chondroitin sulfate to the culture medium before the inoculation of the bacteria. The biomimetic process with heterogeneous calcium phosphate precipitation of biological interest was studied for the guided regeneration purposes on bacterial cellulose. FTIR results showed the incorporation of the chondroitin sulfate in the bacterial cellulose, SEM images confirmed the deposition of the calcium phosphate on the bacterial cellulose surface, XPS analysis showed a selective chemical group influences which change calcium phosphate deposition, besides, the calcium phosphate phase with different Ca/P ratios on bacterial cellulose surface influences wettability. XTT results concluded that these materials did not affect significantly in the cell viability, being non-cytotoxic. Thus, it was produced one biomaterial with the surface charge changes for calcium phosphate deposition, besides different wettability which builds new membranes for Guided Tissue Regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescence interference contrast based approach to study real time interaction of melittin with plasma membranes

Science.gov (United States)

Gupta, Sharad; Gui, Dong; Zandi, Roya; Gill, Sarjeet; Mohideen, Umar

2014-03-01

Melittin is an anti-bacterial and hemolytic toxic peptide found in bee venom. Cell lysis behavior of peptides has been widely investigated, but the exact interaction mechanism of lytic peptides with lipid membranes and its constituents has not been understood completely. In this paper we study the melittin interaction with lipid plasma membranes in real time using non-invasive and non-contact fluorescence interference contrast microscopy (FLIC). Particularly the interaction of melittin with plasma membranes was studied in a controlled molecular environment, where these plasma membrane were composed of saturated lipid, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and unsaturated lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) with and without cholesterol. We found out that melittin starts to form nanometer size pores in the plasma membranes shortly after interacting with membranes. But the addition of cholesterol in plasma membrane slows down the pore formation process. Our results show that inclusion of cholesterol to the plasma membranes make them more resilient towards pore formation and lysis of membrane.

Towards structural and functional analysis of the plant plasma membrane proton pump

DEFF Research Database (Denmark)

Justesen, Bo Højen

The plasma membrane H+-ATPase is a proton pump essential for several physiological important processes in plants. Through the extrusion of protons from the cell, the PM H+-ATPase establishes and maintains a proton gradient used by proton coupled transporters and secondary active transport...... of nutrients and metabolites across the plasma membrane. Additional processes involving the PM H+-ATPase includes plant growth, development, and response to biotic and abiotic stresses. Extensive efforts have been made in attempts to elucidate the detailed physiological role and biochemical characteristics...... of plasma membrane H+-ATPases. Studies on the plasma membrane H+-ATPases have involved both in vivo and in vitro approaches, with the latter employing either solubilisation by detergent micelles, or reconstitution into lipid vesicles. Despite resulting in a large body of information on structure, function...

Identification of lipopolysaccharide-interacting plasma membrane-type proteins in Arabidopsis thaliana.

Science.gov (United States)

Vilakazi, Cornelius S; Dubery, Ian A; Piater, Lizelle A

2017-02-01

Lipopolysaccharide (LPS) is an amphiphatic bacterial glycoconjugate found on the external membrane of Gram-negative bacteria. This endotoxin is considered as a microbe-associated molecular pattern (MAMP) molecule and has been shown to elicit defense responses in plants. Here, LPS-interacting proteins from Arabidopsis thaliana plasma membrane (PM)-type fractions were captured and identified in order to investigate those involved in LPS perception and linked to triggering of innate immune responses. A novel proteomics-based affinity-capture strategy coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for the enrichment and identification of LPS-interacting proteins. As such, LPS isolated from Burkholderia cepacia (LPS B.cep. ) was immobilized on three independent and distinct affinity-based matrices to serve as bait for interacting proteins from A. thaliana leaf and callus tissue. These were resolved by 1D electrophoresis and identified by mass spectrometry. Proteins specifically bound to LPS B.cep. have been implicated in membrane structure (e.g. COBRA-like and tubulin proteins), membrane trafficking and/or transport (e.g. soluble NSF attachment protein receptor (SNARE) proteins, patellin, aquaporin, PM instrinsic proteins (PIP) and H + -ATPase), signal transduction (receptor-like kinases and calcium-dependent protein kinases) as well as defense/stress responses (e.g. hypersensitive-induced response (HIR) proteins, jacalin-like lectin domain-containing protein and myrosinase-binding proteins). The novel affinity-capture strategy for the enrichment of LPS-interacting proteins proved to be effective, especially in the binding of proteins involved in plant defense responses, and can thus be used to elucidate LPS-mediated molecular recognition and disease mechanism(s). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Transport proteins of the plant plasma membrane

Science.gov (United States)

Assmann, S. M.; Haubrick, L. L.; Evans, M. L. (Principal Investigator)

1996-01-01

Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

Phospholipid-binding protein EhC2A mediates calcium-dependent translocation of transcription factor URE3-BP to the plasma membrane of Entamoeba histolytica.

Science.gov (United States)

Moreno, Heriberto; Linford, Alicia S; Gilchrist, Carol A; Petri, William A

2010-05-01

The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) is a transcription factor that binds DNA in a Ca(2+)-inhibitable manner. The protein is located in both the nucleus and the cytoplasm but has also been found to be enriched in the plasma membrane of amebic trophozoites. We investigated the reason for the unusual localization of URE3-BP at the amebic plasma membrane. Here we identify and characterize a 22-kDa Ca(2+)-dependent binding partner of URE3-BP, EhC2A, a novel member of the C2-domain superfamily. Immunoprecipitations of URE3-BP and EhC2A showed that the proteins interact and that such interaction was enhanced in the presence of Ca(2+). Recombinant and native EhC2A bound phospholipid liposomes in a Ca(2+)-dependent manner, with half-maximal binding occurring at 3.4 muM free Ca(2+). A direct interaction between EhC2A and URE3-BP was demonstrated by the ability of recombinant EhC2A to recruit recombinant URE3-BP to phospholipid liposomes in a Ca(2+)-dependent manner. URE3-BP and EhC2A were observed to translocate to the amebic plasma membrane upon an increase in the intracellular Ca(2+) concentration of trophozoites, as revealed by subcellular fractionation and immunofluorescent staining. Short hairpin RNA-mediated knockdown of EhC2A protein expression significantly modulated the mRNA levels of URE3-BP-regulated transcripts. Based on these results, we propose a model for EhC2A-mediated regulation of the transcriptional activities of URE3-BP via Ca(2+)-dependent anchoring of the transcription factor to the amebic plasma membrane.

Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

International Nuclear Information System (INIS)

Wong, K.Y.; Hawley, D.; Vigneri, R.; Goldfine, I.D.

1988-01-01

Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor α subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-[γ- 32 P]triphosphate, a protein of 95 kilodaltons representing the insulin receptor β subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins

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

Science.gov (United States)

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

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

The plant plasma membrane H+-ATPase

DEFF Research Database (Denmark)

Ekberg, Kira

of plants and fungi to generate electrochemical proton gradients. A recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Together with biochemical and structural data presented in this thesis we are now able...

Fibrinogen Reduction During Selective Plasma Exchange due to Membrane Fouling.

Science.gov (United States)

Ohkubo, Atsushi; Okado, Tomokazu; Miyamoto, Satoko; Hashimoto, Yurie; Komori, Shigeto; Yamamoto, Motoki; Maeda, Takuma; Itagaki, Ayako; Yamamoto, Hiroko; Seshima, Hiroshi; Kurashima, Naoki; Iimori, Soichiro; Naito, Shotaro; Sohara, Eisei; Uchida, Shinichi; Rai, Tatemitsu

2017-06-01

Fibrinogen is substantially reduced by most plasmapheresis modalities but retained in selective plasma exchange using Evacure EC-4A10 (EC-4A). Although EC-4A's fibrinogen sieving coefficient is 0, a session of selective plasma exchange reduced fibrinogen by approximately 19%. Here, we investigated sieving coefficient in five patients. When the mean processed plasma volume was 1.15 × plasma volume, the mean reduction of fibrinogen during selective plasma exchange was approximately 15%. Fibrinogen sieving coefficient was 0 when the processed plasma volume was 1.0 L, increasing to 0.07 when the processed plasma volume was 3.0 L, with a mean of 0.03 during selective plasma exchange. When fibrinogen sieving coefficient was 0, selective plasma exchange reduced fibrinogen by approximately 10%. Scanning electron microscopy images revealed internal fouling of EC-4A's hollow fiber membrane by substances such as fibrinogen fibrils. Thus, fibrinogen reduction by selective plasma exchange may be predominantly caused by membrane fouling rather than filtration. © 2017 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.

Calcium titanate (CaTiO{sub 3}) dielectrics prepared by plasma spray and post-deposition thermal treatment

Energy Technology Data Exchange (ETDEWEB)

Ctibor, Pavel [Materials Engineering Department, Institute of Plasma Physics ASCR, v.v.i., Za Slovankou 3, Prague 8 (Czech Republic); Kotlan, Jiri, E-mail: kotlan@ipp.cas.cz [Materials Engineering Department, Institute of Plasma Physics ASCR, v.v.i., Za Slovankou 3, Prague 8 (Czech Republic); Department of Electrotechnology, Faculty of Electrical Engineering, Czech Technical University in Prague, Technicka 2, Prague 6 (Czech Republic); Pala, Zdenek [Materials Engineering Department, Institute of Plasma Physics ASCR, v.v.i., Za Slovankou 3, Prague 8 (Czech Republic); Sedlacek, Josef [Department of Electrotechnology, Faculty of Electrical Engineering, Czech Technical University in Prague, Technicka 2, Prague 6 (Czech Republic); Hajkova, Zuzana; Grygar, Tomas Matys [Institute of Inorganic Chemistry ASCR, v.v.i., Husinec-Rez 1001, Rez (Czech Republic)

2015-12-15

Highlights: • Calcium titanate was sprayed by two different plasma spray systems. • Significant improvement of dielectric properties after annealing was observed. • Calcium titanate self-supporting parts can be fabricated by plasma spraying. - Abstract: This paper studies calcium titanate (CaTiO{sub 3}) dielectrics prepared by plasma spray technology. A water stabilized plasma gun (WSP) as well as a widely used gas stabilized plasma gun (GSP) were employed in this study to deposit three sample sets at different spray conditions. Prepared specimens were annealed in air at atmospheric pressure for 2 h at various temperatures from 530 to 1170 °C. X-ray diffraction (XRD), Raman spectroscopy and porosity measurements were used for sample characterization. Dielectric spectroscopy was applied to obtain relative permittivity, conductivity and loss factor frequency dependence. Band gap energy was estimated from reflectance measurements. The work is focused on the explanation of changes in microstructure and properties of a plasma sprayed deposit after thermal annealing. Obtained results show significant improvement of dielectric properties after thermal annealing.

Calcium and Mitosis

Science.gov (United States)

Hepler, P.

1983-01-01

Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.

Effect of therapeutic concentration of lithium on live HEK293 cells; increase of Na+/K+-ATPase, change of overall protein composition and alteration of surface layer of plasma membrane.

