Sample records for basolateral plasma membranes

  1. Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting. (United States)

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


    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.

  2. Hepatic taurine transport: a Na+-dependent carrier on the basolateral plasma membrane

    International Nuclear Information System (INIS)

    Bucuvalas, J.C.; Goodrich, A.L.; Suchy, F.J.


    Highly purified rat basolateral liver plasma membrane vesicles were used examine the mechanism and the driving forces for hepatic uptake of the β-amino acid, taurine. An inwardly directed 100 mM NaCl gradient stimulated the initial rate of taurine uptake and energized a transient twofold accumulation of taurine above equilibrium (overshoot). In contrast, uptake was slower and no overshoot was detected in the presence of a KCl gradient. A negative intravesicular electrical potential generated by the presence of permeant anions or an outwardly directed K + gradient with valinomycin increased Na + -stimulated taurine uptake. External Cl - stimulated Na + -dependent taurine uptake independent of effects on the transmembrane electrical potential difference. Na + -dependent taurine uptake showed a sigmoidal dependence on extravesicular Na + concentration, suggesting multiple Na + ions are involved in the translocation of each taurine molecule. Na + -dependent taurine uptake demonstrated Michaelis-Menten kinetics with a maximum velocity of 0.537 nmol x mg protein -1 x min -1 and an apparent K/sub m/ of 174 μM. [ 3 H]taurine uptake was inhibited by the presence of excess unlabeled taurine, β-alanine, or hypotaurine but not by L-glutamine or L-alanine. In summary, using basolateral liver plasma membrane vesicles, the authors have shown that hepatic uptake of taurine occurs by a carrier-mediated, secondary active transport process specific for β-amino acids. Uptake is electrogenic, stimulated by external Cl - , and requires multiple Na + ions for the translocation of each taurine molecule

  3. Growth hormone activates phospholipase C in proximal tubular basolateral membranes from canine kidney

    International Nuclear Information System (INIS)

    Rogers, S.A.; Hammerman, M.R.


    To delineate pathways for signal transduction by growth hormone (GH) in proximal tubule, the authors incubated basolateral membranes isolated from canine kidney with human growth hormone (hGH) or human prolactin (hPrl) and measured levels of inositol trisphosphate (InsP 3 ) in suspensions and of diacylglycerol extractable from the membranes. Incubation with hGH, but not hPrl, increased levels of InsP 3 and diacylglycerol in a concentration-dependent manner. Half-maximal effects occurred between 0.1 and 1 nM hGH. Increased levels of InsP 3 were measured after as little as 5 sec of incubation with 1 nM hGH, and increase was maximal after 15 sec. Increases were no longer detectable after 60 sec because of dephosphorylation of InsP 3 in membrane suspensions. hGH did not affect rates of dephosphorylation. hGH-stimulated increases in InsP 3 were detectable in membranes suspended in 0, 0.1, and 0.2 μM calcium but not in 0.3 or 1.0 μM calcium. 125 I-labeled hGH-receptor complexes with M r values of 66,000 and 140,000 were identified in isolated basolateral membranes. The findings establish that GH activates phospholipase C in isolated canine renal proximal tubular basolateral membranes, potentially after binding to a specific receptor. This process could mediate signal transmission by GH across the plasma membrane of the proximal tubular cell and elsewhere

  4. NKCC1 and NHE1 are abundantly expressed in the basolateral plasma membrane of secretory coil cells in rat, mouse, and human sweat glands

    DEFF Research Database (Denmark)

    Nejsum, Lene Niemann; Prætorius, Jeppe; Nielsen, Søren


    1 (NHE1) protein has been localized to both the duct and secretory coil of human sweat duct; however, the NHE1 abundance in the duct was not compared with that in the secretory coil. The aim of this study was to test whether mRNA encoding NKCC1, NKCC2, and Na(+)-coupled acid-base transporters...... and the corresponding proteins are expressed in rodent sweat glands and, if expressed, to determine the cellular and subcellular localization in rat, mouse, and human eccrine sweat glands. NKCC1 mRNA was demonstrated in rat palmar tissue, including sweat glands, using RT-PCR, whereas NKCC2 mRNA was absent. Also, NHE1 m...... palmar skin by immunoblotting, whereas NKCC2, NHE2, and NHE3 proteins were not detected. Immunohistochemistry was performed using sections from rat, mouse, and human palmar tissue. Immunoperoxidase labeling revealed abundant expression of NKCC1 and NHE1 in the basolateral domain of secretory coils of rat...

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

    Energy Technology Data Exchange (ETDEWEB)

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


    To define the mechanism by which Pi exists from the renal proximal tubular cell across the basolateral membrane, we measured 32Pi uptake in basolateral membrane vesicles from dog kidney in the absence of Na+. Preloading of basolateral vesicles with 2 mM Pi transstimulated 32Pi uptake, which is consistent with counterflow. We used measurements of transstimulation to quantitate the transport component of 32Pi uptake. Transstimulation of 32Pi uptake was inhibited less than 30% by concentrations of probenecid as high as 50 mM. In contrast, transstimulation of 35SO4(2-) uptake by intravesicular SO4(2-) was inhibited 92% by 5 mM probenecid. Preloading basolateral vesicles with SO4(2-) did not result in transstimulation of 32Pi uptake. Accumulation of 32Pi in basolateral vesicles above steady state was driven by a membrane potential (intravesicular positive), consistent with Na+-independent Pi transport being accompanied by the net transfer of negative charge across the membrane. We conclude that carrier-mediated, electrogenic Na+-independent 32Pi transport can be demonstrated in basolateral vesicles from dog kidney. This process appears to be mediated, at least in part, via a mechanism different from that by which SO4(2-) is transported. Electrogenic Na+-independent Pi transport may reflect one means by which Pi reabsorbed across the luminal membrane exists from the proximal tubular cell down an electrochemical gradient.

  6. Influence of CO2 on electrophysiology and ionic permeability of the basolateral membrane of frog skin

    International Nuclear Information System (INIS)

    Stoddard, J.S.


    When short-circuited epithelia of frog skin bathed in an alkaline Ringer solution equilibrated with room air, are exposed to a Ringer solution equilibrated with 5% CO 2 , inhibition of transepithelial Na + transport is observed accompanied by a marked depolarization of the basolateral membrane voltage as measured with intracellular microelectrodes. To study further the mechanisms involved, basolateral membrane influxes and effluxes of 24 Na, 42 K, and 36 Cl were measured in control and CO 2 -treated isolated epithelia. In control epithelia, studies of the bidirectional 24 Na fluxes confirmed the existence of an important basolateral membrane permeability to Na + . In control epithelia, the apical membranes of the cells were found to be virtually impermeable to Cl - , while basolateral membranes were highly permeable to Cl - . Although CO 2 caused a partial inhibition of pump activity as assessed from decreases of pump-mediated Na + efflux and K + influx, CO 2 caused little or no change of the leak influx of Na + or K + . K + efflux was increased markedly with CO 2 resulting in a net loss of K + from the cells. Cl - influx was increased and Cl - efflux was decreased by CO 2 leading to a net influx of Cl - . Analysis of the data according to criteria involving changes of flux, ionic equilibrium potentials, mass and charge balance restrictions indicated that the principle changes involve a transient decrease in electrical conductance to K + with a concurrent increase in electrical conductance to HCO 3 - (OH - or H + ) of the basolateral membranes of the cells

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

    International Nuclear Information System (INIS)

    Cheung, P.T.; Hammerman, M.R.


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

  8. Carrier-mediated ¿-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger


    and the anticancer prodrug d-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least...

  9. Volume regulatory responses of basolateral membrane vesicles from Necturus enterocytes: Role of the cytoskeleton


    Dubinsky, William P.; Mayorga-Wark, Otilia; Schultz, Stanley G.


    Previous studies from this laboratory have demonstrated that basolateral membrane vesicles isolated from Necturus maculosus small intestinal epithelial cells possess a K+ channel that is inhibited by ATP. In the present studies, we demonstrate that these vesicles, which are essentially devoid of soluble cytoplasmic contaminants, exhibit volume regulatory responses that parallel those of intact epithelial cells. Thus, suspension of these vesicles in a solution that is hypotonic to the intraves...

  10. Bicarbonate-dependent transport of acetate and butyrate across the basolateral membrane of sheep rumen epithelium. (United States)

    Dengler, F; Rackwitz, R; Benesch, F; Pfannkuche, H; Gäbel, G


    This study aimed to assess the role of HCO₃⁻ in the transport of acetate and butyrate across the basolateral membrane of rumen epithelium and to identify transport proteins involved. The effects of basolateral variation in HCO₃⁻ concentrations on acetate and butyrate efflux out of the epithelium and the transepithelial flux of these short-chain fatty acids were tested in Ussing chamber experiments using (14)C-labelled substrates. HCO₃⁻-dependent transport mechanisms were characterized by adding specific inhibitors of candidate proteins to the serosal side. Effluxes of acetate and butyrate out of the epithelium were higher to the serosal side than to the mucosal side. Acetate and butyrate effluxes to both sides of rumen epithelium consisted of HCO₃⁻-independent and -dependent parts. HCO₃⁻-dependent transport across the basolateral membrane was confirmed in studies of transepithelial fluxes. Mucosal to serosal fluxes of acetate and butyrate decreased with lowering serosal HCO₃⁻ concentrations. In the presence of 25 mm HCO₃⁻, transepithelial flux of acetate was inhibited effectively by p-hydroxymercuribenzoic acid or α-cyano-4-hydroxycinnamic acid, while butyrate flux was unaffected by the blockers. Fluxes of both acetate and butyrate from the serosal to the mucosal side were diminished largely by the addition of NO₃⁻ to the serosal side, with this effect being more pronounced for acetate. Our results indicate the existence of a basolateral short-chain fatty acid/HCO₃⁻ exchanger, with monocarboxylate transporter 1 as a primary candidate for acetate transfer. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  11. Evidence for carrier-mediated Cl-SO4 exchange in rabbit ileal basolateral membrane vesicles

    International Nuclear Information System (INIS)

    Schron, C.M.; Knickelbein, R.G.; Aronson, P.S.; Dobbins, J.W.


    In rabbit ileal basolateral membrane (BLM) vesicles, an outwardly directed Cl gradient ([Cl] in/out = 60/6 mM) stimulated the initial velocity of 35 SO 4 uptake compared with uptake in the absence of Cl. Under Cl gradient conditions, 35 SO 4 was transiently accumulated at a concentration twice that found at equilibrium (overshoot). Chloride gradient-stimulated SO 4 uptake was markedly reduced by inhibitors of anion exchange and was saturable. SO 4 uptake by BLM vesicles was not stimulated by imposition of an inside-positive electrical potential, suggesting that the stimulation by a Cl gradient was not due to an induced electrical potential. Oxalate, nitrate, iodide, and bromide inhibited the initial velocity of Cl gradient-stimulated SO 4 uptake, whereas phosphate, β-hydroxybutyrate, lactate, and p-aminohippurate had not effect. When 35 SO 4 uptake by BLM vesicles was compared with that of brush-border membrane vesicles, Cl gradient-stimulated SO 4 uptake was found predominantly in the BLM preparation. In conclusion, these findings provide evidence for a carrier on the ileal basolateral membrane that mediates Cl-SO 4 exchange

  12. Plasma membrane ATPases

    DEFF Research Database (Denmark)

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


    membrane include ABC transporters, vacuolar (V-type) H+ pumps, and P-type pumps. These pumps all utilize ATP as a fuel for energizing pumping. This review focuses on the physiological roles of plasma membrane P-type pumps, as they represent the major ATP hydrolytic activity in this membrane....

  13. Measuring localization and diffusion coefficients of basolateral proteins in lateral versus basal membranes using functionalized substrates and kICS analysis

    DEFF Research Database (Denmark)

    Marlar, Saw; Christensen, Eva Arnspang; Pedersen, Gitte Albinus


    Micropatterning enabled semiquantitation of basolateral proteins in lateral and basal membranes of the same cell. Lateral diffusion coefficients of basolateral aquaporin-3 (AQP3-EGFP) and EGFP-AQP4 were extracted from “lateral” and “basal” membranes using identical live-cell imaging and k...

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

    International Nuclear Information System (INIS)

    Hammerman, M.R.; Rogers, S.


    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

  15. Effect of Probenecid on Tetraethyl Ammonium (TEA) Transport Across Basolateral Membrane of Rabbit Proximal Tubule (United States)

    Choi, Tae Lyong; Kim, Yong Keun


    The effect of probenecid on the transport of tetraethylammonium (TEA) was investigated in rabbit reanal cortical slices in an attempt to ascertain the interaction of organic anion with the organic cation transport system in proximal tubule. Probenecid reversibly inhibited TEA uptake by cortical slices in a dose-dependent manner over the concentration range of 1 and 5 mM. The efflux of TEA was not affected by the presence of 3 mM probenecid. Kinetic analysis indicated that probenecid decreased Vmax without a significant change in Km. Probenecid inhibited significantly tissue oxgen consumption at concentrations of 3 and 5 mM. However, probenecid did not significantly reduce TEA uptake in brush border and basolateral membrane vesicles prepared from renal cortex even at higher concentration of 10 mM. These results indicate that probenecid reduces TEA uptake in cortical slices by inhibiting the tissue metabolism rather than by the interaction with the organic cation transporter. PMID:1306074

  16. Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane

    DEFF Research Database (Denmark)

    Novak, I; Greger, R


    short circuit current (Isc) under similar conditions was 26 and 50 microA . cm-2. The specific transepithelial resistance (Rte) was 88 omega cm2. In control solutions the PD across the basolateral membrane (PDbl) was -63 +/- 1 mV (n = 314). Ouabain (3 mmol/l) depolarized PDbl by 4.8 +/- 1.1 mV (n = 6.......4 +/- 1.4 mV (n = 19), while another K+ channel blocker TEA+ (50 mmol/l) depolarized PDbl only by 7.7 +/- 2.0 mV (n = 9). Addition of amiloride (1 mmol/l) to the bath caused 3-4 mV depolarization of PDbl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PDbl. An increase in the bath HCO3...

  17. Coupled transport of p-aminohippurate by rat kidney basolateral membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Pritchard, J.B. (National Institutes of Health, Research Triangle Park, NC (USA))


    p-Aminohippuric acid (PAH) transport by basolateral membrane (BLM) vesicles isolated from rat renal cortex was stimulated very little by a Na{sup +} gradient (out > in). However, when micromolar concentrations of glutaric acid or {alpha}-ketoglutaric acid were added in the presence of a out > in Na{sup +} gradient, PAH uptake was accelerated >20-fold and an overshoot of greater than fivefold was produced. Other anions, e.g., fumarate, stimulated PAH uptake very modestly under these conditions, and that stimulation was totally prevented by short circuiting, i.e., with K{sup +} (in = out) and valinomycin. Glutarate-stimulated uptake was inhibited by 4-acetamide-4{prime}-({sup 14}C)-isothiocyanostilbene-2,2{prime}-disulfonic acid (SITS) and probenecid and was slightly stimulated by the imposition of an inside-negative membrane potential. Furthermore, even in the absence of a Na{sup +} gradient, glutarate-loaded vesicles exhibited a marked acceleration of ({sup 3}H)-PAH uptake (5-fold) and a modest overshoot (2.5-fold). These results suggest an indirect coupling of BLM PAH uptake to the Na{sup +} gradient by a cyclic accumulation (Na{sup +}-dependent) of glutarate followed by its efflux from the vesicle in exchange for PAH. This coupled system was absent in apical membranes. Thus net secretory transport of PAH may entail Na{sup +}-dependent, glutarate-driven PAH uptake at the BLM, followed by the exit of PAH into the lumen down its electrochemical gradient, probably in exchange for other anions, e.g., {sup 36}Cl{sup {minus}}, HCO{sub 3}{sup {minus}}, or OH{sup {minus}}.

  18. Structural lipid changes and Na(+)/K(+)-ATPase activity of gill cells' basolateral membranes during saltwater acclimation in sea lamprey (Petromyzon marinus, L.) juveniles. (United States)

    Lança, Maria João; Machado, Maria; Ferreira, Ana Filipa; Quintella, Bernardo Ruivo; de Almeida, Pedro Raposo


    Seawater acclimation is a critical period for anadromous species and a process yet to be understood in lampreys. Considering that changes in lipid composition of the gill cells' basolateral membranes may disrupt the major transporter Na(+)K(+)-ATPase, the goal of this study was to detect changes at this level during juvenile sea lamprey seawater acclimation. The results showed that saltwater acclimation has a direct effect on the fatty acid composition of gill cells basolateral membrane's phospholipids. When held in full-strength seawater, the fatty acid profile of basolateral membrane's phospholipids suffered a restructure by increasing either saturation or the ratio between oleic acid and eicosapentaenoic acid. Simultaneously, the activity of Na(+)K(+)-ATPase revealed a significant and positive correlation with basolateral membrane's cholesterol content in the presence of highest salinity. Our results pointed out for lipid adjustments involving the functional transporter present on the gill cell basolateral membranes to ensure the role played by branchial Na(+)K(+)-ATPase in ion transport during saltwater acclimation process. The responses observed contributed to the strategy adopted by gill cell's basolateral membranes to compensate for osmotic and ionic stressors, to ensure the success of the process of seawater acclimation associated with the downstream trophic migration of juvenile sea lamprey. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Identification and characterization of insulin receptors in basolateral membranes of dog intestinal mucosa

    International Nuclear Information System (INIS)

    Gingerich, R.L.; Gilbert, W.R.; Comens, P.G.; Gavin, J.R. III


    Little is known about hormonal regulation of substrate transport and metabolism in the mucosal lining of the small intestine. Because insulin regulates these functions in other tissues by binding to its receptor, we have investigated the presence of insulin receptors in canine small intestinal mucosa with basolateral membranes (BLM) and brush border membranes (BBM) prepared by sorbitol density centrifugation. A14-[ 125 I]iodoinsulin was used to study binding and structural characteristics of specific insulin receptors in BLM. Analysis of receptors in BLM identified binding sites with high affinity (Kd 88 pM) and low capacity (0.4 pmol/mg protein) as well as with low affinity (Kd 36 nM) and high capacity (4.7 pmol/mg protein). Binding was time, temperature, and pH dependent, and 125 I-labeled insulin dissociation was enhanced in the presence of unlabeled insulin. Cross-reactivity of these receptors to proinsulin, IGF-II, and IGF-I was 4, 1.8, and less than 1%, respectively. Covalent cross-linking of labeled insulin to BLM insulin receptors with disuccinimidyl suberate revealed a single 135,000-Mr band that was completely inhibited by unlabeled insulin. There was a 16-fold greater specific binding of insulin to BLM (39.0 +/- 2.4%) than to BBM (2.5 +/- 0.6%). These results demonstrate the presence of a highly specific receptor for insulin on the vascular, but not the luminal, surface of the small intestinal mucosa in dogs, and suggest that insulin may play an important role in the regulation of gastrointestinal physiology

  20. Inwardly rectifying K+ channels in the basolateral membrane of rat pancreatic acini. (United States)

    Kim, S J; Kerst, G; Schreiber, R; Pavenstädt, H; Greger, R; Hug, M J; Bleich, M


    Previous studies of the whole-cell K+ conductance suggest the presence of inwardly rectifying K+ channels (Kir) in rat pancreatic acini (RPAs). Here we investigate the properties of Kir of RPAs using patch-clamp techniques. The whole-cell current-to-voltage relationship of freshly isolated RPAs was steeper for inward currents than for outward currents when the extracellular K+ concentration ([K+]o) was raised. With a high [K+]o (145 mM), external application of Ba2+ and Cs+ blocked the inward K+ current in a voltage-dependent manner. The apparent IC50 of Ba2+ was 8.5+/-1.9 microM and 1.1+/-0.2 microM at -70 mV and -130 mV, respectively (n=5). The IC50 of Cs+ was 3.5+1.1 mM and 0.2+0.1 mM at -60 mV and -120 mV, respectively (n=4). Application of Ba2+ (0.1 mM) to the extracellular solution reversibly depolarized RPAs from -43+1.1 mV to -37+/-1.2 mV (n=20). In the cell-attached configuration with 145 mM KC1 in the pipette solution, we observed inwardly rectifying channels with a high open probability (PO) of 0.85+/-0.02 (n=6) and a slope conductance (Gs) of 30+/-2.8 pS (n=13). The same type of channel was observed in the outside-out patch. We could also observe a very small conductance K+ channel which was resistant to 0.1 mM Ba2+ and did not show inward rectification (n=11). RT-PCR analysis of RPA confirmed the presence of transcripts for Kir2.1, Kir2.3 and Kir7.1 subfamilies as molecular candidates for the observed channels. The above results demonstrate the presence of Kir channels in the basolateral membrane of the RPA, which may be important for the K+ recycling process during electrolyte secretion as well as for maintaining a hyperpolarized membrane.

  1. Biogenesis of plasma membrane cholesterol

    International Nuclear Information System (INIS)

    Lange, Y.


    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

  2. Layered plasma polymer composite membranes (United States)

    Babcock, Walter C.


    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.

  3. Liver plasma membranes: an effective method to analyze membrane proteome. (United States)

    Cao, Rui; Liang, Songping


    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.

  4. The Organic Anion-Transporting Peptide 2B1 Is Localized in the Basolateral Membrane of the Human Jejunum and Caco-2 Monolayers. (United States)

    Keiser, Markus; Kaltheuner, Lars; Wildberg, Charlotte; Müller, Janett; Grube, Markus; Partecke, Lars Ivo; Heidecke, Claus-Dieter; Oswald, Stefan


    The organic anion-transporting polypeptide (OATP) 2B1 which is ubiquitously expressed in the human body is assumed to play an important role in the cellular uptake of many drugs. Although the expression and function of this solute carrier transporter is well characterized in the human liver and other tissues, little is known about its localization and functional relevance in the intestine. Thus, it was the aim of this study to investigate its localization and function in the human jejunum and in the frequently used intestinal Caco-2 cell line. The basolateral membrane of jejunal tissue from 6 individuals showed a significant enrichment of OATP2B1 (17-fold) and the known basolateral proteins ABCC3 and Na/K-ATPase compared to the apical membrane as derived from targeted proteomics analysis. On the contrary, apical localization could be confirmed for ABCB1, ABCC2, and PEPT1. Basolateral localization of OATP2B1 could also be verified in Caco-2 cells. Bidirectional transport studies with established OATP2B1 substrates (sulfasalazine and pravastatin) across freshly exercised human jejunum and Caco-2 cell monolayers demonstrated a markedly higher transport from the basal to the apical compartment than in the opposite direction. Our data provide evidence for a basolateral localization of OATP2B1 which may improve our understanding of intestinal drug absorption. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  5. Plasma membranes from insect midgut cells

    Directory of Open Access Journals (Sweden)

    Walter R. Terra


    Full Text Available Plasma membranes from insect midgut cells are separated into apical and basolateral domains. The apical domain is usually modified into microvilli with a molecular structure similar to other animals. Nevertheless, the microvillar structure should differ in some insects to permit the traffic inside them of secretory vesicles that may budd laterally or pinch-off from the tips of microvilli. Other microvillar modifications are associated with proton-pumping or with the interplay with an ensheathing lipid membrane (the perimicrovilllar membrane observed in the midgut cells of hemipterans (aphids and bugs. The perimicrovillar membranes are thought to be involved in amino acid absorption from diluted diets. The microvillar and perimicrovillar membranes have densities (and protein content that depend on the insect taxon. The role played by the microvillar and perimicrovillar proteins in insect midgut physiology is reviewed here trying to provide a coherent picture of data and highlighting further research areas.As membranas plasmáticas das células intestinais dos insetos apresentam um domínio apical e outro basal. O domínio apical é geralmente modificado em microvilosidades com organização molecular similar a de outros animais, embora possam diferir naqueles insetos que apresentam vesículas secretoras em trânsito que brotam lateralmente ou destacam-se das extremidades das microvilosidades. Outras modificações microvilares estão associadas a bombeamento de prótons ou a interrelações com uma membrana lipídica (a membrana perimicrovilar que reveste as microvilosidades de células intestinais de hemípteros (pulgões e percevejos. Admite-se que as membranas perimicrovilares estejam envolvidas na absorção de aminoácidos a partir de dietas diluídas. As membranas microvilares e perimicrovilares tem densidades distintas (e conteúdo protéico que dependem do táxon do inseto. O papel desempenhado pelas proteínas microvilares e

  6. IGF-II receptors in luminal and basolateral membranes isolated from pars convoluta and pars recta of rabbit proximal tubule

    DEFF Research Database (Denmark)

    Jacobsen, Christian; Jessen, H; Flyvbjerg, A


    The binding of 125I-labeled insulin-like growth factor-II (125I-IGF-II) to luminal and basolateral membrane vesicles isolated from pars convoluta and the straight part (pars recta) of rabbit proximal tubule was investigated. Analyses of the binding data by use of the general stoichiometric binding...... equation revealed, that in all preparations IGF-II was bound to one high-affinity binding site and other sites with lower affinities. The specificity of the high-affinity 125I-IGF-II binding to the membrane vesicles assessed by displacement by unlabeled IGF-II, IGF-I and insulin showed that IGF-I displaced...... 125I-IGF-II in the range 22.5-47.9 nM (IC50) whereas insulin did not effect 125I-IGF-II binding at all. beta-Galactosidase inhibited the 125I-IGF-II binding with half-maximal inhibition of 20-30 nM beta-galactosidase. D-Mannose 6-phosphate increased the binding of 125I-IGF-II and reversed...

  7. The Plasma Membrane Calcium Pump (United States)

    Rasmussen, H.


    Three aspect of cellular calcium metabolism in animal cells was discussed including the importance of the plasma membrane in calcium homeostasis, experiments dealing with the actual mechanism of the calcium pump, and the function of the pump in relationship to the mitochondria and to the function of calmodulin in the intact cell.

  8. Proteomics and the dynamic plasma membrane

    DEFF Research Database (Denmark)

    Sprenger, Richard R; Jensen, Ole Nørregaard


    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....

  9. Recycling from endosomes to the plasma membrane

    NARCIS (Netherlands)

    Dam, E.M. van


    Summary V Chapter?Summary Many membrane proteins are, after endocytic uptake, efficiently recycled back to the plasma membrane. The aim of the studies presented in this thesis was to determine pathways and molecular mechanisms that are involved in recycling. Plasma membrane-derived clathrin-coated

  10. 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


    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

  11. Membrane order in the plasma membrane and endocytic recycling compartment. (United States)

    Iaea, David B; Maxfield, Frederick R


    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.

  12. Autophagosomal membranes assemble at ER-plasma membrane contact sites. (United States)

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


    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.

  13. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

      The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded H+-ATPases extrude protons from cells...... 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...... to describe the basic molecular components that allow the plasma membrane proton H+-ATPase to carry out proton transport against large membrane potentials. Moreover, a completely new paradigm for post-translational activation of these proteins is presented. The talk will focus on the following themes...

  14. Giant plasma membrane vesicles: models for understanding membrane organization. (United States)

    Levental, Kandice R; Levental, Ilya


    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.

  15. Plasma membrane changes during programmed cell deaths. (United States)

    Zhang, Yingying; Chen, Xin; Gueydan, Cyril; Han, Jiahuai


    Ruptured and intact plasma membranes are classically considered as hallmarks of necrotic and apoptotic cell death, respectively. As such, apoptosis is usually considered a non-inflammatory process while necrosis triggers inflammation. Recent studies on necroptosis and pyroptosis, two types of programmed necrosis, revealed that plasma membrane rupture is mediated by MLKL channels during necroptosis but depends on non-selective gasdermin D (GSDMD) pores during pyroptosis. Importantly, the morphology of dying cells executed by MLKL channels can be distinguished from that executed by GSDMD pores. Interestingly, it was found recently that secondary necrosis of apoptotic cells, a previously believed non-regulated form of cell lysis that occurs after apoptosis, can be programmed and executed by plasma membrane pore formation like that of pyroptosis. In addition, pyroptosis is associated with pyroptotic bodies, which have some similarities to apoptotic bodies. Therefore, different cell death programs induce distinctive reshuffling processes of the plasma membrane. Given the fact that the nature of released intracellular contents plays a crucial role in dying/dead cell-induced immunogenicity, not only membrane rupture or integrity but also the nature of plasma membrane breakdown would determine the fate of a cell as well as its ability to elicit an immune response. In this review, we will discuss recent advances in the field of apoptosis, necroptosis and pyroptosis, with an emphasis on the mechanisms underlying plasma membrane changes observed on dying cells and their implication in cell death-elicited immunogenicity.

  16. Aquaporin-3 expressed in the basolateral membrane of gill chloride cells in Mozambique tilapia Oreochromis mossambicus adapted to freshwater and seawater. (United States)

    Watanabe, Soichi; Kaneko, Toyoji; Aida, Katsumi


    We have cloned a homologue of mammalian aquaporin-3 (AQP3) from gills of Mozambique tilapia using a reverse transcription-polymerase chain reaction (RT-PCR). The deduced amino acid sequence shared 64-75% homology with other vertebrate AQP3 homologues. RT-PCR revealed that tilapia AQP3 was expressed in the brain, pituitary, kidney, spleen, intestine, skin, eye and gill in tilapia adapted to freshwater (FW) and seawater (SW). We also examined functional characteristics of tilapia AQP3 using Xenopus oocytes as an in vitro transcribed cRNA expression system. Osmotic water permeability (Pf) of Xenopus oocytes expressing tilapia AQP3 was about 30-fold higher than that of control oocytes, and was 80% inhibited by treatment with 0.3 mmol l(-1) HgCl2. Light-microscopic immunocytochemistry of branchial epithelia revealed that tilapia AQP3 was expressed in gill chloride cells of FW- and SW-adapted tilapia. Electron-microscopic immunocytochemistry further demonstrated that tilapia AQP3 was localized in the basolateral membrane of gill chloride cells. Basolateral localization of AQP3 in gill chloride cells suggests that AQP3 is involved in regulatory volume changes and osmoreception, which could trigger functional differentiation of chloride cells.

  17. EBIO, an agent causing maintained epithelial chloride secretion by co-ordinate actions at both apical and basolateral membranes. (United States)

    MacVinish, L J; Keogh, J; Cuthbert, A W


    The effect of 1-ethyl-2-benzimidazolone (EBIO) on electrogenic chloride secretion in murine colonic and nasal epithelium was investigated by the short-circuit technique. In the colon, EBIO produces a sustained current increase in the presence of amiloride, which is sensitive to furosemide. In nasal epithelium EBIO causes only a small, transient current increase. Sustained increases in current were obtained in response to forskolin in both epithelia. To examine the mechanisms by which EBIO increases chloride secretion, the effects on intracellular mediators were measured in colonic crypts. There was no effect on [Ca(2+)]i but cAMP content was increased, more so in the presence of IBMX, indicating a direct effect on adenylate cyclase. In colonic epithelia in which the apical surface was permeabilized by nystatin, and the tissue subjected to an apical to basolateral K(+) gradient, EBIO caused a current increase that was entirely sensitive to charybdotoxin (ChTX). In similarly permeabilized colons Br-cAMP caused a current increase that was entirely sensitive to 293B. Thus EBIO increases chloride secretion in the colon by coordinated actions at both the apical and basolateral faces of the cells. These include direct and indirect actions on Ca(2+)-sensitive and cAMP-sensitive K(+) channels respectively, and indirect actions on the basolateral cotransporter and apical CFTR chloride channels via cAMP. In CF colonic epithelia EBIO did not evoke chloride secretion. It is not clear why the nasal epithelium responds poorly to EBIO whereas it gives a sustained response to the related compound chlorzoxazone.

  18. Transport proteins of the plant plasma membrane (United States)

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


    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.

  19. Reverse-osmosis membranes by plasma polymerization (United States)

    Hollahan, J. R.; Wydeven, T.


    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.

  20. Plasma membrane disruption: repair, prevention, adaptation (United States)

    McNeil, Paul L.; Steinhardt, Richard A.


    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.

  1. Polarized Human Retinal Pigment Epithelium Exhibits Distinct Surface Proteome on Apical and Basal Plasma Membranes. (United States)

    Khristov, Vladimir; Wan, Qin; Sharma, Ruchi; Lotfi, Mostafa; Maminishkis, Arvydas; Bharti, Kapil


    Surface proteins localized on the apical and basal plasma membranes are required for a cell to sense its environment and relay changes in ionic, cytokine, chemokine, and hormone levels to the inside of the cell. In a polarized cell, surface proteins are differentially localized on the apical or the basolateral sides of the cell. The retinal pigment epithelium (RPE) is an example of a polarized cell that performs a variety of functions that are dependent on its polarized state including trafficking of ions, fluid, and metabolites across the RPE monolayer. These functions are absolutely crucial for maintaining the health and integrity of adjacent photoreceptors, the photosensitive cells of the retina. Here we present a series of approaches to identify and validate the polarization state of cultured primary human RPE cells using immunostaining for RPE apical/basolateral markers, polarized cytokine secretion, electrophysiology, fluid transport, phagocytosis, and identification of plasma membrane proteins through cell surface capturing technology. These approaches are currently being used to validate the polarized state and the epithelial phenotype of human induced pluripotent stem (iPS) cell derived RPE cells. This work provides the basis for developing an autologous cell therapy for age-related macular degeneration using patient specific iPS cell derived RPE.