Science.gov (United States)

Vosahlikova, Miroslava; Ujcikova, Hana; Chernyavskiy, Oleksandr; Brejchova, Jana; Roubalova, Lenka; Alda, Martin; Svoboda, Petr

2017-05-01

The effect of long-term exposure of live cells to lithium cations (Li) was studied in HEK293 cells cultivated in the presence of 1mM LiCl for 7 or 21days. The alteration of Na + /K + -ATPase level, protein composition and biophysical state of plasma membrane was determined with the aim to characterize the physiological state of Li-treated cells. Na + /K + -ATPase level was determined by [ 3 H]ouabain binding and immunoblot assays. Overall protein composition was determined by 2D electrophoresis followed by proteomic analysis by MALDI-TOF MS/MS and LFQ. Li interaction with plasma membrane was characterized by fluorescent probes DPH, TMA-DPH and Laurdan. Na + /K + -ATPase was increased in plasma membranes isolated from cells exposed to Li. Identification of Li-altered proteins by 2D electrophoresis, MALDI-TOF MS/MS and LFQ suggests a change of energy metabolism in mitochondria and cytosol and alteration of cell homeostasis of calcium. Measurement of Laurdan generalized polarization indicated a significant alteration of surface layer of isolated plasma membranes prepared from both types of Li-treated cells. Prolonged exposure of HEK293 cells to 1mM LiCl results in up-regulation of Na + /K + -ATPase expression, reorganization of overall cellular metabolism and alteration of the surface layer/polar head-group region of isolated plasma membranes. Our findings demonstrate adaptation of live HEK293 cell metabolism to prolonged exposure to therapeutic concentration of Li manifested as up-regulation of Na + /K + -ATPase expression, alteration of protein composition and change of the surface layer of plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

Morphology-properties relationship of gas plasma treated hydrophobic meso-porous membranes and their improved performance for desalination by membrane distillation

International Nuclear Information System (INIS)

Dumée, Ludovic F.; Alglave, Hortense; Chaffraix, Thomas; Lin, Bao; Magniez, Kevin; Schütz, Jürg

2016-01-01

Graphical abstract: - Highlights: • Systematic surface modifications by gas plasma treatment of hydrophobic polymers. • Correlation between plasma parameters and materials surface energy and morphology. • Spectral analysis of the formation of functional groups across the membranes surface. • Relationship between wettability, roughness and performance. - Abstract: The impact on performance of the surface energy and roughness of membrane materials used for direct contact membrane distillation are critical but yet poorly investigated parameters. The capacity to alter the wettability of highly hydrophobic materials such as poly(tetra-fluoro-ethylene) (PTFE) by gas plasma treatments is reported in this paper. An equally important contribution from this investigation arises from illustrating how vaporized material from the treated sample participates after a short while in the composition of the plasma and fundamentally changes the result of surface chemistry processes. The water contact angle across the hydrophobic membranes is generally controlled by varying the plasma gas conditions, such as the plasma power, chamber pressure and irradiation duration. Changes to surface porosity and roughness of the bulk material as well as the surface chemistry, through specific and partial de-fluorination of the surface were detected and systematically studied by Fourier transform infra-red analysis and scanning electron microscopy. It was found that the rupture of fibrils, formed during membrane processing by thermal-stretching, led to the formation of a denser surface composed of nodules similar to these naturally acting as bridging points across the membrane material between fibrils. This structural change has a profound and impart a permanent effect on the permeation across the modified membranes, which was found to be enhanced by up to 10% for long plasma exposures while the selectivity of the membranes was found to remain unaffected by the treatment at a level higher

Morphology-properties relationship of gas plasma treated hydrophobic meso-porous membranes and their improved performance for desalination by membrane distillation

Energy Technology Data Exchange (ETDEWEB)

Dumée, Ludovic F., E-mail: ludovic.dumee@deakin.edu.au [Deakin University, Geelong Victoria–Australia - Institute for Frontier Materials (Australia); Alglave, Hortense; Chaffraix, Thomas; Lin, Bao; Magniez, Kevin [Deakin University, Geelong Victoria–Australia - Institute for Frontier Materials (Australia); Schütz, Jürg [CSIRO, Manufacturing Flagship, 75 Pigdons Road, 3216 Waurn Ponds, Victoria (Australia)

2016-02-15

Graphical abstract: - Highlights: • Systematic surface modifications by gas plasma treatment of hydrophobic polymers. • Correlation between plasma parameters and materials surface energy and morphology. • Spectral analysis of the formation of functional groups across the membranes surface. • Relationship between wettability, roughness and performance. - Abstract: The impact on performance of the surface energy and roughness of membrane materials used for direct contact membrane distillation are critical but yet poorly investigated parameters. The capacity to alter the wettability of highly hydrophobic materials such as poly(tetra-fluoro-ethylene) (PTFE) by gas plasma treatments is reported in this paper. An equally important contribution from this investigation arises from illustrating how vaporized material from the treated sample participates after a short while in the composition of the plasma and fundamentally changes the result of surface chemistry processes. The water contact angle across the hydrophobic membranes is generally controlled by varying the plasma gas conditions, such as the plasma power, chamber pressure and irradiation duration. Changes to surface porosity and roughness of the bulk material as well as the surface chemistry, through specific and partial de-fluorination of the surface were detected and systematically studied by Fourier transform infra-red analysis and scanning electron microscopy. It was found that the rupture of fibrils, formed during membrane processing by thermal-stretching, led to the formation of a denser surface composed of nodules similar to these naturally acting as bridging points across the membrane material between fibrils. This structural change has a profound and impart a permanent effect on the permeation across the modified membranes, which was found to be enhanced by up to 10% for long plasma exposures while the selectivity of the membranes was found to remain unaffected by the treatment at a level higher

Reorganization of plasma membrane lipid domains during conidial germination.

Science.gov (United States)

Santos, Filipa C; Fernandes, Andreia S; Antunes, Catarina A C; Moreira, Filipe P; Videira, Arnaldo; Marinho, H Susana; de Almeida, Rodrigo F M

2017-02-01

Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted. Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30°C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth. Copyright © 2016 Elsevier B.V. All rights reserved.

Method for plasma surface treating and preparation of membrane layers

NARCIS (Netherlands)

1992-01-01

The invention relates to an apparatus suitable for plasma surface treating (e.g. forming a membrane layer on a substrate) which comprises a plasma generation section (2) which is in communication via at least one plasma inlet means (4) (e.g. a nozzle) with an enclosed plasma treating section (3)

Plasma deposition of silver nanoparticles on ultrafiltration membranes: antibacterial and anti-biofouling properties.

Science.gov (United States)

Cruz, Mercedes Cecilia; Ruano, Gustavo; Wolf, Marcus; Hecker, Dominic; Vidaurre, Elza Castro; Schmittgens, Ralph; Rajal, Verónica Beatriz

2015-02-01

A novel and versatile plasma reactor was used to modify Polyethersulphone commercial membranes. The equipment was applied to: i) functionalize the membranes with low-temperature plasmas, ii) deposit a film of poly(methyl methacrylate) (PMMA) by Plasma Enhanced Chemical Vapor Deposition (PECVD) and, iii) deposit silver nanoparticles (SNP) by Gas Flow Sputtering. Each modification process was performed in the same reactor consecutively, without exposure of the membranes to atmospheric air. Scanning electron microscopy and transmission electron microscopy were used to characterize the particles and modified membranes. SNP are evenly distributed on the membrane surface. Particle fixation and transport inside membranes were assessed before- and after-washing assays by X-ray photoelectron spectroscopy depth profiling analysis. PMMA addition improved SNP fixation. Plasma-treated membranes showed higher hydrophilicity. Anti-biofouling activity was successfully achieved against Gram-positive ( Enterococcus faecalis ) and -negative ( Salmonella Typhimurium) bacteria. Therefore, disinfection by ultrafiltration showed substantial resistance to biofouling. The post-synthesis functionalization process developed provides a more efficient fabrication route for anti-biofouling and anti-bacterial membranes used in the water treatment field. To the best of our knowledge, this is the first report of a gas phase condensation process combined with a PECVD procedure in order to deposit SNP on commercial membranes to inhibit biofouling formation.

Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

International Nuclear Information System (INIS)

Wheeler, J.J.; Boss, W.F.

1987-01-01

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-[2- 3 H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [ 3 H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP 2 ). An additional [ 3 H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP 2 on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitchrondrial-rich fraction

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs

OpenAIRE

Laat, S.W. de; Tetteroo, P.A.T.; Bluemink, J.G.; Dictus, W.J.A.G.; Zoelen, E.J.J. van

1984-01-01

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xaopus Levis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein (“HEDAF”) and 5-(N-tetradecanoyl)aminofluorescein (“TEDAF”) as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FP...

The Role of the Plasma Membrane H+-ATPase in Plant Responses to Aluminum Toxicity

Directory of Open Access Journals (Sweden)

Jiarong Zhang

2017-10-01

Full Text Available Aluminum (Al toxicity is a key factor limiting plant growth and crop production on acid soils. Increasing the plant Al-detoxification capacity and/or breeding Al-resistant cultivars are a cost-effective strategy to support crop growth on acidic soils. The plasma membrane H+-ATPase plays a central role in all plant physiological processes. Changes in the activity of the plasma membrane H+-ATPase through regulating the expression and phosphorylation of this enzyme are also involved in many plant responses to Al toxicity. The plasma membrane H+-ATPase mediated H+ influx may be associated with the maintenance of cytosolic pH and the plasma membrane gradients as well as Al-induced citrate efflux mediated by a H+-ATPase-coupled MATE co-transport system. In particular, modulating the activity of plasma membrane H+-ATPase through application of its activators (e.g., magnesium or IAA or using transgenics has effectively enhanced plant resistance to Al stress in several species. In this review, we critically assess the available knowledge on the role of the plasma membrane H+-ATPase in plant responses to Al stress, incorporating physiological and molecular aspects.

Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

Science.gov (United States)

Liu, Zan; Xu, Bo; Nameta, Masaaki; Zhang, Ying; Magdeldin, Sameh; Yoshida, Yutaka; Yamamoto, Keiko; Fujinaka, Hidehiko; Yaoita, Eishin; Tasaki, Masayuki; Nakagawa, Yuki; Saito, Kazuhide; Takahashi, Kota; Yamamoto, Tadashi

2013-06-01

Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

Simulations of simple linoleic acid-containing lipid membranes and models for the soybean plasma membranes.

Science.gov (United States)

Zhuang, Xiaohong; Ou, Anna; Klauda, Jeffery B

2017-06-07

The all-atom CHARMM36 lipid force field (C36FF) has been tested with saturated, monounsaturated, and polyunsaturated lipids; however, it has not been validated against the 18:2 linoleoyl lipids with an unsaturated sn-1 chain. The linoleoyl lipids are common in plants and the main component of the soybean membrane. The lipid composition of soybean plasma membranes has been thoroughly characterized with experimental studies. However, there is comparatively less work done with computational modeling. Our molecular dynamics (MD) simulation results show that the pure linoleoyl lipids, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0/18:2) and 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (di-18:2), agree very well with the experiments, which demonstrates the accuracy of the C36FF for the computational study of soybean membranes. Based on the experimental composition, the soybean hypocotyl and root plasma membrane models are developed with each containing seven or eight types of linoleoyl phospholipids and two types of sterols (sitosterol and stigmasterol). MD simulations are performed to characterize soybean membranes, and the hydrogen bonds and clustering results demonstrate that the lipids prefer to interact with the lipids of the same/similar tail unsaturation. All the results suggest that these two soybean membrane models can be used as a basis for further research in soybean and higher plant membranes involving membrane-associated proteins.

Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

Science.gov (United States)

Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2015-10-20

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation

Science.gov (United States)

Fujimoto, Toyoshi; Parmryd, Ingela

2017-01-01

The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence. PMID:28119914

Grafting of molecularly imprinted polymer to porous polyethylene filtration membranes by plasma polymerization.

Science.gov (United States)

Cowieson, D; Piletska, E; Moczko, E; Piletsky, S

2013-08-01

An application of plasma-induced grafting of polyethylene membranes with a thin layer of molecularly imprinted polymer (MIP) was presented. High-density polyethylene (HDPE) membranes, "Vyon," were used as a substrate for plasma grafting modification. The herbicide atrazine, one of the most popular targets of the molecular imprinting, was chosen as a template. The parameters of the plasma treatment were optimized in order to achieve a good balance between polymerization and ablation processes. Modified HDPE membranes were characterized, and the presence of the grafted polymeric layer was confirmed based on the observed weight gain, pore size measurements, and infrared spectrometry. Since there was no significant change in the porosity of the modified membranes, it was assumed that only a thin layer of the polymer was introduced on the surface. The experiments on the re-binding of the template atrazine to the membranes modified with MIP and blank polymers were performed. HDPE membranes which were grafted with polymer using continuous plasma polymerization demonstrated the best result which was expressed in an imprinted factor equal to 3, suggesting that molecular imprinting was successfully achieved.

Interactions of Ras proteins with the plasma membrane and their roles in signaling.

Science.gov (United States)

Eisenberg, Sharon; Henis, Yoav I

2008-01-01

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

Protein kinase C interaction with calcium: a phospholipid-dependent process.

LENUS (Irish Health Repository)

Bazzi, M D

1990-08-21

The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol\\/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

Science.gov (United States)

Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

2008-10-01

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans.

Science.gov (United States)

Douglas, Lois M; Konopka, James B

2016-03-01

Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans.

Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans

Science.gov (United States)

Douglas, Lois M.; Konopka, James. B.

2017-01-01

Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans. PMID:26920878

Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

International Nuclear Information System (INIS)

Hayashi, Masami; Shimada, Yukiko; Inomata, Mitsushi; Ohno-Iwashita, Yoshiko

2006-01-01

The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with β-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

Expression of membrane targeted aequorin in Xenopus laevis oocytes.

Science.gov (United States)

Daguzan, C; Nicolas, M T; Mazars, C; Leclerc, C; Moreau, M

1995-08-01

We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved.

Plasma membrane lipids and their role in fungal virulence.

Science.gov (United States)

Rella, Antonella; Farnoud, Amir M; Del Poeta, Maurizio

2016-01-01

There has been considerable evidence in recent years suggesting that plasma membrane lipids are important regulators of fungal pathogenicity. Various glycolipids have been shown to impart virulent properties in several fungal species, while others have been shown to play a role in host defense. In addition to their role as virulence factors, lipids also contribute to other virulence mechanisms such as drug resistance, biofilm formation, and release of extracellular vesicles. In addition, lipids also affect the mechanical properties of the plasma membrane through the formation of packed microdomains composed mainly of sphingolipids and sterols. Changes in the composition of lipid microdomains have been shown to disrupt the localization of virulence factors and affect fungal pathogenicity. This review gathers evidence on the various roles of plasma membrane lipids in fungal virulence and how lipids might contribute to the different processes that occur during infection and treatment. Insight into the role of lipids in fungal virulence can lead to an improved understanding of the process of fungal pathogenesis and the development of new lipid-mediated therapeutic strategies. Published by Elsevier Ltd.

Regulation of glycolytic oscillations by mitochondrial and plasma membrane H+-ATPases

DEFF Research Database (Denmark)

Olsen, Lars Folke; Andersen, Ann Zahle; Lunding, Anita

2009-01-01

,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol....../ATP antiporter and the mitochondrial F(0)F(1)-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F(0)F(1)-ATPase and plasma membrane H(+)-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations...

Crystal structure of the plasma membrane proton pump

DEFF Research Database (Denmark)

Pedersen, Bjørn P.; Buch-Pedersen, Morten Jeppe; Morth, J. Preben

2007-01-01

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H1-ATPase (the proton pump) in plants and fungi1......-3, and Na1,K1-ATPase (the sodium-potassium pump) in animals4. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis5.The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na1,K1-ATPase and Ca21......- ATPase are type II6. Electron microscopy has revealed the overall shape of proton pumps7, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define...

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

International Nuclear Information System (INIS)

Hasan, Nazarul; Hu, Chuan

2010-01-01

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of 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.

Multi-protein assemblies underlie the mesoscale organization of the plasma membrane

Science.gov (United States)

Saka, Sinem K.; Honigmann, Alf; Eggeling, Christian; Hell, Stefan W.; Lang, Thorsten; Rizzoli, Silvio O.

2014-01-01

Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity. PMID:25060237

REAL INTAKE AND PROVISION WITH VITAMINS AND CALCIUM IN OSTEOPOROSIS: ASSESSMENT BY MEASURING INTAKE AND PLASMA CONCENTRATIONS

Directory of Open Access Journals (Sweden)

V. N. Khodyrev

2014-01-01

Full Text Available Aim:  To assess the providing an organism with vitamins (А, В2, С, Е, β-carotene, and calcium in osteoporosis patients by measuring their intake and plasma concentrations. Materials and methods: 108 pairs “osteoporosis patient – healthy volunteer” aged 64.29±9.73 years were formed using a “case-control” method. Real intake of vitamins and calcium was studied defining the frequency of food taking for a month using questionnaire method. Daily intake of vitamins А, В2, С, Е, β-carotene, and calcium was calculated based on the analysis of the frequency of food taking. Among the basic group, using randomized method, 60% of osteoporosis patients were selected who underwent definition of plasma levels of vitamins А, В2, С, and Е. The same study was performed in 60 control volunteers. Results: Correlation of the intake of vitamins A, C, and β-carotene with their plasma levels wasn’t noted. It may reveal an increased need in these nutrients in osteoporosis. According to the calcium intake, every osteoporosis patient can be attributed to a population category with deep insufficiency of calcium intake (less than 500 mg a day. Conclusion: The data obtained need further investigation and, first of all, in clinical and biochemical fields (enzymatic non-provision for calcium uptake, clinical manifestations of insufficient provision with nutrients. Solution of these problems would enable regulation of food intake concerning calcium uptake in osteoporosis.

Apparatus for plasma surface treating and preparation of membrane layers

NARCIS (Netherlands)

1990-01-01

An apparatus suitable for plasma surface treating (e.g., forming a membrane layer on a substrate surface) comprises a plasma generation section which is operable at least at substantially atmospheric pressure and is in communication via at least one plasma inlet (e.g., a nozzle) with an enclosed

Response of plasma membrane H+-ATPase in rice (Oryza sativa) seedlings to simulated acid rain.

Science.gov (United States)

Liang, Chanjuan; Ge, Yuqing; Su, Lei; Bu, Jinjin

2015-01-01

Understanding the adaptation of plants to acid rain is important to find feasible approaches to alleviate such damage to plants. We studied effects of acid rain on plasma membrane H(+)-ATPase activity and transcription, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate during stress and recovery periods. Simulated acid rain at pH 5.5 did not affect plasma membrane H(+)-ATPase activity, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate. Plasma membrane H(+)-ATPase activity and transcription in leaves treated with acid rain at pH 3.5 was increased to maintain ion homeostasis by transporting excessive H(+) out of cells. Then intracellular H(+) was close to the control after a 5-day recovery, alleviating damage on membrane and sustaining photosynthetic efficiency and growth. Simulated acid rain at pH 2.5 inhibited plasma membrane H(+)-ATPase activity by decreasing the expression of H(+)-ATPase at transcription level, resulting in membrane damage and abnormal intracellular H(+), and reduction in photosynthetic efficiency and relative growth rate. After a 5-day recovery, all parameters in leaves treated with pH 2.5 acid rain show alleviated damage, implying that the increased plasma membrane H(+)-ATPase activity and its high expression were involved in repairing process in acid rain-stressed plants. Our study suggests that plasma membrane H(+)-ATPase can play a role in adaptation to acid rain for rice seedlings.

Plasma-deposited hybrid silica membranes with a controlled retention of organic bridges

Energy Technology Data Exchange (ETDEWEB)

Ngamou, P.H.T.; Creatore, M. [Department of Applied Physics, Eindhoven University of Technology, 5600 MB Eindhoven (Netherlands); Overbeek, J.P.; Kreiter, R.; Van Veen, H.M.; Vente, J.F. [ECN, Energy research Centre of the Netherlands, Petten (Netherlands); Wienk, I.M.; Cuperus, P.F. [SolSep BV, Apeldoorn (Netherlands)

2013-03-05

Hybrid organically bridged silica membranes are suitable for energy-efficient molecular separations under harsh industrial conditions. Such membranes can be useful in organic solvent nanofiltration if they can be deposited on flexible, porous and large area supports. Here, we report the proof of concept for applying an expanding thermal plasma to the synthesis of perm-selective hybrid silica films from an organically bridged monomer, 1,2-bis(triethoxysilyl)ethane. This membrane is the first in its class to be produced by plasma enhanced chemical vapor deposition. By tuning the plasma and process parameters, the organic bridging groups could be retained in the separating layer. This way, a defect free film could be made with pervaporation performances of an n-butanol-water mixture comparable with those of conventional ceramic supported membranes made by sol-gel technology (i.e. a water flux of [similar]1.8 kg m'-{sup 2} h{sup -1}, a water concentration in the permeate higher than 98% and a separation factor of >1100). The obtained results show the suitability of expanding thermal plasma as a technology for the deposition of hybrid silica membranes for molecular separations.

14C leucine chloromethylketone interaction with sarcoma 37 cell plasma membrane components

International Nuclear Information System (INIS)

Matthews, R.H.; Milo, G.E.; McMichael, T.L.; Lewis, N.J.

1982-01-01

Leucine chloromethylketone labelling of viable S37 cells was preferential for the plasma membrane fraction. The pattern of radiolabelling of the plasma membrane proteins was time-dependent. After 5 min the radiolabel was localized with glutamyl transpeptidase, and subsequently, with other physiologically active proteins as a function of time after incubation. Labelling of proteins was temperature-dependent and incubation of viable S37 cells with the radiolabelled substrate at 0 0 C yielded little or no radioactivity localized in the plasma membrane. The molecular weight of one radiolabelled substratemembrane protein complex was estimated on sodium dodecyl sulfate polyacrylamide gel electrophoresis to be between 100,000-200,000. (author)

[Biocompatibility of poly-L-lactic acid/Bioglass-guided bone regeneration membranes processed with oxygen plasma].