  2. Isolation of plasma membrane-associated membranes from rat liver. (United States)

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


    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.

  3. Relationship between sperm plasma membrane integrity and ...

    African Journals Online (AJOL)

    Sperm quality plays an important role in determining fertility. The aim of the study was to examine the relationship between sperm plasma membrane integrity and morphology, and fertility following artificial insemination (AI). A total of 16 ejaculates were collected from three Large White boars using the gloved hand ...

  4. 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: [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)


    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.

  5. Aquaporin-4 Is Downregulated in the Basolateral Membrane of Ileum Epithelial Cells during Enterotoxigenic Escherichia coli-Induced Diarrhea in Mice (United States)

    Zhang, Di; Yang, Longfei; Su, Weiheng; Zhao, Yuan; Ma, Xin; Zhou, Haizhu; Xu, Bo; Zhang, Kaiqi; Ma, Hongxia


    Enterotoxigenic Escherichia coli (ETEC) are opportunistic pathogens that colonize the small intestine, produce enterotoxins and induce diarrhea. Some aquaporins (AQPs), such as AQP3 and AQP8, have been reported to participate in diarrhea by decreasing cellular influx in the gastrointestinal (GI) tract. AQP4 is another important water channel in the GI tract, but its role in ETEC-induced diarrhea has not been reported. Here, we demonstrated the potential roles of AQP4 in ETEC-induced diarrhea. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting showed that AQP4 was expressed in the mouse ileum, but not in the duodenum or jejunum while immunohistochemical staining showed that AQP4 localized to the basolateral membrane of ileum epithelial cells. Using an ETEC-induced mice diarrhea model, we demonstrated that both AQP4 mRNA level and the AQP4 protein level in the ileum decreased gradually over a time course of 7 days. These results suggest that AQP4 plays a role in the pathogenesis of ETEC-induced diarrhea by mediating water transport. PMID:29375520

  6. Aquaporin-4 Is Downregulated in the Basolateral Membrane of Ileum Epithelial Cells during Enterotoxigenic Escherichia coli-Induced Diarrhea in Mice

    Directory of Open Access Journals (Sweden)

    Di Zhang


    Full Text Available Enterotoxigenic Escherichia coli (ETEC are opportunistic pathogens that colonize the small intestine, produce enterotoxins and induce diarrhea. Some aquaporins (AQPs, such as AQP3 and AQP8, have been reported to participate in diarrhea by decreasing cellular influx in the gastrointestinal (GI tract. AQP4 is another important water channel in the GI tract, but its role in ETEC-induced diarrhea has not been reported. Here, we demonstrated the potential roles of AQP4 in ETEC-induced diarrhea. Reverse transcription-polymerase chain reaction (RT-PCR and western blotting showed that AQP4 was expressed in the mouse ileum, but not in the duodenum or jejunum while immunohistochemical staining showed that AQP4 localized to the basolateral membrane of ileum epithelial cells. Using an ETEC-induced mice diarrhea model, we demonstrated that both AQP4 mRNA level and the AQP4 protein level in the ileum decreased gradually over a time course of 7 days. These results suggest that AQP4 plays a role in the pathogenesis of ETEC-induced diarrhea by mediating water transport.

  7. N-linked glycans do not affect plasma membrane localization of multidrug resistance protein 4 (MRP4) but selectively alter its prostaglandin E2 transport activity. (United States)

    Miah, M Fahad; Conseil, Gwenaëlle; Cole, Susan P C


    Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. MRP4 mediates the ATP-dependent efflux of many endogenous and exogenous solutes across the plasma membrane, and in polarized cells, it localizes to the apical or basolateral plasma membrane depending on the tissue type. MRP4 is a 170 kDa glycoprotein and here we show that MRP4 is simultaneously N-glycosylated at Asn746 and Asn754. Furthermore, confocal immunofluorescence studies showed that N-glycans do not affect MRP4's apical membrane localization in polarized LLC-PK1 cells or basolateral membrane localization in polarized MDCKI cells. However, vesicular transport assays showed that N-glycans differentially affect MRP4's ability to transport prostaglandin E2, but not estradiol glucuronide. Together these data indicate that N-glycosylation at Asn746 and Asn754 is not essential for plasma membrane localization of MRP4 but cause substrate-selective effects on its transport activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia. (United States)

    Yaffe, Yakey; Shepshelovitch, Jeanne; Nevo-Yassaf, Inbar; Yeheskel, Adva; Shmerling, Hedva; Kwiatek, Joanna M; Gaus, Katharina; Pasmanik-Chor, Metsada; Hirschberg, Koret


    Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60-269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

  9. Angiotensin II-induced hypertension increases plasma membrane Na pump activity by enhancing Na entry in rat thick ascending limbs. (United States)

    Gonzalez-Vicente, Agustin; Garvin, Jeffrey L


    Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na-K-2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (ANG II) infusion increases net TAL Na transport and Na apical entry; however, little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by ANG II-infusion, a model of ANG II-induced hypertension. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 7 days, and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from ANG II-infused rats was 12.8 ± 0.4 arbitrary fluorescent units (AFU)·mg(-1)·min(-1) compared with only 9.9 ± 1.1 AFU·mg(-1)·min(-1) in controls (P Na pump expression, the number of Na pumps in the plasma membrane, or the affinity for Na. When furosemide (1.1 mg·kg(-1)·day(-1)) was coinfused with ANG II, no increase in plasma membrane Na pump activity was observed. We concluded that in ANG II-induced hypertension Na pump activity is increased in the plasma membrane of TALs and that this increase is caused by the chronically enhanced Na entry occurring in this model.

  10. Mechanotransduction through the plasma membrane & cytoskeleton (United States)

    Haase, Kristina; Pelling, Andrew


    Mechanical forces initiate immediate and long-term changes in cells; however the exact mechanisms remain unclear, albeit crucial for understanding the pathology of disease. We used combined confocal and atomic force microscopy (AFM) to investigate changes in cell morphology and elasticity in response to a mechanical stimulus. The AFM was used as a nano-indentor to gauge the response of the membrane and cytoskeleton (CSK) of HeLa cells. We observed their viscoelastic nature by probing cells transfected with a green fluorescent protein localized at the plasma membrane. Inhibition of acto-myosin contractility (AMc) resulted in a significant decrease of cellular elasticity, and a corresponding increase in mean deformation. We also investigated the rate at which the membrane and CSK deform and relax in response to a local force. The response to a local perturbation is nearly instantaneous for control cells and shows no statistical difference when compared to cells treated with CSK-inhibiting drugs. Inhibition of AMc affects the rate of recovery, in comparison to control cells which recover quite quickly (30-60s). Overall, we demonstrated short and long-term deformation and subsequent recovery of both the cell membrane and actin network in response to a local force.

  11. Plasma membrane isolation using immobilized concanavalin A magnetic beads. (United States)

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


    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.

  12. Hydrogen Production from Ammonia Using a Plasma Membrane Reactor

    Directory of Open Access Journals (Sweden)

    Shinji Kambara


    Full Text Available In this study, an efficient method for using pulsed plasma to produce hydrogen from ammonia was developed. An original pulsed plasma reactor with a hydrogen separation membrane was developed for efficient hydrogen production, and its hydrogen production performance was investigated. Hydrogen production in the plasma was affected by the applied voltage and flow rate of ammonia gas. The maximum hydrogen production flow rate of a typical plasma reactor was 8.7 L/h, whereas that of the plasma membrane reactor was 21.0 L/h. We found that ammonia recombination reactions in the plasma controlled hydrogen production in the plasma reactor. In the plasma membrane reactor, a significant increase in hydrogen production was obtained because ammonia recombination reactions were inhibited by the permeation of hydrogen radicals generated in the plasma through a palladium alloy membrane. The energy efficiency was 4.42 mol-H2/kWh depending on the discharge power.

  13. Selective photosensitizer delivery into plasma membrane for effective photodynamic therapy. (United States)

    Kim, Jiyoung; Santos, Olavo Amorim; Park, Ji-Ho


    Subcellular localization of photosensitizers (PSs) determines the therapeutic efficacy in the photodynamic therapy. However, among the subcellular compartments, there has been little effort to deliver the PSs selectively into the plasma membrane and examine the phototherapeutic efficacy of membrane-localized PSs. Here, we developed a liposomal delivery system to localize the hydrophobic PSs selectively into the plasma membrane. The membrane fusogenic liposomes (MFLs), the membrane of which is engineered to fuse with the plasma membrane, was prepared for the membrane localization of PSs. The phototherapeutic efficacy of cells treated with ZnPc-loaded MFLs was superior over that of cells treated with ZnPc-loaded non-fusogenic liposomes, which is the conventional liposomal formulation that delivers the PSs into the intracellular compartments via endocytosis. The membrane localization of ZnPc molecules led to rapid membrane disruption upon irradiation and subsequent necrosis-like cell death. The membrane-localized generation of reactive oxygen species in the cells treated with ZnPc-loaded MFLs was likely to account for the effective disruption of plasma membrane. Thus, this work provides a novel delivery method to localize the PSs selectively into the plasma membrane with the enhanced phototherapeutic efficacy. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Vesicular and Plasma Membrane Transporters for Neurotransmitters (United States)

    Blakely, Randy D.; Edwards, Robert H.


    The regulated exocytosis that mediates chemical signaling at synapses requires mechanisms to coordinate the immediate response to stimulation with the recycling needed to sustain release. Two general classes of transporter contribute to release, one located on synaptic vesicles that loads them with transmitter, and a second at the plasma membrane that both terminates signaling and serves to recycle transmitter for subsequent rounds of release. Originally identified as the target of psychoactive drugs, these transport systems have important roles in transmitter release, but we are only beginning to understand their contribution to synaptic transmission, plasticity, behavior, and disease. Recent work has started to provide a structural basis for their activity, to characterize their trafficking and potential for regulation. The results indicate that far from the passive target of psychoactive drugs, neurotransmitter transporters undergo regulation that contributes to synaptic plasticity. PMID:22199021

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

    African Journals Online (AJOL)

    The expression pattern of a germination specific plasma membrane H+-ATPase was analyzed by RTPCR and in situ RNA hybridization methods. RT-PCR results revealed that germination specific plasma membrane H+-ATPase accumulation was detectable in all organs and tissues of germinating wheat embryos.

  16. A plasma membrane H ATPase gene is germination- induced in ...

    African Journals Online (AJOL)



    Jan 18, 2010 ... The expression pattern of a germination specific plasma membrane H+-ATPase was analyzed by RT-. PCR and in situ RNA hybridization methods. RT-PCR results revealed that germination specific plasma membrane H+-ATPase accumulation was detectable in all organs and tissues of germinating wheat.

  17. Channelopathies linked to plasma membrane phosphoinositides (United States)

    Logothetis, Diomedes E.; Petrou, Vasileios I.; Adney, Scott K.; Mahajan, Rahul


    The plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) controls the activity of most ion channels tested thus far through direct electrostatic interactions. Mutations in channel proteins that change their apparent affinity to PIP2 can lead to channelopathies. Given the fundamental role that membrane phosphoinositides play in regulating channel activity, it is surprising that only a small number of channelopathies have been linked to phosphoinositides. This review proposes that for channels whose activity is PIP2-dependent and for which mutations can lead to channelopathies, the possibility that the mutations alter channel-PIP2 interactions ought to be tested. Similarly, diseases that are linked to disorders of the phosphoinositide pathway result in altered PIP2 levels. In such cases, it is proposed that the possibility for a concomitant dysregulation of channel activity also ought to be tested. The ever-growing list of ion channels whose activity depends on interactions with PIP2 promises to provide a mechanism by which defects on either the channel protein or the phosphoinositide levels can lead to disease. PMID:20396900

  18. Large Deformation Mechanics of Plasma Membrane Chained Vesicles in Cells (United States)

    Kosawada, Tadashi; Sanada, Kouichi; Takano, Tetsuo

    The clathrin-coated pits, vesicles and chained vesicles on the inner surface of the plasma membrane facilitate the cell to transport specific extracellular macromolecules. This cellular process is strongly involved with large mechanical deformations of the plasma membrane accompanied by changes in membrane curvature. The assembly of the clathrin coat is thought to provide curvature into the membrane. Hence, effects of in-plane shear elasticity due to these coat structure may be significant on the vesicular mechanics. In this study, large deformation mechanics of plasma membrane chained vesicles in cells have been formulated based on minimization of bending and in-plane shear strain energy of the membrane. Effects of outer surrounding cytoplasmic flat membrane upon mechanically stable shapes of the vesicles were revealed, while effects of in-plane shear elasticity were partly discussed.

  19. Fatty acid profiles from the plasma membrane and detergent resistant membranes of two plant species. (United States)

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


    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.

  20. Basolateral sorting of the coxsackie and adenovirus receptor through interaction of a canonical YXXPhi motif with the clathrin adaptors AP-1A and AP-1B. (United States)

    Carvajal-Gonzalez, Jose Maria; Gravotta, Diego; Mattera, Rafael; Diaz, Fernando; Perez Bay, Andres; Roman, Angel C; Schreiner, Ryan P; Thuenauer, Roland; Bonifacino, Juan S; Rodriguez-Boulan, Enrique


    The coxsackie and adenovirus receptor (CAR) plays key roles in epithelial barrier function at the tight junction, a localization guided in part by a tyrosine-based basolateral sorting signal, (318)YNQV(321). Sorting motifs of this type are known to route surface receptors into clathrin-mediated endocytosis through interaction with the medium subunit (μ2) of the clathrin adaptor AP-2, but how they guide new and recycling membrane proteins basolaterally is unknown. Here, we show that YNQV functions as a canonical YxxΦ motif, with both Y318 and V321 required for the correct basolateral localization and biosynthetic sorting of CAR, and for interaction with a highly conserved pocket in the medium subunits (μ1A and μ1B) of the clathrin adaptors AP-1A and AP-1B. Knock-down experiments demonstrate that AP-1A plays a role in the biosynthetic sorting of CAR, complementary to the role of AP-1B in basolateral recycling of this receptor. Our study illustrates how two clathrin adaptors direct basolateral trafficking of a plasma membrane protein through interaction with a canonical YxxΦ motif.

  1. The plasma membrane proteome of germinating barley embryos

    DEFF Research Database (Denmark)

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


    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 amphiphilic...... 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....

  2. Macroscopic domain formation in the platelet plasma membrane

    DEFF Research Database (Denmark)

    Bali, Rachna; Savino, Laura; Ramirez, Diego A.


    phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents......There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large...

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

    International Nuclear Information System (INIS)

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


    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

  4. Gravity Responsive NADH Oxidase of the Plasma Membrane (United States)

    Morre, D. James (Inventor)


    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.

  5. Colchicine blocks the effects of secretin on bile duct cell tubulovesicles and plasma membrane geometry and impairs ductular HCO3- secretion in the pig. (United States)

    Veel, T; Buanes, T; Grotmol, T; Engeland, E; Raeder, M G


    Secretin causes the bile duct cells to secrete HCO3-. To examine whether the transformation of duct cell ultrastructure that follows secretin stimulation depends on microtubules and is important for ductular HCO3- secretion, we examined the effect of colchicine on ductular HCO3- secretion and on the morphology of cells lining bile ductules of anaesthetized pigs. Colchicine blocked secretin-dependent cytoplasmic clearance of tubulovesicles and prevented expansion of the basolateral plasma membrane in duct cells and reduced the ductular HCO3- secretory response from 132 +/- 25 mumol min-1 to 97 +/- 14 mumol min-1. In contrast, lumicolchicine did not affect secretin-dependent tubulovesicle clearance or plasma membrane geometry or ductular HCO3- secretion. Accordingly, secretin-dependent cytoplasmic clearance of tubulovesicles in bile duct cells appears to depend on microtubules and to be important for ductular HCO3- secretion.

  6. 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


    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

  7. 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...

  8. Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins. (United States)

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


    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.

  9. Preparation of artificial plasma membrane mimicking vesicles with lipid asymmetry.

    Directory of Open Access Journals (Sweden)

    Qingqing Lin

    Full Text Available Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM and/or phosphatidylcholine (PC outside/phosphatidylethanolamine (PE and phosphatidylserine (PS inside, and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes "artificial plasma membrane mimicking" ("PMm" vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.

  10. Preparation of artificial plasma membrane mimicking vesicles with lipid asymmetry. (United States)

    Lin, Qingqing; London, Erwin


    Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes "artificial plasma membrane mimicking" ("PMm") vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.

  11. There Is No Simple Model of the Plasma Membrane Organization (United States)

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


    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

  12. Evidence for carrier-mediated chloride/bicarbonate exchange in canalicular rat liver plasma membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Meier, P.J.; Knickelbein, R.; Moseley, R.H.; Dobbins, J.W.; Boyer, J.L.


    To determine whether anion exchangers might play a role in hepatic bile formation, the authors looked for the presence of Cl/sup -/:OH/sup -/ and Cl/sup -/:HCO3/sup -/ exchange in highly purified canalicular (c) and basolateral (bl) rat liver plasma membrane (LPM) vesicles. In cLPM vesicles, a pH gradient stimulated /sup 36/Cl- uptake twofold above values obtained during pH-equilibrated conditions. When 50 mM HCO3/sup -/ was also present inside the vesicles, the same pH gradient resulted in Cl/sup -/ uptake to levels fourfold above pH- and HCO3--equilibrated controls and two- to threefold above Cl- equilibrium. Initial rates of both pH and HCO3/sup -/ gradient-stimulated Cl/sup -/ uptake were completely inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). A valinomycin-induced K/sup +/ diffusion potential (inside positive) also stimulated Cl/sup -/ uptake in cLPM, but this conductive Cl- pathway was insensitive to DIDS. The DIDS-sensitive, pH and HCO3- gradient-stimulated Cl/sup -/ uptake demonstrated: saturation with Cl/sup -/; partial inhibition by bumetanide (26%), furosemide (33%), probenecid (37%), and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (49%); cis-inhibition by chloride and nitrate but not by sulfate and various organic anions, and independence from the membrane potential. These data demonstrate the presence of an electroneutral Cl/sup -/:OH/sup -/ and Cl/sup -/:HCO3/sup -/ exchanger in rat liver canalicular membranes that favors Cl/sup -/:HCO3/sup -/ exchange. In contrast, no evidence was found for the presence of a Cl/sup -/:HCO3/sup -/ (OH/sup -/) exchange system in blLPM vesicles.

  13. Miscibility Critical Points in Plasma Membranes (United States)

    Machta, Benjamin; Veatch, Sarah; Papanikolaou, Stefanos; Sethna, James P.


    Lipid bilayers surround all cells and are home to a host of proteins and lipids that mediate interactions between the cell and its environment. Recent experimental work has shown that simple membranes composed of three lipid components show complex phase behavior at temperatures in the physiological range. For example, two liquid phases and a gel or solid phase are seen, and a second order phase transition with Ising critical behavior can be reached at a boundary of the liquid-liquid coexistence region [1]. Surprisingly, membrane vesicles isolated from living cells can be tuned with a single parameter (temperature) to criticality [1]. This suggests that cell membranes in vivo sit near miscibility critical points, and may help explain some of the paradoxes associated with putative lipid rafts proposed in other experiments. Here we report on work mapping phase diagrams for the simple membranes utilizing NMR and microscopy data. In addition, we use canonical models of phase transitions to understand the qualitative features of the membranes. Finally we explore ideas from information theory and self organized criticality to understand how and why real cells maintain a membrane near criticality. [1] Honerkamp-Smith, Veatch, and Keller, Biochim Biophys Acta. 2008 (in press)

  14. Effect of Plasma Membrane Semipermeability in Making the Membrane Electric Double Layer Capacitances Significant. (United States)

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


    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.

  15. Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry? (United States)

    Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R


    A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

  16. NADH-Ferricyanide Reductase of Leaf Plasma Membranes 1 (United States)

    Askerlund, Per; Laurent, Pascal; Nakagawa, Hiroki; Kader, Jean-Claude


    Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b5 reductase, EC, purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber

  17. Detection of glycoproteins in the Acanthamoeba plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Paatero, G.I.L. (Abo Akademi (Finland)); Gahmberg, C.G. (Univ. of Helsinki (Finland))


    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.

  18. Detection of glycoproteins in the Acanthamoeba plasma membrane

    International Nuclear Information System (INIS)

    Paatero, G.I.L.; Gahmberg, C.G.


    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by 125 I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB 3 H 4 and galactose oxidase/NaB 3 H 4 labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M r of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with [ 35 S]methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis

  19. Plasma treatment of polyethersulfone membrane for benzene removal from water by air gap membrane distillation. (United States)

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


    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.

  20. Magnetic apatite for structural insights on the plasma membrane. (United States)

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


    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.

  1. 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


    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

  2. Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Bonekamp, Nina A. [Centre for Cell Biology and Department of Biology, University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro (Portugal); Vormund, Kerstin; Jacob, Ralf [Department of Cell Biology and Cell Pathology, University of Marburg, Robert-Koch-Str. 6, 35037 Marburg (Germany); Schrader, Michael, E-mail: [Centre for Cell Biology and Department of Biology, University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro (Portugal)


    The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.

  3. Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

    International Nuclear Information System (INIS)

    Bonekamp, Nina A.; Vormund, Kerstin; Jacob, Ralf; Schrader, Michael


    The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.

  4. A plasma membrane H ATPase gene is germination- induced in ...

    African Journals Online (AJOL)



    Jan 18, 2010 ... Ewing and Bennet (1994) identi- fied at least 7 genes in tomato and Harper et al. (1994) identified 10 genes in Arabidopsis thaliana, indicating the presence of large families of H+-ATPase genes. In this report, we determine and localize the expression pattern of germination specific plasma membrane ...

  5. Mammalian gamete plasma membranes re-assessments and reproductive implications (United States)

    Establishment of the diploid status occurs with the fusion of female and male gametes. Both the mammalian oocyte and spermatozoa are haploid cells surrounded with plasma membranes that are rich in various proteins playing a crucial role during fertilization. Fertilization is a complex and ordered st...

  6. Plants and fungi in the era of heterogeneous plasma membranes

    Czech Academy of Sciences Publication Activity Database

    Opekarová, Miroslava; Malínský, Jan; Tanner, W.


    Roč. 12, č. 1 (2010), s. 94-98 ISSN 1435-8603 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50390512 Keywords : Confocal microscopy * plasma membrane * raft-like micro domains Subject RIV: EE - Microbiology, Virology Impact factor: 2.409, year: 2010

  7. 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.


    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

  8. Uniform Structure of Eukaryotic Plasma Membrane: Lateral Domains in Plants

    Czech Academy of Sciences Publication Activity Database

    Malínská, Kateřina; Zažímalová, Eva


    Roč. 12, č. 2 (2011), s. 148-155 ISSN 1389-2037 R&D Projects: GA MŠk(CZ) LC06034 Institutional research plan: CEZ:AV0Z50380511 Keywords : Plasma membrane * microdomains * lateral segregation Subject RIV: ED - Physiology Impact factor: 2.886, year: 2011

  9. A plasma membrane association module in yeast amino acid transporters

    NARCIS (Netherlands)

    Popov-Čeleketić, Dušan; Bianchi, Frans; Ruiz, Stephanie J; Meutiawati, Febrina; Poolman, Bert


    Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in

  10. Lateral mobility of plasma membrane lipids in dividing Xenopus eggs

    NARCIS (Netherlands)

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


    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

  11. Calcium pumps of plasma membrane and cell interior

    DEFF Research Database (Denmark)

    Strehler, Emanuel E; Treiman, Marek


    /endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue...

  12. 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.

  13. Plasma membrane aquaporins mediates vesicle stability in broccoli. (United States)

    Martínez-Ballesta, Maria Del Carmen; García-Gomez, Pablo; Yepes-Molina, Lucía; Guarnizo, Angel L; Teruel, José A; Carvajal, Micaela


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

  14. Simulations of simple linoleic acid-containing lipid membranes and models for the soybean plasma membranes (United States)

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


    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.

  15. 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)


    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)

  16. Reorganization of plasma membrane lipid domains during conidial germination. (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


    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.

  17. Measurement of autoantibodies against human eye muscle plasma membranes in Graves' ophthalmopathy.


    Faryna, M; Nauman, J; Gardas, A


    Antibodies that reacted with plasma membranes of human eye muscle but showed no binding to plasma membranes of human skeletal muscle were identified in serum of patients with Graves' ophthalmopathy. Rabbit antithyroglobulin serum at a dilution of 1 X 10(-3) to 20 X 10(-3) had no effect on the binding of these antibodies to eye muscle membrane antigens. There was no correlation between antihuman eye muscle plasma membrane antibodies and antihuman thyroid membrane antibodies or antibodies again...

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

    International Nuclear Information System (INIS)

    Dung Thi Tran; Mori, Shinsuke; Suzuki, Masaaki


    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

  19. Advancements in the analysis of the Arabidopsis plasma membrane proteome

    Directory of Open Access Journals (Sweden)

    Koste eYadeta


    Full Text Available The plasma membrane (PM regulates diverse processes essential to plant growth, development, and survival in an ever-changing environment. In addition to maintaining normal cellular homeostasis and plant nutrient status, PM proteins perceive and respond to a myriad of environmental cues. Here we review recent advances in the analysis of the plant PM proteome with a focus on the model plant Arabidopsis thaliana. Due to membrane heterogeneity, hydrophobicity, and low relative abundance, analysis of the PM proteome has been a special challenge. Various experimental techniques to enrich PM proteins and different protein and peptide separation strategies have facilitated the identification of thousands of integral and membrane-associated proteins. Numerous classes of proteins are present at the PM with diverse biological functions. Plasma membrane microdomains have attracted much attention. However, it still remains a challenge to characterize these cell membrane compartments. Dynamic changes in the PM proteome in response to different biotic and abiotic stimuli are highlighted. Future prospects for PM proteomics research are also discussed.

  20. Manufacturing of metal supported BSCF membranes by spark plasma sintering


    Laptev, Alexander; Bram, Martin; Zivcec, Maria; Baumann, Stefan; Jarligo, Maria Ophelia; Sebold, Doris; Pfaff, Ewald; Broeckmann, Christoph


    Spark plasma sintering (SPS), also known as field assisted sintering technique (FAST), is a relatively new method for rapid consolidation of metallic or ceramic powders. In the present work, its suitability for the manufacturing of metal supported Ba0.5Sr0.5Co0.8Fe0.2O3-δ (BSCF) based membrane by co-sintering of functional ceramic BSCF layer and porous metallic support has been investigated. The BSCF based membranes are highly attractive for oxygen separation from air due to mixed ionic and e...

  1. Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes. (United States)

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


    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.

  2. Remodeling of the postsynaptic plasma membrane during neural development. (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


    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 (

  3. Modulation of Erythrocyte Plasma Membrane Redox System Activity by Curcumin

    Directory of Open Access Journals (Sweden)

    Prabhakar Singh


    Full Text Available Plasma membrane redox system (PMRS is an electron transport chain system ubiquitously present throughout all cell types. It transfers electron from intracellular substrates to extracellular acceptors for regulation of redox status. Curcumin, isolated from Curcuma longa, has modulatory effects on cellular physiology due to its membrane interaction ability and antioxidant potential. The present study investigates the effect of curcumin on PMRS activity of erythrocytes isolated from Wistar rats in vitro and in vivo and validated through an in silico docking simulation study using Molegro Virtual Docker (MVD. Effects of curcumin were also evaluated on level of glutathione (GSH and the oxidant potential of plasma measured in terms of plasma ferric equivalent oxidative potentials (PFEOP. Results show that curcumin significantly (p<0.01 downregulated the PMRS activity in a dose-dependent manner. Molecular docking results suggest that curcumin interacts with amino acids at the active site cavity of cytochrome b5 reductase, a key constituent of PMRS. Curcumin also increased the GSH level in erythrocytes and plasma while simultaneously decreasing the oxidant potential (PFEOP of plasma. Altered PMRS activity and redox status are associated with the pathophysiology of several health complications including aging and diabetes; hence, the above finding may explain part of the role of curcumin in health beneficial effects.

  4. Role of plasma membrane surface charges in dictating the feasibility of membrane-nanoparticle interactions (United States)

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


    Receptor-ligand (R-L) binding mediated interactions between the plasma membrane (PM) and a nanoparticle (NP) require the ligand-functionalized NPs to come to a distance of separation (DOS) of at least dRL (length of the R-L complex) from the receptor-bearing membranes. In this letter, we establish that the membrane surface charges and the surrounding ionic environment dictate whether or not the attainment of such a critical DOS is possible. The negatively charged membrane invariably induces a negative electrostatic potential at the NP surface, repelling the NP from the membrane. This is countered by the attractive influences of the thermal fluctuations and van der Waals (vdw) interactions that drive the NP close to the membrane. For a NP approaching the membrane from a distance, the ratio of the repulsive (electrostatic) and attractive (thermal and vdW) effects balances at a critical NP-membrane DOS of dg,c. For a given set of parameters, there can be two possible values of dg,c, namely, dg,c,1 and dg,c,2 with dg,c,1 ≫ dg,c,2. We establish that any R-L mediated NP-membrane interaction is possible only if dRL > dg,c,1. Therefore, our study proposes a design criterion for engineering ligands for a NP that will ensure the appropriate length of the R-L complex in order to ensure the successful membrane-NP interaction in the presence of a given electrostatic environment. Finally, we discuss the manner in which our theory can help designing ligand-grafted NPs for targeted drug delivery, design biomimetics NPs, and also explain various experimental results.

  5. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation. (United States)

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


    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.

  6. An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes. (United States)

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


    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.

  7. Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes. (United States)

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


    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

  8. Artificial plasma membrane models based on lipidomic profiling. (United States)

    Essaid, Donia; Rosilio, Véronique; Daghildjian, Katia; Solgadi, Audrey; Vergnaud, Juliette; Kasselouri, Athena; Chaminade, Pierre


    Phospholipid monolayers are often described as membrane models for analyzing drug-lipid interactions. In many works, a single phosphatidylcholine is chosen, sometimes with one or two additional components. Drug penetration is studied at 30mN/m, a surface pressure considered as corresponding to the pressure in bilayers, independently of the density of lipid molecular packing. In this work, we have extracted, identified, and quantified the major lipids constituting the lipidome of plasma and mitochondrial membranes of retinoblastoma (Y79) and retinal pigment epithelium cells (ARPE-19), using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The results obtained from this lipidomic analysis were used in an attempt to build an artificial lipid monolayer with a composition mimicking that of the plasma membrane of Y79 cells, better than a single phospholipid. The variety and number of lipid classes and species in cell extracts monolayers exceeding by far those of the phospholipids chosen to mimic them, the π-A isotherms of model monolayers differed from those of lipid extracts in shape and apparent packing density. We propose a model monolayer based on the most abundant species identified in the extracts, with a surface compressional modulus at 30mN/m close to the one of the lipid extracts. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Crystal structure of the plasma membrane proton pump

    DEFF Research Database (Denmark)

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


    -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...... the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane...

  10. Solid polymer electrolyte composite membrane comprising plasma etched porous support (United States)

    Liu, Han; LaConti, Anthony B.


    A solid polymer electrolyte composite membrane and method of manufacturing the same. According to one embodiment, the composite membrane comprises a rigid, non-electrically-conducting support, the support preferably being a sheet of polyimide having a thickness of about 7.5 to 15 microns. The support has a plurality of cylindrical pores extending perpendicularly between opposing top and bottom surfaces of the support. The pores, which preferably have a diameter of about 0.1 to 5 microns, are made by plasma etching and preferably are arranged in a defined pattern, for example, with fewer pores located in areas of high membrane stress and more pores located in areas of low membrane stress. The pores are filled with a first solid polymer electrolyte, such as a perfluorosulfonic acid (PFSA) polymer. A second solid polymer electrolyte, which may be the same as or different than the first solid polymer electrolyte, may be deposited over the top and/or bottom of the first solid polymer electrolyte.

  11. Autoinhibitory Regulation of Plasma Membrane H+-ATPases

    DEFF Research Database (Denmark)

    Pedersen, Jesper Torbøl

    Electrochemical gradients across cell membranes are essential for nutrient uptake. In plant and fungal cells the electrochemical gradient across the plasma membrane (PM) can build much higher than in mammalian cells. The protein responsible for this gradient is the essential PM H+-ATPase that uses...... a huge amount of energy in form of ATP, to pump out protons. To avoid complete energy depletion in the cells, tight regulation of the PM H+-ATPase is a necessity. The proteins two terminal domains have been identified as autoinhibitory domains that regulate the pumping activity, but due to lack of a high...... in mammalian cells and it has been speculated if they have a similar function in plants. In this thesis we show, that plant PM H+-ATPases are receptors for lysophospholipids and the autoinhibitory terminal inhibition is released upon lysophospholipid binding. Finally, we have used a group of stabilizing...