Science.gov (United States)

Fang, Wei; Zeng, Shu-Guang; Gao, Wen-Feng

2015-04-01

To prepare and characterize a nano-scale fibrous hydrophilic poly-L-lactic acid/ Bioglass (PLLA/BG) composite membrane and evaluate its biocompatibility as a composite membrane for guiding bone regeneration (GBR). PLLA/BG-guided bone regeneration membrane was treated by oxygen plasma to improved its hydrophilicity. The growth of MG-63 osteoblasts on the membrane was observed using Hoechst fluorescence staining, and the biocompatibility of the membrane was evaluated by calculating the cells adhesion rate and proliferation rate. Osteogenesis of MG-63 cells was assessed by detecting alkaline phosphatase (ALP), and the formation of calcified nodules and cell morphology changes were observed using scanning electron microscope (SEM). The cell adhesion rates of PLLA/BG-guided bone regeneration membrane treated with oxygen plasma were (30.570±0.96)%, (47.27±0.78)%, and (66.78±0.69)% at 1, 3, and 6 h, respectively, significantly higher than those on PLLA membrane and untreated PLLA/BG membrane (Pmembranes increased with time, but highest on oxygen plasma-treated PLLA/BG membrane (Pplasma treatment of the PLLA/BG membrane promoted cell adhesion. The membranes with Bioglass promoted the matrix secretion of the osteoblasts. Under SEM, the formation of calcified nodules and spindle-shaped cell morphology were observed on oxygen plasma-treated PLLA/BG membrane. Oxygen plasma-treated PLLA/BG composite membrane has good biocompatibility and can promote adhesion, proliferation and osteogenesis of the osteoblasts.

Surface characterization of the chitosan membrane after oxygen plasma treatment and its aging effect

International Nuclear Information System (INIS)

Wang Yingjun; Yin Shiheng; Ren Li; Zhao Lianna

2009-01-01

Chitosan has received considerable attention for biomedical applications in recent years because of its biocompatibility and biodegradability. In this paper, angle-resolved x-ray photoelectron spectroscopy (ARXPS) was carried out to investigate the chemical groups' spatial orientation on the chitosan membrane surface. Oxygen plasma treatment was also employed to improve the surface hydrophilicity of the chitosan membrane. The results of ARXPS revealed the distribution of surface polar groups, such as-OH and O=CNH 2 toward the membrane bulk, which was the origin of the chitosan membrane surface hydrophobicity. The contact angle measurements and XPS results indicated that oxygen plasma treatment can markedly improve the surface hydrophilicity and surface energy of the chitosan membrane by incorporating oxygen-containing polar groups. With the existence of the aging process, the influence of plasma treatment was not permanent, it faded with storage time. The ARXPS result discovered that the reorientation of polar functional groups generated by plasma treatment toward the membrane bulk was primarily responsible for the aging effect.

Opposing effects of cAMP and T259 phosphorylation on plasma membrane diffusion of the water channel aquaporin-5 in Madin-Darby canine kidney cells

DEFF Research Database (Denmark)

Koffman, Jennifer Skaarup; Christensen, Eva Arnspang; Marlar, Saw

2015-01-01

Aquaporin-5 (AQP5) facilitates passive water transport in glandular epithelia in response to secretory stimuli via intracellular pathways involving calcium release, cAMP and protein kinase A (PKA). In epithelial plasma membranes, AQP5 may be acutely regulated to facilitate water transport...... in the plasma membrane diffusion coefficient of AQP5. We aimed to test the short-term regulatory effects of the above pathways, by measuring lateral diffusion of AQP5 and an AQP5 phospho-mutant, T259A, using k-space Image Correlation Spectroscopy of quantum dot- and EGFP-labeled AQP5. Elevated cAMP and PKA...... inhibition significantly decreased lateral diffusion of AQP5, whereas T259A mutation showed opposing effects; slowing diffusion without stimulation and increasing diffusion to basal levels after cAMP elevation. Thus, lateral diffusion of AQP5 is significantly regulated by cAMP, PKA, and T259 phosphorylation...

Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

Science.gov (United States)

Yoshida, Aiko; Sakai, Nobuaki; Uekusa, Yoshitsugu; Imaoka, Yuka; Itagaki, Yoshitsuna; Suzuki, Yuki

2018-01-01

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open–close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME. PMID:29723197

The functions of store-operated calcium channels.

Science.gov (United States)

Putney, James W; Steinckwich-Besançon, Natacha; Numaga-Tomita, Takuro; Davis, Felicity M; Desai, Pooja N; D'Agostin, Diane M; Wu, Shilan; Bird, Gary S

2017-06-01

Store-operated calcium channels provide calcium signals to the cytoplasm of a wide variety of cell types. The basic components of this signaling mechanism include a mechanism for discharging Ca 2+ stores (commonly but not exclusively phospholipase C and inositol 1,4,5-trisphosphate), a sensor in the endoplasmic reticulum that also serves as an activator of the plasma membrane channel (STIM1 and STIM2), and the store-operated channel (Orai1, 2 or 3). The advent of mice genetically altered to reduce store-operated calcium entry globally or in specific cell types has provided important tools to understand the functions of these widely encountered channels in specific and clinically important physiological systems. This review briefly discusses the history and cellular properties of store-operated calcium channels, and summarizes selected studies of their physiological functions in specific physiological or pathological contexts. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Published by Elsevier B.V.

Distribution of IGF receptors in the plasma membrane of proximal tubular cells

International Nuclear Information System (INIS)

Hammerman, M.R.; Rogers, S.

1987-01-01

To characterize the distribution of receptors for insulin-like growth factors I and II (IGF I and II) in the plasma membrane of the renal proximal tubular cell, the authors measured binding of 125 I-labeled IGF I and 125 I-labeled IGF II to proximal tubular basolateral and brush-border membranes and characterized IGF I-stimulated phosphorylation of detergent-solubilized membranes. 125 I-IGF bound primarily to a 135,000 relative molecular weight (M r ) protein and IGF II to a 260,000 M r protein in isolated membranes. Binding of 125 I-IGF I was severalfold greater in basolateral than in brush-border membranes. IGF I-stimulated phosphorylation of the 92,000 M r β-subunit of its receptors could be demonstrated only in basolateral membranes. These findings are consistent with an asymmetrical distribution of receptors for IGF I in the plasma membrane of the renal proximal tubular cell, localization being primary on the basolateral side. In contrast, binding of 125 I-IGF II to isolated basolateral and brush-border membranes was equivalent, suggesting that receptors for this peptide are distributed more symmetrically in the plasma membrane. The findings suggest that the action of IGF I in proximal tubule are mediated via interaction of circulating peptide with specific receptors in the basolateral membrane. However, the findings established the potential for actions of IGF II to be exerted in proximal tubule via interaction with both basolateral and/or brush-border membrane receptors

Glycine Receptor Activation Impairs ATP-Induced Calcium Transients in Cultured Cortical Astrocytes

Directory of Open Access Journals (Sweden)

Tatiana P. Morais

2018-01-01

Full Text Available In central nervous system, glycine receptor (GlyR is mostly expressed in the spinal cord and brainstem, but glycinergic transmission related elements have also been identified in the brain. Astrocytes are active elements at the tripartite synapse, being responsible for the maintenance of brain homeostasis and for the fine-tuning of synaptic activity. These cells communicate, spontaneously or in response to a stimulus, by elevations in their cytosolic calcium (calcium transients, Ca2+T that can be propagated to other cells. How these Ca2+T are negatively modulated is yet poorly understood. In this work, we evaluated GlyR expression and its role on calcium signaling modulation in rat brain astrocytes. We first proved that GlyR, predominantly subunits α2 and β, was expressed in brain astrocytes and its localization was confirmed in the cytoplasm and astrocytic processes by immunohistochemistry assays. Calcium imaging experiments in cultured astrocytes showed that glycine (500 μM, a GlyR agonist, caused a concentration-dependent reduction in ATP-induced Ca2+T, an effect abolished by the GlyR antagonist, strychnine (0.8 μM, as well as by nocodazole (1 μM, known to impair GlyR anchorage to the plasma membrane. This effect was mimicked by activation of GABAAR, another Cl--permeable channel. In summary, we demonstrated that GlyR activation in astrocytes mediates an inhibitory effect upon ATP induced Ca2+T, which most probably involves changes in membrane permeability to Cl- and requires GlyR anchorage at the plasma membrane. GlyR in astrocytes may thus be part of a mechanism to modulate astrocyte-to-neuron communication.

(poly)Phosphoinositide phosphorylation is a marker for plasma membrane in Friend erythroleukaemic cells

NARCIS (Netherlands)

Rawyler, A.J.; Roelofsen, B.; Wirtz, K.W.A.; Kamp, J.A.F. op den

1982-01-01

Upon subcellular fractionation of (murine) Friend erythroleukaemic cells (FELCs), purified plasma membranes were identified by their high enrichment in specific marker enzymes and typical plasma membrane lipids. When FELCs were incubated for short periods with 32Pi before cell fractionation, the

Detecting subtle plasma membrane perturbation in living cells using second harmonic generation imaging.

Science.gov (United States)

Moen, Erick K; Ibey, Bennett L; Beier, Hope T

2014-05-20

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ~50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

There Is No Simple Model of the Plasma Membrane Organization

Czech Academy of Sciences Publication Activity Database

de la serna, J. B.; Schütz, G.; Eggeling, Ch.; Cebecauer, Marek

2016-01-01

Roč. 4, SEP 2016 (2016), 106 ISSN 2296-634X R&D Projects: GA ČR GA15-06989S Institutional support: RVO:61388955 Keywords : plasma membrane * membrane organization models * heterogeneous distribution Subject RIV: CF - Physical ; Theoretical Chemistry

The C-terminal domain of TRPV4 is essential for plasma membrane localization.

Science.gov (United States)

Becker, Daniel; Müller, Margarethe; Leuner, Kristina; Jendrach, Marina

2008-02-01

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.

Endoplasmic Reticulum-Plasma Membrane Contact Sites.

Science.gov (United States)

Saheki, Yasunori; De Camilli, Pietro

2017-06-20

The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca 2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

Data on final calcium concentration in native gel reagents determined accurately through inductively coupled plasma measurements

Directory of Open Access Journals (Sweden)

Jeffrey Viviano

2016-03-01

Full Text Available In this article we present data on the concentration of calcium as determined by Inductively Coupled Plasma (ICP measurements. Calcium was estimated in the reagents used for native gel electrophoresis of Neuronal Calcium Sensor (NCS proteins. NCS proteins exhibit calcium-dependent mobility shift in native gels. The sensitivity of this shift to calcium necessitated a precise determination of calcium concentrations in all reagents used. We determined the calcium concentrations in different components used along with the samples in the native gel experiments. These were: 20 mM Tris pH 7.5, loading dye and running buffer, with distilled water as reference. Calcium determinations were through ICP measurements. It was found that the running buffer contained calcium (244 nM over the blank. Keywords: Neuronal calcium sensor proteins, Electrophoresis, Mobility shift, Calcium, Magnesium

Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting.

Science.gov (United States)

Moeller, Hanne B; Fuglsang, Cecilia Hvitfeldt; Pedersen, Cecilie Nøhr; Fenton, Robert A

2018-01-01

Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2. Copyright © 2017 Elsevier Inc. All rights reserved.

Copper-induced activation of TRP channels promotes extracellular calcium entry and activation of CaMs and CDPKs leading to copper entry and membrane depolarization in Ulva compressa

Directory of Open Access Journals (Sweden)

Melissa eGómez

2015-03-01

Full Text Available In order to identify channels involved in membrane depolarization, Ulva compressa was incubated with agonists of TRP channels C5, A1 and V1 and the level of intracellular calcium was detected. Agonists of TRPC5, A1 and V1 induced increases in intracellular calcium at 4, 9 and 12 min of exposure, respectively, and antagonists of TRPC5, A1 and V1 corresponding to SKF-96365 (SKF, HC-030031 (HC and capsazepin (CPZ, respectively, inhibited calcium increases indicating that functional TRPs exist in U. compressa. In addition, copper excess induced increases in intracellular calcium at 4, 9 and 12 min which were inhibited by SKF, HC and CPZ, respectively, indicating that copper activate TRPC5, A1 and V1 channels. Moreover, copper-induced calcium increases were inhibited by EGTA, a non-permeable calcium chelating agent, but not by thapsigargin, an inhibitor of endoplasmic reticulum (ER calcium ATPase, indicating that activation of TRPs leads to extracellular calcium entry. Furthermore, copper-induced calcium increases were not inhibited by W-7, an inhibitor of CaMs, and staurosporine, an inhibitor of CDPKs, indicating that extracellular calcium entry did not require CaMs and CDPKs activation. In addition, copper induced membrane depolarization events at 4, 8 and 11 min and these events were inhibited by SKF, HC, CPZ and bathocuproine, a specific copper chelating agent, indicating copper entry through TRP channels leading to membrane depolarization. Moreover, membrane depolarization events were inhibited by W-7 and staurosporine, indicating that CaMs and CDPKs are required in order to activate TRPs to allow copper entry. Thus, light-dependent copper-induced activation TRPC5, A1 and V1 promotes extracellular calcium entry leading to activation of CaMs and CDPKs which, in turn, promotes copper entry through these TRP channels leading to membrane depolarization.

A cell-free assay to determine the stoichiometry of plasma membrane proteins.

Science.gov (United States)

Trigo, Cesar; Vivar, Juan P; Gonzalez, Carlos B; Brauchi, Sebastian

2013-04-01

Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.

The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

Science.gov (United States)

Cihil, Kristine M; Swiatecka-Urban, Agnieszka

2013-12-13

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

Science.gov (United States)

Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung

2013-08-06

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

Plasma Membrane Protein Profiling in Beta-Amyloid-Treated Microglia Cell Line.

Science.gov (United States)

Correani, Virginia; Di Francesco, Laura; Mignogna, Giuseppina; Fabrizi, Cinzia; Leone, Stefano; Giorgi, Alessandra; Passeri, Alessia; Casata, Roberto; Fumagalli, Lorenzo; Maras, Bruno; Schininà, M Eugenia

2017-09-01

In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

Science.gov (United States)

Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

Surface-enhanced Raman scattering reveals adsorption of mitoxantrone on plasma membrane of living cells

International Nuclear Information System (INIS)

Breuzard, G.; Angiboust, J.-F.; Jeannesson, P.; Manfait, M.; Millot, J.-M.

2004-01-01

Surface-enhanced Raman scattering (SERS) spectroscopy was applied to analyze mitoxantrone (MTX) adsorption on the plasma membrane microenvironment of sensitive (HCT-116 S) or BCRP/MXR-type resistant (HCT-116 R) cells. The addition of silver colloid to MTX-treated cells revealed an enhanced Raman scattering of MTX. Addition of extracellular DNA induced a total extinction of MTX Raman intensity for both cell lines, which revealed an adsorption of MTX on plasma membrane. A threefold higher MTX Raman intensity was observed for HCT-116 R, suggesting a tight MTX adsorption in the plasma membrane microenvironment. Fluorescence confocal microscopy confirmed a relative MTX emission around plasma membrane for HCT-116 R. After 30 min at 4 deg. C, a threefold decrease of the MTX Raman scattering was observed for HCT-116 R, contrary to HCT-116 S. Permeation with benzyl alcohol revealed a threefold decrease of membrane MTX adsorption on HCT-116 R, exclusively. This additional MTX adsorption should correspond to the drug bound to an unstable site on the HCT-116 R membrane. This study showed that SERS spectroscopy could be a direct method to reveal drug adsorption to the membrane environment of living cells

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

International Nuclear Information System (INIS)

Hicks, G.R.; Rayle, D.L.; Jones, A.M.; Lomax, T.L.

1989-01-01

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7- 3 H]IAA([ 3 H]N 3 IAA), in a manner similar to the accumulation of [ 3 H]IAA. The association of the [ 3 H]N 3 IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [ 3 H]N 3 IAA to plasma membrane vesicles prior to exposure to UV light and detected by subsequent NaDodSO 4 /PAGE and fluorography. When the reaction temperature was lowered to -196 degree C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors

Molecular cloning and characterization of the plasma membrane ...

African Journals Online (AJOL)

User

2011-05-09

May 9, 2011 ... Southern blot analysis indicated that OvPIP gene was present in O. ... Key words: Orychophragmus violaceus, plasma membrane, tonoplast aquaporins .... fractionated by 0.8% agarose gel electrophoresis and transferred to.

β-Microseminoprotein endows post coital seminal plasma with potent candidacidal activity by a calcium- and pH-dependent mechanism

DEFF Research Database (Denmark)

Edström Hägerwall, Anneli; Rydengård, Victoria; Fernlund, Per

2012-01-01

is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did...... not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as β-microseminoprotein. At neutral pH, the fungicidal activity of β......-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E(71) was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of β...

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

Science.gov (United States)

Leser, George P; Lamb, Robert A

2017-05-01

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships

[Does a lateral gradient of membrane potential on the plasma membrane of growing pollen tube of germinating pollen grain exist?].

Science.gov (United States)

Andreev, I M

2011-01-01

The data presented in the article by Breigina et al. (2009) "Changes in the membrane potential during pollen grain germination and pollen tube growth" (Tsitologiya. 51 (10): 815-823) and concerning the measurement of electric membrane potential (Delta Psi) on the plasma membrane of growing pollen tube of germinating pollen grain with the use of fluorescent potential-sensitive dye, di-4-ANEPPS, were critically analyzed in order to clarify whether a lateral gradient of Delta Psi on this membrane indeed exists. This analysis showed that the main conclusion of the authors of the above article on the existence of polar distribution of Delta Psi along the pollen tube plasma membrane is not in accordance with a number of known peculiarities of di-4-ANEPPS behavior in biological membranes and requires a significant revision. The findings in question reported by the authors, in my opinion, might be interpreted as evidence for the presence on the plasma membrane of growing pollen tube not only the membrane potential Delta Psi but also lateral gradient of so called intra-membrane dipole potential. Based on the comments made, another interpretation of the experimental results described by Breigina et al. has been offered. In addition, some drawbacks in the methodology used by the authors for measurement of Delta Psi with other fluorescent potential-sensitive dye, DiBAC3(3), are also shortly considered.

Possible evidence that dehydroepiandrosterone sulfate (DHA-S) stimulates cervical ripening by a membrane-mediated process: Specific binding-sites in plasma membrane from human uterine cervix

International Nuclear Information System (INIS)

Ohno, T.; Imai, A.; Tamaya, T.

1991-01-01

Fetal adrenal steroid, dehydroepiandrosterone sulfate (DHA-S) is well known to promote cervical ripening in late pregnancy. The presence of sites specifically binding the DHA-S in plasma membrane was studied in human cervical fibroblasts prepared from pregnant uterus. The fibroblasts were incubated with 3 H DHA-S and then fractionated into plasma membranes, cytosol, nuclei, and other organella debris. The specific activity of 3H-count in the plasma membrane fraction was enriched ∼ 7-fold compared with the whole homogenate. When the isolated plasma membrane preparations from the fibroblasts were exposed to 3 H DHA-S, the binding showed saturation kinetics; an apparent equilibrium dissociation constant (Kd) of 12 nM, and the binding capacity (Bmax) of 1.25 pmol/mg protein. The present results suggest that DHA is bound to and recognized by components in plasma membrane, and may exert its action on cervical ripening through the membrane-mediated processes

Receptor kinase-mediated control of primary active proton pumping at the plasma membrane

DEFF Research Database (Denmark)

Fuglsang, Anja Thoe; Kristensen, Astrid; Cuin, Tracey A.

2014-01-01

Acidification of the cell wall space outside the plasma membrane is required for plant growth and is the result of proton extrusion by the plasma membrane-localized H+-ATPases. Here we show that the major plasma membrane proton pumps in Arabidopsis, AHA1 and AHA2, interact directly in vitro...... and in planta with PSY1R, a receptor kinase of the plasma membrane that serves as a receptor for the peptide growth hormone PSY1. The intracellular protein kinase domain of PSY1R phosphorylates AHA2/AHA1 at Thr-881, situated in the autoinhibitory region I of the C-terminal domain. When expressed in a yeast...... heterologous expression system, the introduction of a negative charge at this position caused pump activation. Application of PSY1 to plant seedlings induced rapid in planta phosphorylation at Thr-881, concomitant with an instantaneous increase in proton efflux from roots. The direct interaction between AHA2...

Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

Science.gov (United States)

Di Giovanni, Jerome; Iborra, Cécile; Maulet, Yves; Lévêque, Christian; El Far, Oussama; Seagar, Michael

2010-07-30

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.

14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

Directory of Open Access Journals (Sweden)

Michael P Grant

Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

Rapid, directed transport of DC-SIGN clusters in the plasma membrane.

Science.gov (United States)

Liu, Ping; Weinreb, Violetta; Ridilla, Marc; Betts, Laurie; Patel, Pratik; de Silva, Aravinda M; Thompson, Nancy L; Jacobson, Ken

2017-11-01

C-type lectins, including dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), are all-purpose pathogen receptors that exist in nanoclusters in plasma membranes of dendritic cells. A small fraction of these clusters, obvious from the videos, can undergo rapid, directed transport in the plane of the plasma membrane at average speeds of more than 1 μm/s in both dendritic cells and MX DC-SIGN murine fibroblasts ectopically expressing DC-SIGN. Surprisingly, instantaneous speeds can be considerably greater. In MX DC-SIGN cells, many cluster trajectories are colinear with microtubules that reside close to the ventral membrane, and the microtubule-depolymerizing drug, nocodazole, markedly reduced the areal density of directed movement trajectories, suggesting a microtubule motor-driven transport mechanism; by contrast, latrunculin A, which affects the actin network, did not depress this movement. Rapid, retrograde movement of DC-SIGN may be an efficient mechanism for bringing bound pathogen on the leading edge and projections of dendritic cells to the perinuclear region for internalization and processing. Dengue virus bound to DC-SIGN on dendritic projections was rapidly transported toward the cell center. The existence of this movement within the plasma membrane points to an unexpected lateral transport mechanism in mammalian cells and challenges our current concepts of cortex-membrane interactions.

Targeting the plasma membrane of neoplastic cells through alkylation: a novel approach to cancer chemotherapy.