  12. Plant Phosphoproteomics: Analysis of Plasma Membrane Transporters by Mass Spectrometry

    DEFF Research Database (Denmark)

    Ye, Juanying; Rudashevskaya, Elena; Young, Clifford

      Phosphorylation is a key regulatory factor in all aspects of eukaryotic biology including the regulation of plant membrane-bound transport proteins. To date, mass spectrometry (MS) has been introduced as powerful technology for study of post translational modifications (PTMs), including protein...... phosphorylation. Due to the low abundance of phosphoprotein, the specific enrichment prior to MS analysis is necessary. Plant proton pump (H+-ATPase) is an enzyme controls the major transport processes in the plant, such as root nutrient uptake. Moreover, this pump has been proposed to be involved in other...... important physiological functions, such as stomata aperture, cell elongation, or cellular pH regulation. It is known that the activity of plant plasma membrane H+-ATPase is regulated by phosphorylation. Therefore, we first investigated the phosphorylation profile of plant H+-ATPase by enriching...

  13. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities (United States)

    Caldwell, C.


    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  14. Specific interaction of postsynaptic densities with membrane rafts isolated from synaptic plasma membranes. (United States)

    Liu, Qian; Yao, Wei-Dong; Suzuki, Tatsuo


    Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. We recently demonstrated the presence, at the electron microscopic level, of complexes consisting of membrane rafts and postsynaptic densities (PSDs) in detergent-resistant membranes (DRMs) prepared from synaptic plasma membranes (SPMs) ( Suzuki et al., 2011 , J Neurochem, 119, 64-77). To further explore these complexes, here we investigated the nature of the binding between purified SPM-DRMs and PSDs in vitro. In binding experiments, we used SPM-DRMs prepared after treating SPMs with n-octyl-β-d-glucoside, because at concentrations of 1.0% or higher it completely separates SPM-DRMs and PSDs, providing substantially PSD-free unique SPM-DRMs as well as DRM-free PSDs. PSD binding to PSD-free DRMs was identified by mass spectrometry, Western blotting, and electron microscopy. PSD proteins were not incorporated into SPMs, and significantly less PSD proteins were incorporated into DRMs prepared from liver membranes, providing in vitro evidence that binding of PSDs to DRMs is specific and suggestion of the presence of specific interacting molecules. These specific interactions may have important roles in synaptic development, function, and plasticity in vivo. In addition, the binding system we developed may be a good tool to search for binding molecules and binding mechanisms between PSDs and rafts.

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

    Energy Technology Data Exchange (ETDEWEB)

    Steponkus, P.L.


    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.

  16. Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It. (United States)

    Kraft, Mary L


    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Meier, P.J.; Valantinas, J.; Hugentobler, G.; Rahm, I. (University Hospital, Zurich (Switzerland))


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

  18. Glycine transporter dimers: evidence for occurrence in the plasma membrane. (United States)

    Bartholomäus, Ingo; Milan-Lobo, Laura; Nicke, Annette; Dutertre, Sébastien; Hastrup, Hanne; Jha, Alok; Gether, Ulrik; Sitte, Harald H; Betz, Heinrich; Eulenburg, Volker


    Different Na(+)/Cl(-)-dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuT(Aa), as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs.

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

    International Nuclear Information System (INIS)

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


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

  20. Dynamics of glycine receptor insertion in the neuronal plasma membrane. (United States)

    Rosenberg, M; Meier, J; Triller, A; Vannier, C


    The exocytosis site of newly synthesized glycine receptor was defined by means of a morphological assay to characterize its export from the trans-Golgi Network to the plasma membrane. This was achieved by expressing in transfected neurons an alpha1 subunit bearing an N-terminal tag selectively cleavable from outside the cell by thrombin. This was combined with a transient temperature-induced block of exocytic transport that creates a synchronized exocytic wave. Immunofluorescence microscopy analysis of the cell surface appearance of newly synthesized receptor revealed that exocytosis mainly occurred at nonsynaptic sites in the cell body and the initial portion of dendrites. At the time of cell surface insertion, the receptors existed as discrete clusters. Quantitative analysis showed that glycine receptor clusters are stable in size and subsequently appeared in more distal dendritic regions. This localization resulted from diffusion in the plasma membrane and not from exocytosis of transport vesicles directed to dendrites. Kinetic analysis established a direct substrate-product relationship between pools of somatic and dendritic receptors. This indicated that clusters represent intermediates between newly synthesized and synaptic receptors. These results support a diffusion-retention model for the formation of receptor-enriched postsynaptic domains and not that of a vectorial intracellular targeting to synapses.

  1. Purification of plant plasma membranes by two-phase partitioning and measurement of H+ pumping. (United States)

    Lund, Anette; Fuglsang, Anja Thoe


    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.

  2. Induction of stable ER-plasma-membrane junctions by Kv2.1 potassium channels. (United States)

    Fox, Philip D; Haberkorn, Christopher J; Akin, Elizabeth J; Seel, Peter J; Krapf, Diego; Tamkun, Michael M


    Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER-plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K(+) channel in the mammalian brain, induces the formation of ER-plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER-plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER-plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca(2+) signaling. © 2015. Published by The Company of Biologists Ltd.

  3. Bile acids modulate signaling by functional perturbation of plasma membrane domains. (United States)

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


    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.

  4. Thymocyte plasma membrane of the rainbow trout, Salmo gairdneri: Associated immunoglobulin and heteroantigens (United States)

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


    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.

  5. Closure of Oroantral Communication With Plasma-Rich Fibrin Membrane. (United States)

    Demetoglu, Umut; Ocak, Hakan; Bilge, Suheyb


    Oroantral communication (OAC) is the opening between the maxillary sinus and oral cavity. It may cause oroantral fistula or maxillary sinusitis if left untreated. The surgical closure of the OAC within 48 hours was recommended to avoid the complications like sinus infections. The aim of this study is to evaluate the treatment of OACs with plasma-rich fibrin (PRF) which is safe and easy to implement in the OACs.This study was conducted with the patients, who required the treatment of the OAC, which was developed after the posterior maxillary tooth extraction in the Dental and Maxillofacial Department of the Faculty of Dentistry in Adnan Menderes University.Plasma-rich fibrin membranes were inserted in layers into the tooth socket so that they covered the OAC. Then these membranes were fixated with the sutures to the surrounding gingiva. Antibiotic (amoxicillin/clavulanic acid 1000 mg), analgesic (dexketoprofen trometamol and/or paracetamol), and oral rinse (0.2% chlorhexidine digluconate) agents were prescribed to all patients. The patients were examined in the 3rd and 7th days and 2 months after the operation.All patients tolerated PRF perfectly, and the soft tissue recovery was completed without any problem. Full epithelization was observed in the defect area in all patients. The OAC did not relapse in any patient.Plasma-rich fibrin technique is a simple and effective method, which can be used in the treatment of OACs with a diameter of 5 mm or less with a low risk of complications.

  6. Isolation and characterization of the plasma membrane from the yeast Pichia pastoris. (United States)

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


    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.

  7. Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

    International Nuclear Information System (INIS)

    Witt, P.L.; Bownds, M.D.


    Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger

  8. Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane

    Directory of Open Access Journals (Sweden)

    Thomas Graham H


    Full Text Available Abstract Background The plasma membrane (PM is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP, based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila. Results We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments. Conclusion A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this

  9. Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy. (United States)

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


    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.

  10. Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry. (United States)

    Kanno, Chihiro; Kang, Sung-Sik; Kitade, Yasuyuki; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi


    The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE-PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE-PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE-PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE-PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously.

  11. Calcium pumps of plasma membrane and cell interior

    DEFF Research Database (Denmark)

    Strehler, Emanuel E; Treiman, Marek


    Calcium entering the cell from the outside or from intracellular organelles eventually must be returned to the extracellular milieu or to intracellular storage organelles. The two major systems capable of pumping Ca2+ against its large concentration gradient out of the cell or into the sarco....../endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue......- and cell type-specific patterns of expression. Different PMCA and SERCA isoforms are characterized by different regulatory and kinetic properties that likely are optimized for the distinct functional tasks fulfilled by each pump in setting resting cytosolic or intra-organellar Ca2+ levels, and in shaping...

  12. Influence of plasma modification on hygienic properties of textile fabrics with nonporous membrane coating (United States)

    Voznesensky, E. F.; Ibragimov, R. G.; Vishnevskaya, O. V.; Sisoev, V. A.; Lutfullina, G. G.; Tihonova, N. V.


    The work investigated the possibility of using plasma modification to improve the hygienic properties of textile materials with nonporous membrane coating to improve vapor-, air-permeability and water-resistant. Determined that, after plasma modification changes degree of supramolecular orderliness of the polymers nonporous membrane coating and the base fabric.

  13. (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


    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

  14. pH-induced proton permeability changes of plasma membrane vesicles

    NARCIS (Netherlands)

    Miedema, H; Prins, HBA; Staal, H.

    In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles.

  15. 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


    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 membran...

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

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


    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.

  17. Study on surface adhesion of Plasma modified Polytetrafluoroethylene hollow fiber membrane (United States)

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


    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.

  18. Production of selective membranes using plasma deposited nanochanneled thin films

    Directory of Open Access Journals (Sweden)

    Rodrigo Amorim Motta Carvalho


    Full Text Available The hydrolization of thin films obtained by tetraethoxysilane plasma polymerization results in the formation of a nanochanneled silicone like structure that could be useful for the production of selective membranes. Therefore, the aim of this work is to test the permeation properties of hydrolyzed thin films. The films were tested for: 1 permeation of polar organic compounds and/or water in gaseous phase and 2 permeation of salt in liquid phase. The efficiency of permeation was tested using a quartz crystal microbalance (QCM technique in gas phase and conductimetric analysis (CA in liquid phase. The substrates used were: silicon for characterization of the deposited films, piezoelectric quartz crystals for tests of selective membranes and cellophane paper for tests of permeation. QCM analysis showed that the nanochannels allow the adsorption and/or permeation of polar organic compounds, such as acetone and 2-propanol, and water. CA showed that the films allow salt permeation after an inhibition time needed for hydrolysis of the organic radicals within the film. Due to their characteristics, the films can be used for grains protection against microorganism proliferation during storage without preventing germination.

  19. Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA. (United States)

    Fan, Yang-Yi; Fuentes, Natividad R; Hou, Tim Y; Barhoumi, Rola; Li, Xian C; Deutz, Nicolaas E P; Engelen, Marielle P K J; McMurray, David N; Chapkin, Robert S


    Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietary n-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conducted in vitro and in vivo experiments to examine the effects of n-3 PUFA on CD4+ T cell membrane order, mitochondrial bioenergetics and lymphoproliferation. n-3 PUFA were incorporated into human primary CD4+ T cells phospholipids in vitro in a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses, n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 g n-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4+ T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4+ T cell plasma membrane organisation by n-3 PUFA may modulate down-stream clonal expansion.

  20. 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


    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.

  1. Serial plasma concentrations of atrial natriuretic peptide, plasma renin activity, aldosterone, and antidiuretic hormone in neonates on extracorporeal membrane oxygenation.

    NARCIS (Netherlands)

    Semmekrot, B.A.; Pesman, G.J.; Span, P.N.; Sweep, C.G.J.; Heyst, A.F.J. van; Monnens, L.A.H.; Bor, M. van de; Tanke, R.B.; Staak, F.H.J.M. van der


    To obtain information on water and salt regulating hormones and volume homeostasis during neonatal extracorporeal membrane oxygenation (ECMO), serial determinations of atrial natriuretic peptide (ANP), plasma renin activity (PRA), aldosterone (Aldo), antidiuretic hormone (ADH), colloid-osmotic

  2. Red wine activates plasma membrane redox system in human erythrocytes. (United States)

    Tedesco, Idolo; Moccia, Stefania; Volpe, Silvestro; Alfieri, Giovanna; Strollo, Daniela; Bilotto, Stefania; Spagnuolo, Carmela; Di Renzo, Massimo; Aquino, Rita P; Russo, Gian Luigi


    In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13-73 μg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70-100 μg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity.

  3. Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes. (United States)

    Subczynski, Witold Karol; Widomska, Justyna; Mainali, Laxman


    Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

  4. Preparation and characteristics of acrylic acid/styrene composite plasma polymerized membranes (United States)

    Jiang, Zhongqing; Jiang, Zhong-jie; Shi, Yicai; Meng, Yuedong


    Plasma polymerization has gained increasing interest for the deposition of functional plasma-polymerized membranes suitable for a wide range of applications on account of its advantageous features. In this work, acrylic acid/styrene composite plasma polymerized membranes were synthesized by plasma polymerization of a mixture of acrylic acid and styrene monomers in a low-frequency after-glow capacitively coupled plasma (CCP) discharge process. The structure and composition of the plasma polymerized membranes were characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The results showed that the partial pressure ratio between acrylic acid (AA) and styrene (St), applied discharge power and the energy of the extracted particles have considerable effects on the structure and the content of functional groups of the deposited membranes.

  5. Characterization of auxin-binding proteins from zucchini plasma membrane (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.


    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  6. Fluorescence interference contrast based approach to study real time interaction of melittin with plasma membranes (United States)

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


    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.

  7. Plasma membranes modified by plasma treatment or deposition as solid electrolytes for potential application in solid alkaline fuel cells. (United States)

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


    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.

  8. Plasma Membranes Modified by Plasma Treatment or Deposition as Solid Electrolytes for Potential Application in Solid Alkaline Fuel Cells (United States)

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


    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

  9. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets. (United States)

    Prada, Ilaria; Meldolesi, Jacopo


    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.

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

    International Nuclear Information System (INIS)

    Pal, Dipankar; Neogi, Sudarsan; De, Sirshendu


    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.

  11. 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:


    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.

  12. Review of low pressure plasma processing of proton exchange membrane fuel cell electrocatalysts


    Brault , Pascal


    Review article; International audience; The present review is describing recent advances in plasma deposition and treatment of low temperature proton exchange membrane fuel cells electrocatalysts. Interest of plasma processing for growth of platinum based, non-precious and metal free electrocatalysts is highlighted. Electrocatalysts properties are tentatively correlated to plasma parameters.

  13. A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking

    DEFF Research Database (Denmark)

    Aguilar, Pablo S; Fröhlich, Florian; Rehman, Michael


    The plasma membrane delimits the cell and controls material and information exchange between itself and the environment. How different plasma-membrane processes are coordinated and how the relative abundance of plasma-membrane lipids and proteins is homeostatically maintained are not yet understood...... for Rho1 and Rho2, in the regulation of sphingolipid metabolism....

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


    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

  15. Tailoring the properties of asymmetric cellulose acetate membranes by gas plasma etching. (United States)

    Olde Riekerink, M B; Engbers, G H M; Wessling, M; Feijen, J


    Cellulose triacetate (CTA) ultrafilters and cellulose acetate blend (CAB) desalination membranes were treated with a radiofrequency gas plasma (tetrafluoromethane (CF(4)) or carbon dioxide (CO(2)), 47-49 W, 0.04-0.08 mbar). Treatment times were varied between 15 s and 120 min. The plasma-treated top layer of the membranes was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and contact angle measurements to obtain information about surface structure, chemistry, and wettability, respectively. The membrane properties (e.g., permeability, selectivity, fouling) were studied by waterflux measurements, molecular weight cutoff measurements, and fouling experiments with bovine serum albumin. CO(2) plasma treatment resulted in gradual etching of the membrane's dense top layer. Permeation and selectivity changed significantly for treatment times of 0-15 min for CTA and 5-60 min for CAB membranes. Moreover, CTA membranes were hydrophilized during CO(2) plasma treatment whereas CF(4) plasma treatment led to hydrophobic surfaces due to strong fluorination of the top layer. This study shows that gas plasma etching can tailor the properties of asymmetric cellulose acetate membranes by simultaneously modifying the chemistry and structure of the top layer. The low fouling properties of CTA membranes were thereby largely maintained.

  16. Alterations in lipid composition and fluidity of liver plasma membranes in copper-deficient rats

    Energy Technology Data Exchange (ETDEWEB)

    Lei, K.Y.; Rosenstein, F.; Shi, F.; Hassel, C.A.; Carr, T.P.; Zhang, J. (Univ. of Arizona, Tucson (USA))


    In view of the importance of membrane fluidity on cell functions, the influence of phospholipid acyl groups on membrane fluidity, and the changes in lipid metabolism induced by copper (Cu) deficiency, this study was designed to examine the influence of dietary Cu on the lipid composition and fluidity of liver plasma membranes. Male Sprague-Dawley rats were divided into two dietary treatments, namely Cu deficient and Cu adequate. After 8 weeks of treatment, liver plasma membranes were isolated by sucrose density gradient centrifugation. The lipid fluidity of plasma membranes, as assessed by the intramolecular eximer fluorescence of 1,3-di(1-pyrenyl) propane, was significantly depressed by Cu deficiency. In addition, Cu deficiency significantly reduced the content of arachidonic and palmitoleic acids but increased the docosatetraenoic acids of membrane phospholipids. This alteration in unsaturated phospholipid fatty acid composition, especially the large reduction in arachidonic acid, may have contributed to the depressed membrane fluidity. Furthermore, Cu deficiency also markedly altered the fatty acid composition of the triacylglycerols associated with the plasma membranes. Thus, the lipid composition and fluidity of liver plasma membranes are responsive to the animal's Cu status.

  17. A microfluidic platform for probing single cell plasma membranes using optically trapped Smart Droplet Microtools (SDMs). (United States)

    Lanigan, Peter M P; Ninkovic, Tanja; Chan, Karen; de Mello, Andrew J; Willison, Keith R; Klug, David R; Templer, Richard H; Neil, Mark A A; Ces, Oscar


    We recently introduced a novel platform based upon optically trapped lipid coated oil droplets (Smart Droplet Microtools-SDMs) that were able to form membrane tethers upon fusion with the plasma membrane of single cells. Material transfer from the plasma membrane to the droplet via the tether was seen to occur. Here we present a customised version of the SDM approach based upon detergent coated droplets deployed within a microfluidic format. These droplets are able to differentially solubilise the plasma membrane of single cells with spatial selectivity and without forming membrane tethers. The microfluidic format facilitates separation of the target cells from the bulk SDM population and from downstream analysis modules. Material transfer from the cell to the SDM was monitored by tracking membrane localized EGFP.

  18. 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


    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

  19. 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.


    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

  20. 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.

  1. Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane.

    Directory of Open Access Journals (Sweden)

    Laura Paparelli


    Full Text Available Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH. We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided.

  2. 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.


    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...... 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...

  3. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens. (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K


    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. A method to modify PVDF microfiltration membrane via ATRP with low-temperature plasma pretreatment

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yu [School of Marine Science, Ningbo University, Fenghua Road 818, Ningbo, 315211 (China); Ningbo University of Technology, Fenghua Road 201, Ningbo, 315211 (China); Song, Shuijun [School of Marine Science, Ningbo University, Fenghua Road 818, Ningbo, 315211 (China); Zhejiang University of Science Technology, Liuhe Road 318, Hangzhou, 310023 (China); Lu, Yin, E-mail: [School of Marine Science, Ningbo University, Fenghua Road 818, Ningbo, 315211 (China); Zhu, Dongfa [School of Marine Science, Ningbo University, Fenghua Road 818, Ningbo, 315211 (China)


    Highlights: • We report a simple method to modify hydrophobic PVDF modification membrane. • Surface modification of PVDF membrane via ATRP with plasma pre-treatment. • ATRP grafting of SBMA onto the PVDF membrane surface form PVDF-g-SBMA membrane. • PVDF-g-SBMA membrane shows superior antifouling properties and hydrophilic. - Abstract: The hydrophilic modification of a polyvinylidene fluoride (PVDF) microfiltration membrane via pretreatment with argon plasma and direct surface-initiated atom transfer radical polymerization (ATRP) was studied. Both modified and unmodified PVDF membranes were characterized by Fourier transform infrared spectroscopy (FTIR), water contact angle, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and pore size distribution measurements. FTIR and XPS spectra confirmed that sulfobetaine methacrylate (SBMA) had been grafted onto the membrane surface. The initial contact angle decreased from 87.0° to 29.8° and a water drop penetrated into the modified membrane completely in 8 s. The pore size distribution of the modified membrane exhibited a smaller mean value than that of the original membrane. The antifouling properties of the modified PVDF membrane were evaluated by a filtration test using bovine serum albumin (BSA) solution. The results showed that the initial flux of the modified membrane increased from 2140.1 L/m{sup 2} h to 2812.7 L/m{sup 2} h and the equilibrium flux of BSA solution increased from 31 L/m{sup 2} h to 53 L/m{sup 2} h.

  5. Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Hasan, Nazarul [Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 319 Abraham Flexner Way, Room 515, Louisville, KY 40202 (United States); Hu, Chuan, E-mail: [Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 319 Abraham Flexner Way, Room 515, Louisville, KY 40202 (United States)


    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 {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 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 {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

  6. Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4. (United States)

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


    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.

  7. Application of Higher Density Iron Oxide Nanoparticle Pellicles to Enrich the Plasma Membrane and Its Proteome from Cells in Suspension. (United States)

    Rose, Rebecca L; Choksawangkarn, Waeowalee; Fenselau, Catherine


    Enrichment of the plasma membrane represents one valuable method to characterize the surfaceome, along with other plasma membrane and structural proteins. Currently, the overlapping densities of many subcellular organelles hinder enrichment of the plasma membrane by centrifugation. However, external access to the plasma membrane of intact cells allows the attachment of a nanoparticle pellicle to enhance its density and facilitate enrichment. We describe the synthesis of iron oxide nanoparticles, attachment of the pellicle to suspended cells, and recovery of plasma membrane proteins for proteomic analysis.

  8. Plasma deposition of silver nanoparticles on ultrafiltration membranes: antibacterial and anti-biofouling properties. (United States)

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


    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.

  9. The influence of hemodialysis membranes on the plasma insulin level of diabetic patients on maintenance hemodialysis. (United States)

    Abe, M; Kikuchi, F; Kaizu, K; Matsumoto, K


    The aim of the present study was to evaluate the alteration in plasma immunoreactive insulin (IRI) concentrations due to hemodialysis (HD) treatment by using three types of membranes in diabetic HD patients. We recruited 20 outpatients on maintenance HD with diabetes for this crossover study. HD was performed using membranes made of cellulose triacetate (CTA), polyester-polymer alloy (PEPA), and polysulphone (PS). These membranes were used for 2 weeks (6 HD sessions) in each patient in a randomized order decided by drawing lots. Blood samples were obtained at the beginning and end of the HD session from the blood tubing at the arterial (A) site. At 60 min after the initiation of dialysis, blood samples were obtained from the blood tubing at both the A and venous (V) sites of the dialyzer. The plasma IRI levels decreased significantly at the sites an hour after initiating HD in all membranes. The clearance of IRI was significantly higher in the case of the PS membrane when compared with the CTA and PEPA membranes. It was concluded that plasma insulin is cleared by HD, and the rate differs for each membrane. Plasma insulin clearance with the PS membrane is higher than that with the PEPA and CTA membranes.

  10. Atmospheric-pressure plasma activation and surface characterization on polyethylene membrane separator (United States)

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


    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.

  11. Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans (United States)

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


    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

  12. Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane. (United States)

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


    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.

  13. New saliva secretion model based on the expression of Na+-K+ pump and K+ channels in the apical membrane of parotid acinar cells. (United States)

    Almássy, János; Siguenza, Elias; Skaliczki, Marianna; Matesz, Klara; Sneyd, James; Yule, David I; Nánási, Péter P


    The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl - efflux and the subsequent paracellular Na + transport. In this model, the Na + -K + pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl - transport via basolateral Na + -K + -2Cl - cotransport is generated by the Na + -K + pump. In addition, the continuous electrochemical gradient for Cl - flow during acinar cell stimulation is maintained by the basolateral K + efflux. However, using a combination of single-cell electrophysiology and Ca 2+ -imaging, we demonstrate that photolysis of Ca 2+ close to the apical membrane of parotid acinar cells triggered significant K + current, indicating that a substantial amount of K + is secreted into the lumen during stimulation. Nevertheless, the K + content of the primary saliva is relatively low, suggesting that K + might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na + -K + pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K + from and secretes Na + to the lumen, which can partially supplement the paracellular Na + pathway.

  14. Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity

    Directory of Open Access Journals (Sweden)

    L.S.L.S. Reis


    Full Text Available ABSTRACT This study evaluated the plasma membrane integrity, acrosomal membrane integrity, and mitochondrial membrane potential of Nelore bull sperm from early puberty to early sexual maturity and their associations with sperm motility and vigor, the mass motility of the spermatozoa (wave motion, scrotal circumference, and testosterone. Sixty Nelore bulls aged 18 to 19 months were divided into four lots (n=15 bulls/lot and evaluated over 280 days. Semen samples, collected every 56 days by electroejaculation, were evaluated soon after collection for motility, vigor and wave motion under an optical microscope. Sperm membrane integrity, acrosomal integrity, and mitochondrial activity were evaluated under a fluorescent microscope using probe association (FITC-PSA, PI, JC-1, H342. The sperm were classified into eight integrity categories depending on whether they exhibited intact or damaged membranes, an intact or damaged acrosomal membrane, and high or low mitochondrial potential. The results show that bulls have a low amount of sperm with intact membranes at puberty, and the sperm show low motility, vigor, and wave motion; however, in bulls at early sexual maturity, the integrity of the sperm membrane increased significantly. The rate of sperm membrane damage was negatively correlated with motility, vigor, wave motion, and testosterone in the bulls, and a positive correlation existed between sperm plasma membrane integrity and scrotal circumference. The integrity of the acrosomal membrane was not influenced by puberty. During puberty and into early sexual maturity, bulls show low sperm mitochondrial potential, but when bulls reached sexual maturity, high membrane integrity with high mitochondrial potential was evident.

  15. Photostable bipolar fluorescent probe for video tracking plasma membranes related cellular processes. (United States)

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


    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.

  16. Directing membrane chromatography to manufacture α1-antitrypsin from human plasma fraction IV. (United States)

    Fan, Jinxin; Luo, Jianquan; Song, Weijie; Chen, Xiangrong; Wan, Yinhua


    The surging demand for plasma proteins, mainly driven by the growing market and the development of new therapeutic indications, is promoting manufacturers to improve the throughput of plasma proteins. Due to the inherent convective mass transfer, membrane chromatography has been proved to be an efficient approach for extracting a small amount of target proteins from large-volume feed. In this study, α1-antitrypsin (AAT) was extracted from human plasma fraction IV by a two-step membrane chromatography. An anion-exchange membrane chromatography (AEMC) was used to capture the plasma proteins in bind/elute mode, and the obtained effluent was further polished by a hydrophobic interaction membrane chromatography (HIMC) in flow-through mode. Under optimal conditions, the recovery and purity of AAT achieved 87.0% and 0.58 AAT/protein (g/g) by AEMC, respectively. After the precise polishing by HIMC, the purity of AAT was 1.22 AAT/protein (g/g). The comparison results showed that membrane chromatography outperformed column chromatography in both steps because of its high throughput. This two-step membrane chromatography could obtain an AAT recovery of 83.3% and an activity recovery of 91.4%. The outcome of this work not only offers an alternative process for protein purification from plasma, but also provides guidelines for manufacturing product from a large-volume feed with multi-components by membrane chromatography. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. LPS-Induced Macrophage Activation and Plasma Membrane Fluidity Changes are Inhibited Under Oxidative Stress. (United States)

    de la Haba, Carlos; Morros, Antoni; Martínez, Paz; Palacio, José R


    Macrophage activation is essential for a correct and efficient response of innate immunity. During oxidative stress membrane receptors and/or membrane lipid dynamics can be altered, leading to dysfunctional cell responses. Our aim is to analyze membrane fluidity modifications and cell function under oxidative stress in LPS-activated macrophages. Membrane fluidity of individual living THP-1 macrophages was evaluated by the technique two-photon microscopy. LPS-activated macrophage function was determined by TNFα secretion. It was shown that LPS activation causes fluidification of macrophage plasma membrane and production of TNFα. However, oxidative stress induces rigidification of macrophage plasma membrane and inhibition of cell activation, which is evidenced by a decrease of TNFα secretion. Thus, under oxidative conditions macrophage proinflammatory response might develop in an inefficient manner.

  18. Targeting the plasma membrane of neoplastic cells through alkylation: a novel approach to cancer chemotherapy. (United States)

    Trendowski, Matthew; Fondy, Thomas P


    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.

  19. Imaging of blood plasma coagulation at supported lipid membranes. (United States)

    Faxälv, Lars; Hume, Jasmin; Kasemo, Bengt; Svedhem, Sofia


    The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO(2) and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of ∼6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Localization of plasma membrane t-SNAREs syntaxin 2 and 3 in intracellular compartments

    Directory of Open Access Journals (Sweden)

    Kuismanen Esa


    Full Text Available Abstract Background Membrane fusion requires the formation of a complex between a vesicle protein (v-SNARE and the target membrane proteins (t-SNAREs. Syntaxin 2 and 3 are t-SNAREs that, according to previous over-expression studies, are predominantly localized at the plasma membrane. In the present study we investigated localization of the endogenous syntaxin 2 and 3. Results Endogenous syntaxin 2 and 3 were found in NRK cells in intracellular vesicular structures in addition to regions of the plasma membrane. Treatment of these cells with N-ethylmaleimide (NEM, which is known to inactivate membrane fusion, caused syntaxin 3 to accumulate in the trans-Golgi network and syntaxin 2 in perinuclear membrane vesicles. Kinetic analysis in the presence of NEM indicated that this redistribution of syntaxin 2 and 3 takes place via actin containing structures. Conclusion Our data suggest that syntaxin 2 cycles between the plasma membrane and the perinuclear compartment whereas syntaxin 3 cycles between the plasma membrane and the trans-Golgi network. It is possible that this cycling has an important role in the regulation of t-SNARE function.


    NARCIS (Netherlands)


    In leaves of Elodea densa the membrane potential measured in light equals the equilibrium potential of H+ on the morphological upper plasma membrane. The apoplastic pH on the upper side of the leaf is as high as 10.5-11.0, which indicates that alkaline pH induces an increased H+ permeability of the

  2. 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


    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...

  3. Normal chemotaxis in Dictyostelium discoideum cells with a depolarized plasma membrane potential

    NARCIS (Netherlands)

    Duijn, Bert van; Vogelzang, Sake A.; Ypey, Dirk L.; Molen, Loek G. van der; Haastert, Peter J.M. van


    We examined a possible role for the plasma membrane potential in signal transduction during cyclic AMP-induced chemotaxis in the cellular slime mold Dictyostelium discoideum. Chemotaxis, cyclic GMP and cyclic AMP responses in cells with a depolarized membrane potential were measured. Cells can be

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


    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

  5. Parallel artificial liquid membrane extraction of acidic drugs from human plasma

    DEFF Research Database (Denmark)

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


    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...

  6. Tailoring the properties of asymmetric cellulose acetate membranes by gas plasma etching

    NARCIS (Netherlands)

    Olde riekerink, M.B.; Engbers, G.H.M.; Wessling, Matthias; Feijen, Jan


    Cellulose triacetate (CTA) ultrafilters and cellulose acetate blend (CAB) desalination membranes were treated with a radiofrequency gas plasma (tetrafluoromethane (CF4) or carbon dioxide (CO2), 47¿49 W, 0.04¿0.08 mbar). Treatment times were varied between 15 s and 120 min. The plasma-treated top

  7. Characterization of the intracellular and the plasma membrane Ca2+-ATPases in fractionated pig brain membranes using calcium pump inhibitors. (United States)

    Salvador, J M; Mata, A M


    The Ca2+-ATPase activity of isolated membranes and purified plasma membrane ATPase from pig brain was measured in the presence of specific inhibitors. The inhibition of the enzymatic activity by vanadate presents a lower affinity in microsomes than in the synaptic plasma membrane vesicles, showing K0.5 of 0.4 and 0.2 microM, respectively. The purified enzyme showed a higher sensitivity to vanadate with a K0.5 of 0.10 microM. Thapsigargin (Tg) and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) were stronger inhibitors of the Ca2+-ATPase activity in microsomes than in the synaptic membrane vesicles. The activity of the purified enzyme was not affected by Tg and only partially by BHQ. Cyclopiazonic acid inhibited the enzymatic activity in all fractions, being more sensitive in microsomes. The microsome preparation incorporated 32P from [gamma-32P]ATP into two main proteins that appear at approx 110,000 and 140,000. According to the inhibition pattern, the lower phosphorylated band was identified as the sarco(endo)plasmic reticulum Ca2+-ATPase, being in a higher percentage than the upper band. Synaptic membrane vesicles also incorporated radioactive 32P into two protein bands. The 140,000 protein (upper band) shows the typical behavior of the purified plasma membrane Ca2+-ATPase, being more abundant in this preparation than the organellar Ca2+-pump (lower band). This study highlights the heterogeneous nature of the Ca2+-ATPase activity measured in brain membrane fractions. Copyright 1998 Academic Press.



    Savitsky, A. A.; Pankov, V. V.


    Atomic hydrogen interaction with metals processes introduces major interest concern for atomic and hydrogen energetic, and also space materials technology. In this paper the data on permeability of hydrogen through palladium membranes in a wide temperature range are cited, and also is informed about the effect of atomic hydrogen superpermeability through palladium membranes

  9. Molecular characterization of a cold-induced plasma membrane protein gene from wheat. (United States)

    Koike, Michiya; Sutoh, Keita; Kawakami, Akira; Torada, Atsushi; Oono, Kiyoharu; Imai, Ryozo


    As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Deltapmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat.

  10. Novel determinants of H-Ras plasma membrane localization and transformation

    DEFF Research Database (Denmark)

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


    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....