Science.gov (United States)

Trendowski, Matthew; Fondy, Thomas P

2015-08-01

Although DNA-directed alkylating agents and related compounds have been a mainstay in chemotherapeutic protocols due to their ability to readily interfere with the rapid mitotic progression of malignant cells, their clinical utility is limited by DNA repair mechanisms and immunosuppression. However, the same destructive nature of alkylation can be reciprocated at the cell surface using novel plasma membrane alkylating agents. Plasma membrane alkylating agents have elicited long term survival in mammalian models challenged with carcinomas, sarcomas, and leukemias. Further, a specialized group of plasma membrane alkylating agents known as tetra-O-acetate haloacetamido carbohydrate analogs (Tet-OAHCs) potentiates a substantial leukocyte influx at the administration and primary tumor site, indicative of a potent immune response. The effects of plasma membrane alkylating agents may be further potentiated through the use of another novel class of chemotherapeutic agents, known as dihydroxyacetone phosphate (DHAP) inhibitors, since many cancer types are known to rely on the DHAP pathway for lipid synthesis. Despite these compelling data, preliminary clinical trials for plasma membrane-directed agents have yet to be considered. Therefore, this review is intended for academics and clinicians to postulate a novel approach of chemotherapy; altering critical malignant cell signaling at the plasma membrane surface through alkylation, thereby inducing irreversible changes to functions needed for cell survival.

Calcium transport into the cells of the sea urchin larva in relation to spicule formation.

Science.gov (United States)

Vidavsky, Netta; Addadi, Sefi; Schertel, Andreas; Ben-Ezra, David; Shpigel, Muki; Addadi, Lia; Weiner, Steve

2016-10-24

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium.

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

Science.gov (United States)

Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

2016-12-06

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

Purification of a large molecular weight transglutaminase substrate from liver plasma membranes

International Nuclear Information System (INIS)

Slife, C.W.; Morris, G.S.; Tyrrell, D.J.

1986-01-01

Transglutaminases are enzymes which catalyze the covalent crosslinking of proteins by forming epsilon(γ-glutamyl)lysine isopeptide linkages. In earlier studies, the authors reported that a large molecular weight protein aggregate in rat liver plasma membranes served as a substrate for a plasma membrane-associated transglutaminase. The enzyme specifically incorporated a lysine analog, [ 3 H]putrescine, into a protein complex which remained at the top of an acrylamide gel upon electrophoresis in SDS and reducing agents. The complex has now been isolated by extracting the plasma membranes with detergent (octylglucoside) resuspending the detergent insoluble residues in 6 M guanidine HCl and chromatographing the residue on a 4% agarose column in 6 M guanidine HCl. Most of the radioactivity is found in the void volume fractions from the column. SDS polyacrylamide gel electrophoresis shows that these fractions contain mostly proteins that do not enter the acrylamide gel. Since this purification procedure is essentially the same as that used to isolate a rat hepatocyte adhesion factor from rat liver plasma membranes it is possible that the large molecular weight transglutaminase substrate and the adhesion factor are contained in the same protein aggregate

Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

Science.gov (United States)

Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

2014-01-01

The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

Plasma-chemical modification of the structure and properties of poly(ethylene terephthalate) track membranes

International Nuclear Information System (INIS)

Kravets, L I; Dmitriev, S N; Dinescu, G; Lazea, A; Sleptsov, V V; Elinson, V M

2007-01-01

A process of extraction of the low-molecular products of the synthesis from the poly(ethylene terephthalate) track membranes modified by plasma has been investigated. It is shown that the deposition of a thin polymeric hydrocarbon film by cyclohexane plasma on the membrane surface with preliminary treatment in a plasma of non-polymerizing gases, for example oxygen, allows one to produce membranes possessing a high productivity. Their advantages are much better hydrodynamic properties and a small amount of the low-molecular products of the synthesis extracted by organic solvents

Effect of ethionine on hepatic mitochondrial and microsomal calcium uptake

International Nuclear Information System (INIS)

Agarwal, A.K.; Zinermon, W.D.; Latoni, L.

1988-01-01

Ethionine, an ethyl analog of methionine, produces a variety of physiological and pathological effects in animals. These range from acute effects in the liver, kidney, pancreas, and other organs to liver carcinogenesis. Female rats when injected with ethionine exhibit a rapid decrease in hepatic adenosine triphosphate levels followed by a marked inhibition of RNA and protein synthesis and accumulation of triglycerides. Since calcium transport in mitochondria and microsomes is ATP dependent, it becomes interesting to find out if ethionine administration has any effect on subcellular calcium transport. Calcium has recently gained an increased controversy regarding its role in chemical induced lethal cell damage. Certain groups believe that influx of extracellular calcium across the damaged plasma membrane might actually mediate the irreversible damage to the cell, whereas according to other, entry of calcium into the cell is secondary to the damage. The present study was carried out to investigate the calcium [ 45 Ca] transport in mitochondria and microsomes following ethionine administration. The effect of carbon tetrachloride on calcium uptake in ethionine treated rats was also studied

Accumulation of raft lipids in T-cell plasma membrane domains engaged in TCR signalling

DEFF Research Database (Denmark)

Zech, Tobias; Ejsing, Christer S.; Gaus, Katharina

2009-01-01

Activating stimuli for T lymphocytes are transmitted through plasma membrane domains that form at T-cell antigen receptor (TCR) signalling foci. Here, we determined the molecular lipid composition of immunoisolated TCR activation domains. We observed that they accumulate cholesterol, sphingomyelin...... and saturated phosphatidylcholine species as compared with control plasma membrane fragments. This provides, for the first time, direct evidence that TCR activation domains comprise a distinct molecular lipid composition reminiscent of liquid-ordered raft phases in model membranes. Interestingly, TCR activation...... domains were also enriched in plasmenyl phosphatidylethanolamine and phosphatidylserine. Modulating the T-cell lipidome with polyunsaturated fatty acids impaired the plasma membrane condensation at TCR signalling foci and resulted in a perturbed molecular lipid composition. These results correlate...

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

Science.gov (United States)

Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W

2014-11-01

Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.

Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

DEFF Research Database (Denmark)

Schindler, J.; Ye, J. Y.; Jensen, Ole Nørregaard

2013-01-01

Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane...

Assembly of fission yeast eisosomes in the plasma membrane of budding yeast: Import of foreign membrane microdomains

Czech Academy of Sciences Publication Activity Database

Vaškovičová, Katarína; Strádalová, Vendula; Efenberk, Aleš; Opekarová, Miroslava; Malínský, Jan

2015-01-01

Roč. 94, č. 1 (2015), s. 1-11 ISSN 0171-9335 R&D Projects: GA ČR(CZ) GAP302/11/0146 Institutional support: RVO:68378041 Keywords : plasma membrane * membrane microdomain * MCC Subject RIV: EA - Cell Biology Impact factor: 4.011, year: 2015

Radio-iodination of plasma membranes of toad bladder epithelium

Energy Technology Data Exchange (ETDEWEB)

Rodriguez, H J; Edelman, I S [California Univ., San Francisco (USA). Cardiovascular Research Inst.; California Univ., San Francisco (USA). Dept. of Medicine; California Univ., San Francisco (USA). Dept. of Biochemistry and Biophysics)

1979-01-01

The present report describes high yield enzymatic radio-iodination of the apical and basal-lateral plasma membranes of toad bladder epithelium with /sup 125/I-Na, by a procedure that does not breach the functional integrity of the epithelium, as assessed by the basal and vasopressin-sensitive short-circuit current (SCC). Iodination of basal-lateral plasma membranes, at a yield comparable to that obtained with apical labelling, was attained after about 30 min of exposure of the intact bladder to the labelling solutions. Approximately 25% of the basal-lateral labeling was lost when the epithelial cells were harvested after collagenase treatment, implying that some iodination of the basement membrane had taken place. Less than 10% of iodination of the apical or basal-lateral surfaces was accounted for by lipid-labeling. Analysis of the labeled apical and basal-lateral species by enzymatic digestion and thin layer chromatography disclosed that virtually all the radioactivity was present as mono-iodotyrosine (MIT). (orig./AJ).

Remodeling of the postsynaptic plasma membrane during neural development.

Science.gov (United States)

Tulodziecka, Karolina; Diaz-Rohrer, Barbara B; Farley, Madeline M; Chan, Robin B; Di Paolo, Gilbert; Levental, Kandice R; Waxham, M Neal; Levental, Ilya

2016-11-07

Neuronal synapses are the fundamental units of neural signal transduction and must maintain exquisite signal fidelity while also accommodating the plasticity that underlies learning and development. To achieve these goals, the molecular composition and spatial organization of synaptic terminals must be tightly regulated; however, little is known about the regulation of lipid composition and organization in synaptic membranes. Here we quantify the comprehensive lipidome of rat synaptic membranes during postnatal development and observe dramatic developmental lipidomic remodeling during the first 60 postnatal days, including progressive accumulation of cholesterol, plasmalogens, and sphingolipids. Further analysis of membranes associated with isolated postsynaptic densities (PSDs) suggests the PSD-associated postsynaptic plasma membrane (PSD-PM) as one specific location of synaptic remodeling. We analyze the biophysical consequences of developmental remodeling in reconstituted synaptic membranes and observe remarkably stable microdomains, with the stability of domains increasing with developmental age. We rationalize the developmental accumulation of microdomain-forming lipids in synapses by proposing a mechanism by which palmitoylation of the immobilized scaffold protein PSD-95 nucleates domains at the postsynaptic plasma membrane. These results reveal developmental changes in lipid composition and palmitoylation that facilitate the formation of postsynaptic membrane microdomains, which may serve key roles in the function of the neuronal synapse. © 2016 Tulodziecka et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Synthesis and Characterization of Calcium Phosphate Powders for Biomedical Applications by Plasma Spray Coating

OpenAIRE

Sasidharan Pillai, Rahul

2015-01-01

This PhD work mainly focus on the synthesis and characterization of calcium phosphate powders for plasma spray coating. The preparation of high temperature phase stabilized βTCP and HA/βTCP powders for plasma spray coating applications has been the topic of investigation. Nowadays plasma sprayed coatings are widely used for biomedical applications especially in the dental and orthopaedic implantation field. Previously Ti based alloys were widely used for the orthopaedic and dental implant ap...

Interactions of sugar-based bolaamphiphiles with biomimetic systems of plasma membranes.

Science.gov (United States)

Nasir, Mehmet Nail; Crowet, Jean-Marc; Lins, Laurence; Obounou Akong, Firmin; Haudrechy, Arnaud; Bouquillon, Sandrine; Deleu, Magali

2016-11-01

Glycolipids constitute a class of molecules with various biological activities. Among them, sugar-based bolaamphiphiles characterized by their biocompatibility, biodegradability and lower toxicity, became interesting for the development of efficient and low cost lipid-based drug delivery systems. Their activity seems to be closely related to their interactions with the lipid components of the plasma membrane of target cells. Despite many works devoted to the chemical synthesis and characterization of sugar-based bolaamphiphiles, their interactions with plasma membrane have not been completely elucidated. In this work, two sugar-based bolaamphiphiles differing only at the level of their sugar residues were chemically synthetized. Their interactions with membranes have been investigated using model membranes containing or not sterol and with in silico approaches. Our findings indicate that the nature of sugar residues has no significant influence for their membrane interacting properties, while the presence of sterol attenuates the interactions of both bolaamphiphiles with the membrane systems. The understanding of this distinct behavior of bolaamphiphiles towards sterol-containing membrane systems could be useful for their applications as drug delivery systems. Copyright © 2016. Published by Elsevier B.V.