  11. Leucine-based receptor sorting motifs are dependent on the spacing relative to the plasma membrane

    DEFF Research Database (Denmark)

    Geisler, C; Dietrich, J; Nielsen, B L


    Many integral membrane proteins contain leucine-based motifs within their cytoplasmic domains that mediate internalization and intracellular sorting. Two types of leucine-based motifs have been identified. One type is dependent on phosphorylation, whereas the other type, which includes an acidic...... amino acid, is constitutively active. In this study, we have investigated how the spacing relative to the plasma membrane affects the function of both types of leucine-based motifs. For phosphorylation-dependent leucine-based motifs, a minimal spacing of 7 residues between the plasma membrane...... and the phospho-acceptor was required for phosphorylation and thereby activation of the motifs. For constitutively active leucine-based motifs, a minimal spacing of 6 residues between the plasma membrane and the acidic residue was required for optimal activity of the motifs. In addition, we found that the acidic...

  12. Emerging role of chemoprotective agents in the dynamic shaping of plasma membrane organization. (United States)

    Fuentes, Natividad R; Salinas, Michael L; Kim, Eunjoo; Chapkin, Robert S


    In the context of an organism, epithelial cells by nature are designed to be the defining barrier between self and the outside world. This is especially true for the epithelial cells that form the lining of the digestive tract, which absorb nutrients and serve as a barrier against harmful substances. These cells are constantly bathed by a complex mixture of endogenous (bile acids, mucus, microbial metabolites) and exogenous (food, nutrients, drugs) bioactive compounds. From a cell biology perspective, this type of exposure would directly impact the plasma membrane, which consists of a myriad of complex lipids and proteins. The plasma membrane not only functions as a barrier but also as the medium in which cellular signaling complexes form and function. This property is mediated by the organization of the plasma membrane, which is exquisitely temporally (nanoseconds to minutes) and spatially (nanometers to micrometers) regulated. Since numerous bioactive compounds found in the intestinal lumen can directly interact with lipid membranes, we hypothesize that the dynamic reshaping of plasma membrane organization underlies the chemoprotective effect of select membrane targeted dietary bioactives (MTDBs). This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments. (United States)

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


    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.

  14. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids. (United States)

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


    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.

  15. Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation (United States)

    Fujimoto, Toyoshi; Parmryd, Ingela


    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

  16. Characterization of phospholipid composition and its control in the plasma membrane of developing soybean root

    International Nuclear Information System (INIS)

    Whitman, C.E.


    The phospholipid composition of plasma membrane enriched fractions from developing soybean root and several mechanisms which may regulate it have been examined. Plasma membrane vesicles were isolated from meristematic and mature sections of four-day-old dark grown soybean roots (Glycine max [L.] Merr. Cult. Wells II). Analysis of lipid extracts revealed two major phospholipid classes: phosphatidylcholine and phosphatidylethanolamine. Minor phospholipid classes were phosphatidylinositol, phosphatidylserine, phosphatidylgylcerol and diphosphatidylgylcerol. Phospholipid composition was similar at each developmental stage. Fatty acids of phosphatidylcholine and phosphatidylethanolamine were 16:0, 18:0, 18:2, and 18:3. Fatty acid composition varied with both phospholipid class and the developmental stage of the root. The degradation of phosphatidylcholine by endogenous phospholipase D during membrane isolation indicated that this enzyme might be involved in phospholipid turnover within the membrane. Phospholipase D activity was heat labile and increasing the pH of the enzyme assay from 5.3 to 7.8 resulted in 90% inhibition of activity. The turnover of fatty acids within the phospholipids of the plasma membrane was studied. Mature root sections were incubated with [1- 14 C] acetate, 1 mM Na acetate and 50 μg/ml chloramphenicol. Membrane lipid extracts analyzed for phospholipid class and acyl chain composition revealed that the long incubation times did not alter the phospholipid composition of the plasma membrane enriched fraction

  17. 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.


    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

  18. Suicidal behaviour is associated with decreased esterified cholesterol in plasma and membrane fluidity of platelets. (United States)

    Mathew, Boby; Srinivasan, Krishnamachari; Pradeep, Johnson; Thomas, Tinku; Mandal, Amit Kumar


    Altered cholesterol levels in body fluids and brain tissues have been shown to be associated with suicidal behaviour, violence and aggression. But the biological underpinnings of this association in the pathophysiology of suicide are not clear. Cholesterol plays a crucial role in maintaining the cellular membrane fluidity and alterations in cellular membrane fluidity may impair serotonergic neurotransmission in the central nervous system. We measured plasma esterified cholesterol and platelet membrane fluidity using fluorescence anisotropy and estimated flow activation energy which is a measure of order of membrane lipid bilayer in patients with recent suicidal attempt and compared with age and gender matched controls. The plasma esterified cholesterol, platelet membrane fluidity and flow activation energy was found to be significantly lower in patients with recent suicidal attempts compared to controls. Altered levels of plasma esterified cholesterol which is in equilibrium with membrane cholesterol might have resulted in decreased membrane fluidity and an increase in the order of membrane lipid bilayer. This might impair the serotonergic neurotransmission, which has been implicated in the pathophysiology of suicide. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. The plasma membrane as a capacitor for energy and metabolism (United States)

    Ray, Supriyo; Kassan, Adam; Busija, Anna R.; Rangamani, Padmini


    When considering which components of the cell are the most critical to function and physiology, we naturally focus on the nucleus, the mitochondria that regulate energy and apoptotic signaling, or other organelles such as the endoplasmic reticulum, Golgi, ribosomes, etc. Few people will suggest that the membrane is the most critical element of a cell in terms of function and physiology. Those that consider the membrane critical will point to its obvious barrier function regulated by the lipid bilayer and numerous ion channels that regulate homeostatic gradients. What becomes evident upon closer inspection is that not all membranes are created equal and that there are lipid-rich microdomains that serve as platforms of signaling and a means of communication with the intracellular environment. In this review, we explore the evolution of membranes, focus on lipid-rich microdomains, and advance the novel concept that membranes serve as “capacitors for energy and metabolism.” Within this framework, the membrane then is the primary and critical regulator of stress and disease adaptation of the cell. PMID:26771520

  20. Does Increased Expression of the Plasma Membrane Calcium-ATPase Isoform 2 Confer Resistance to Apoptosis on Breast Cancer Cells?

    National Research Council Canada - National Science Library

    VanHouten, Joshua N


    The plasma membrane calcium ATPase isoform 2 (PMCA2) is highly expressed on the apical membrane of mammary epithelial cells during lactation, and is the predominant pump responsible for calcium transport into milk...

  1. Free-cholesterol loading does not trigger phase separation of the fluorescent sterol dehydroergosterol in the plasma membrane of macrophages

    DEFF Research Database (Denmark)

    Wüstner, Daniel


    membrane distribution of the fluorescent cholesterol-mimicking sterol dehydroergosterol (DHE) was investigated in FC-loaded J774 macrophages. Wide field fluorescence and deconvolution microscopy were combined with quantitative assessment of sterol distribution in straightened plasma membrane image segments...

  2. 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


    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.

  3. Towards Enhanced Performance Thin-film Composite Membranes via Surface Plasma Modification. (United States)

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


    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.

  4. Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

    Directory of Open Access Journals (Sweden)

    Sokolowska Izabela


    Full Text Available Abstract Liver infection with hepatitis B virus (HBV, a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (− and differentiated (+ cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (− and (+ cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.

  5. Plasma modified PLA electrospun membranes for actinorhodin production intensification in Streptomyces coelicolor immobilized-cell cultivations. (United States)

    Scaffaro, Roberto; Lopresti, Francesco; Sutera, Alberto; Botta, Luigi; Fontana, Rosa Maria; Gallo, Giuseppe


    Most of industrially relevant bioproducts are produced by submerged cultivations of actinomycetes. The immobilization of these Gram-positive filamentous bacteria on suitable porous supports may prevent mycelial cell-cell aggregation and pellet formation which usually negatively affect actinomycete submerged cultivations, thus, resulting in an improved biosynthetic capability. In this work, electrospun polylactic acid (PLA) membranes, subjected or not to O 2 -plasma treatment (PLA-plasma), were used as support for immobilized-cell submerged cultivations of Streptomyces coelicolor M145. This strain produces different bioactive compounds, including the blue-pigmented actinorhodin (ACT) and red-pigmented undecylprodigiosin (RED), and constitutes a model for the study of antibiotic-producing actinomycetes. Wet contact angles and X-ray photoelectron spectroscopy analysis confirmed the increased wettability of PLA-plasma due to the formation of polar functional groups such as carboxyl and hydroxyl moieties. Scanning electron microscope observations, carried out at different incubation times, revealed that S. coelicolor immobilized-cells created a dense "biofilm-like" mycelial network on both kinds of PLA membranes. Cultures of S. coelicolor immobilized-cells on PLA or PLA-plasma membranes produced higher biomass (between 1.5 and 2 fold) as well as higher levels of RED and ACT than planktonic cultures. In particular, cultures of immobilized-cells on PLA and PLA-plasma produced comparable levels of RED that were approximatively 4 and 5 fold higher than those produced by planktonic cultures, respectively. In contrast, levels of ACT produced by immobilized-cell cultures on PLA and PLA-plasma were different, being 5 and 10 fold higher than those of planktonic cultures, respectively. Therefore, this is study demonstrated the positive influence of PLA membrane on growth and secondary metabolite production in S. coelicolor and also revealed that O 2 -plasma treated PLA membranes

  6. Fluorinated carboxylic membranes deposited by plasma enhanced chemical vapour deposition for fuel cell applications

    Energy Technology Data Exchange (ETDEWEB)

    Thery, J.; Faucheux, V.; Truffier-Boutry, D.; Martinent, A.; Laurent, J.-Y. [Laboratory of Printed Component, LITEN, CEA Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 09 (France); Martin, S.; Le Van Jodin, L. [Laboratory of Components for the Micro-storage of Energy, LITEN, CEA Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 09 (France)


    Among the fuel cell technologies, the polymer electrolyte membrane fuel cells (PEMFCs) are particularly promising because they are energy-efficient, clean, and fuel-flexible (i.e., can use hydrogen or methanol). The great majority of PEM fuel cells rely on a polymer electrolyte from the family of perfluorosulfonic acid membranes, nevertheless alternative materials are currently being developed, mainly to offer the alternative workout techniques which are required for the portable energy sources. Plasma polymerization represents a good solution, as it offers the possibility to deposit thin layer with an accurate and homogeneous thickness, even on 3D surfaces. In this paper, we present the results for the growth of proton conductive fluoro carboxylic membranes elaborated by plasma enhanced chemical vapour deposition. These membranes present conductivity values of the same order than the one of Nafion {sup registered}. The properties of the membrane, such as the chemical composition, the ionic conductivity, the swelling behaviour and the permeability were correlated to the plasma process parameters. The membranes were integrated in fuel cells on porous substrates and we present here the results regarding the barrier effect and the power output. Barrier effect similar to those of 40 {mu}m Nafion {sup registered} layers was reached for 10 {mu}m thick carboxylic membranes. Power outputs around 3 mW cm{sup -2} were measured. We discuss the results regarding the gas barrier effect and the power outputs. (author)

  7. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas. (United States)

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu


    Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

  8. Fluorinated carboxylic membranes deposited by plasma enhanced chemical vapour deposition for fuel cell applications (United States)

    Thery, J.; Martin, S.; Faucheux, V.; Le Van Jodin, L.; Truffier-Boutry, D.; Martinent, A.; Laurent, J.-Y.

    Among the fuel cell technologies, the polymer electrolyte membrane fuel cells (PEMFCs) are particularly promising because they are energy-efficient, clean, and fuel-flexible (i.e., can use hydrogen or methanol). The great majority of PEM fuel cells rely on a polymer electrolyte from the family of perfluorosulfonic acid membranes, nevertheless alternative materials are currently being developed, mainly to offer the alternative workout techniques which are required for the portable energy sources. Plasma polymerization represents a good solution, as it offers the possibility to deposit thin layer with an accurate and homogeneous thickness, even on 3D surfaces. In this paper, we present the results for the growth of proton conductive fluoro carboxylic membranes elaborated by plasma enhanced chemical vapour deposition. These membranes present conductivity values of the same order than the one of Nafion ®. The properties of the membrane, such as the chemical composition, the ionic conductivity, the swelling behaviour and the permeability were correlated to the plasma process parameters. The membranes were integrated in fuel cells on porous substrates and we present here the results regarding the barrier effect and the power output. Barrier effect similar to those of 40 μm Nafion ® layers was reached for 10 μm thick carboxylic membranes. Power outputs around 3 mW cm -2 were measured. We discuss the results regarding the gas barrier effect and the power outputs.

  9. Basolateral Cl- channels in the larval bullfrog skin epithelium

    DEFF Research Database (Denmark)

    Hillyard, Stanley D.; Rios, K.; Larsen, Erik Hviid


    The addition of 150 U/ml nystatin to the mucosal surface of isolated skin from larval bullfrogs increases apical membrane permeability and allows a voltage clamp to be applied to the basolateral membrane. With identical Ringer's solutions bathing either side of the tissue the short-circuit current......, respectively. The Lorentzian plateau was minimal at the lowest ISC and increased as the ISC became greater in the positive or negative direction. Current-voltage plots with identical Ringer's on either side of the tissue showed a pattern of outward rectification. Cell attached patches of cells isolated from...... the skin with collagenase-trypsin treatment showed spontaneous channel activity with a conductance of 20.9 pS at a pipette potential, -Vp=20 mV. Current-voltage plots of single channels showed a similar pattern of rectification to that of the intact skin, and partial replacement of Cl- by gluconate...

  10. Surface modification of PTMSP membranes by plasma treatment: Asymmetry of transport in organic solvent nanofiltration. (United States)

    Volkov, A V; Tsarkov, S E; Gilman, A B; Khotimsky, V S; Roldughin, V I; Volkov, V V


    For the first time, the effect of asymmetry of the membrane transport was studied for organic solvents and solutes upon their nanofiltration through the plasma-modified membranes based on poly(1-trimethylsilyl-1-propyne) (PTMSP). Plasma treatment is shown to provide a marked hydrophilization of the hydrophobic PTMSP surface (the contact angle of water decreases from 88 down to 20°) and leads to the development of a negative charge of -5.2 nC/cm(2). The XPS measurements prove the formation of the oxygen-containing groups (Si-O and C-O) due to the surface modification. The AFM images show that the small-scale surface roughness of the plasma-treated PTMSP sample is reduced but the large-scale surface heterogeneities become more pronounced. The modified membranes retain their hydrophilic surface properties even after the nanofiltration tests and 30-day storage under ambient conditions. The results of the filtration tests show that when the membrane is oriented so that its modified layer contacts the feed solution, the membrane permeability for linear alcohols (methanol-propanol) and acetone decreases nearly two times. When the modified membrane surface faces the permeate, the membrane is seen to regain its transport characteristics: the flux becomes equal to that of the unmodified PTMSP. The well-pronounced effect of the transport asymmetry is observed for the solution of the neutral dye Solvent Blue 35 in methanol, ethanol, and acetone. For example, the initial membrane shows the negative retention for the Solvent Blue 35 dye (-16%) upon its filtration from the ethanol solution whereas, for the modified PTMSP membrane, the retention increases up to 17%. Various effects contributing to the asymmetry of the membrane transport characteristics are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Plasma membrane proteomics and its application in clinical cancer biomarker discovery

    DEFF Research Database (Denmark)

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


    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...... of this research is to improve individualized treatment and increase cancer patient survival, further development is crucial to direct analysis of clinically relevant patient samples. These efforts include optimized specimen handling and preparation as well as improved proteomics platforms. Identification......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...

  12. Plasma membrane organization and dynamics is probe and cell line dependent. (United States)

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


    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

  13. Surface Modification of Polyvinylidene Fluoride (PVDF) Membranes by Low-Temperature Plasma with Grafting Styrene (United States)

    Chen, Jian; Li, Jiding; Chen, Cuixian


    In order to control the surface pore sizes of polyvinylidene fluoride membranes and their distribution, low temperature plasma-induced grafting modifications of PVDF were studied to prepare hydrophobe membranes. By argon (Ar) treating and subsequent grafting reaction, a hydrophobe monomer, styrene, was introduced into the PVDF membrane. Fourier transform infrared attenuated total reflection (FTIR-ATR) was utilized to characterize the chemical and physical changes in the Ar plasma modified membrane. The surface modifications of PVDF membranes were investigated by using scanning electron microscopy (SEM), atomic force microscopy (AFM) and differential scanning calorimeter (DSC). The water permeability and the solute rejection were measured by PVDF membrane modified in different graft conditions. Results demonstrated that the pores in the modified membranes get smaller and the distribution of pores gets narrowed with the increase in grafting reaction duration. Longer graft time caused the water flux of PVDF membrane to decrease from 578 kg/(m2·h) to 23 kg/(m2·h) and the solute rejection to increase from 73% to 92%.

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

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


    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.

  15. Effect of gamma-radiation on the plasma and vacuolar membranes of cultured spinach cells

    International Nuclear Information System (INIS)

    Mitsuhashi, N.; Koshiba, T.; Sato, M.


    By the use of 31 P NMR spectroscopy, a higher radiosensitivity of the vacuolar membrane (tonoplast) than the plasma membrane was inferred for cultured spinach cells irradiated with 0-7.5 kGy and immediately transferred to fresh medium. In non-irradiated cells, inorganic phosphate (Pi) was rapidly taken up from the medium and transported into the vacuole; the content of cytoplasmic Pi was maintained at a constant level throughout the incubation period (7 days), whilst the Pi in the vacuole was exhausted. In irradiated cells, Pi uptake was not greatly altered up to 5 kGy, and fluorescein diacetate, a substance used to determine the integrity of the plasma membrane, was readily absorbed. However, movement of the Pi taken up into the vacuole across the tonoplast was markedly reduced with increasing dose. This membrane impairment was not amplified during subsequent incubation. A crucial question as to the fate of the Pi is critically discussed. (author)


    Grzegorzewicz, Anna E.; Pham, Ha; Gundi, Vijay A. K. B.; Scherman, Michael S.; North, Elton J.; Hess, Tamara; Jones, Victoria; Gruppo, Veronica; Born, Sarah E. M.; Korduláková, Jana; Chavadi, Sivagami Sundaram; Morisseau, Christophe; Lenaerts, Anne J.; Lee, Richard E.; McNeil, Michael R.; Jackson, Mary


    New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane. PMID:22344175

  17. ABBA regulates plasma-membrane and actin dynamics to promote radial glia extension. (United States)

    Saarikangas, Juha; Hakanen, Janne; Mattila, Pieta K; Grumet, Martin; Salminen, Marjo; Lappalainen, Pekka


    Radial glia play key roles in neuronal migration, axon guidance, and neurogenesis during development of the central nervous system. However, the molecular mechanisms regulating growth and morphology of these extended cells are unknown. We show that ABBA, a novel member of the IRSp53-MIM protein family, is enriched in different types of radial glia. ABBA binds ATP-actin monomers with high affinity and deforms PtdIns(4,5)P(2)-rich membranes in vitro through its WH2 and IM domains, respectively. In radial-glia-like C6-R cells, ABBA localises to the interface between the actin cytoskeleton and plasma membrane, and its depletion by RNAi led to defects in lamellipodial dynamics and process extension. Together, this study identifies ABBA as a novel regulator of actin and plasma membrane dynamics in radial glial cells, and provides evidence that membrane binding and deformation activity is critical for the cellular functions of IRSp53-MIM-ABBA family proteins.

  18. Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane (United States)

    Golan, Yonatan; Sherman, Eilon


    The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy. Protein mobility is highly heterogeneous, subdiffusive and ergodic-like. Using mobility characteristics, we segment individual trajectories into subpopulations with distinct Gaussian step-size distributions. Particles of low-to-medium mobility consist of clusters, diffusing in a viscoelastic and fractal-like medium and are enriched at the centre of the cell footprint. Particles of high mobility undergo weak confinement and are more evenly distributed. This study presents a methodological approach to resolve simultaneous mixed subdiffusion mechanisms acting on polydispersed samples and complex media such as cell membranes.

  19. Numerical calculation on a two-step subdiffusion behavior of lateral protein movement in plasma membranes (United States)

    Sumi, Tomonari; Okumoto, Atsushi; Goto, Hitoshi; Sekino, Hideo


    A two-step subdiffusion behavior of lateral movement of transmembrane proteins in plasma membranes has been observed by using single-molecule experiments. A nested double-compartment model where large compartments are divided into several smaller ones has been proposed in order to explain this observation. These compartments are considered to be delimited by membrane-skeleton "fences" and membrane-protein "pickets" bound to the fences. We perform numerical simulations of a master equation using a simple two-dimensional lattice model to investigate the heterogeneous diffusion dynamics behavior of transmembrane proteins within plasma membranes. We show that the experimentally observed two-step subdiffusion process can be described using fence and picket models combined with decreased local diffusivity of transmembrane proteins in the vicinity of the pickets. This allows us to explain the two-step subdiffusion behavior without explicitly introducing nested double compartments.

  20. Plasma-induced Styrene Grafting onto the Surface of Polytetrafluoroethylene Powder for Proton Exchange Membrane Application (United States)

    Lan, Yan; Cheng, Cheng; Zhang, Suzhen; Ni, Guohua; Chen, Longwei; Yang, Guangjie; Nagatsu, M.; Meng, Yuedong


    Low-temperature plasma treatment was adopted to graft styrene onto polytetrafluoroethylene (PTFE) powder, which is widely used in the fabrication of proton exchange membrane (PEM). The grafted PTFE powder was sulfonated in chlorosulfonic acid and fabricated into a membrane, which was used as inexpensive PEM material for a proton exchange membrane fuel cell (PEMFC). Fourier transform infrared spectroscopy attenuated total reflection spectroscopy (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) analysis were used to characterize the structure of the sulfonated PTFE powder. The results showed that all the PTFE powders were successfully grafted by nitrogen plasma and then sulfonated under such experimental conditions. A scanning electron microscopy (SEM) image indicated that the fabricated membrane exhibits flat morphology and homogenous structure. The ion exchange capacity (IEC) of this kind of PEM was also investigated.

  1. Phosphate depletion in opossum kidney cells: apical but not basolateral or transepithelial adaptions of Pi transport. (United States)

    Barac-Nieto, M; Alfred, M; Spitzer, A


    Monolayers of opossum kidney (OK) cells are widely used as models for the renal proximal tubule. OK cells adapt to phosphate (Pi) depletion by increasing their capacity for apical and basolateral Na+-dependent Pi uptake. Because NMR-visible cell Pi was found to be decreased in Pi-deprived kidney cells, we suggested that up-regulation of basolateral Pi efflux also occurs during adaptation to Pi deprivation [American Journal of Physiol 1994;267:C915-919]. In order to test this hypothesis, we measured the cell Pi pool, basolateral Pi efflux and transepithelial Pi fluxes in OK cells grown on permeable plastic filters, exposed overnight to solutions containing either 0.5 mM (deprived) or 2.0 mM (replete) Pi or 32Pi. Following steady state or acute loading with 32Pi, the specific activity (SA) of cell Pi, the cell Pi pool and the basolateral efflux of 32Pi were measured. In the steady state, a 2-fold increase in Pi uptake sustained the intracellular Pi pool at 85% of the control level (30 +/- 5 nmol/mg) in spite of a decrease in extracellular Pi from 2 to 0.5 mM. When the extracellular Pi was acutely (1 h) reduced to 0.1 mM, the cell Pi pool decreased (to 3 +/- 1 nmol/mg) both in cells previously adapted overnight to either 0.5 or to 2 mM Pi (p >0.3). The rates of absolute and fractional basolateral washout of cell 32Pi after 1 h loading with 0.1 mM 32Pi were similar in cells adapted to 0.5 compared to 2 mM Pi. This indicates that Pi depletion did not affect the effective permeability of the basolateral membranes to Pi. Adaptation for 16 h to 0.5 compared to 2 mM Pi did not alter the rate of net transepithelial transport of 0.1 mM Pi from the apical to the basal compartment but reduced (p OK cells grown on plastic support there are no adaptive increases in either basolateral Pi efflux, or in transcellular and paracellular Pi transport, in response to Pi depletion. Adaptations are limited to increases in apical and basolateral sodium-dependent Pi uptakes that can maintain

  2. Duration of ultrasound-mediated enhanced plasma membrane permeability

    NARCIS (Netherlands)

    Lammertink, Bart; Deckers, Roel; Storm, Gerrit; Moonen, Chrit; Bos, Clemens


    Ultrasound (US) induced cavitation can be used to enhance the intracellular delivery of drugs by transiently increasing the cell membrane permeability. The duration of this increased permeability, termed temporal window, has not been fully elucidated. In this study, the temporal window was

  3. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.


    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This "protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in sig...

  4. Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes (United States)

    Bag, Nirmalya; Ng, Xue Wen; Sankaran, Jagadish; Wohland, Thorsten


    Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7  ×  7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSDACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSDACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

  5. Cellulose microfibril deposition: coordinated activity at the plant plasma membrane

    NARCIS (Netherlands)

    Lindeboom, J.J.; Mulder, B.; Vos, J.W.; Ketelaar, M.J.; Emons, A.M.C.


    Plant cell wall production is a membrane-bound process. Cell walls are composed of cellulose microfibrils, embedded inside a matrix of other polysaccharides and glycoproteins. The cell wall matrix is extruded into the existing cell wall by exocytosis. This same process also inserts the cellulose

  6. 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.


    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

  7. Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane. (United States)

    Iaea, David B; Mao, Shu; Lund, Frederik W; Maxfield, Frederick R


    Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and nonvesicular sterol transport processes. Using the fluorescent cholesterol analogue dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a t 1/2 =12-15 min. Approximately 70% of sterol transport is ATP independent and therefore is nonvesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. A soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of nonvesicular sterol transport between the plasma membrane and ERC. This study shows that nonvesicular sterol transport mechanisms and STARD4 in particular account for a large fraction of sterol transport between the plasma membrane and the ERC. © 2017 Iaea 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 (

  8. Studies on the postnatal development of the rat liver plasma membrane following maternal ethanol ingestion

    Energy Technology Data Exchange (ETDEWEB)

    Rovinski, B.


    Studies on the developing rat liver and on the structure and function of the postnatal rat liver plasma membrane were carried out following maternal consumption of alcohol during pregnancy and lactation. A developmental study of alcohol dehydrogenase (ADH) indicated that both the activity and certain kinetic properties of the enzyme from the progeny of alcohol-fed and pair-fed mothers were similar. Fatty liver, however, developed in the alcoholic progeny only after ADH appeared on a day 19 of gestation. Further studies on structural and functional changes were then undertaken on the postnatal development of the rat liver plasma membrane. Radioligand binding studies performed using the hapatic alpha{sub 1}-adrenergic receptor as a plasma membrane probe demonstrated a significant decrease in receptor density in the alcoholic progeny, but no changes in binding affinity. Finally, the fatty acid composition of constituent phospholipids and the cholesterol content of rat liver plasma membranes were determined. All these observations suggest that membrane alterations in the newborn may be partially responsible for the deleterious action(s) of maternal alcoholism at the molecular level.

  9. Surface Modification of Asymmetric Polysulfone/Polyethylene Glycol Membranes by DC Ar-Glow Discharge Plasma

    Directory of Open Access Journals (Sweden)

    Chalad Yuenyao


    Full Text Available Polysulfone/polyethylene glycol (PSF/PEG membranes were prepared by dry/wet phase inversion method. Effects of direct current glow discharge plasma using argon as working gas on morphological structures and gas separation properties of membranes were studied. Alteration of membrane characteristics were analyzed by various techniques like contact angle, scanning electron microscope, Fourier transform infrared spectroscopy, and dynamic mechanical thermal analysis. Gas separation properties were measured in terms of permeation and ideal O2/N2 selectivity. Results showed that hydrophilic and gas separation properties of PSF/PEG membranes increased by plasma surface modification. It was also shown that the dosage of PEG and plasma treatment affected the morphological structures and mechanical and gas separation properties. The macro voids and transmembrane structure disappeared with a little amount of PEG dosage. Pore size and mechanical strength tend to decrease with increasing PEG dosage up to 10 wt%. Glass transition temperature (Tg receded from 201.8 to 143.7°C for pure PSF and PSF/PEG with PEG dosage of 10 wt%. O2 and N2 gases permeation through the 10-minute plasma treated membranes tend to increase. However, the permeation strongly dispersed when treatment time was more extended.

  10. Amine Enrichment of Thin-Film Composite Membranes via Low Pressure Plasma Polymerization for Antimicrobial Adhesion. (United States)

    Reis, Rackel; Dumée, Ludovic F; He, Li; She, Fenghua; Orbell, John D; Winther-Jensen, Bjorn; Duke, Mikel C


    Thin-film composite membranes, primarily based on poly(amide) (PA) semipermeable materials, are nowadays the dominant technology used in pressure driven water desalination systems. Despite offering superior water permeation and salt selectivity, their surface properties, such as their charge and roughness, cannot be extensively tuned due to the intrinsic fabrication process of the membranes by interfacial polymerization. The alteration of these properties would lead to a better control of the materials surface zeta potential, which is critical to finely tune selectivity and enhance the membrane materials stability when exposed to complex industrial waste streams. Low pressure plasma was employed to introduce amine functionalities onto the PA surface of commercially available thin-film composite (TFC) membranes. Morphological changes after plasma polymerization were analyzed by SEM and AFM, and average surface roughness decreased by 29%. Amine enrichment provided isoelectric point changes from pH 3.7 to 5.2 for 5 to 15 min of plasma polymerization time. Synchrotron FTIR mappings of the amine-modified surface indicated the addition of a discrete 60 nm film to the PA layer. Furthermore, metal affinity was confirmed by the enhanced binding of silver to the modified surface, supported by an increased antimicrobial functionality with demonstrable elimination of E. coli growth. Essential salt rejection was shown minimally compromised for faster polymerization processes. Plasma polymerization is therefore a viable route to producing functional amine enriched thin-film composite PA membrane surfaces.

  11. Regulation of plant plasma membrane H+- and Ca2+-ATPases by terminal domains

    DEFF Research Database (Denmark)

    Bækgaard, Lone; Fuglsang, Anja Thoe; Palmgren, Michael Gjedde


    In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H(+)-and Ca(2+)-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulator...... mechanisms of these pumps. In plant plasma membrane H(+)- and Ca(2+)-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed....

  12. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma

    DEFF Research Database (Denmark)

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine


    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic...... solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation...

  13. Spatial cycles mediated by UNC119 solubilisation maintain Src family kinases plasma membrane localisation. (United States)

    Konitsiotis, Antonios D; Roßmannek, Lisaweta; Stanoev, Angel; Schmick, Malte; Bastiaens, Philippe I H


    The peripheral membrane proto-oncogene Src family protein tyrosine kinases relay growth factor signals to the cytoplasm of mammalian cells. We unravel the spatial cycles of solubilisation, trapping on perinuclear membrane compartments and vesicular transport that counter entropic equilibration to endomembranes for maintaining the enrichment and activity of Src family protein tyrosine kinases at the plasma membrane. The solubilising factor UNC119 sequesters myristoylated Src family protein tyrosine kinases from the cytoplasm, enhancing their diffusion to effectively release Src family protein tyrosine kinases on the recycling endosome by localised Arl2/3 activity. Src is then trapped on the recycling endosome via electrostatic interactions, whereas Fyn is quickly released to be kinetically trapped on the Golgi by palmitoyl acyl-transferase activity. Vesicular trafficking from these compartments restores enrichment of the Src family protein tyrosine kinases to the plasma membrane. Interference with these spatial cycles by UNC119 knockdown disrupts Src family protein tyrosine kinase localisation and signalling activity, indicating that UNC119 could be a drug target to affect oncogenic Src family protein tyrosine kinase signalling.The peripheral membrane proto-oncogene Src family protein tyrosine kinases (SFKs) transmit growth factor signals to the cytoplasm. Here the authors show that the solubilising factor UNC119 sequesters myristoylated SFKs to maintain its enrichment at the plasma membrane to enable signal transduction.

  14. 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.


    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

  15. Effect of oxidative stress on plasma membrane fluidity of THP-1 induced macrophages. (United States)

    de la Haba, Carlos; Palacio, José R; Martínez, Paz; Morros, Antoni


    Plasma membrane is one of the preferential targets of reactive oxygen species which cause lipid peroxidation. This process modifies membrane properties such as membrane fluidity, a very important physical feature known to modulate membrane protein localization and function. The aim of this study is to evaluate the effect of oxidative stress on plasma membrane fluidity regionalization of single living THP-1 macrophages. These cells were oxidized with H(2)O(2) at different concentrations, and plasma membrane fluidity was analyzed by two-photon microscopy in combination with the environment-sensitive probe Laurdan. Results show a significant H(2)O(2) concentration dependent increase in the frequency of rigid lipid regions, mainly attributable to lipid rafts, at the expense of the intermediate fluidity regions. A novel statistical analysis evaluated changes in size and number of lipid raft domains under oxidative stress conditions, as lipid rafts are platforms aiding cell signaling and are thought to have relevant roles in macrophage functions. It is shown that H(2)O(2) causes an increase in the number, but not the size, of raft domains. As macrophages are highly resistant to H(2)O(2), these new raft domains might be involved in cell survival pathways. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools

    International Nuclear Information System (INIS)

    Jacobs, D.B.; Berenski, C.J.; Spangler, R.A.; Jung, C.Y.


    The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size

  17. Membrane-based, sedimentation-assisted plasma separator for point-of-care applications. (United States)

    Liu, Changchun; Mauk, Michael; Gross, Robert; Bushman, Frederic D; Edelstein, Paul H; Collman, Ronald G; Bau, Haim H


    Often, high-sensitivity, point-of-care (POC) clinical tests, such as HIV viral load, require large volumes of plasma. Although centrifuges are ubiquitously used in clinical laboratories to separate plasma from whole blood, centrifugation is generally inappropriate for on-site testing. Suitable alternatives are not readily available to separate the relatively large volumes of plasma from milliliters of blood that may be needed to meet stringent limit-of-detection specifications for low-abundance target molecules. We report on a simple-to-use, low-cost, pump-free, membrane-based, sedimentation-assisted plasma separator capable of separating a relatively large volume of plasma from undiluted whole blood within minutes. This plasma separator consists of an asymmetric, porous, polysulfone membrane housed in a disposable chamber. The separation process takes advantage of both gravitational sedimentation of blood cells and size exclusion-based filtration. The plasma separator demonstrated a "blood in-plasma out" capability, consistently extracting 275 ± 33.5 μL of plasma from 1.8 mL of undiluted whole blood within less than 7 min. The device was used to separate plasma laden with HIV viruses from HIV virus-spiked whole blood with recovery efficiencies of 95.5% ± 3.5%, 88.0% ± 9.5%, and 81.5% ± 12.1% for viral loads of 35,000, 3500, and 350 copies/mL, respectively. The separation process is self-terminating to prevent excessive hemolysis. The HIV-laden plasma was then injected into our custom-made microfluidic chip for nucleic acid testing and was successfully subjected to reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP), demonstrating that the plasma is sufficiently pure to support high-efficiency nucleic acid amplification.

  18. Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes. (United States)

    Hrynevich, Sviatlana V; Pekun, Tatyana G; Waseem, Tatyana V; Fedorovich, Sergei V


    Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

  19. The influence of blood plasma of irradiated animals on activity of Ca2+ - ATPase and Mg2+ - ATPase in plasma membrane of thymocytes

    International Nuclear Information System (INIS)

    Dreval', V.I.


    Rats were irradiated at doses 1.5, 4.0, 7.0 and 10 Gy. After 1, 8, 15, 22 and 30 days the effect of blood plasma on activity of Ca 2+ -ATPase and Mg 2+ -ATPase in plasma membrane of thymocytes was investigated. It was found that the raise of irradiation dose leads to increasing of blood plasma effect on membrane-bound enzymes

  20. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs. (United States)

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan


    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  1. Coiled-coil oligomerization controls localization of the plasma membrane REMORINs. (United States)

    Martinez, Denis; Legrand, Anthony; Gronnier, Julien; Decossas, Marion; Gouguet, Paul; Lambert, Olivier; Berbon, Mélanie; Verron, Loris; Grélard, Axelle; Germain, Veronique; Loquet, Antoine; Mongrand, Sébastien; Habenstein, Birgit


    REMORINs are nanodomain-organized proteins located in the plasma membrane and involved in cellular responses in plants. The dynamic assembly of the membrane nanodomains represents an essential tool of the versatile membrane barriers to control and modulate cellular functions. Nevertheless, the assembly mechanisms and protein organization strategies of nanodomains are poorly understood and many structural aspects are difficult to visualize. Using an ensemble of biophysical approaches, including solid-state nuclear magnetic resonance, cryo-electron microscopy and in vivo confocal imaging, we provide first insights on the role and the structural mechanisms of REMORIN trimerization. Our results suggest that the formation of REMORIN coiled-coil trimers is essential for membrane recruitment and promotes REMORIN assembly in vitro into long filaments by trimer-trimer interactions that might participate in nanoclustering into membrane domains in vivo. Copyright © 2018. Published by Elsevier Inc.

  2. Interactions of sugar-based bolaamphiphiles with biomimetic systems of plasma membranes. (United States)

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


    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.

  3. Androgen Receptor Localizes to Plasma Membrane by Binding to Caveolin-1 in Mouse Sertoli Cells

    Directory of Open Access Journals (Sweden)

    Qiong Deng


    Full Text Available The nonclassical androgen signaling pathway translates signals into alterations in cellular function within minutes, and this action is proposed to be mediated by an androgen receptor (AR localized to the plasma membrane. This study was designed to determine the mechanism underlying the membrane association of androgen receptor in TM4 cells, a mouse Sertoli cell line. Western blot analysis indicated testosterone-induced AR translocation to the cell membrane. Data from coimmunoprecipitation indicated that AR is associated with caveolin-1, and testosterone enhanced this association. Knockdown of caveolin-1 by shRNA decreased the amount of AR localized to membrane fraction and prevented AR membrane trafficking after being exposed to testosterone at physiological concentration. The palmitoylation inhibitor 2-bromopalmitate decreased AR membrane localization in basal condition and completely blocked testosterone-induced AR translocation to membrane fraction. These data suggested that AR localized to membrane fraction by binding with caveolin-1 through palmitoylation of the cysteine residue. This study provided a new evidence for AR membrane localization and its application for clarifying the nonclassical signaling pathway of androgens.

  4. Interaction pathways between soft lipid nanodiscs and plasma membranes: A molecular modeling study. (United States)

    Li, Shixin; Luo, Zhen; Xu, Yan; Ren, Hao; Deng, Li; Zhang, Xianren; Huang, Fang; Yue, Tongtao


    Lipid nanodisc, a model membrane platform originally synthesized for study of membrane proteins, has recently been used as the carrier to deliver amphiphilic drugs into target tumor cells. However, the central question of how cells interact with such emerging nanomaterials remains unclear and deserves our research for both improving the delivery efficiency and reducing the side effect. In this work, a binary lipid nanodisc is designed as the minimum model to investigate its interactions with plasma membranes by using the dissipative particle dynamics method. Three typical interaction pathways, including the membrane attachment with lipid domain exchange of nanodiscs, the partial membrane wrapping with nanodisc vesiculation, and the receptor-mediated endocytosis, are discovered. For the first pathway, the boundary normal lipids acting as ligands diffuse along the nanodisc rim to gather at the membrane interface, repelling the central bola lipids to reach a stable membrane attachment. If bola lipids are positioned at the periphery and act as ligands, they diffuse to form a large aggregate being wrapped by the membrane, leaving the normal lipids exposed on the membrane exterior by assembling into a vesicle. Finally, by setting both central normal lipids and boundary bola lipids as ligands, the receptor-mediated endocytosis occurs via both deformation and self-rotation of the nanodiscs. All above pathways for soft lipid nanodiscs are quite different from those for rigid nanoparticles, which may provide useful guidelines for design of soft lipid nanodiscs in widespread biomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Measuring the Viscosity of the Escherichia coli Plasma Membrane Using Molecular Rotors. (United States)

    Mika, Jacek T; Thompson, Alexander J; Dent, Michael R; Brooks, Nicholas J; Michiels, Jan; Hofkens, Johan; Kuimova, Marina K


    The viscosity is a highly important parameter within the cell membrane, affecting the diffusion of small molecules and, hence, controlling the rates of intracellular reactions. There is significant interest in the direct, quantitative assessment of membrane viscosity. Here we report the use of fluorescence lifetime imaging microscopy of the molecular rotor BODIPY C10 in the membranes of live Escherichia coli bacteria to permit direct quantification of the viscosity. Using this approach, we investigated the viscosity in live E. coli cells, spheroplasts, and liposomes made from E. coli membrane extracts. For live cells and spheroplasts, the viscosity was measured at both room temperature (23°C) and the E. coli growth temperature (37°C), while the membrane extract liposomes were studied over a range of measurement temperatures (5-40°C). At 37°C, we recorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than that previously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillus vegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid ordering within their liquid-phase plasma membranes. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. Plasma lipid pattern and red cell membrane structure in β-thalassemia patients in Jakarta

    Directory of Open Access Journals (Sweden)

    Seruni K.U. Freisleben


    Full Text Available Background: Over the last 10 years, we have investigated thalassemia patients in Jakarta to obtain a comprehensive picture of iron overload, oxidative stress, and cell damage.Methods: In blood samples from 15 transfusion-dependent patients (group T, 5 non-transfused patients (group N and 10 controls (group C, plasma lipids and lipoproteins, lipid-soluble vitamin E, malondialdehyde (MDA and thiol status were measured. Isolated eryhtrocyte membranes were investigated with electron paramagnetic resonance (EPR spectroscopy using doxyl-stearic acid and maleimido-proxyl spin lables. Data were analyzed statistically with ANOVA.Results: Plasma triglycerides were higher and cholesterol levels were lower in thalassemic patients compared to controls. Vitamin E, group C: 21.8 vs T: 6.2 μmol/L and reactive thiols (C: 144 vs. T: 61 μmol/L were considerably lower in transfused patients, who exert clear signs of oxidative stress (MDA, C: 1.96 vs T: 9.2 μmol/L and of tissue cell damage, i.e., high transaminases plasma levels. Non-transfused thalassemia patients have slight signs of oxidative stress, but no significant indication of cell damage. Erythrocyte membrane parameters from EPR spectroscopy differ considerably between all groups. In transfusion-dependent patients the structure of the erythrocyte membrane and the gradients of polarity and fluidity are destroyed in lipid domains; binding capacity of protein thiols in the membrane is lower and immobilized.Conclusion: In tranfusion-dependent thalassemic patients, plasma lipid pattern and oxidative stress are associated with structural damage of isolated erythrocyte membranes as measured by EPR spectroscopy with lipid and proteinthiol spin labels. (Med J Indones 2011; 20:178-84Keywords: electron paramagnetic resonance spectroscopy, erythrocyte membrane, lipoproteins, oxidative stress, thalassemia, plasma lipids.

  7. 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.


    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

  8. GLUT4 expression at the plasma membrane is related to fibre volume in human skeletal muscle fibres

    DEFF Research Database (Denmark)

    Gaster, M; Vach, W; Beck-Nielsen, H


    In this study we examined the relationship between GLUT4 expression at the plasma membrane and muscle fibre size in fibre-typed human muscle fibres by immunocytochemistry and morphometry in order to gain further insight into the regulation of GLUT4 expression. At the site of the plasma membrane...

  9. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin (United States)

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


    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 (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. 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.

  10. Effect of saline stress on plasma membrane structure and function of barley roots

    International Nuclear Information System (INIS)

    Rahmani, F. H.


    Barely (Hordeum vulgare L. c v. Black Local) plants were grown hydroponic ally under different saline stresses (50, 100, 150 And 200 mm NaCI. The adverse effect of each saline stress on the structure and function of root cells plasma membrane was studied in terms of root surface ATPase activation by NaCI in the reaction mixture. Was 0, 50, 100. 150 and 200mM. ATPase activity was found to be increased gradually at certain concentrations of NaCI. For control and 50mM stressed plants, the increase in root surface ATPase activity was started at 150mM NaCI. For 100mM stressed plants it was started at 100mM NaCI. For 150 and 200mM stressed plants it was stated at 50mM NaCI Results indicated that the adverse effect of the growth medium saline stresses on the integrity of the plasma membrane was started at 100mM saline stress. Accordingly the role of plasma membrane bound ATPase in active ion transport was disturbed at 100mM saline stress and may be impaired at 150 and 200mM saline stresses. It was suggested that the lipid environment of the plasma membrane surrounding ATPase was modified by the saline stresses 100-200mM. (author). 38 refs., 2 figs., 2 tabs

  11. Eisosomes promote the ability of Sur7 to regulate plasma membrane organization in Candida albicans.

    Czech Academy of Sciences Publication Activity Database

    Wang, H.X.; Douglas, L.M.; Veselá, Petra; Rachel, R.; Malínský, Jan; Konopka, J.B.


    Roč. 27, č. 10 (2016), s. 1663-1675 ISSN 1059-1524 R&D Projects: GA ČR(CZ) GA15-10641S Institutional support: RVO:68378041 Keywords : plasma membrane * eisosome * PIL1 * SUR7 Subject RIV: EA - Cell Biology Impact factor: 3.685, year: 2016

  12. Potassium as an intrinsic uncoupler of the plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Palmgren, Michael Gjedde; Buch-Pedersen, Morten Jeppe

    The plant plasma membrane proton pump (H(+)-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K(+) is bound to the proton pump at a site involving Asp(617) in the cytoplasmic...

  13. Video Views and Reviews: Golgi Export, Targeting, and Plasma Membrane Caveolae (United States)

    Watters, Christopher


    In this article, the author reviews videos from "Molecular Biology of the Cell (MBC)" depicting various aspects of plasma membrane (PM) dynamics, including the targeting of newly synthesized components and the organization of those PM invaginations called caveolae. The papers accompanying these videos describe, respectively, the constitutive…

  14. Plasma membrane surface potential: dual effects upon ion uptake and toxicity (United States)

    Electrical properties of plasma membranes (PMs), partially controlled by the ionic composition of the bathing medium, play significant roles in the distribution of ions at the exterior surface of PMs and in the transport of ions across PMs. The effects of coexistent cations (commonly Al3+, Ca2+, Mg...

  15. The SCN9A channel and plasma membrane depolarization promote cellular senescence through Rb pathway. (United States)

    Warnier, Marine; Flaman, Jean-Michel; Chouabe, Christophe; Wiel, Clotilde; Gras, Baptiste; Griveau, Audrey; Blanc, Elena; Foy, Jean-Philippe; Mathot, Pauline; Saintigny, Pierre; Van Coppenolle, Fabien; Vindrieux, David; Martin, Nadine; Bernard, David


    Oncogenic signals lead to premature senescence in normal human cells causing a proliferation arrest and the elimination of these defective cells by immune cells. Oncogene-induced senescence (OIS) prevents aberrant cell division and tumor initiation. In order to identify new regulators of OIS, we performed a loss-of-function genetic screen and identified that the loss of SCN9A allowed cells to escape from OIS. The expression of this sodium channel increased in senescent cells during OIS. This upregulation was mediated by NF-κB transcription factors, which are well-known regulators of senescence. Importantly, the induction of SCN9A by an oncogenic signal or by p53 activation led to plasma membrane depolarization, which in turn, was able to induce premature senescence. Computational and experimental analyses revealed that SCN9A and plasma membrane depolarization mediated the repression of mitotic genes through a calcium/Rb/E2F pathway to promote senescence. Taken together, our work delineates a new pathway, which involves the NF-κB transcription factor, SCN9A expression, plasma membrane depolarization, increased calcium, the Rb/E2F pathway and mitotic gene repression in the regulation of senescence. This work thus provides new insight into the involvement of ion channels and plasma membrane potential in the control of senescence. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  16. Purification of the synaptosomal plasma membrane (Ca(2+) + Mg(2+))-ATPase from pig brain. (United States)

    Salvador, J M; Mata, A M


    The Ca(2+)-ATPase from the synaptosomal plasma membrane has been purified nearly to homogeneity from pig brain by a new procedure involving the calmodulin-affinity-chromatography technique. This is a convenient alternative to the standard methods for the purification of the plasma membrane Ca(2+)-ATPase from different sources that were unsuitable to purify the enzyme from pig brain. The main feature of this procedure is the use of 15% (v/v) glycerol as stabilizing agent, instead of acidic phospholipid. By using this protocol the enzyme was purified 36-fold with respect to the plasma membrane vesicle fraction, showing a specific activity of 2.3 i.u. in the presence of acidic phospholipid. In SDS/PAGE, it appears as a single protein band around Mr140 000 that can be phosphorylated by [gamma-(32)P]ATP in the presence of La(3+) and recognized by specific antibodies against the plasma membrane Ca(2+)-ATPase from pig antral smooth muscle. Calmodulin activates the enzyme 1.5-1.8-fold in the presence of phosphatidylcholine but not in the presence of phosphatidylserine.

  17. Visualization of plasma membrane compartmentalization by high-speed quantum dot tracking

    DEFF Research Database (Denmark)

    Clausen, M. P.; Lagerholm, B. C.


    In this study, we have imaged plasma membrane molecules labeled with quantum dots in live cells using a conventional wide-field microscope with high spatial precision at sampling frequencies of 1.75 kHz. Many of the resulting single molecule trajectories are sufficiently long (up to several...

  18. Lectin receptor kinase LecRK-b2 localizes to plasma membrane and ...

    African Journals Online (AJOL)



    -b2, has been characterized. Confocal microscopy images showed that the LecRK-b2-GFP fusion protein is localized to plasma membrane. The results of yeast 2 hybrid showed that lectin domain of LecRK-b2 had self-.

  19. Lectin receptor kinase LecRK-b2 localizes to plasma membrane and ...

    African Journals Online (AJOL)

    -b2, has been characterized. Confocal microscopy images showed that the LecRK-b2-GFP fusion protein is localized to plasma membrane. The results of yeast 2 hybrid showed that lectin domain of LecRK-b2 had selfinteraction, while the ...

  20. Membranes produced by plasma enhanced chemical vapor deposition technique for low temperature fuel cell applications (United States)

    Ennajdaoui, Aboubakr; Roualdes, Stéphanie; Brault, Pascal; Durand, Jean

    A plasma polymerization process using a continuous glow discharge has been implemented for preparing proton conducting membranes from trifluoromethane sulfonic acid and styrene. The chemical and physical structure of plasma membranes has been investigated using FTIR and SEM. The films are homogeneous with a good adhesion on commercial gas diffusion layer (E-Tek ®). Their deposition rate can be increased with increasing flow rate and input power. The thermogravimetric analysis under air of plasma polymers has showed a thermal stability up to 140 °C. Compared to the pulsed glow discharge studied in a previous paper, the continuous glow discharge has enabled to enhance the proton conductivity of membranes by a factor 3 (up to 1.7 mS cm -1). Moreover, the low methanol permeability (methanol diffusion coefficient down to 5 × 10 -13 m 2 s -1) of membranes has been confirmed by this study. In an industrial context, a reactor prototype has been developed to manufacture by plasma processes all active layers of fuel cell cores to be integrated in original compact PEMFC or DMFC.

  1. Method of preparing water purification membranes. [polymerization of allyl amine as thin films in plasma discharge (United States)

    Hollahan, J. R.; Wydeven, T. J., Jr. (Inventor)


    Allyl amine and chemically related compounds are polymerized as thin films in the presence of a plasma discharge. The monomer compound can be polymerized by itself or in the presence of an additive gas to promote polymerization and act as a carrier. The polymerized films thus produced show outstanding advantages when used as reverse osmosis membranes.

  2. Effect of washing on the plasma membrane and on stress reactions of cultured rose cells

    International Nuclear Information System (INIS)

    Qian, Y.C.; Nguyen, T.; Murphy, T.M.


    Cultured cells of Rosa damascena have been used as a model for studies of responses of plant cells to various stresses, including UV radiation, protein-synthesis inhibitors, and elicitors from pathogens. Many of the responses involve reactions at the plasma membrane: efflux of K + , changes in the acid balance between cytoplasm and external medium, synthesis of H 2 O 2 , and inhibition of ferricyanide reduction. In previous studies, the cells have typically been washed with a solution of low ionic strength. We now show that this washing procedure results in changes in the protein composition of the plasma membrane, in the labeling of the proteins in the plasma membrane, and in the specific activity of ATPase in purified plasma membrane vesicles. Also, compared to the unwashed cells, the washed cells show less net K + efflux after UV-C and Phytophthora elicitor treatments; more synthesis of H 2 O 2 after UV-C and a pattern of accumulation of H 2 O 2 after elicitor treatment that shows a delayed but higher peak; and more inhibition of ferricyanide reduction after UV-C, but not after elicitor treatment. The results suggest that washing has differential effects on the mechanisms by which cultured plant cells perceive or respond to two stresses, UV-C and elicitor

  3. A Hydrophobic Filter Confers the Cation Selectivity of Zygosaccharomyces rouxii Plasma-Membrane Na (+)/H (+) Antiporter

    Czech Academy of Sciences Publication Activity Database

    Kinclová-Zimmermannová, Olga; Falson, P.; Cmunt, Denis; Sychrová, Hana


    Roč. 427, č. 8 (2015), s. 1681-1697 ISSN 0022-2836 R&D Projects: GA ČR(CZ) GAP503/10/0307; GA MŠk(CZ) LD13037 Institutional support: RVO:67985823 Keywords : yeast * plasma membrane * sodium proton exchanger * substrate specificity * potassium transport Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.517, year: 2015

  4. Plasma membrane microdomains regulate turnover of transport proteins in yeast

    Czech Academy of Sciences Publication Activity Database

    Grossmann, G.; Malínský, Jan; Stahlschmidt, W.; Loibl, M.; Weig-Meckl, I.; Frommer, W.B.; Opekarová, Miroslava; Tanner, W.


    Roč. 183, č. 6 (2008), s. 1075-1088 ISSN 0021-9525 R&D Projects: GA ČR GA204/06/0009; GA ČR GA204/07/0133; GA ČR GC204/08/J024 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50200510 Keywords : Lithium acetate * Membrane compartment of Can1 * Monomeric red fluorescent protein Subject RIV: EA - Cell Biology Impact factor: 9.120, year: 2008

  5. Elevated cAMP increases aquaporin-3 plasma membrane diffusion

    DEFF Research Database (Denmark)

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


    Regulated urine concentration takes place in the renal collecting duct upon arginine vasopressin (AVP) stimulation, where subapical vesicles containing aquaporin-2 (AQP2) are inserted into the apical membrane instantly increasing water reabsorption and urine concentration. The reabsorped water...... be short-term regulated via changes in protein-protein interactions, incorporation into lipid rafts, and/or changes in steady-state turnover, which could result in changes in the diffusion behavior of AQP3. Thus we measured AQP3 diffusion coefficients upon stimulation with the AVP mimic forskolin to reveal...

  6. Rapid, directed transport of DC-SIGN clusters in the plasma membrane. (United States)

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


    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.

  7. Effects of intense noise exposure on the outer hair cell plasma membrane fluidity. (United States)

    Chen, Guang-Di; Zhao, Hong-Bo


    Outer hair cells (OHCs) play an important role in cochlear amplification via their length changes (electromotility). A noise-induced cochlear amplification loss leading to a permanent threshold shift (PTS) was observed without a significant hair cell loss in rats [Chen, G.D., Liu, Y., 2005. Mechanisms of noise-induced hearing loss potentiation by hypoxia. Hear. Res. 200, 1-9.]. Since motor proteins are inserted in the OHC lateral membrane, any change in the OHC plasma membrane may result in a loss of OHC electromotility, leading to a loss of cochlear amplification. In this study, the lateral diffusion in the OHC plasma membrane was determined in vitro in guinea pigs by fluorescent recovery after photobleaching (FRAP) after an in vivo noise exposure. The lateral diffusion in the OHC plasma membrane demonstrated a length-dependence, which increased as OHC length increased. A reduction in the lateral diffusion was observed in those OHCs with lengths of 50-70 microm after exposure to an 8-kHz octave band noise at 110 dB SPL for 3h. This membrane fluidity change was associated with the selective PTS at frequencies around 8 kHz. The reduction of the lateral diffusion in the OHC lateral wall indicated that noise could impair the micromechanics of the OHC lateral wall and might consequently impair OHC electromotility to induce threshold shift.

  8. Model lipid bilayers mimic non-specific interactions of gold nanoparticles with macrophage plasma membranes. (United States)

    Montis, Costanza; Generini, Viola; Boccalini, Giulia; Bergese, Paolo; Bani, Daniele; Berti, Debora


    Understanding the interaction between nanomaterials and biological interfaces is a key unmet goal that still hampers clinical translation of nanomedicine. Here we investigate and compare non-specific interaction of gold nanoparticles (AuNPs) with synthetic lipid and wild type macrophage membranes. A comprehensive data set was generated by systematically varying the structural and physicochemical properties of the AuNPs (size, shape, charge, surface functionalization) and of the synthetic membranes (composition, fluidity, bending properties and surface charge), which allowed to unveil the matching conditions for the interaction of the AuNPs with macrophage plasma membranes in vitro. This effort directly proved for the first time that synthetic bilayers can be set to mimic and predict with high fidelity key aspects of nanoparticle interaction with macrophage eukaryotic plasma membranes. It then allowed to model the experimental observations according to classical interface thermodynamics and in turn determine the paramount role played by non-specific contributions, primarily electrostatic, Van der Waals and bending energy, in driving nanoparticle-plasma membrane interactions. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. 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


    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

  10. Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana (United States)

    Tischner, R.; Ward, M. R.; Huffaker, R. C.


    Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

  11. Assay of Plasma Membrane H+-ATPase in Plant Tissues under Abiotic Stresses. (United States)

    Janicka, Małgorzata; Wdowikowska, Anna; Kłobus, Grażyna


    Plasma membrane (PM) H + -ATPase, which generates the proton gradient across the outer membrane of plant cells, plays a fundamental role in the regulation of many physiological processes fundamental for growth and development of plants. It is involved in the uptake of nutrients from external solutions, their loading into phloem and long-distance transport, stomata aperture and gas exchange, pH homeostasis in cytosol, cell wall loosening, and cell expansion. The crucial role of the enzyme in resistance of plants to abiotic and biotic stress factors has also been well documented. Such great diversity of physiological functions linked to the activity of one enzyme requires a suitable and complex regulation of H + -ATPase. This regulation comprises the transcriptional as well as post-transcriptional levels. Herein, we describe the techniques that can be useful for the analysis of the plasma membrane proton pump modifications at genetic and protein levels under environmental factors.

  12. Glia plasma membrane transporters: Key players in glutamatergic neurotransmission. (United States)

    Flores-Méndez, Marco; Mendez-Flores, Orquidia G; Ortega, Arturo


    Glutamate, the main excitatory amino acid in the central nervous system, elicits its functions through the activation of specific membrane receptors that are expressed in neurons and glial cells. The re-cycling of this amino acid is carried out mostly through a continuous interplay between neurons and glia cells, given the fact that the removal of glutamate from the synaptic cleft depends mainly on glial glutamate transporters. Therefore, a functional and physical interaction between membrane transporters links glutamate uptake, transformation to glutamine and its release to the extra-synaptic space and its uptake to the pre-synaptic terminal. This sequence of events, best known as the glutamate/glutamine shuttle is central to glutamatergic transmission. In this sense, the uptake process triggers a complex series of biochemical cascades that modify the physiology of glial cells in the immediate, short and long term so as to be capable to take up, transform and release these amino acids in a regulated amount and in an appropriate time frame to sustain glutamatergic neurotransmission. Among the signaling cascades activated in glial cells by glutamate transporters, a sustained Na(+) and Ca(2+) influx, protein posttranslational modifications and gene expression regulation at the transcriptional and translational levels are present. Therefore, it is clear that the pivotal role of glial cells in the context of excitatory transmission has been constantly underestimated. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Positive zeta potential of a negatively charged semi-permeable plasma membrane (United States)

    Sinha, Shayandev; Jing, Haoyuan; Das, Siddhartha


    The negative charge of the plasma membrane (PM) severely affects the nature of moieties that may enter or leave the cells and controls a large number of ion-interaction-mediated intracellular and extracellular events. In this letter, we report our discovery of a most fascinating scenario, where one interface (e.g., membrane-cytosol interface) of the negatively charged PM shows a positive surface (or ζ) potential, while the other interface (e.g., membrane-electrolyte interface) still shows a negative ζ potential. Therefore, we encounter a completely unexpected situation where an interface (e.g., membrane-cytosol interface) that has a negative surface charge density demonstrates a positive ζ potential. We establish that the attainment of such a property by the membrane can be ascribed to an interplay of the nature of the membrane semi-permeability and the electrostatics of the electric double layer established on either side of the charged membrane. We anticipate that such a membrane property can lead to such capabilities of the cell (in terms of accepting or releasing certain kinds of moieties as well regulating cellular signaling) that was hitherto inconceivable.

  14. Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes. (United States)

    Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan


    Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

  15. Lipid components in the detergent-resistant membrane microdomain (DRM) obtained from the synaptic plasma membrane of rat brain. (United States)

    Matsuura, Daisuke; Taguchi, Katsutoshi; Yagisawa, Hitoshi; Maekawa, Shohei


    Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as "lipid rafts" obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP(2) is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP(2) in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP(2) in SPM-derived DRM, we measured the amount of PIP(2) in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cdelta1 as a PIP(2) binding probe. About 10% of the PIP(2) in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP(2) in DRM prepared from SPM.

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

    International Nuclear Information System (INIS)

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


    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

  17. Polyethylene glycol acrylate-grafted polysulphone membrane for artificial lungs: plasma modification and haemocompatibility improvement. (United States)

    Wang, Weiping; Huang, Xin; Yin, Haiyan; Fan, Wenling; Zhang, Tao; Li, Lei; Mao, Chun


    In this study, polyethylene glycol acrylate (PEGA) was introduced onto the surface of polysulphone (PSF) membrane to prepare PSF-PEGA membranes through low-temperature plasma technology for haemocompatibility improvement of artificial lungs. The effects of plasma power, PEGA solution concentration and dipcoating temperature on surface modification were systematically investigated. Results of Fourier transform infrared spectroscopy, x-ray photoelectron spectroscopy and PEGA grafting degree confirmed that PEGA was successfully grafted onto the PSF membranes. Contact angle values showed that the hydrophilicity of the PSF-PEGA membrane surface increased by 21.5%. The results of the protein adsorption, platelet adhesion and coagulation tests further showed the excellent haemocompatibility of the modified membrane. Gas exchange tests also revealed that at a porcine blood flow rate of 5 l min(-1), O2 and CO2 exchange rates through the PSF-PEGA membrane were 198.6 and 170.9 ml min(-1), respectively; approximately this is the gas exchange capacity of commercial respiratory assistance devices.

  18. Glycine transporter dimers: evidence for occurrence in the plasma membrane

    DEFF Research Database (Denmark)

    Bartholomäus, Ingo; Milan-Lobo, Laura; Nicke, Annette


    by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization......Different Na(+)/Cl(-)-dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma...

  19. Putting together a plasma membrane NADH oxidase: a tale of three laboratories. (United States)

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


    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.

  20. Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung-Min [Department of Biological Sciences, University of Columbia, NY (United States); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Attieh, Zouhair K. [Department of Laboratory Science and Technology, American University of Science and Technology, Ashrafieh (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Son, Hee Sook [Department of Food Science and Human Nutrition, College of Human Ecology, Chonbuk National University (Korea, Republic of); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Chen, Huijun [Medical School, Nanjing University, Nanjing 210008, Jiangsu Province (China); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Bacouri-Haidar, Mhenia [Department of Biology, Faculty of Sciences (I), Lebanese University, Hadath (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Vulpe, Chris D., E-mail: [Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States)


    Highlights: Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in non-polarized cells. Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in iron deficient and polarized cells. Black-Right-Pointing-Pointer Hephaestin with apical iron moves near to basolateral membrane of polarized cells. Black-Right-Pointing-Pointer Peri-basolateral location of hephaestin is accessible to the extracellular space. Black-Right-Pointing-Pointer Hephaestin is involved in iron mobilization from the intestine to circulation. -- Abstract: While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO{sub 4}) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a

  1. The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins. (United States)

    Cihil, Kristine M; Swiatecka-Urban, Agnieszka


    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.

  2. Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants. (United States)

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane bound sodium/proton (Sodium/Hydrogen) antiporter that transports sodium into the vacuole and exports hydrogen into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane bound sodium/hydrogen antiporter that exports sodium to the ex...

  3. Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins. (United States)

    Yamashita, Tetsuji; Hakizimana, Pierre; Wu, Siva; Hassan, Ahmed; Jacob, Stefan; Temirov, Jamshid; Fang, Jie; Mellado-Lagarde, Marcia; Gursky, Richard; Horner, Linda; Leibiger, Barbara; Leijon, Sara; Centonze, Victoria E; Berggren, Per-Olof; Frase, Sharon; Auer, Manfred; Brownell, William E; Fridberger, Anders; Zuo, Jian


    Nature's fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5's active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.

  4. Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins.

    Directory of Open Access Journals (Sweden)

    Tetsuji Yamashita


    Full Text Available Nature's fastest motors are the cochlear outer hair cells (OHCs. These sensory cells use a membrane protein, Slc26a5 (prestin, to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5's active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.