Parallel artificial liquid membrane extraction of acidic drugs from human plasma

DEFF Research Database (Denmark)

Roldan-Pijuan, Mercedes; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

2015-01-01

The new sample preparation concept “Parallel artificial liquid membrane extraction (PALME)” was evaluated for extraction of the acidic drugs ketoprofen, fenoprofen, diclofenac, flurbiprofen, ibuprofen, and gemfibrozil from human plasma samples. Plasma samples (250 μL) were loaded into individual......-performance liquid chromatography-ultraviolet detection of the individual acceptor solutions. Important PALME parameters including the chemical composition of the liquid membrane, extraction time, and sample pH were optimized, and the extraction performance was evaluated. Except for flurbiprofen, exhaustive...

Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

Science.gov (United States)

Löw, Hans; Crane, Frederick L; Morré, D James

2012-11-01

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

Magnetic apatite for structural insights on the plasma membrane

International Nuclear Information System (INIS)

Stanca, Sarmiza E; Müller, Robert; Dellith, Jan; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph

2015-01-01

The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications. (paper)

Magnetic apatite for structural insights on the plasma membrane

Science.gov (United States)

Stanca, Sarmiza E.; Müller, Robert; Dellith, Jan; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang

2015-01-01

The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.

Effects of spontaneously hypertensive rat plasma on blood pressure and tail artery calcium uptake in normotensive rats

International Nuclear Information System (INIS)

Lewanczuk, R.Z.; Wang, J.; Zhang, Z.R.; Pang, P.K.

1989-01-01

Previous studies have described the presence of humoral hypertensive factors in spontaneously hypertensive rats (SHR). Other studies have described factors that increase calcium uptake in vascular tissue. In this study, we attempted to confirm, and thereby correlate, the presence of both types of factors in SHR plasma. Intravenous infusion or bolus administration of dialyzed SHR plasma consistently induced an increase in blood pressure in normotensive rats. This hypertensive response was somewhat delayed, with peak blood pressures occurring 45 minutes after bolus administration and 90 minutes after infusion of SHR plasma. Spontaneously hypertensive rat plasma also increased 45 Ca uptake in isolated normotensive rat tail arteries in a dose-dependent manner, with a time course similar to that for the hypertensive response to bolus administration. These findings suggest, therefore, that a substance exists in SHR plasma that can increase intracellular calcium in vascular tissues and thereby increase blood pressure

The molecular mechanisms of plant plasma membrane intrinsic proteins trafficking and stress response.

Science.gov (United States)

Wang, Xing; Zhang, Ji-long; Feng, Xiu-xiu; Li, Hong-jie; Zhang, Gen-fa

2017-04-20

Plasma membrane intrinsic proteins (PIPs) are plant channel proteins located on the plasma membrane. PIPs transfer water, CO 2 and small uncharged solutes through the plasma membrane. PIPs have high selectivity to substrates, suggestive of a central role in maintaining cellular water balance. The expression, activity and localization of PIPs are regulated at the transcriptional and post-translational levels, and also affected by environmental factors. Numerous studies indicate that the expression patterns and localizations of PIPs can change in response to abiotic stresses. In this review, we summarize the mechanisms of PIP trafficking, transcriptional and post-translational regulations, and abiotic stress responses. Moreover, we also discuss the current research trends and future directions on PIPs.

Membrane raft association is a determinant of plasma membrane localization.

Science.gov (United States)

Diaz-Rohrer, Blanca B; Levental, Kandice R; Simons, Kai; Levental, Ilya

2014-06-10

The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.

Characterization of cadmium plasma membrane transport in gills of a mangrove crab Ucides cordatus

International Nuclear Information System (INIS)

Ortega, P.; Custódio, M.R.; Zanotto, F.P.

2014-01-01

Highlights: • Cd 2+ gill cell transport, a non-essential toxic metal, was characterized in a hypo-hyper-regulating mangrove crab Ucides cordatus. • Cd 2+ enter gill cells through Ca 2+ channels and is dependent of intracellular Ca 2+ levels. • Route of entry in gill cells also involves a Cd 2+ /Ca 2+ (2Na) exchanger. • Cd transport depends on Na + /K + -ATPase and gill cell electrochemical gradient. • Vanadate inhibits gill Cd 2+ transport and ouabain increase gill Cd 2+ transport. - Abstract: Membrane pathway for intracellular cadmium (Cd 2+ ) accumulation is not fully elucidated in many organisms and has not been studied in crab gill cells. To characterize membrane Cd 2+ transport of anterior and posterior gill cells of Ucides cordatus, a hypo-hyper-regulating crab, a change in intracellular Cd 2+ concentration under various experimental conditions was examined by using FluoZin, a fluorescent probe. The membrane Cd 2+ transport was estimated by the augmentation of FluoZin fluorescence induced by extracellular application of CdCl 2 and different inhibitors. Addition of extracellular calcium (Ca 2+ ) to the cells affected little the fluorescence of FluoZin, confirming that Cd 2+ was the main ion increasing intracellular fluorescence. Ca 2+ channels blockers (nimodipine and verapamil) decreased Cd 2+ influx as well as vanadate, a Ca 2+ -ATPase blocker. Chelating intracellular Ca 2+ (BAPTA) decreased Cd 2+ influx in gill cells, while increasing intracellular Ca 2+ (caffeine) augmented Cd influx. Cd 2+ and ATP added at different temporal conditions were not effective at increasing intracellular Cd 2+ accumulation. Ouabain (Na + /K + -ATPase inhibitor) increased Cd 2+ influx probably through a change in intracellular Na and/or a change in cell membrane potential. Routes of Cd 2+ influx, a non-essential metal, through the gill cell plasma membrane of crabs are suggested

The Enzymology of Protein Translocation across the Escherichia coli Plasma Membrane

NARCIS (Netherlands)

Wickner, William; Driessen, Arnold J.M.; Hartl, Franz-Ulrich

1991-01-01

Converging physiological, genetic, and biochemical studies have established the salient features of preprotein translocation across the plasma membrane of Escherichia coli. Translocation is catalyzed by two proteins, a soluble chaperone and a membrane-bound translocase. SecB, the major chaperone for

Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

Science.gov (United States)

Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

2016-05-01

Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. © 2016 American Society of Plant Biologists. All Rights Reserved.

Apparatus suitable for plasma surface treating and process for preparing membrane layers

NARCIS (Netherlands)

1988-01-01

The invention relates to an apparatus suitable for plasma surface treating (e.g. forming a membrane layer on a substrate) which comprises a plasma generation section (2) which is in communication via at least one plasma inlet means (4) (e.g. a nozzle) with an enclosed plasma treating section (3)

GLUT-4 content in plasma membrane of muscle from patients with non-insulin-dependent diabetes mellitus

DEFF Research Database (Denmark)

Lund, S; Vestergaard, H; Andersen, P H

1993-01-01

The abundance of GLUT-4 protein in both total crude membrane and plasma membrane fractions of vastus lateralis muscle from 13 obese non-insulin-dependent diabetes mellitus (NIDDM) patients and 14 healthy subjects were examined in the fasting state and after supraphysiological hyperinsulinemia....... In the basal state the immunoreactive mass of GLUT-4 protein both in the crude membrane preparation and in the plasma membrane fraction was similar in NIDDM patients and control subjects. Moreover, in vivo insulin exposure neither for 30 min nor for 4 h had any impact on the content of GLUT-4 protein in plasma...... membranes. With the use of the same methodology, antibody, and achieving the same degree of plasma membrane purification and recovery, we found, however, that intraperitoneal administration of insulin to 7-wk-old rats within 30 min increased the content of GLUT-4 protein more than twofold (P

Effect of propionyl-L-carnitine on L-type calcium channels in human heart sarcolemma

International Nuclear Information System (INIS)

Bevilacqua, M.; Vago, T.; Norbiato, G.

1991-01-01

Propionyl-L-carnitine (PC) protects perfused rat hearts against damage by ischemia-reperfusion. Activation of L-type calcium channel play a role on ischemia-reperfusion damage. Therefore, we studied the effect of PC on some properties of L-type calcium channels in an in vitro preparation from human myocardium sarcolemma (from patients with idiopathic dilated cardiomyopathy). Binding of the L-type calcium channel blockers isradipine [ 3 H]-PN 200-110 (PN) to plasma membrane preparations revealed a single population of binding sites (total number: Bmax = 213 +/- 34 fM/mg protein and affinity: Kd = 152 +/- 19 nM; n = 6). The characteristics of these binding sites were evaluated in the presence and in the absence of Ca 2+ and of calcium blockers (D-888, a verapamillike drug, and diltiazem). Incubation in a Ca 2+ -containing buffer increased the affinity of PN binding sites. Binding sites for PN were modulated by organic calcium channel blockers; in competition isotherms at 37 degree C, D-888 (desmethoxyverapamil) decreased the PN binding, whereas diltiazem increased it. These results strongly suggest that the site labelled by PN is the voltage-operated calcium channel of the human myocardium. The addition of PC (1 mM) to plasma membranes labelled with PN at 37 degree C decreased the affinity of the binding; this effect was counteracted by the addition of Ca 2+ to the medium. This result was consistent with a competition between Ca 2+ and PC. The effect of PC incubation at 4 degree C was the opposite; at this temperature PC increased the affinity of the binding sites and the effect was obscured by Ca 2+

Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

Science.gov (United States)

Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

2017-08-01

While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

The Road not Taken: Less Traveled Roads from the TGN to the Plasma Membrane.

Science.gov (United States)

Spang, Anne

2015-03-10

The trans-Golgi network functions in the distribution of cargo into different transport vesicles that are destined to endosomes, lysosomes and the plasma membrane. Over the years, it has become clear that more than one transport pathway promotes plasma membrane localization of proteins. In spite of the importance of temporal and spatial control of protein localization at the plasma membrane, the regulation of sorting into and the formation of different transport containers are still poorly understood. In this review different transport pathways, with a special emphasis on exomer-dependent transport, and concepts of regulation and sorting at the TGN are discussed.

In vitro sealing of iatrogenic fetal membrane defects by a collagen plug imbued with fibrinogen and plasma.

Science.gov (United States)

Engels, A C; Hoylaerts, M F; Endo, M; Loyen, S; Verbist, G; Manodoro, S; DeKoninck, P; Richter, J; Deprest, J A

2013-02-01

We aimed to demonstrate local thrombin generation by fetal membranes, as well as its ability to generate fibrin from fibrinogen concentrate. Furthermore, we aimed to investigate the efficacy of collagen plugs, soaked with plasma and fibrinogen, to seal iatrogenic fetal membrane defects. Thrombin generation by homogenized fetal membranes was measured by calibrated automated thrombography. To identify the coagulation caused by an iatrogenic membrane defect, we analyzed fibrin formation by optical densitometry, upon various concentrations of fibrinogen. The ability of a collagen plug soaked with fibrinogen and plasma was tested in an ex vivo model for its ability to seal an iatrogenic fetal membrane defect. Fetal membrane homogenates potently induced thrombin generation in amniotic fluid and diluted plasma. Upon the addition of fibrinogen concentrate, potent fibrin formation was triggered. Measured by densiometry, fibrin formation was optimal at 1250 µg/mL fibrinogen in combination with 4% plasma. A collagen plug soaked with fibrinogen and plasma sealed an iatrogenic membrane defect about 35% better than collagen plugs without these additives (P = 0.037). These in vitro experiments suggest that the addition of fibrinogen and plasma may enhance the sealing efficacy of collagen plugs in closing iatrogenic fetal membrane defects. © 2013 John Wiley & Sons, Ltd.