  5. The Chemical Potential of Plasma Membrane Cholesterol: Implications for Cell Biology. (United States)

    Ayuyan, Artem G; Cohen, Fredric S


    Cholesterol is abundant in plasma membranes and exhibits a variety of interactions throughout the membrane. Chemical potential accounts for thermodynamic consequences of molecular interactions, and quantifies the effective concentration (i.e., activity) of any substance participating in a process. We have developed, to our knowledge, the first method to measure cholesterol chemical potential in plasma membranes. This was accomplished by complexing methyl-β-cyclodextrin with cholesterol in an aqueous solution and equilibrating it with an organic solvent containing dissolved cholesterol. The chemical potential of cholesterol was thereby equalized in the two phases. Because cholesterol is dilute in the organic phase, here activity and concentration were equivalent. This equivalence allowed the amount of cholesterol bound to methyl-β-cyclodextrin to be converted to cholesterol chemical potential. Our method was used to determine the chemical potential of cholesterol in erythrocytes and in plasma membranes of nucleated cells in culture. For erythrocytes, the chemical potential did not vary when the concentration was below a critical value. Above this value, the chemical potential progressively increased with concentration. We used standard cancer lines to characterize cholesterol chemical potential in plasma membranes of nucleated cells. This chemical potential was significantly greater for highly metastatic breast cancer cells than for nonmetastatic breast cancer cells. Chemical potential depended on density of the cancer cells. A method to alter and fix the cholesterol chemical potential to any value (i.e., a cholesterol chemical potential clamp) was also developed. Cholesterol content did not change when cells were clamped for 24-48 h. It was found that the level of activation of the transcription factor STAT3 increased with increasing cholesterol chemical potential. The cholesterol chemical potential may regulate signaling pathways. Copyright © 2018. Published by

  6. The effect of a genetically reduced plasma membrane protonmotive force on vegetative growth of Arabidopsis. (United States)

    Haruta, Miyoshi; Sussman, Michael R


    The plasma membrane proton gradient is an essential feature of plant cells. In Arabidopsis (Arabidopsis thaliana), this gradient is generated by the plasma membrane proton pump encoded by a family of 11 genes (abbreviated as AHA, for Arabidopsis H(+)-ATPase), of which AHA1 and AHA2 are the two most predominantly expressed in seedlings and adult plants. Although double knockdown mutant plants containing T-DNA insertions in both genes are embryonic lethal, under ideal laboratory growth conditions, single knockdown mutant plants with a 50% reduction in proton pump concentration complete their life cycle without any observable growth alteration. However, when grown under conditions that induce stress on the plasma membrane protonmotive force (PMF), such as high external potassium to reduce the electrical gradient or high external pH to reduce the proton chemical gradient, aha2 mutant plants show a growth retardation compared with wild-type plants. In this report, we describe the results of studies that examine in greater detail AHA2's specific role in maintaining the PMF during seedling growth. By comparing the wild type and aha2 mutants, we have measured the effects of a reduced PMF on root and hypocotyl growth, ATP-induced skewed root growth, and rapid cytoplasmic calcium spiking. In addition, genome-wide gene expression profiling revealed the up-regulation of potassium transporters in aha2 mutants, indicating, as predicted, a close link between the PMF and potassium uptake at the plasma membrane. Overall, this characterization of aha2 mutants provides an experimental and theoretical framework for investigating growth and signaling processes that are mediated by PMF-coupled energetics at the cell membrane.

  7. Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

    Directory of Open Access Journals (Sweden)

    de la Vega Michelle


    Full Text Available Abstract Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

  8. Plasma membrane calcium channels in cancer: Alterations and consequences for cell proliferation and migration. (United States)

    Déliot, Nadine; Constantin, Bruno


    The study of calcium channels in molecular mechanisms of cancer transformation is still a novel area of research. Several studies, mostly conducted on cancer cell lines, however support the idea that a diversity of plasma membrane channels participates in the remodeling of Ca2+ homeostasis, which regulates various cancer hallmarks such as uncontrolled multiplication and increase in migration and invasion abilities. However few is still understood concerning the intracellular signaling cascades mobilized by calcium influx participating to cancer cell behavior. This review intends to gather some of these pathways dependent on plasma membrane calcium channels and described in prostate, breast and lung cancer cell lines. In these cancer cell types, the calcium channels involved in calcium signaling pathways promoting cancer behaviors are mostly non-voltage activated calcium channels and belong to the TRP superfamily (TRPC, TPRPV and TRPM families) and the Orai family. TRP and Orai channels are part of many signaling cascades involving the activation of transmembrane receptors by extracellular ligand from the tumor environment. TRPV can sense changes in the physical and chemical environment of cancer cells and TRPM7 are stretch activated and sensitive to cholesterol. Changes in activation and or expression of plasma-membrane calcium channels affect calcium-dependent signaling processes relevant to tumorigenesis. The studies cited in this review suggest that an increase in plasma membrane calcium channel expression and/or activity sustain an elevated calcium entry (constitutive or under the control of extracellular signals) promoting higher cell proliferation and migration in most cases. A variety of non-voltage-operated calcium channels display change expression and/or activity in a same cancer type and cooperate to the same process relevant to cancer cell behavior, or can be involved in a different sequence of events during the tumorigenesis. This article is part of a

  9. Basolateral potassium channel in turtle colon. Evidence for single-file ion flow



    Treatment of the apical surface of the isolated, ouabain-inhibited turtle colon with the polyene antibiotic amphotericin B permitted the properties of a barium-sensitive potassium conductance in the basolateral membrane to be discerned from the measurements of transepithelial fluxes and electrical currents. Simultaneous measurements of potassium currents and 42K fluxes showed that the movement of potassium was not in accord with simple diffusion. Two other cations, thallium and rubidium, were...

  10. Steric exclusion and protein conformation determine the localization of plasma membrane transporters. (United States)

    Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert


    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

  11. Navigation through the Plasma Membrane Molecular Landscape Shapes Random Organelle Movement. (United States)

    Dun, Alison R; Lord, Gabriel J; Wilson, Rhodri S; Kavanagh, Deirdre M; Cialowicz, Katarzyna I; Sugita, Shuzo; Park, Seungmee; Yang, Lei; Smyth, Annya M; Papadopulos, Andreas; Rickman, Colin; Duncan, Rory R


    Eukaryotic plasma membrane organization theory has long been controversial, in part due to a dearth of suitably high-resolution techniques to probe molecular architecture in situ and integrate information from diverse data streams [1]. Notably, clustered patterning of membrane proteins is a commonly conserved feature across diverse protein families (reviewed in [2]), including the SNAREs [3], SM proteins [4, 5], ion channels [6, 7], and receptors (e.g., [8]). Much effort has gone into analyzing the behavior of secretory organelles [9-13], and understanding the relationship between the membrane and proximal organelles [4, 5, 12, 14] is an essential goal for cell biology as broad concepts or rules may be established. Here we explore the generally accepted model that vesicles at the plasmalemma are guided by cytoskeletal tracks to specific sites on the membrane that have clustered molecular machinery for secretion [15], organized in part by the local lipid composition [16]. To increase our understanding of these fundamental processes, we integrated nanoscopy and spectroscopy of the secretory machinery with organelle tracking data in a mathematical model, iterating with knockdown cell models. We find that repeated routes followed by successive vesicles, the re-use of similar fusion sites, and the apparently distinct vesicle "pools" are all fashioned by the Brownian behavior of organelles overlaid on navigation between non-reactive secretory protein molecular depots patterned at the plasma membrane. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  12. Confining domains lead to reaction bursts: reaction kinetics in the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Ziya Kalay

    Full Text Available Confinement of molecules in specific small volumes and areas within a cell is likely to be a general strategy that is developed during evolution for regulating the interactions and functions of biomolecules. The cellular plasma membrane, which is the outermost membrane that surrounds the entire cell, was considered to be a continuous two-dimensional liquid, but it is becoming clear that it consists of numerous nano-meso-scale domains with various lifetimes, such as raft domains and cytoskeleton-induced compartments, and membrane molecules are dynamically trapped in these domains. In this article, we give a theoretical account on the effects of molecular confinement on reversible bimolecular reactions in a partitioned surface such as the plasma membrane. By performing simulations based on a lattice-based model of diffusion and reaction, we found that in the presence of membrane partitioning, bimolecular reactions that occur in each compartment proceed in bursts during which the reaction rate is sharply and briefly increased even though the asymptotic reaction rate remains the same. We characterized the time between reaction bursts and the burst amplitude as a function of the model parameters, and discussed the biological significance of the reaction bursts in the presence of strong inhibitor activity.

  13. In vitro sealing of iatrogenic fetal membrane defects by a collagen plug imbued with fibrinogen and plasma. (United States)

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


    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.

  14. Focal junctions retard lateral movement and disrupt fluid phase connectivity in the plasma membrane

    DEFF Research Database (Denmark)

    Vind-Kezunovic, D.; Wojewodzka, U.; Gniadecki, R.


    -p-cyclodextrin further decreased the recovery of DiI-C-18:0. We propose a model in which focal junctions impose lateral heterogeneity in the plasma membrane by entrapment of lipid microdomains between dense arrays of immobilized transmembrane molecules which can enmesh otherwise freely percolating L-d phase lipids. (c...... containing liquid-ordered (L-o) lipids. Indeed, values of maximal fluorescence recovery after photobleaching revealed that the long-range mobility of cholera toxin B subunit (CTB, marker of L-o) was similar to 1.5-fold retarded within the focal junctions compared to the surrounding membrane. However, 1...

  15. Morphology-properties relationship of gas plasma treated hydrophobic meso-porous membranes and their improved performance for desalination by membrane distillation (United States)

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


    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 than 99.99%. This is the first time that an investigation demonstrates how the permeation characteristics of these membranes is directly related to data from spectral, morphological and surface charge analyses, which provide new insights on the impact of plasma treatments on both, the surface charge and roughness, of PTFE porous materials.

  16. Effect of PAF on polyrnorphonuclear leucocyte plasma membrane polarity: a fluorescence study

    Directory of Open Access Journals (Sweden)

    A. Kantar


    Full Text Available The effect of PAF on the plasma membrane polarity of polymorphonuclear leukocytes (PMNs was investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-1auroyl naphthalene (Laurdan, which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surrounding. Laurdan shows a marked steady-state emission blue-shift in non-polar solvents, with respect to polar solvents. Our results demonstrate that PAF (10−7 M induces a blue shift of the fluorescence emission spectra of Laurdan. These changes are blocked in the presence of the PAF antagonist, L-659,989. Our data indicate that the interaction between PAF and PMNs is accompanied by a decrease in polarity in the hydrophobic-hydrophilic interface of the plasma membrane.

  17. A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization

    DEFF Research Database (Denmark)

    Schiøtt, Morten; Romanowsky, Shawn M; Bækgaard, Lone


    Ca(2+) signals are thought to play important roles in plant growth and development, including key aspects of pollen tube growth and fertilization. The dynamics of a Ca(2+) signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). The Arabidopsis genome...... encodes 14 Ca(2+) pumps, 10 of which belong to a family of autoinhibited Ca(2+) ATPases (ACA) that are predicted to be activated by Ca(2+)/calmodulin. Here, we show that isoform ACA9 is expressed primarily in pollen and localized to the plasma membrane. Three independent T-DNA [portion of the Ti (tumor......-inducing) plasmid that is transferred to plant cells] gene disruptions of ACA9 were found to result in partial male sterility. Complementation was observed by using a ACA9-yellow fluorescence protein (YFP) fusion that displayed plasma membrane localization. Mutant aca9 pollen displayed a reduced growth potential...

  18. Plasma membrane reorganization induced by tumor promoters in an epithelial cell line

    Energy Technology Data Exchange (ETDEWEB)



    The effects of phorbol ester tumor promoters on the lateral diffusion in plasma membrane lipid environments were examined by the technique of fluorescence recovery after photobleaching. To this end, the probe collarein, a fluorescent lipid analog that has the property of exclusive localization in the plasma membrane, was synthesized. Measured decreases in three parameters [percentage of fluorescence bleached (30%), percentage of recovery (52%), and half-time for recovery (52%)] connoted the appearance of an immobile fraction upon exposure to tumor promoters. These data are consistent with lipid reorganization in response to a reorganization of the intra- and perimembranous macromolecular scaffolding upon the interaction of cells with tumor promoters. The idea of induced reorganization is supported by experiments in which cell shape change, brought about by either exposure to cytochalasin B or growth on matrices of collagen, fibronectin, or laminin, resulted in values in the fluorescence recovery after photobleaching technique similar to those with active phorbol esters.

  19. Arabidopsis TWISTED DWARF1 functionally interacts with Auxin Exporter ABCB1 on the root plasma membrane

    DEFF Research Database (Denmark)

    Wang, Bangjun; Bailly, Aurélien; Zwiewka, Marta


    Plant architecture is influenced by the polar, cell-to-cell transport of auxin that is primarily provided and regulated by plasma membrane efflux catalysts of the PIN-FORMED and B family of ABC transporter (ABCB) classes. The latter were shown to require the functionality of the FK506 binding...... protein42 TWISTED DWARF1 (TWD1), although underlying mechanisms are unclear. By genetic manipulation of TWD1 expression, we show here that TWD1 affects shootward root auxin reflux and, thus, downstream developmental traits, such as epidermal twisting and gravitropism of the root. Using immunological....... In planta bioluminescence resonance energy transfer analysis was used to verify specific ABC transporter B1 (ABCB1)-TWD1 interaction. Our data support a model in which TWD1 promotes lateral ABCB-mediated auxin efflux via protein-protein interaction at the plasma membrane, minimizing reflux from the root...

  20. Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate. (United States)

    von Erlach, Thomas C; Bertazzo, Sergio; Wozniak, Michele A; Horejs, Christine-Maria; Maynard, Stephanie A; Attwood, Simon; Robinson, Benjamin K; Autefage, Hélène; Kallepitis, Charalambos; Del Río Hernández, Armando; Chen, Christopher S; Goldoni, Silvia; Stevens, Molly M


    Cell size and shape affect cellular processes such as cell survival, growth and differentiation 1-4 , thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

  1. Surface functionalization dependent subcellular localization of Superparamagnetic nanoparticle in plasma membrane and endosome. (United States)

    Thimiri Govinda Raj, Deepak B; Khan, Niamat Ali


    In this article, we elaborate the application of thermal decomposition based synthesis of Fe 3 O 4 superparamagnetic nanoparticle (SPMNP) in subcellular fractionation context. Here, we performed surface functionalization of SPMNP with phospholipids and dimercaptosuccinic acid. Surprisingly, we observed surface functionalization dependent SPMNP localization in subcellular compartments such as plasma membrane, endosomes and lysosomes. By using SPMNP based subcellular localization with pulse-chase methodology, we could use SPMNP for high pure-high yield organelle (plasma membrane, endosomes and lysosome) fractionation. Further, SPMNP that are distinctly localized in subcellular compartments can be used as technology for subcellular fractionation that can complement existing tools for cell biology research. As a future perspective, isolated magnetic organelles can be extended to protein/protein complex purification for biochemical and structural biology studies.

  2. Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals. (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi


    A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

  3. Proliferation and differentiation of stem cells in contact with eluate from fibrin-rich plasma membrane. (United States)

    Souza, Fernanda Gimenez de; Fernandes, Beatriz Luci; Rebelatto, Carmen Lucia Kuniyoshi; Aguiar, Alessandra Melo de; Fracaro, Letícia; Brofman, Paulo Roberto Slud


    To evaluate the ability of the eluate from fibrin-rich plasma (FRP) membrane to induce proliferation and differentiation of isolated human adipose-derived stem cells (ASCs) into chondrocytes. FRP membranes were obtained by centrifugation of peripheral blood from two healthy donors, cut, and maintained in culture plate wells for 48 h to prepare the fibrin eluate. The SCATh were isolated from adipose tissue by collagenase digestion solution, and expanded in vitro . Cells were expanded and treated with DMEM-F12 culture, a commercial media for chondrogenic differentiation, and eluate from FRP membrane for three days, and labeled with BrdU for quantitative assessment of cell proliferation using the High-Content Operetta ® imaging system. For the chondrogenic differentiation assay, the SCATh were grown in micromass for 21 days and stained with toluidine blue and aggrecan for qualitative evaluation by light microscopy. The statistical analysis was performed using ANOVA and Tukey's test. There was a greater proliferation of cells treated with the eluate from FRP membrane compared to the other two treatments, where the ANOVA test showed significance ( p  membrane stimulated cell proliferation and induced differentiation of the stem cells into chondrocytes, suggesting a potential application of FRP membranes in hyaline cartilage regeneration therapies.

  4. Alteration in Fluidity of Cell Plasma Membrane in Huntington Disease Revealed by Spectral Phasor Analysis. (United States)

    Sameni, Sara; Malacrida, Leonel; Tan, Zhiqun; Digman, Michelle A


    Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN. Using phasor approach, we characterized the fluorescence of LAURDAN that is sensitive to the polarity of the immediate environment. LAURDAN is affected by the physical order of phospholipids (lipid order) and reports the membrane fluidity. We also validated our results using a different fluorescent membrane probe, Nile Red (NR). The plasma membrane in the cells expressing expanded polyQ shows a shift toward increased membrane fluidity revealed by both LAURDAN and NR spectral phasors. This finding brings a new perspective in the understanding of the early stages of HD that can be used as a target for drug screening.

  5. Myelin basic protein induces neuron-specific toxicity by directly damaging the neuronal plasma membrane.

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    Full Text Available The central nervous system (CNS insults may cause massive demyelination and lead to the release of myelin-associated proteins including its major component myelin basic protein (MBP. MBP is reported to induce glial activation but its effect on neurons is still little known. Here we found that MBP specifically bound to the extracellular surface of the neuronal plasma membrane and induced neurotoxicity in vitro. This effect of MBP on neurons was basicity-dependent because the binding was blocked by acidic lipids and competed by other basic proteins. Further studies revealed that MBP induced damage to neuronal membrane integrity and function by depolarizing the resting membrane potential, increasing the permeability to cations and other molecules, and decreasing the membrane fluidity. At last, artificial liposome vesicle assay showed that MBP directly disturbed acidic lipid bilayer and resulted in increased membrane permeability. These results revealed that MBP induces neurotoxicity through its direct interaction with acidic components on the extracellular surface of neuronal membrane, which may suggest a possible contribution of MBP to the pathogenesis in the CNS disorders with myelin damage.

  6. Proton-sensing transistor systems for detecting ion leakage from plasma membranes under chemical stimuli. (United States)

    Imaizumi, Yuki; Goda, Tatsuro; Schaffhauser, Daniel F; Okada, Jun-Ichi; Matsumoto, Akira; Miyahara, Yuji


    The membrane integrity of live cells is routinely evaluated for cytotoxicity induced by chemical or physical stimuli. Recent progress in bioengineering means that high-quality toxicity validation is required. Here, we report a pH-sensitive transistor system developed for the continuous monitoring of ion leakage from cell membranes upon challenge by toxic compounds. Temporal changes in pH were generated with high reproducibility via periodic flushing of HepG2 cells on a gate insulator of a proton-sensitive field-effect transistor with isotonic buffer solutions with/without NH 4 Cl. The pH transients at the point of NH 4 Cl addition/withdrawal originated from the free permeation of NH 3 across the semi-permeable plasma membranes, and the proton sponge effect produced by the ammonia equilibrium. Irreversible attenuation of the pH transient was observed when the cells were subjected to a membrane-toxic reagent. Experiments and simulations proved that the decrease in the pH transient was proportional to the area of the ion-permeable pores on the damaged plasma membranes. The pH signal was correlated with the degree of hemolysis produced by the model reagents. The pH assay was sensitive to the formation of molecularly sized pores that were otherwise not measurable via detection of the leakage of hemoglobin, because the hydrodynamic radius of hemoglobin was greater than 3.1nm in the hemolysis assay. The pH transient was not disturbed by inherent ion-transporter activity. The ISFET assay was applied to a wide variety of cell types. The system presented here is fast, sensitive, practical and scalable, and will be useful for validating cytotoxins and nanomaterials. The plasma membrane toxicity and hemolysis are widely and routinely evaluated in biomaterials science and biomedical engineering. Despite the recent development of a variety of methods/materials for efficient gene/drug delivery systems to the cytosol, the methodologies for safety validation remain unchanged in

  7. Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane. (United States)

    Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji


    Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of

  8. Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation. (United States)

    Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer


    By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

  9. Silymarin protects plasma membrane and acrosome integrity in sperm treated with sodium arsenite

    Directory of Open Access Journals (Sweden)

    Farzaneh Eskandari


    Full Text Available Background: Exposure to arsenic is associated with impairment of male reproductive function by inducing oxidative stress. Silymarin with an antioxidant property scavenges free radicals. Objective: The aim of this study was to investigate if silymarin can prevent the adverse effects of sodium arsenite on ram sperm plasma membrane and acrosome integrity. Materials and Methods: Ram epidydimal spermatozoa were divided into five groups: spermatozoa at 0 hr, spermatozoa at 180 min (control, spermatozoa treated with silymarin (20 μM + sodium arsenite (10 μM for 180 min, spermatozoa treated with sodium arsenite (10 μM for 180 min and spermatozoa treated with silymarin (20 μM for 180 min. Double staining of Hoechst and propidium iodide was performed to evaluate sperm plasma membrane integrity, whereas comassie brilliant blue staining was used to assess acrosome integrity. Results: Plasma membrane (p< 0.001 and acrosome integrity (p< 0.05 of the spermatozoa were significantly reduced in sodium arsenite group compared to the control. In silymarin + sodium arsenite group, silymarin was able to significantly (p< 0.001 ameliorate the adverse effects of sodium arsenite on these sperm parameters compared to sodium arsenite group. The incubation of sperm for 180 min (control group showed a significant (p< 0.001 decrease in acrosome integrity compared to the spermatozoa at 0 hour. The application of silymarin alone for 180 min could also significantly (p< 0.05 increase sperm acrosome integrity compared to the control. Conclusion: Silymarin as a potent antioxidant could compensate the adverse effects of sodium arsenite on the ram sperm plasma membrane and acrosome integrity.

  10. Origin and development of plasma membrane derived invaginations in Vinca rosea l. (United States)

    Mahlberg, P.; Walkinshaw, C.; Olson, K.


    The occurrence, morphology, and possible ontogeny of plasma-membrane-related structures are described which can develop into invaginations or intravacuolar formations. An underlying study of meristematic tissues from the shoot of Vinca rosea supports the interpretation that endocytosis does occur in plant cells and that it is appropriate to refer to these structures as endocytoses. The function of these invaginations or their content remains to be elucidated.

  11. Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase. (United States)

    Haruta, Miyoshi; Tan, Li Xuan; Bushey, Daniel B; Swanson, Sarah J; Sussman, Michael R


    A P-type H + -ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis ( Arabidopsis thaliana ) plant expressing H + -ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H + secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H + -ATPase. © 2018 American Society of Plant Biologists. All Rights Reserved.

  12. Biotransformation of endorphins by a synaptosomal plasma membrane preparation of rat brain and by human serum

    NARCIS (Netherlands)

    Burbach, J.P.H.; Loeber, J.G.; Verhoef, J.; Kloet, E.R. de; Wied, D. de


    β-Endorphin (β-LPH 61–91), γ-endorphin (61–77), des-tyrosine-γ-endorphin (62–77), α-endorphin (61–76), and β-LPH 61–69 either labeled with [125I] at the N-terminal 61-tyrosine residue or unlabeled were incubated with a crude synaptosomal plasma membrane fraction of rat brain or in human serum. At

  13. Plasma surface modification of chitosan membranes : characterization and preliminary cell response studies


    Silva, Simone Santos; Luna, Sandra M.; Gomes, Manuela E.; Benesch, Johan; Pashkuleva, I.; Mano, J. F.; Reis, R. L.


    Surface modification of biomaterials is a way to tailor cell responses whilst retaining the bulk properties. In this work, chitosan membranes were prepared by solvent casting and treated with nitrogen or argon plasma at 20Wfor 10–40 min. AFM indicated an increase in the surface roughness as a result of the ongoing etching process. XPS and contact angle measurements showed different surface elemental compositions and higher surface free energy. The MTS test and direct contact...

  14. Simultaneous AFM topography and recognition imaging at the plasma membrane of mammalian cells. (United States)

    Chtcheglova, Lilia A; Hinterdorfer, Peter


    Elucidation the nano-organization of membrane proteins at/within the plasma membrane is probably the most demanding and still challenging task in cell biology since requires experimental approaches with nanoscale resolution. During last decade, atomic force microscopy (AFM)-based simultaneous topography and recognition imaging (TREC) has become a powerful tool to quickly obtain local receptor nano-maps on complex heterogeneous biosurfaces such as cells and membranes. Here we emphasize the TREC technique and explain how to unravel the nano-landscape of mammalian cells. We describe the procedures for all steps of the experiment including tip functionalization with ligand molecules, sample preparation, and localization of key molecules on the cell surface. We also discuss the current limitations and future perspectives of this technique. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Plasma Membrane H(+)-ATPase Regulation in the Center of Plant Physiology. (United States)

    Falhof, Janus; Pedersen, Jesper Torbøl; Fuglsang, Anja Thoe; Palmgren, Michael


    The plasma membrane (PM) H(+)-ATPase is an important ion pump in the plant cell membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the PM, which is a prerequisite for growth. Modification of the autoinhibitory terminal domains activates PM H(+)-ATPase activity, and on this basis it has been hypothesized that these regulatory termini are targets for physiological factors that activate or inhibit proton pumping. In this review, we focus on the posttranslational regulation of the PM H(+)-ATPase and place regulation of the pump in an evolutionary and physiological context. The emerging picture is that multiple signals regulating plant growth interfere with the posttranslational regulation of the PM H(+)-ATPase. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  16. Plasma membrane is the site of productive HIV-1 particle assembly.

    Directory of Open Access Journals (Sweden)

    Nolwenn Jouvenet


    Full Text Available Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 assembly is initiated and completed at the PM, and not at endosomal membranes.

  17. Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner (United States)

    Bejarano, Eloy; Girao, Henrique; Yuste, Andrea; Patel, Bindi; Marques, Carla; Spray, David C.; Pereira, Paulo; Cuervo, Ana Maria


    Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins. PMID:22496425

  18. Identification of chitosan oligosaccharides binding proteins from the plasma membrane of wheat leaf cell. (United States)

    Liu, Dongdong; Jiao, Siming; Cheng, Gong; Li, Xueming; Pei, Zhichao; Pei, Yuxin; Yin, Heng; Du, Yuguang


    Chitosan oligosaccharides (COS) have the ability to improve plant resistance to pests and diseases through activating plant immune system. However, it remains unclear whether stimulating reason of COS was associated with the plasma membrane proteins. Here, the interaction of COS with wheat leaf cell demonstrated that fluorescence-labeled COS were enriched on the cell surface and the interaction of COS with plasma membrane proteins was confirmed by quartz crystal microbalance (QCM) biosensor. What's more, HPLC and SDS-PAGE analysis showed that COS binding proteins exhibited more than three peaks and the molecular weight were 66 kDa to 97 kDa, where the COS binding proteins were fished out from wheat plasma membrane proteins by the COS affinity column. More importantly, LC-MS/MS analysis demonstrated that several candidates, including W5G2U8_WHEAT (a potential wall-associated receptor kinase protein), W5HY42_WHEAT and W5I0R4_WHEAT (potential G-type lectin S-receptor-like serine/threonine-protein kinase), have the potential to be COS receptors. Copyright © 2018. Published by Elsevier B.V.

  19. ACA12 Is a Deregulated Isoform of Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana (United States)

    Limonta, Margherita; Romanowsky, Shawn; Olivari, Claudio; Bonza, Maria Cristina; Luoni, Laura; Rosenberg, Alexa; Harper, Jeffrey F.; De Michelis, Maria Ida


    Plant auto-inhibited Ca2+-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca2+-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues - highly conserved in other ACA isoforms - localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation. PMID:24101142

  20. A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization. (United States)

    Schiøtt, Morten; Romanowsky, Shawn M; Baekgaard, Lone; Jakobsen, Mia Kyed; Palmgren, Michael G; Harper, Jeffrey F


    Ca(2+) signals are thought to play important roles in plant growth and development, including key aspects of pollen tube growth and fertilization. The dynamics of a Ca(2+) signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). The Arabidopsis genome encodes 14 Ca(2+) pumps, 10 of which belong to a family of autoinhibited Ca(2+) ATPases (ACA) that are predicted to be activated by Ca(2+)/calmodulin. Here, we show that isoform ACA9 is expressed primarily in pollen and localized to the plasma membrane. Three independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] gene disruptions of ACA9 were found to result in partial male sterility. Complementation was observed by using a ACA9-yellow fluorescence protein (YFP) fusion that displayed plasma membrane localization. Mutant aca9 pollen displayed a reduced growth potential and a high frequency of aborted fertilization, resulting in a >80% reduction in seed set. These findings identify a plasma membrane Ca(2+) transporter as a key regulator of pollen development and fertilization in flowering plants.

  1. Alteration of plasma membrane-bound redox systems of iron deficient pea roots by chitosan. (United States)

    Meisrimler, Claudia-Nicole; Planchon, Sebastien; Renaut, Jenny; Sergeant, Kjell; Lüthje, Sabine


    Iron is essential for all living organisms and plays a crucial role in pathogenicity. This study presents the first proteome analysis of plasma membranes isolated from pea roots. Protein profiles of four different samples (+Fe, +Fe/Chitosan, -Fe, and -Fe/Chitosan) were compared by native IEF-PAGE combined with in-gel activity stains and DIGE. Using DIGE, 89 proteins of interest were detected in plasma membrane fractions. Data revealed a differential abundance of several spots in all samples investigated. In comparison to the control and -FeCh the abundance of six protein spots increased whereas 56 spots decreased in +FeCh. Altered protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. Besides stress-related proteins, transport proteins and redox enzymes were identified. Activity stains after native PAGE and spectrophotometric measurements demonstrated induction of a ferric-chelate reductase (-Fe) and a putative respiratory burst oxidase homolog (-FeCh). However, the activity of the ferric-chelate reductase decreased in -Fe plants after elicitor treatment. The activity of plasma membrane-bound class III peroxidases increased after elicitor treatment and decreased under iron-deficiency, whereas activity of quinone reductases decreased mostly after elicitor treatment. Possible functions of proteins identified and reasons for a weakened pathogen response of iron-deficient plants were discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals. (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi


    The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.

  3. [Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na+, K+-ATPase]. (United States)

    Danylovych, H V; Danylovych, Iu V; Kolomiiets', O V; Kosterin, S O; Rodik, R V; Cherenok, S O; Kal'chenko, V I; Chunikhin, O Iu; Horchev, V F; Karakhim, S O


    The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

  4. Macroscopic domain formation during cooling in the platelet plasma membrane: an issue of low cholesterol content. (United States)

    Bali, Rachna; Savino, Laura; Ramirez, Diego A; Tsvetkova, Nelly M; Bagatolli, Luis; Tablin, Fern; Crowe, John H; Leidy, Chad


    There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large domains. In contrast, some polarizable cells do show large regions with qualitative differences in lipid fluidity. It is important to ask more precisely, based on the current phase diagrams, under what conditions would large domains be expected to form in cells. In this work we study the thermotropic phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents inhomogeneous distribution of DiI18:0 at 24 degrees C, but not at 37 degrees C, which suggests the formation of macroscopic lipid domains at low temperatures. We show by fluorescence microscopy, and by comparison with published phase diagrams, that the outer leaflet model system enters the macroscopic domain region only at the lower temperature. In addition, the low cholesterol content in platelets ( approximately 15 mol%), appears to be crucial for the formation of large domains during cooling.

  5. One-step extraction of polar drugs from plasma by parallel artificial liquid membrane extraction. (United States)

    Pilařová, Veronika; Sultani, Mumtaz; Ask, Kristine Skoglund; Nováková, Lucie; Pedersen-Bjergaard, Stig; Gjelstad, Astrid


    The new microextraction technique named parallel artificial liquid membrane extraction (PALME) was introduced as an alternative approach to liquid-liquid extraction of charged analytes from aqueous samples. The concept is based on extraction of analytes across a supported liquid membrane sustained in the pores of a thin polymeric membrane, a well-known extraction principle also used in hollow fiber liquid-phase microextraction (HF-LPME). However, the new PALME technique offers a more user-friendly setup in which the supported liquid membrane is incorporated in a 96 well plate system. Thus, high-throughput is achievable, in addition to the green chemistry offered by using PALME. The consumption of organic solvent is minimized to 3-5μL per sample. With a sample volume of 250μL and acceptor solution volume of 50μL, a maximal enrichment factor of five is achievable. Based on these parameters, a new method for extraction of polar basic drugs was developed in the present work. The basic drugs hydralazine, ephedrine, metaraminol, salbutamol, and cimetidine were used as model analytes, and were extracted from alkalized human plasma into an aqueous solution via the supported liquid membrane. The extraction was promoted by a carrier dissolved in the membrane, creating a temporary ion-pair complex between the hydrophilic drug and the carrier. As the model analytes were extracted directly into an aqueous solution, there was no need for evaporation of the extract before injection into LC-MS. Hence, the sample preparation is performed in one step. With optimized conditions, the extraction recoveries were in the range 50-89% from human plasma after 45min extraction. The data from the method evaluation were satisfactory and in line with current guidelines, and revealed an extraction method with substantial potential for high throughput bioanalysis of polar basic drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

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


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

  7. Easy measurement of diffusion coefficients of EGFP-tagged plasma membrane proteins using k-space Image Correlation Spectroscopy

    DEFF Research Database (Denmark)

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


    Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1 was developed to enable...... routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely...... to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS. First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope Then...

  8. Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

    DEFF Research Database (Denmark)

    Hartwig Petersen, Nicole; Færgeman, Nils J; Yu, Liqing


    fluorescent protein (NPC1L1-EGFP) and cholesterol analogues in hepatoma cells. At steady state about 42% of NPC1L1 resided in the transferrin (Tf) positive, sterol enriched endocytic recycling compartment (ERC), while time-lapse microscopy demonstrated NPC1L1 traffic between plasma membrane and ERC...... the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells NPC1L1 resided almost...... exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1 mediated cellular sterol uptake....

  9. Solute removal capacity of high cut-off membrane plasma separators. (United States)

    Ohkubo, Atsushi; Kurashima, Naoki; Nakamura, Ayako; Miyamoto, Satoko; Iimori, Soichiro; Rai, Tatemitsu


    In vitro blood filtration was performed by a closed circuit using high cut-off membrane plasma separators, EVACURE EC-2A10 (EC-2A) and EVACURE EC-4A10 (EC-4A). Samples were obtained from sampling sites before the plasma separator, after each plasma separator, and from the ultrafiltrate of each separator. The sieving coefficient (S.C.) of total protein (TP), albumin (Alb), IgG, interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), fibrinogen (Fib), antithrombin III (AT-III), and coagulation factor XIII (FXIII) were calculated. The S.C. of each solute using EC-2A and EC-A4 were as follows; TP: 0.25 and 0.56, Alb: 0.32 and 0.73, IgG: 0.16 and 0.50, IL-6:0.73 and 0.95, IL-8:0.85 and 0.82, TNF-α: 1.07 and 0.99, Fib: 0 and 0, FXIII: 0.07 and 0.17, respectively. When compared with the conventional type of membrane plasma separators, EVACURE could efficiently remove cytokines while retaining coagulation factors such as fibrinogen. Moreover, EC-2A prevented protein loss, whereas EC-4A could remove approximately 50% of IgG. © 2013 The Authors. Therapeutic Apheresis and Dialysis © 2013 International Society for Apheresis.

  10. Reduction in lateral lipid mobility of lipid bilayer membrane by atmospheric pressure plasma irradiation (United States)

    Suda, Yoshiyuki; Tero, Ryugo; Yamashita, Ryuma; Yusa, Kota; Takikawa, Hirofumi


    Plasma medicine is an emerging research field in which various applications of electrical discharge, especially in the form of nonequilibrium plasma at atmospheric pressure, are examined, for example, the application of plasma to biological targets for various purposes such as selective killing of tumor cells and blood stanching. We have focused on the behavior of an artificial cell membrane system at the solid-liquid interface. To evaluate the lateral lipid mobility, we measured the diffusion coefficient of the supported lipid bilayer (SLB) composed of dioleoylphosphatidylcholine with fluorescence recovery after photobleaching by confocal laser scanning microscopy. It was found that the diffusion coefficient was decreased by plasma irradiation and that the diffusion coefficient decreasing rate proceeded with increasing plasma power. We investigated the effects of stimulation with an equilibrium chemical, H2O2, on the SLB and confirmed that the diffusion coefficient did not change at least up to a H2O2 concentration of 5 mM. These results indicate that transient active species generated by plasma play critical roles in the reduction in SLB fluidity. The effects of the two generated major oxidized lipid species, hydroxyl- or hydroperoxy-phosphatidylcholine (PC) and acyl-chain-truncated PCs terminated with aldehyde or carboxyl group, on lateral lipid mobility are discussed.

  11. Large basolateral processes on type II hair cells comprise a novel processing unit in mammalian vestibular organs (United States)

    Pujol, Rémy; Pickett, Sarah B.; Nguyen, Tot Bui; Stone, Jennifer S.


    Sensory receptors in the vestibular system (hair cells) encode head movements and drive central motor reflexes that control gaze, body movements, and body orientation. In mammals, type I and II vestibular hair cells are defined by their shape, contacts with vestibular afferent nerves, and membrane conductance. Here, we describe unique morphological features of type II vestibular hair cells in mature rodents (mice and gerbils) and bats. These features are cytoplasmic processes that extend laterally from the hair cell’s base and project under type I hair cells. Closer analysis of adult mouse utricles demonstrated that the basolateral processes of type II hair cells range in shape, size, and branching, with the longest processes extending 3–4 hair cell widths. The hair cell basolateral processes synapse upon vestibular afferent nerves and receive inputs from vestibular efferent nerves. Further, some basolateral processes make physical contacts with the processes of other type II hair cells, forming some sort of network amongst type II hair cells. Basolateral processes are rare in perinatal mice and do not attain their mature form until 3–6 weeks of age. These observations demonstrate that basolateral processes are significant signaling regions of type II vestibular hair cells, and they suggest type II hair cells may directly communicate with each other, which has not been described in vertebrates. PMID:24825750

  12. The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner

    DEFF Research Database (Denmark)

    Bojsen, Rasmus; Torbensen, Rasmus; Larsen, Camilla Eggert


    that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma...... observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids....

  13. Plasma membrane proteome analysis identifies a role of barley Membrane Steroid Binding Protein in root architecture response to salinity. (United States)

    Witzel, Katja; Matros, Andrea; Møller, Anders L B; Ramireddy, Eswarrayya; Finnie, Christine; Peukert, Manuela; Rutten, Twan; Herzog, Andreas; Kunze, Gotthard; Melzer, Michael; Kaspar-Schoenefeld, Stephanie; Schmülling, Thomas; Svensson, Birte; Mock, Hans-Peter


    Although physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane (PM) proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 PM proteins were identified by mass spectrometry, of which 182 were either cultivar- or salinity stress-responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol-binding (a GTPase-activating protein for the ADP ribosylation factor, ZIGA2, and a membrane steroid binding protein, MSBP) or in phospholipid synthesis (phosphoethanolamine methyltransferase, PEAMT). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, while knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity and root tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity. This article is protected by copyright. All rights reserved.

  14. Two stage binding of glucagon to receptors in rat liver plasma membranes

    International Nuclear Information System (INIS)

    Wyborski, R.J.; Horwitz, E.M.; Gurd, R.S.


    A homogeneous class of noncooperative receptors in isolated rat hepatocytes undergoes a time- and temperature-dependent conformation change with glucagon binding. A comparable system exists in rat liver plasma membranes. Dissociation assays (30 0 C) quantify the number of receptors in each conformational state. Membranes incubated without GTP demonstrated two dissociation rates. The fraction of hormone bound to the high affinity state increases with incubation time to a limiting value. With isolated membranes and a concentration of 0.2 nM [( 125 I)Iodotyrosyl 10 ]glucagon, the fraction of the high affinity form is significantly greater than that found in isolated hepatocytes. Previous work without GTP indicated that a lack of cooperativity characterized the liver membrane system. Incubation of membranes with 0.1 mM GTP increases the K/sub D/ as determined by competition assays while the slope factor (.98 +/- 0.04) indicated noncooperativity. Furthermore, in the presence of GTP a significantly greater proportion of receptors is in the low affinity state while in the absence of GTP more are in the high affinity state. The data are consistent with a mechanism by which GTP diminishes the conversion of the low affinity state to the high affinity state

  15. Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings. (United States)

    de Michele, Roberto; McFarlane, Heather E; Parsons, Harriet T; Meents, Miranda J; Lao, Jeemeng; González Fernández-Niño, Susana M; Petzold, Christopher J; Frommer, Wolf B; Samuels, A Lacey; Heazlewood, Joshua L


    The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.

  16. Dehydroepiandrosterone inhibits intracellular calcium release in beta-cells by a plasma membrane-dependent mechanism. (United States)

    Liu, Dongmin; Ren, Min; Bing, Xinyu; Stotts, Corey; Deorah, Sundeep; Love-Homan, Laurie; Dillon, Joseph S


    Both dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) affect glucose stimulated insulin secretion, though their cellular mechanisms of action are not well characterized. We tested the hypothesis that human physiological concentrations of DHEA alter insulin secretion by an action initiated at the plasma membrane of beta-cells. DHEA alone had no effect on intracellular calcium concentration ([Ca(2+)](i)) in a rat beta-cell line (INS-1). However, it caused an immediate and dose-dependent inhibition of carbachol-induced Ca(2+) release from intracellular stores, with a 25% inhibition at zero. One nanometer DHEA. DHEA also inhibited the Ca(2+) mobilizing effect of bombesin (29% decrease), but did not inhibit the influx of extracellular Ca(2+) evoked by glyburide (100 microM) or glucose (15 mM). The steroids (androstenedione, 17-alpha-hydroxypregnenolone, and DHEAS) had no inhibitory effect on carbachol-induced intracellular Ca(2+) release. The action of DHEA depended on a signal initiated at the plasma membrane, since membrane impermeant DHEA-BSA complexes also inhibited the carbachol effect on [Ca(2+)](i) (39% decrease). The inhibition of carbachol-induced Ca(2+) release by DHEA was blocked by pertussis toxin (PTX). DHEA also inhibited the carbachol induction of phosphoinositide generation, with a maximal inhibition at 0.1 nM DHEA. Furthermore, DHEA inhibited insulin secretion induced by carbachol in INS-1 cells by 25%, and in human pancreatic islets by 53%. Taken together, this is the first report showing that human physiological concentrations of DHEA decrease agonist-induced Ca(2+) release by a rapid, non-genomic mechanism in INS-1 cells. Furthermore, these data provide evidence consistent with the existence of a specific plasma membrane DHEA receptor, mediating this signal transduction pathway by pertussis toxin-sensitive G-proteins.

  17. Practical techniques for bovine sperm simultaneous fluorimetric assessment of plasma, acrosomal and mitochondrial membranes. (United States)

    Celeghini, E C C; de Arruda, R P; de Andrade, A F C; Nascimento, J; Raphael, C F


    This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and rhodamine 123; protocol 2: PI, FITC-PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC-PSA and CMXRos; and protocol 4: PI, H342, FITC-PSA and JC-1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility >/=80% and abnormal morphology plasma membrane integrity (R(2) = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R(2) = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R(2) = 0.84, 0.93 and R(2) = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC-1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential.

  18. Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells (United States)

    Morre, D. M.; Morre, D. J.


    Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum Salt concentrations and temperature affect partitioning behavior and must be precisely standardized. In some cases, it is more fortuitous to combine aqueous two-phase partition with other procedures to obtain a more highly purified preparation. A procedure is described for preparation of Golgi apparatus from transformed mammalian cells that combines aqueous two-phase partition and centrifugation. Also described is a periodic NADH oxidase, a new enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions for measurement of activity.

  19. The C-terminal hypervariable domain targets Aradopsis ROP9 to the invaginated pollen tube plasma membrane (United States)

    Rop9 is a small GTPase of the Type II class, whereas the often studied type I Rops play roles during pollen tube growth. In pollen, Rop9 is located at the invaginated plasma membrane that surrounds the sperm cells, whereas type I Rops are located at the apical membrane of the pollen tube. The C-ter...

  20. 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


    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...

  1. Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells. (United States)

    Andoh, Yoshimichi; Okazaki, Susumu; Ueoka, Ryuichi


    Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes. Copyright © 2013

  2. High-protein-PUFA supplementation, red blood cell membranes, and plasma antioxidant activity in volleyball athletes. (United States)

    Malaguti, Marco; Baldini, Marta; Angeloni, Cristina; Biagi, Pierluigi; Hrelia, Silvana


    The authors evaluated the role of a high-protein, low-calorie, polyunsaturated fatty-acid (PUFA) -supplemented diet on anthropometric parameters, erythrocyte-membrane fatty-acid composition, and plasma antioxidant defenses of nonprofessional volleyball athletes. The athletes were divided in two groups: One (n = 5) followed the Mediterranean diet, and the other (n = 6) followed a high-protein, low-calorie diet with a 3-g/day fish-oil supplementation. All the athletes had anthropometric measurements taken, both at the beginning and at the end of the study, which lasted for 2 months. Body-mass index and total body fat were significantly diminished in the second group, while they remained unchanged in the first. Plasma total antioxidant activity (TAA) was significantly increased in the plasma of both groups, with no differences between the groups, suggesting that physical activity, not the different diets, is the main contributor to the increase of plasma TAA. The second group showed a significant increase in erythrocyte-membrane PUFA content and in the unsaturation index value (UI) because of the fish-oil supplementation.A high-protein, low-carbohydrate, fish-oil-supplemented diet seems to be useful only when the aim of the diet is to obtain weight loss in a short-term period. The significant increase in the UI of erythrocyte membranes indicates the potential for harm, because a high intake of PUFA might increase susceptibility to lipid peroxidation not counterbalanced by a higher increase in TAA. Adherence to the Mediterranean diet seems to be the better choice.

  3. In Vitro Dialysis of Cytokine-Rich Plasma With High and Medium Cut-Off Membranes Reduces Its Procalcific Activity. (United States)

    Willy, Kevin; Hulko, Michael; Storr, Markus; Speidel, Rose; Gauss, Julia; Schindler, Ralf; Zickler, Daniel


    Recently developed high-flux (HF) dialysis membranes with extended permeability provide better clearance of middle-sized molecules such as interleukins (ILs). Whether this modulation of inflammation influences the procalcific effects of septic plasma on vascular smooth muscle cells (VSMCs) is not known. To assess the effects of high cut-off (HCO) and medium cut-off (MCO) membranes on microinflammation and in vitro vascular calcification we developed a miniature dialysis model. Plasma samples from lipopolysaccharide-spiked blood were dialyzed with HF, HCO, and MCO membranes in an in vitro miniature dialysis model. Afterwards, IL-6 concentrations were determined in dialysate and plasma. Calcifying VSMCs were incubated with dialyzed plasma samples and vascular calcification was assessed. Osteopontin (OPN) and matrix Gla protein (MGP) were measured in VSMC supernatants. IL-6 plasma concentrations were markedly lower with HCO and MCO dialysis. VSMC calcification was significantly lower after incubation with MCO- and HCO-serum compared to HF plasma. MGP and OPN levels in supernatants were significantly lower in the MCO but not in the HCO group compared to HF. In vitro dialysis of cytokine-enriched plasma samples with MCO and HCO membranes reduces IL-6 levels. The induction of vascular calcification by cytokine-enriched plasma is reduced after HCO and MCO dialysis. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

  4. Electron Pathways through Erythrocyte Plasma Membrane in Human Physiology and Pathology: Potential Redox Biomarker?

    Directory of Open Access Journals (Sweden)

    Elena Matteucci


    Full Text Available Erythrocytes are involved in the transport of oxygen and carbon dioxide in the body. Since pH is the influential factor in the Bohr-Haldane effect, pHi is actively maintained via secondary active transports Na+/H+ exchange and HC3 -/Cl- anion exchanger. Because of the redox properties of the iron, hemoglobin generates reactive oxygen species and thus, the human erythrocyte is constantly exposed to oxidative damage. Although the adult erythrocyte lacks protein synthesis and cannot restore damaged proteins, it is equipped with high activity of protective enzymes. Redox changes in the cell initiate various signalling pathways. Plasma membrane oxido-reductases (PMORs are transmembrane electron transport systems that have been found in the membranes of all cells and have been extensively characterized in the human erythrocyte. Erythrocyte PMORs transfer reducing equivalents from intracellular reductants to extracellular oxidants, thus their most important role seems to be to enable the cell respond to changes in intra- and extra-cellular redox environments.So far the activity of erythrocyte PMORs in disease states has not been systematically investigated. This review summarizes present knowledge on erythrocyte electron transfer activity in humans (health, type 1 diabetes, diabetic nephropathy, and chronic uremia and hypothesizes an integrated model of the functional organization of erythrocyte plasma membrane where electron pathways work in parallel with transport metabolons to maintain redox homeostasis.

  5. Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

    International Nuclear Information System (INIS)

    Ebner, Andreas; Hinterdorfer, Peter; Nikova, Dessy; Lange, Tobias; Bruns, Reimer; Oberleithner, Hans; Schillers, Hermann; Haeberle, Johannes; Falk, Sabine; Duebbers, Angelika


    CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl - ) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients

  6. Enquiry into the Topology of Plasma Membrane-Localized PIN Auxin Transport Components. (United States)

    Nodzyński, Tomasz; Vanneste, Steffen; Zwiewka, Marta; Pernisová, Markéta; Hejátko, Jan; Friml, Jiří


    Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Insulin stimulates the tyrosine phosphorylation of a Mr = 160,000 glycoprotein in adipocyte plasma membranes

    International Nuclear Information System (INIS)

    Yu, K.T.; Khalaf, N.; Czech, M.P.


    In an attempt to identify putative substrates for the insulin receptor kinase, adipocyte plasma membranes were incubated with [γ- 32 P]ATP in the presence and absence of insulin. Insulin stimulates the tyrosine phosphorylation of its receptor β subunit but does not detectably alter the phosphorylation of other membrane proteins. In contrast, when plasma membranes from insulin-treated adipocytes are phosphorylated, the 32 P-labeling of a Mr=160,000 species (p160) and insulin receptor β subunit are markedly increased when compared to controls. p160 exhibits a rapid response (max. at 1 min) and high sensitivity (ED 50 = 2 x 10 -10 M) to insulin. The stimulatory effect of insulin on the phosphorylation of p160 is rapidly reversed following the addition of anti-insulin serum. Cold chase experiments indicate that insulin promotes the phosphorylation of p160 rather than inhibiting its dephosphorylation. p160 is a glycoprotein as evidenced by its adsorption to immobilized lectins and does not represent the insulin receptor precursor. The action of insulin on p160 tyrosine phosphorylation is mimicked by concanavalin A but not by EGF and other insulin-like agents such as hydrogen peroxide and vanadate. These results suggest that p160 tyrosine phosphorylation is an insulin receptor-mediated event and may participate in signalling by the insulin receptor

  8. Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls (United States)

    Spalding, E. P.; Cosgrove, D. J.


    A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H(+)-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca(2+)-channel blockers (verapamil, La3+) or by reducing extracellular free Ca2+ by treatment with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca2+ is involved. No effects of the K(+)-channel blocker tetraethylammonium (TEA+) were detected. Vanadate and KCN, used to inhibit the H(+)-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11-26% after 1-2 min of BL. Input resistance of trichrome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H(+)-ATPase with subsequent transient activation of one or more types of ion channels.

  9. DC-SIGN and Influenza Hemagglutinin Dynamics in Plasma Membrane Microdomains Are Markedly Different (United States)

    Itano, Michelle S.; Neumann, Aaron K.; Liu, Ping; Zhang, Feng; Gratton, Enrico; Parak, Wolfgang J.; Thompson, Nancy L.; Jacobson, Ken


    DC-SIGN, a Ca2+-dependent transmembrane lectin, is found assembled in microdomains on the plasma membranes of dendritic cells. These microdomains bind a large variety of pathogens and facilitate their uptake for subsequent antigen presentation. In this study, DC-SIGN dynamics in microdomains were explored with several fluorescence microscopy methods and compared with dynamics for influenza hemagglutinin (HA), which is also found in plasma membrane microdomains. Fluorescence imaging indicated that DC-SIGN microdomains may contain other C-type lectins and that the DC-SIGN cytoplasmic region is not required for microdomain formation. Fluorescence recovery after photobleaching measurements showed that neither full-length nor cytoplasmically truncated DC-SIGN in microdomains appreciably exchanged with like molecules in other microdomains and the membrane surround, whereas HA in microdomains exchanged almost completely. Line-scan fluorescence correlation spectroscopy indicated an essentially undetectable lateral mobility for DC-SIGN but an appreciable mobility for HA within their respective domains. Single-particle tracking with defined-valency quantum dots confirmed that HA has significant mobility within microdomains, whereas DC-SIGN does not. By contrast, fluorescence recovery after photobleaching indicated that inner leaflet lipids are able to move through DC-SIGN microdomains. The surprising stability of DC-SIGN microdomains may reflect structural features that enhance pathogen uptake either by providing high-avidity platforms and/or by protecting against rapid microdomain endocytosis. PMID:21641311

  10. ABCA1, ABCG1, and ABCG4 are distributed to distinct membrane meso-domains and disturb detergent-resistant domains on the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Osamu Sano

    Full Text Available ATP-binding cassette A1 (ABCA1, ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.

  11. Influence of phospholipid environment on the phosphatidylethanolamine: ceramide-phosphorylethanolamine transferase activity in rat liver plasma membranes. (United States)

    Nikolova, M N; Petkova, D H; Koumanov, K S


    1. Plasma membranes were treated with phospholipase A2, phospholipase C or phospholipase D. The phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was deactivated by phospholipase C treatment, whereas phospholipase A2 and phospholipase D did not affect the enzyme. 2. Incorporation of phosphatidylethanolamine and phosphatidylglycerol into partially delipidated plasma membranes resulted in significant stimulation of the transferase, whereas inclusion of sphingomyelin and phosphatidylserine suppressed the enzyme activity. Our results suggest that phosphatidylserine is a regulator of sphingomyelin level in membranes. 3. The activity of phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was not influenced by the fluidity of its lipid environment.

  12. Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages. (United States)

    Vega, Virginia L; Rodríguez-Silva, Monica; Frey, Tiffany; Gehrmann, Mathias; Diaz, Juan Carlos; Steinem, Claudia; Multhoff, Gabriele; Arispe, Nelson; De Maio, Antonio


    Heat shock proteins (hsps) are intracellular chaperones that play a key role in the recovery from stress. Hsp70, the major stress-induced hsp, has been found in the extracellular medium and is capable of activating immune cells. The mechanism involved in Hsp70 release is controversial because this protein does not present a consensual secretory signal. In this study, we have shown that Hsp70 integrates into artificial lipid bilayer openings of ion conductance pathways. In addition, this protein was found inserted into the plasma membrane of cells after stress. Hsp70 was released into the extracellular environment in a membrane-associated form, sharing the characteristics of this protein in the plasma membrane. Extracellular membranes containing Hsp70 were at least 260-fold more effective than free recombinant protein in inducing TNF-alpha production as an indicator of macrophage activation. These observations suggest that Hsp70 translocates into the plasma membrane after stress and is released within membranous structures from intact cells, which could act as a danger signal to activate the immune system.

  13. Cholesterol Oxidase/Triton X-100 Parked Microelectrodes for the Detection of Cholesterol in Plasma Membrane at Single Cells. (United States)

    Xu, Haiyan; Zhou, Shuai; Jiang, Dechen; Chen, Hong-Yuan


    The classic electrochemical analysis of plasma membrane cholesterol at single cells utilizes a cholesterol oxidase modified microelectrode that oxidizes local cholesterol efflux from the plasma membrane to generate hydrogen peroxide for the electrochemical quantification. In this letter, a mixture of cholesterol oxidase and Triton X-100 was filled in the microcapillary that could park at the Pt layer coated tip due to slow hydrodynamic flow. During the contact of the tip with the cellular membrane, Triton X-100 at the tip permeabilized the contacted membrane to release cholesterol for the reaction with cholesterol oxidase. As compared with the linkage of cholesterol oxidase at the electrode surface, the oxidase parked in aqueous solution at the tip had a higher turnover rate resulting in larger electrochemical signal for single cell analysis. More charge collected at acyl-coA:cholesterol acyltransferase (ACAT) inhibited cells supported that this novel detection strategy could monitor the flunctation of membrane cholesterol at single cells. The successful detection of plasma membrane cholesterol at single cells using the oxidase parked microelectrode will provide a special strategy for the fabrication of biosensor that permits the integration of more molecules without functional groups at the electrode to measure active and inactive molecules in the plasma membrane. Moreover, the larger electrochemical signals collected could further increase the spatial resolution for single cell electrochemical analysis.

  14. Hemocompatible control of sulfobetaine-grafted polypropylene fibrous membranes in human whole blood via plasma-induced surface zwitterionization. (United States)

    Chen, Sheng-Han; Chang, Yung; Lee, Kueir-Rarn; Wei, Ta-Chin; Higuchi, Akon; Ho, Feng-Ming; Tsou, Chia-Chun; Ho, Hsin-Tsung; Lai, Juin-Yih


    In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.

  15. The effect of propofol on plasma membrane ultrastructure in the intact cells (United States)

    Jin, Weixiang; Pralle, Arnd

    The mechanism of general anesthesia is still unknown. One drug used for human anesthesia, propofol, has been shown to interact with some ligand gated ion-channels, but also easily dissolves in the lipid bilayer and alters fluidity. Which mechanism dominates or even how anesthesia arises are unclear. We study the influence of propofol on plasma membrane (PM) ultrastructure in intact cells. In the PM, transient submicroscopic nanodomains form by interactions between lipid-acyl-chains or lipid head groups, stabilized by cholesterol. In addition, membrane cytoskeleton further regulates the nanodomains, which then regulate signaling. We study transient propofol effects on these domains from low to clinically relevant propofol concentrations by analyzing diffusion of GFP-tagged outer leaflet/inner leaflet membrane proteins. Using bimFCS we measure diffusion on multiple length scales simultaneously. We observe that at low propofol concentrations, the nanodomains trap GPI-mGFP less, consistent with studies showing that propofol decreases the phase transition temperature of membrane derived vesicles. Interestingly, at clinical relevant concentrations of propofol, the nanodomains trap GPI-mGFP more strongly. This is only observed at 37C. By inhibiting myosin activity or actin filaments (de-)polymerization, we find that the activity of actin filaments further alters the behavior of cholesterol nanodomains due to propofol. We compare the effect of propofol and its analog confirming specificity.

  16. Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells (United States)

    Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F.


    Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 {\\mu}s. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

  17. Intact transmembrane isoforms of the neural cell adhesion molecule are released from the plasma membrane

    DEFF Research Database (Denmark)

    Olsen, M; Krog, L; Edvardsen, K


    . By density-gradient centrifugation it was shown that shed transmembrane NCAM-B was present in fractions of high, as well as low, density, indicating that a fraction of the shed NCAM is associated with minor plasma membrane fragments. Finally, it was shown that isolated soluble NCAM inhibited cell binding......-s1 and NCAM-s2 and the function of soluble NCAM forms were investigated. It was shown that all three soluble forms could be released from brain membranes with M(r) values identical to the three major membrane-associated forms: the large transmembrane 190,000-M(r) form (NCAM-A), the smaller...... intact soluble form from membranes of cells transfected with this isoform. Thus, NCAM-s1 and NCAM-s2 probably represent intact released transmembrane NCAM-A and NCAM-B. The soluble transmembrane forms are likely to exist in vivo, as NCAM-s1 and NCAM-s2 were readily demonstrated in cerebrospinal fluid...

  18. One-step extraction of polar drugs from plasma by Parallel Artificial Liquid Membrane Extraction

    DEFF Research Database (Denmark)

    Pilařová, Veronika; Sultani, Mumtaz; Ask, Kristine Skoglund


    -throughput is achievable, in addition to the green chemistry offered by using PALME. The consumption of organic solvent is minimized to 3-5μL per sample. With a sample volume of 250μL and acceptor solution volume of 50μL, a maximal enrichment factor of five is achievable. Based on these parameters, a new method...... for extraction of polar basic drugs was developed in the present work. The basic drugs hydralazine, ephedrine, metaraminol, salbutamol, and cimetidine were used as model analytes, and were extracted from alkalized human plasma into an aqueous solution via the supported liquid membrane. The extraction...... was promoted by a carrier dissolved in the membrane, creating a temporary ion-pair complex between the hydrophilic drug and the carrier. As the model analytes were extracted directly into an aqueous solution, there was no need for evaporation of the extract before injection into LC-MS. Hence, the sample...

  19. Visualization of phosphatidic acid fluctuations in the plasma membrane of living cells.

    Directory of Open Access Journals (Sweden)

    José P Ferraz-Nogueira

    Full Text Available We developed genetically-encoded fluorescent sensors based on Förster Resonance Energy Transfer to monitor phosphatidic acid (PA fluctuations in the plasma membrane using Spo20 as PA-binding motif. Basal PA levels and phospholipase D activity varied in different cell types. In addition, stimuli that activate PA phosphatases, leading to lower PA levels, increased lamellipodia and filopodia formation. Lower PA levels were observed in the leading edge than in the trailing edge of migrating HeLa cells. In MSC80 and OLN93 cells, which are stable cell lines derived from Schwann cells and oligodendrocytes, respectively, a higher ratio of diacylglycerol to PA levels was demonstrated in the membrane processes involved in myelination, compared to the cell body. We propose that the PA sensors reported here are valuable tools to unveil the role of PA in a variety of intracellular signaling pathways.

  20. Plasma membrane protein trafficking in plant-microbe interactions: a plant cell point of view

    Directory of Open Access Journals (Sweden)

    Nathalie eLeborgne-Castel


    Full Text Available In order to ensure their physiological and cellular functions, plasma membrane (PM proteins must be properly conveyed from their site of synthesis, i.e. the endoplasmic reticulum, to their final destination, the PM, through the secretory pathway. PM protein homeostasis also relies on recycling and/or degradation, two processes that are initiated by endocytosis. Vesicular membrane trafficking events to and from the PM have been shown to be altered when plant cells are exposed to mutualistic or pathogenic microbes. In this review, we will describe the fine-tune regulation of such alterations, and their consequence in PM protein activity. We will consider the formation of intracellular perimicrobial compartments, the PM protein trafficking machinery of the host, and the delivery or retrieval of signaling and transport proteins such as pattern-recognition receptors, producers of reactive oxygen species, and sugar transporters.

  1. Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Vancini, Ricardo [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States); Kramer, Laura D. [Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, NY (United States); Ribeiro, Mariana; Hernandez, Raquel [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States); Brown, Dennis, E-mail: [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States)


    Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.

  2. Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane

    International Nuclear Information System (INIS)

    Vancini, Ricardo; Kramer, Laura D.; Ribeiro, Mariana; Hernandez, Raquel; Brown, Dennis


    Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.

  3. In vitro and in vivo phosphorylation of polypeptides in plasma membrane and tonoplast-enriched fractions from barley roots

    International Nuclear Information System (INIS)

    Garbarino, J.E.; Hurkman, W.J.; Tanaka, C.K.; DuPont, F.M.


    Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with γ[p 32 P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg 2+ was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46 and 28 kilodaltons required millimolar Mg 2+ concentrations and was greatly enhanced by Ca 2+ . When roots of intact plants were labeled with [ 32 P]orthophosphate, polypeptides at approximately 135, 166, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mM NaCl had no effect

  4. 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


    proteins are major targets of the signalling cascades, we developed a protocol to monitor their phosphorylation state starting from a single mouse cerebellum. An aqueous polymer two-phase system was used to enrich for plasma membrane proteins. Subsequently, calcium phosphate precipitation, immobilized...... metal affinity chromatography, and TiO2 were combined to a sequential extraction procedure prior to mass spectrometric analyses. This strategy resulted in the identification of 1501 different native phosphorylation sites in 507 different proteins. 765 (51%) of these phosphorylation sites were localized...

  5. Redox enzymes in the plant plasma membrane and their possible roles

    DEFF Research Database (Denmark)

    Berczi, A.; Møller, I.M.


    Purified plasma membrane (PM) vesicles from higher plants contain redox proteins with low-molecular-mass prosthetic groups such as flavins (both FMN and FAD), hemes, metals (Cu, Fe and Mn), thiol groups and possibly naphthoquinone (vitamin K-1), all of which are likely to participate in redox...... protein which has been partially purified from plant PM so far is a high-potential and ascorbate-reducible b-type cytochrome. In co-operation with vitamin K-1 and an NAD(P)H-quinone oxidoreductase, it may participate in trans-PM electron transport....

  6. Changes in plasma membrane state of thymocytes during spontaneous and radiation-induced leukemogenesis

    International Nuclear Information System (INIS)

    Gonta-Grabiec, K.


    Changes in plasma membrane properties characteristic for malignant cells were reviewed. Investigations of spontaneous (in AKR mice) and radiation-induced (in C57Bl) leukemogenesis were carried out; changes in properties of Na + , K + ATPase and alkaline phosphatase were characterized. On the basis of the results reported a pre-leukemic stage was distinguished, corresponding to the following features at the cellular level: increase in activity of alkaline phosphatase; decrease in relative activity of Na + , K + ATPase; decrease in efficiency of the Na + K + pump; decrease in cAMP content. 473 refs. (author)

  7. Advanced Fluorescence Microscopy Approaches to Understand the Dynamic Organization of the Plasma Membrane in Eukaryotes

    DEFF Research Database (Denmark)

    Ziomkiewicz, Iwona

    signaling in plants. Furthermore, it was established that ENODL9 clustering affects the organization of the PM and distribution of other PM proteins. Analysis of the phenotype of mutant lines revealed that ENODL9 has an important role for plant development and the adaptation to osmotic stress. This resulted......The plasma membrane (PM) is a physical barrier that defines the boundaries of a cell. It not only isolates the cell interior from the environment, but also enables cell communication and a selective exchange of solutes. To serve those contrasting functions, the PM has a dynamic structure consisting...

  8. Focal junctions retard lateral movement and disrupt fluid phase connectivity in the plasma membrane

    DEFF Research Database (Denmark)

    Vind-Kezunovic, D.; Wojewodzka, U.; Gniadecki, R.


    ,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C-18:0), which specifically partitions to the liquid-disordered (L-d), non-raft phase, was also enriched in focal junctions and its mobility was slightly retarded. Cross-linking of GM(1) by CTB or raft aggregation by methyl......-p-cyclodextrin further decreased the recovery of DiI-C-18:0. We propose a model in which focal junctions impose lateral heterogeneity in the plasma membrane by entrapment of lipid microdomains between dense arrays of immobilized transmembrane molecules which can enmesh otherwise freely percolating L-d phase lipids. (c...