Binding and assembly of actin filaments by plasma membranes from dictyostelium discoideum

International Nuclear Information System (INIS)

Schwartz, M.A.; Luna, E.J.

1986-01-01

The binding of native, 125 I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. The authors conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

Science.gov (United States)

Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

2018-05-01

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

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

Science.gov (United States)

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

2018-01-01

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

LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

Science.gov (United States)

Clayton, R N; Shakespear, R A; Marshall, J C

1978-06-01

Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

Dynamic complexity: plant receptor complexes at the plasma membrane.

Science.gov (United States)

Burkart, Rebecca C; Stahl, Yvonne

2017-12-01

Plant receptor complexes at the cell surface perceive many different external and internal signalling molecules and relay these signals into the cell to regulate development, growth and immunity. Recent progress in the analyses of receptor complexes using different live cell imaging approaches have shown that receptor complex formation and composition are dynamic and take place at specific microdomains at the plasma membrane. In this review we focus on three prominent examples of Arabidopsis thaliana receptor complexes and how their dynamic spatio-temporal distribution at the PM has been studied recently. We will elaborate on the newly emerging concept of plasma membrane microdomains as potential hubs for specific receptor complex assembly and signalling outputs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Derangement of cellular plasma membranes due to non-lethal radiation doses

International Nuclear Information System (INIS)

Koeteles, G.J.; Kubasova, T.; Somosy, Z.; Horvath, L.

1983-01-01

Earlier observations in the laboratory on fibroblasts and various blood cells of animal and human origins pointed to alteration of concanavalin A binding sites of plasma membranes as well as to concomitant morphological changes and scanning electron microscopic appearance of cell surfaces following sub-lethal doses of X-, fission neutron and beta irradiations. The effects appeared early and existed temporarily; their intensities and the restitution of membrane function depended on radiation doses, types and conditions of cells. In the present paper further aspects of structural and functional derangements of plasma membranes are introduced which were provoked by X- and tritium beta irradiation in the dose range up to 2.5 Gy and in the concentration range from 3.7 kBq/mL, respectively. The state of membrane structure was followed by bindings of various ligands of different receptor requirements, concanavalin A, cationized ferritin and polio virus. In the case of X-irradiation the binding conditions suggest the shift of overall negative surface charges to less negative ones. It was also found that radiation-induced phenomena appear on the cell surface unevenly. Long- and short-term treatments of cells with 3 H-thymidine and 3 H-water also perturb the plasma membrane; beta irradiation affects it directly. Membrane structure and function are suggested to offer good biological models to study correlation of energy deposition and biological effects, both restricted to domains of nanometre range. The data give evidence for radiation-induced membrane alterations in the sub-lethal or non-lethal ranges which might have consequences in the development of stochastic and non-stochastic effects. (author)

Resolution of intracellular calcium metabolism in intact segments of rabbit aorta

International Nuclear Information System (INIS)

Phair, R.D.; Hai, C.M.

1986-01-01

A new method, based on computer-assisted kinetic analysis of 45 Ca efflux data, was used to measure calcium contents and fluxes for extracellular and intracellular compartments in intact segments of rabbit aorta. After a 1-hour loading period, efflux data were collected for 8 hours using a flow-through tissue chamber. These long-term effluxes were necessary because information on intracellular calcium metabolism was concentrated in the slow components of the efflux curves while earlier components appeared to be dominated by washout of extracellular calcium. Intracellular compartments were identified as those whose calcium contents were altered by 10 microM phenylephrine. This method complements previous approaches by providing simultaneous estimates of compartmental calcium contents and fluxes without requiring the assumption of isotopic equilibrium and without recourse to standard wash techniques for removal of extracellular calcium. In normal, calcium-containing, bicarbonate-buffered physiological salt solution these compartments contained a total of approximately 300 nmol Ca/g wet aorta. Of this total, 55 nmol/g were associated with the slowest resolvable compartment whose turnover time was 170 minutes and whose exchange flux was 0.32 nmol min-1g-1. Two other intracellular compartments had turnover times of 30 minutes. One of these was phenylephrine releasable and contained 145 nmol/g; it exchanged calcium at 4.9 nmol min-1g-1. In normal physiological salt solution the plasma membrane was, surprisingly, not rate limiting for Ca efflux; and in 10 microM phenylephrine the membrane Ca flux was even greater, increasing 3.5-fold compared to control

Characterization of plasma-induced cell membrane permeabilization: focus on OH radical distribution

International Nuclear Information System (INIS)

Sasaki, Shota; Honda, Ryosuke; Hokari, Yutaro; Takashima, Keisuke; Kaneko, Toshiro; Kanzaki, Makoto

2016-01-01

Non-equilibrium atmospheric-pressure plasma (APP) is used medically for plasma-induced cell permeabilization. However, how plasma irradiation specifically triggers permeabilization remains unclear. In an attempt to identify the dominant factor( s ), the distribution of plasma-produced reactive species was investigated, primarily focusing on OH radicals. A stronger plasma discharge, which produced more OH radicals in the gas phase, also produced more OH radicals in the liquid phase (OH aq ), enhancing the cell membrane permeability. In addition, plasma irradiation-induced enhancement of cell membrane permeability decreased markedly with increased solution thickness (<1 mm), and the plasma-produced OH aq decayed in solution (diffusion length on the order of several hundred micrometers). Furthermore, the horizontally center-localized distribution of OH aq corresponded with the distribution of the permeabilized cells by plasma irradiation, while the overall plasma-produced oxidizing species in solution (detected by iodine-starch reaction) exhibited a doughnut-shaped horizontal distribution. These results suggest that OH aq, among the plasma-produced oxidizing species, represents the dominant factor in plasma-induced cell permeabilization. These results enhance the current understanding of the mechanism of APP as a cell-permeabilization tool. (paper)

Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

Science.gov (United States)

Koldsø, Heidi; Sansom, Mark S P

2015-11-25

The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

Performance enhancement of membrane electrode assemblies with plasma etched polymer electrolyte membrane in PEM fuel cell

Energy Technology Data Exchange (ETDEWEB)

Cho, Yong-Hun; Yoon, Won-Sub [School of Advanced Materials Engineering, Kookmin University, 861-1 Jeongneung-dong, Seongbuk-gu, Seoul 136-702 (Korea); Bae, Jin Woo; Cho, Yoon-Hwan; Lim, Ju Wan; Ahn, Minjeh; Jho, Jae Young; Sung, Yung-Eun [World Class University (WCU) program of Chemical Convergence for Energy and Environment (C2E2), School of Chemical and Biological Engineering, College of Engineering, Seoul National University (SNU), 599 Gwanak-Ro, Gwanak-gu, Seoul 151-744 (Korea); Kwon, Nak-Hyun [Fuel Cell Vehicle Team 3, Advanced Technology Center, Corporate Research and Development Division, Hyundai-Kia Motors, 104 Mabuk-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-912 (Korea)

2010-10-15

In this work, a surface modified Nafion 212 membrane was fabricated by plasma etching in order to enhance the performance of a membrane electrode assembly (MEA) in a polymer electrolyte membrane fuel cell. Single-cell performance of MEA at 0.7 V was increased by about 19% with membrane that was etched for 10 min compared to that with untreated Nafion 212 membrane. The MEA with membrane etched for 20 min exhibited a current density of 1700 mA cm{sup -2} at 0.35 V, which was 8% higher than that of MEA with untreated membrane (1580 mA cm{sup -2}). The performances of MEAs containing etched membranes were affected by complex factors such as the thickness and surface morphology of the membrane related to etching time. The structural changes and electrochemical properties of the MEAs with etched membranes were characterized by field emission scanning electron microscopy, Fourier transform-infrared spectrometry, electrochemical impedance spectroscopy, and cyclic voltammetry. (author)

Effects of fiber density and plasma modification of nanofibrous membranes on the adhesion and growth of HaCaT keratinocytes.

Science.gov (United States)

Bacakova, Marketa; Lopot, Frantisek; Hadraba, Daniel; Varga, Marian; Zaloudkova, Margit; Stranska, Denisa; Suchy, Tomas; Bacakova, Lucie

2015-01-01

It may be possible to regulate the cell colonization of biodegradable polymer nanofibrous membranes by plasma treatment and by the density of the fibers. To test this hypothesis, nanofibrous membranes of different fiber densities were treated by oxygen plasma with a range of plasma power and exposure times. Scanning electron microscopy and mechanical tests showed significant modification of nanofibers after plasma treatment. The intensity of the fiber modification increased with plasma power and exposure time. The exposure time seemed to have a stronger effect on modifying the fiber. The mechanical behavior of the membranes was influenced by the plasma treatment, the fiber density, and their dry or wet state. Plasma treatment increased the membrane stiffness; however, the membranes became more brittle. Wet membranes displayed significantly lower stiffness than dry membranes. X-ray photoelectron spectroscopy (XPS) analysis showed a slight increase in oxygen-containing groups on the membrane surface after plasma treatment. Plasma treatment enhanced the adhesion and growth of HaCaT keratinocytes on nanofibrous membranes. The cells adhered and grew preferentially on membranes of lower fiber densities, probably due to the larger area of void spaces between the fibers. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells

International Nuclear Information System (INIS)

Chubinskiy-Nadezhdin, Vladislav I.; Vasileva, Valeria Y.; Pugovkina, Natalia A.; Vassilieva, Irina O.; Morachevskaya, Elena A.; Nikolsky, Nikolay N.; Negulyaev, Yuri A.

2017-01-01

Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs). Functional expression of native mechanosensitive stretch-activated channels (SACs) and calcium-sensitive potassium channels of different conductances in hMESCs was shown. Single current analysis of stretch-induced channel activity revealed functional coupling of SACs and BK channels in plasma membrane. The combination of cell-attached and inside-out experiments have indicated that highly localized Ca 2+ entry via SACs triggers BK channel activity. At the same time, SK channels are not coupled with SACs despite of high calcium sensitivity as compared to BK. Our data demonstrate novel mechanism controlling BK channel activity in native cells. We conclude that SACs and BK channels are clusterized in functional mechanosensitive domains in the plasma membrane of hMESCs. Co-clustering of ion channels may significantly contribute to mechano-dependent calcium signalling in stem cells. - Highlights: • Stretch-induced channel activity in human mesenchymal stem cells was analyzed. • Functional expression of SACs and Ca 2+ -sensitive BK and SK channels was shown. • Local Ca 2+ influx via stretch-activated channels triggers BK channel activity. • SK channels are not coupled with SACs despite higher sensitivity to [Ca 2+ ] i . • Functional clustering of SACs and BK channels in stem cell membrane is proposed.