  9. Effects of fiber density and plasma modification of nanofibrous membranes on the adhesion and growth of HaCaT keratinocytes. (United States)

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


    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:

  10. Adrenal Chromaffin Cells Exposed to 5-ns Pulses Require Higher Electric Fields to Porate Intracellular Membranes than the Plasma Membrane: An Experimental and Modeling Study. (United States)

    Zaklit, Josette; Craviso, Gale L; Leblanc, Normand; Yang, Lisha; Vernier, P Thomas; Chatterjee, Indira


    Nanosecond-duration electric pulses (NEPs) can permeabilize the endoplasmic reticulum (ER), causing release of Ca 2+ into the cytoplasm. This study used experimentation coupled with numerical modeling to understand the lack of Ca 2+ mobilization from Ca 2+ -storing organelles in catecholamine-secreting adrenal chromaffin cells exposed to 5-ns pulses. Fluorescence imaging determined a threshold electric (E) field of 8 MV/m for mobilizing intracellular Ca 2+ whereas whole-cell recordings of membrane conductance determined a threshold E-field of 3 MV/m for causing plasma membrane permeabilization. In contrast, a 2D numerical model of a chromaffin cell, which was constructed with internal structures representing a nucleus, mitochondrion, ER, and secretory granule, predicted that exposing the cell to the same 5-ns pulse electroporated the plasma and ER membranes at the same E-field amplitude, 3-4 MV/m. Agreement of the numerical simulations with the experimental results was obtained only when the ER interior conductivity was 30-fold lower than that of the cytoplasm and the ER membrane permittivity was twice that of the plasma membrane. A more realistic intracellular geometry for chromaffin cells in which structures representing multiple secretory granules and an ER showed slight differences in the thresholds necessary to porate the membranes of the secretory granules. We conclude that more sophisticated cell models together with knowledge of accurate dielectric properties are needed to understand the effects of NEPs on intracellular membranes in chromaffin cells, information that will be important for elucidating how NEPs porate organelle membranes in other cell types having a similarly complex cytoplasmic ultrastructure.

  11. Plasma Biomarkers of Brain Injury as Diagnostic Tools and Outcome Predictors After Extracorporeal Membrane Oxygenation. (United States)

    Bembea, Melania M; Rizkalla, Nicole; Freedy, James; Barasch, Noah; Vaidya, Dhananjay; Pronovost, Peter J; Everett, Allen D; Mueller, Gregory


    To determine if elevations in plasma brain injury biomarkers are associated with outcome at hospital discharge in children who require extracorporeal membrane oxygenation. Prospective observational study. Single tertiary-care academic center. Eighty children who underwent extracorporeal membrane oxygenation between June 2010 and December 2013. None. We measured six brain injury biomarkers (glial fibrillary acidic protein, monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2, neuron-specific enolase, S100b, intercellular adhesion molecule-5, and brain-derived neurotrophic factor) daily during extracorporeal membrane oxygenation, using an electrochemiluminescent multiplex assay. We recorded clinical, neuroimaging, and extracorporeal membrane oxygenation course data. We analyzed the association of biomarker concentrations with favorable versus unfavorable outcome at hospital discharge. Favorable outcome was defined as Pediatric Cerebral Performance Category 1, 2, or no change from baseline. Patients had a median age of 3 days (interquartile range, 1 d-10 mo), and 56% were male. Thirty-three of 80 (41%) had unfavorable outcome, and 22 of 70 (31%) had abnormal neuroimaging findings during or after extracorporeal membrane oxygenation. Peak concentrations were significantly higher in patients with unfavorable outcome than in those with favorable outcome for glial fibrillary acidic protein (p = 0.002), monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 (p = 0.030), neuron-specific enolase (p = 0.006), and S100b (p = 0.015) and in patients with versus without abnormal neuroimaging findings for glial fibrillary acidic protein (p = 0.001) and intercellular adhesion molecule-5 (p = 0.001). The area under the receiver operator characteristic curve for unfavorable outcome was 0.73 for a noncollinear biomarker combination. After removing collinear biomarkers, the adjusted odds ratios for unfavorable outcome were 2.89 (95% CI, 1.09-7.73) for neuron

  12. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts. Summary progress report, May 16, 1987--June 1, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Steponkus, P.L.


    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.

  13. Plasma membrane cholesterol level and agonist-induced internalization of delta-opioid receptors; colocalization study with intracellular membrane markers of Rab family\

    Czech Academy of Sciences Publication Activity Database

    Brejchová, Jana; Vošahlíková, Miroslava; Roubalová, Lenka; Parenti, M.; Mauri, M.; Chernyavskiy, Oleksandr; Svoboda, Petr


    Roč. 48, č. 4 (2016), s. 375-396 ISSN 0145-479X R&D Projects: GA ČR(CZ) GAP207/12/0919 Institutional support: RVO:67985823 Keywords : cholesterol * plasma membrane * delta-opioid receptor * internalization * Rab proteins Subject RIV: CE - Biochemistry Impact factor: 2.576, year: 2016

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

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.


    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

  15. Analysis of the induction of the myelin basic protein binding to the plasma membrane phospholipid monolayer

    International Nuclear Information System (INIS)

    Zhang Lei; Hao Changchun; Feng Ying; Gao Feng; Lu Xiaolong; Li Junhua; Sun Runguang


    Myelin basic protein (MBP) is an essential structure involved in the generation of central nervous system (CNS) myelin. Myelin shape has been described as liquid crystal structure of biological membrane. The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin. In this paper, we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy (AFM). By analyzing the pressure–area ( π – A ) and pressure–time ( π – T ) isotherms, univariate linear regression equation was obtained. In addition, the elastic modulus, surface pressure increase, maximal insertion pressure, and synergy factor of monolayers were detected. These parameters can be used to modulate the monolayers binding of protein, and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3- phosphoserine (DPPS) monolayer, followed by DPPC/DPPS mixed and 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) monolayers via electrostatic and hydrophobic interactions. AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP (5 nM) show a phase separation texture at the surface pressure of 20 mN/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure. MBP is not an integral membrane protein but, due to its positive charge, interacts with the lipid head groups and stabilizes the membranes. The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value. (special topic)

  16. Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells. (United States)

    Castanheira, Diogo Dias; Santana, Eduardo Perovano; Godoy-Santos, Fernanda; Diniz, Raphael Hermano Santos; Faria-Oliveira, Fábio; Pereira, Renata Rebeca; Trópia, Maria José Magalhães; Castro, Ieso Miranda; Brandão, Rogelio Lopes


    In yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, no protein that could be regulated by calcium in this context has been identified. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, by using different approaches, we obtained many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that maintains the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase accessible for phosphorylation by at least one protein kinase. © FEMS 2017. All rights reserved. For permissions, please e-mail:

  17. In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. (United States)

    Caro, L H; Tettelin, H; Vossen, J H; Ram, A F; van den Ende, H; Klis, F M


    Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment as asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs.

  18. The Superoxide Synthases of Plasma Membrane Preparations from Cultured Rose Cells. (United States)

    Murphy, T. M.; Auh, C. K.


    Preparations of plasma membranes isolated from cultured rose (Rosa damascena Mill. cv Gloire de Guilan) cells synthesized O2- when incubated with either NADH or NADPH, as measured by an O2--specific assay based on the chemiluminescence of lucigenin. The activities were strongly dependent on the presence of Triton X-100. The Km for NADH was 159 [mu]M; that for NADPH was 19 [mu]M. Neither NADH- nor NADPH-dependent activity was inhibited by azide, an inhibitor of peroxidase, nor by antimycin A, an inhibitor of mitochondrial electron transport; both activities were inhibited by 30 to 100 nM diphenylene iodonium, an inhibitor of the mammalian NADPH oxidase. The NADH- and NADPH-dependent activities could be distinguished by detergent solubilization and ultracentrifugation: the NADH-dependent activity sedimented more easily, whereas the NADPH-dependent activity remained in suspension. One or both of these enzymes may provide the O2- seen when plant cells are exposed to pathogens or pathogen-associated elicitors; however, plasma membranes from rose cells treated with a Phytophthora elicitor had the same activity as control cells. PMID:12226208

  19. SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors. (United States)

    Temkin, Paul; Lauffer, Ben; Jäger, Stefanie; Cimermancic, Peter; Krogan, Nevan J; von Zastrow, Mark


    Endocytic sorting of signalling receptors between recycling and degradative pathways is a key cellular process controlling the surface complement of receptors and, accordingly, the cell's ability to respond to specific extracellular stimuli. The β2 adrenergic receptor (β2AR) is a prototypical seven-transmembrane signalling receptor that recycles rapidly and efficiently to the plasma membrane after ligand-induced endocytosis. β2AR recycling is dependent on the receptor's carboxy-terminal PDZ ligand and Rab4. This active sorting process is required for functional resensitization of β2AR-mediated signalling. Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4. Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule. SNX27 does not seem to directly interact with the retromer core complex, but does interact with the retromer-associated Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.

  20. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma. (United States)

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine; Halvorsen, Trine Grønhaug; Øiestad, Elisabeth Leere; Pedersen-Bjergaard, Stig; Gjelstad, Astrid


    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Plasma membrane NADH oxidase of maize roots responds to gravity and imposed centrifugal forces (United States)

    Bacon, E.; Morre, D. J.


    NADH oxidase activities measured with excised roots of dark-grown maize (Zea mays) seedlings and with isolated plasma membrane vesicles from roots of dark-grown maize oscillated with a regular period length of 24 min and were inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic [correction of dichorophenoxyacetic] acid. The activities also responded to orientation with respect to gravity and to imposed centrifugal forces. Turning the roots upside down resulted in stimulation of the activity with a lag of about 10 min. Returning the sections to the normal upright position resulted in a return to initial rates. The activity was stimulated reversibly to a maximum of about 2-fold with isolated plasma membrane vesicles, when subjected to centrifugal forces of 25 to 250 x g for 1 to 4 min duration. These findings are the first report of a gravity-responsive enzymatic activity of plant roots inhibited by auxin and potentially related to the gravity-induced growth response. c2001 Editions scientifiques et medicales Elsevier SAS.

  2. The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux. (United States)

    Sedgwick, Alanna; Olivia Balmert, M; D'Souza-Schorey, Crislyn


    Aberrant cellular cholesterol accumulation contributes to the pathophysiology of many diseases including neurodegenerative disorders such as Niemann-Pick Type C (NPC) and Alzheimer's Disease 1-4 . Many aspects of cholesterol efflux from cells remain elusive. Here we describe the utility of cholesterol-rich giant plasma membrane vesicles (GPMVs) as a means to monitor cholesterol that is translocated to the plasma membrane for secretion. We demonstrate that small molecules known to enhance lipid efflux, including those in clinical trials for lipid storage disorders, enhance this GPMV formation. Conversely, pharmacological inhibition of cholesterol efflux blocks GPMV formation. We show that microtubule stabilization via paclitaxel treatment and increased tubulin acetylation via HDAC6 inhibition promotes the formation of GPMVs with concomitant reduction in cellular cholesterol in a cell model of NPC disease. The pan-deacetylase inhibitor panobinostat, which has been shown to reduce the severity of cholesterol storage in NPC, elicited a similar response. Further, the disruption of actin polymerization inhibits the formation of GPMVs, whereas the small GTP-binding protein Arl4c promotes actin remodeling at sites overlapping with GPMV formation. Thus, monitoring the formation of GPMVs provides a new avenue to better understand diseases whose pathology may be sensitive to alterations in cellular cholesterol. Copyright © 2018. Published by Elsevier Inc.

  3. The plasma membrane calcium pumps-The old and the new. (United States)

    Zaidi, Asma; Adewale, Mercy; McLean, Lauren; Ramlow, Paul


    The plasma membrane Ca 2+ -ATPase (PMCA) pumps play a critical role in the maintenance of calcium (Ca 2+ ) homeostasis, crucial for optimal neuronal function and cell survival. Loss of Ca 2+ homeostasis is a key precursor in neuronal dysfunction associated with brain aging and in the pathogenesis of neurodegenerative disorders. In this article, we review evidence showing age-related changes in the PMCAs in synaptic plasma membranes (SPMs) and lipid raft microdomains isolated from rat brain. Both PMCA activity and protein levels decline progressively with increasing age. However, the loss of activity is disproportionate to the reduction of protein levels suggesting the presence of dysfunctional PMCA molecules in aged brain. PMCA activity is also diminished in post-mortem human brain samples from Alzheimer's disease and Parkinson's disease patients and in cell models of these neurodegenerative disorders. Experimental reduction of the PMCAs not only alter Ca 2+ homeostasis but also have diverse effects on neurons such as reduced neuritic network, impaired release of neurotransmitter and increased susceptibility to stressful stimuli, particularly to agents that elevate intracellular Ca 2+ [Ca 2+ ] i . Loss of PMCA is likely to contribute to neuronal dysfunction observed in the aging brain and in the development of age-dependent neurodegenerative disorders. Therapeutic (pharmacological and/or non-pharmacological) approaches that can enhance PMCA activity and stabilize [Ca 2+ ] i homeostasis may be capable of preventing, slowing, and/or reversing neuronal degeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Large plasma-membrane depolarization precedes rapid blue-light-induced growth inhibition in cucumber (United States)

    Spalding, E. P.; Cosgrove, D. J.


    Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls began after a 30-s lag time, which was halved by increasing the fluence rate from 10 to 100 micromoles m-2 s-1. Prior to the growth suppression, the plasma-membrane of the irradiated cells depolarized by as much as 100 mV, then returned within 2-3 min to near its initial value. The potential difference measured with surface electrodes changed with an identical time course but opposite polarity. The lag time for the change in surface potential showed an inverse dependence on fluence rate, similar to the lag for the growth inhibition. Green light and red light caused neither the electrical response nor the rapid inhibition of growth. The depolarization by BL did not propagate to nonirradiated regions and exhibited a refractory period of about 10 min following a BL pulse. Fluence-response relationships for the electrical and growth responses provide correlational evidence that the plasma-membrane depolarization reflects an event in the transduction chain of this light-growth response.

  5. Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa

    Directory of Open Access Journals (Sweden)

    N. Prapaiwan


    Full Text Available Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05 than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05. In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

  6. PIST regulates the intracellular trafficking and plasma membrane expression of Cadherin 23

    Directory of Open Access Journals (Sweden)

    Oshima Kazuo


    Full Text Available Abstract Background The atypical cadherin protein cadherin 23 (CDH23 is crucial for proper function of retinal photoreceptors and inner ear hair cells. As we obtain more and more information about the specific roles of cadherin 23 in photoreceptors and hair cells, the regulatory mechanisms responsible for the transport of this protein to the plasma membrane are largely unknown. Results PIST, a Golgi-associated, PDZ domain-containing protein, interacted with cadherin 23 via the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI of cadherin 23. By binding to cadherin 23, PIST retained cadherin 23 in the trans-Golgi network of cultured cells. The retention was released when either of the two known cadherin 23-binding proteins MAGI-1 and harmonin was co-expressed. Similar to MAGI-1 and harmonin, PIST was detected in mouse inner ear sensory hair cells. Conclusions PIST binds cadherin 23 via its PDZ domain and retains cadherin 23 in trans-Golgi network. MAGI-1 and harmonin can compete with PIST for binding cadherin 23 and release cadherin 23 from PIST's retention. Our finding suggests that PIST, MAGI-1 and harmonin collaborate in intracellular trafficking of cadherin 23 and regulate the plasma membrane expression of cadherin 23.

  7. Alternative fates of newly formed PrPSc upon prion conversion on the plasma membrane. (United States)

    Goold, Rob; McKinnon, Chris; Rabbanian, Samira; Collinge, John; Schiavo, Giampietro; Tabrizi, Sarah J


    Prion diseases are fatal neurodegenerative diseases characterised by the accumulation of misfolded prion protein (PrP(Sc)) in the brain. They are caused by the templated misfolding of normal cellular protein, PrP(C), by PrP(Sc). We have recently generated a unique cell system in which epitope-tagged PrP(C) competent to produce bona fide PrP(Sc) is expressed in neuroblastoma cells. Using this system we demonstrated that PrP(Sc) forms on the cell surface within minutes of prion exposure. Here, we describe the intracellular trafficking of newly formed PrP(Sc). After formation in GM1-enriched lipid microdomains at the plasma membrane, PrP(Sc) is rapidly internalised to early endosomes containing transferrin and cholera toxin B subunit. Following endocytosis, PrP(Sc) intracellular trafficking diverges: some is recycled to the plasma membrane via Rab11-labelled recycling endosomes; the remaining PrP(Sc) is subject to retromer-mediated retrograde transport to the Golgi. This pathway leads to lysosomal degradation, and we show that this is the dominant PrP(Sc) degradative mechanism in the early stages of prion infection.

  8. The Homer-1 protein Ania-3 interacts with the plasma membrane calcium pump (United States)

    Sgambato-Faure, Véronique; Xiong, Yuning; Berke, Joshua D.; Hyman, Steven E.; Strehler, Emanuel E.


    The Homer family of scaffold proteins couples NMDA receptors to metabotropic glutamate receptors, and links extracellular signals to calcium release from intracellular stores. Ania-3 is a member of the Homer family and is rapidly inducible in brain in response to diverse stimuli. Here we report the identification of the plasma membrane Ca2+ ATPase (PMCA) as a novel Ania-3/Homer-associated protein. Ania-3/Homer interacts with the b-splice forms of all PMCAs (PMCA1b, 2b, 3b, and 4b) via their PDZ domain-binding COOH-terminal tail. Ectopically expressed Ania-3 colocalized with the PMCA at the plasma membrane of polarized MDCK epithelial cells, and endogenous Ania-3/Homer and PMCA2 are co-expressed in the soma and dendrites of primary rat hippocampal neurons. The interaction between Ania-3/Homer and PMCAs may represent a novel mechanism by which local calcium signaling and hence synaptic function can be modulated in neurons. PMID:16554037

  9. Surface Modification of Polypropylene Microporous Membrane by Atmospheric-Pressure Plasma Immobilization of N,N-dimethylamino Ethyl Methacrylate

    International Nuclear Information System (INIS)

    Zhong Shaofeng


    Surface modification of polypropylene microporous membrane (PPMM) was performed by atmospheric pressure dielectric barrier discharge plasma immobilization of N,N-dimethylamino ethyl methacrylate (DMAEMA). Structural and morphological changes on the membrane surface were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (FT-IR/ATR), X-ray photoelectron spectroscope (XPS) and field emission scanning electron microscopy (FE-SEM). Water contact angles of the membrane surfaces were also measured by the sessile drop method. Results reveal that both the plasma-treating conditions and the adsorbed DMAEMA amount have remarkable effects on the immobilization degree of DMAEMA. Peroxide determination by 1,1-diphenyl-2-picrvlhydrazyl (DPPH) method verifies the exsistence of radicals induced by plasma, which activize the immobilization reaction. Pure water contact angle on the membrane surface decreased with the increase of DMAEMA immobilization degree, which indicates an enhanced hydrophilicity for the modified membranes. The effects of immobilization degrees on pure water fluxes were also measured. It is shown that pure water fluxes first increased with immobilization degree and then decreased. Finally, permeation of bovine serum albumin (BSA) and lysozyme solution were measured to evaluate the antifouling property of the DMAEMA-modified membranes, from which it is shown that both hydrophilicity and electrostatic repulsion are beneficial for membrane antifouling.

  10. Antifouling coatings via plasma polymerization and atom transfer radical polymerization on thin film composite membranes for reverse osmosis (United States)

    Hirsch, Ulrike; Ruehl, Marco; Teuscher, Nico; Heilmann, Andreas


    A major drawback to otherwise highly efficient membrane-based desalination techniques like reverse osmosis (RO) is the susceptibility of the membranes to biofouling. In this work, a combination of plasma activation, plasma bromination and surface-initiated atom transfer radical polymerization (si-ATRP) of hydrophilic and zwitterionic monomers, namely hydroxyethyl methacrylate (HEMA), 2-methacryloyloxyethyl phosphorylcholine (MPC) and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA), was applied to generate non-specific, anti-adhesive coatings on thin film composite (TFC) membranes. The antifouling effect of the coatings was shown by short-time batch as well as long-time steady state cultivation experiments with the microorganism Pseudomonas fluorescens. It could be shown that plasma functionalization and polymerization is possible on delicate thin film composite membranes without restricting their filtration performance. All modified membranes showed an increased resistance towards the adhesion of Pseudomonas fluorescens. On average, the biofilm coverage was reduced by 51.4-12.6% (for HEMA, SBMA, and MPC), the highest reduction was monitored for MPC with a biofilm reduction by 85.4%. The hydrophilic coatings applied did not only suppress the adhesion of Pseudomonas fluorescens, but also significantly increase the permeate flux of the membranes relative to uncoated membranes. The stability of the coatings was however not ideal and will have to be improved for future commercial use.

  11. Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non-polarized form MDCK cells and mainly basolaterally from Caco-2 cells

    DEFF Research Database (Denmark)

    Vogel, L K; Suske, G; Beato, M


    A complete cDNA encoding rabbit uteroglobin was constructed and expressed in MDCK and Caco-2 cells. The MDCK cells secrete uteroglobin in approximately equal amounts to the apical and the basolateral side, whereas the Caco-2 cells secrete uteroglobin mainly to the basolateral side. Both MDCK...... and Caco-2 cells thus secrete uteroglobin in a non-sorted manner. It has, however, previously been shown that uteroglobin is secreted exclusively at the apical membrane in primary cell culture of endometrial epithelial cells [S.K. Mani et al. (1991) Endocrinology 128, 1563-1573]. This suggests that either...... the endometrial epithelium has an apical default pathway or recognises a sorting signal not recognised by MDCK cells and Caco-2 cells. Our data thus show that a soluble molecule can be secreted at the apical, the basolateral or both membranes depending on the cell type....

  12. Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties (United States)

    Morre, D. James; Morre, Dorothy M.; Ternes, Philipp


    The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

  13. Proteomic analysis of plasma membrane and tonoplast from the leaves of mangrove plant Avicennia officinalis. (United States)

    Krishnamurthy, Pannaga; Tan, Xing Fei; Lim, Teck Kwang; Lim, Tit-Meng; Kumar, Prakash P; Loh, Chiang-Shiong; Lin, Qingsong


    In order to understand the salt tolerance and secretion in mangrove plant species, gel electrophoresis coupled with LC-MS-based proteomics was used to identify key transport proteins in the plasma membrane (PM) and tonoplast fractions of Avicennia officinalis leaves. PM and tonoplast proteins were purified using two-aqueous-phase partitioning and density gradient centrifugation, respectively. Forty of the 254 PM proteins and 31 of the 165 tonoplast proteins identified were predicted to have transmembrane domains. About 95% of the identified proteins could be classified based on their functions. The major classes of proteins were predicted to be involved in transport, metabolic processes, defense/stress response, and signal transduction, while a few of the proteins were predicted to be involved in other functions such as membrane trafficking. The main classes of transporter proteins identified included H(+) -ATPases, ATP-binding cassette transporters, and aquaporins, all of which could play a role in salt secretion. These data will serve as the baseline membrane proteomic dataset for Avicennia species. Further, this information can contribute to future studies on understanding the mechanism of salt tolerance in halophytes in addition to salt secretion in mangroves. All MS data have been deposited in the ProteomeXchange with identifier PXD000837 ( © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome. (United States)

    Sidhu, Vishaldeep K; Huang, Bill X; Desai, Abhishek; Kevala, Karl; Kim, Hee-Yong


    Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis. We found significant reduction of 15 synaptic plasma membrane proteins in aging brains including fodrin-α, synaptopodin, postsynaptic density protein 95, synaptic vesicle glycoprotein 2B, synaptosomal-associated protein 25, synaptosomal-associated protein-α, N-methyl-D-aspartate receptor subunit epsilon-2 precursor, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, vesicle-associated membrane protein 2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first 9 proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins downregulated during aging. Reduction of 2 of these proteins was reversed by raising the brain DHA level by supplementing aged animals with an omega-3 fatty acid sufficient diet for 2 months. The recognition memory compromised in DHA-depleted animals was also improved. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function. Published by Elsevier Inc.

  15. Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates. (United States)

    Bienert, Gerd Patrick; Bienert, Manuela Désirée; Jahn, Thomas Paul; Boutry, Marc; Chaumont, François


    Major intrinsic proteins (MIPs) transport water and uncharged solutes across membranes in all kingdoms of life. Recently, an uncharacterized MIP subfamily was identified in the genomes of plants and fungi and named X Intrinsic Proteins (XIPs). Here, we describe the genetic features, localization, expression, and functions of a group of Solanaceae XIPs. XIP cDNA and gDNA were cloned from tobacco, potato, tomato, and morning glory. A conserved sequence motif in the first intron of Solanaceae XIPs initiates an RNA-processing mechanism that results in two splice variants (α and β). When transiently or stably expressed in tobacco plants, yellow fluorescent protein-tagged NtXIP1;1α and NtXIP1;1β were both localized in the plasma membrane. Transgenic tobacco lines expressing NtXIP1;1-promoter-GUS constructs and RT-PCR studies showed that NtXIP1;1 was expressed in all organs. The NtXIP1;1 promoter was mainly active in cell layers facing the environment in all above-ground tissues. Heterologous expression of Solanaceae XIPs in Xenopus laevis oocytes and various Saccharomyces cerevisiae mutants demonstrated that these isoforms facilitate the transport of bulky solutes, such as glycerol, urea, and boric acid. In contrast, permeability for water was undetectable. These data suggest that XIPs function in the transport of uncharged solutes across the cell plasma membrane in specific plant tissues, including at the interface between the environment and external cell layers. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  16. Toxoplasma gondii infection induces dendritic retraction in basolateral amygdala accompanied by reduced corticosterone secretion

    Directory of Open Access Journals (Sweden)

    Rupshi Mitra


    Pathological anxiety is thought to reflect a maladaptive state characterized by exaggerated fear. Naturally occurring perturbations that reduce fear can be crucial in the search for new treatments. The protozoan parasite Toxoplasma gondii invades rat brain and removes the fear that rats have of cat odors, a change believed to be parasitic manipulation of host behavior aimed at increasing parasite transmission. It is likely that mechanisms employed by T. gondii can be used as a heuristic tool to understand possible means of fear reduction in clinical settings. Male Long-Evans rats were infected with T. gondii and compared with sham-infected animals 8 weeks after infection. The amount of circulating plasma corticosterone and dendritic arborization of basolateral amygdala principal neurons were quantified. Previous studies have shown that corticosterone, acting within the basolateral amygdala, enhances the fear response to environmental stimuli. Here we show that T. gondii infection causes a dendritic retraction in basolateral amygdala neurons. Such dendritic retraction is accompanied by lower amounts of circulating corticosterone, both at baseline and when induced by an aversive cat odor. The concerted effects of parasitism on two pivotal physiological nodes of the fear response provide an animal model relevant to interactions between stress hormones and amygdalar plasticity.

  17. Convergence of lateral dynamic measurements in the plasma membrane of live cells from single particle tracking and STED-FCS

    DEFF Research Database (Denmark)

    Lagerholm, B. Christoffer; Andrade, Débora M.; Clausen, Mathias P.


    Fluorescence correlation spectroscopy (FCS) in combination with the super-resolution imaging method STED (STED-FCS), and single-particle tracking (SPT) are able to directly probe the lateral dynamics of lipids and proteins in the plasma membrane of live cells at spatial scales much below the diff......Fluorescence correlation spectroscopy (FCS) in combination with the super-resolution imaging method STED (STED-FCS), and single-particle tracking (SPT) are able to directly probe the lateral dynamics of lipids and proteins in the plasma membrane of live cells at spatial scales much below...... the diffraction limit of conventional microscopy. However, a major disparity in interpretation of data from SPT and STED-FCS remains, namely the proposed existence of a very fast (unhindered) lateral diffusion coefficient, ≥5 μm2 s-1, in the plasma membrane of live cells at very short length scales, ≈ 100 nm...

  18. Advanced spinning disk-TIRF microscopy for faster imaging of the cell interior and the plasma membrane. (United States)

    Zobiak, Bernd; Failla, Antonio Virgilio


    Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup. © 2017 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

  19. Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes. (United States)

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


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

  20. Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes (United States)

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


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

  1. Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells. (United States)

    Migocka, Magdalena; Papierniak, Anna; Kosatka, Ewelina; Klobus, Grazyna


    The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

  2. Full-length, glycosylated NSP4 is localized to plasma membrane caveolae by a novel raft isolation technique. (United States)

    Storey, Stephen M; Gibbons, Thomas F; Williams, Cecelia V; Parr, Rebecca D; Schroeder, Friedhelm; Ball, Judith M


    Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, high-mannose glycosylated NSP4. The lack of Golgi network-specific processing of the caveolar NSP4 glycans supports studies showing that NSP4 bypasses the Golgi apparatus. Confocal imaging showed the colocalization of NSP4 with caveolin-1 early and late in infection, elucidating the temporal and spatial NSP4-caveolin-1 association during infection. These data were extended with fluorescent resonance energy transfer analyses that confirmed the NSP4 and caveolin-1 interaction in that the specific fluorescently tagged antibodies were within 10 nm of each other during infection. Cells transfected with NSP4 showed patterns of staining and colocalization with caveolin-1 similar to those of infected cells. This study presents an endoplasmic reticulum contaminant-free caveola isolation protocol; describes the presence of full-length, endoglycosidase H-sensitive NSP4 in plasma membrane caveolae; provides confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins; and provides a final plasma membrane destination for Golgi network-bypassing NSP4 transport.

  3. Silver ions increase plasma membrane permeability through modulation of intracellular calcium levels in tobacco BY-2 cells. (United States)

    Klíma, Petr; Laňková, Martina; Vandenbussche, Filip; Van Der Straeten, Dominique; Petrášek, Jan


    Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides a dramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO 3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO 3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca 2+ ) as shown by comparison of transport assays in Ca 2+ -rich and Ca 2+ -free buffers and upon treatment with inhibitors of plasma membrane Ca 2+ -permeable channels Al 3+ and ruthenium red, both abolishing the effect of AgNO 3 . Confocal microscopy of Ca 2+ -sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca 2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca 2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca 2+ -permeable channels at the plasma membrane.

  4. Properties of Fiber Cell Plasma Membranes Isolated from the Cortex and Nucleus of the Porcine Eye Lens (United States)

    Mainali, Laxman; Raguz, Marija; O’Brien, William J.; Subczynski, Witold K.


    The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eyes lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes—namely, the domain formed by boundary lipids and the domain formed by trapped lipids—were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region. PMID:22326289

  5. [Interaction of FABP4 with plasma membrane proteins of endothelial cells]. (United States)

    Saavedra, Paula; Girona, Josefa; Aragonès, Gemma; Cabré, Anna; Guaita, Sandra; Heras, Mercedes; Masana, Lluís


    Fatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. HUVEC cells were incubated with and without FABP4 (100 ng/ml) for 5 minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker. A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100 ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. The results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33 kDa containing FAPB4 and its putative receptor. The existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular. Copyright © 2014 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

  6. Total internal reflection fluorescence (TIRF) microscopy for real-time imaging of nanoparticle-cell plasma membrane interaction

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Moghimi, Seyed Moien


    fluorescence (TIRF) microscopy allows for real-time monitoring of nanoparticle-membrane interaction events, which can provide vital information in relation to design and surface engineering of therapeutic nanoparticles for cell-specific targeting. In contrast to other microscopy techniques, the bleaching...... effect by lasers in TIRF microscopy is considerably less when using fluorescent nanoparticles and it reduces photo-induced cytotoxicity during visualization of live-cell events since it only illuminates the specific area near or at the plasma membrane....

  7. 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


    . 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....... Phosphopeptide enrichment by titanium dioxide chromatography followed by capillary liquid chromatography-tandem mass spectrometry allowed us to assign 703 unique phosphorylation sites in 376 phosphoproteins. Our experiments revealed that treatment of cell cultures with three different types of protein...

  8. Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes. (United States)

    Sarkar, Mitul; Koland, John G


    The fluorescence recovery after photobleaching (FRAP) method is a straightforward means of assessing the diffusional mobility of membrane-associated proteins that is readily performed with current confocal microscopy instrumentation. We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility. Our FRAP measurements of the diffusional mobility of GFP-tagged HER3 reporters expressed in MCF7 cultured breast cancer cells showed that despite the observed segregation of HER3 receptors within plasma membrane microdomains their diffusion on the macroscopic scale is not spatially restricted. Thus, in FRAP analyses of various HER3 reporters a near-complete recovery of fluorescence after photobleaching was observed, indicating that HER3 receptors are not immobilized by long-lived physical interactions with intracellular species. An examination of HER3 proteins with varying intracellular domain sequence truncations also indicated that a proposed formation of oligomeric HER3 networks, mediated by physical interactions involving specific HER3 intracellular domain sequences, either does not occur or does not significantly reduce HER3 mobility on the macroscopic scale.

  9. Effects of temperature and buffer composition on calcium sequestration by sarcoplasmic reticulum and plasma membrane of rabbit renal artery

    International Nuclear Information System (INIS)

    McGuffee, L.J.; Little, S.A.; Mercure, J.V.; Skipper, B.J.; Wheeler-Clark, E.S.


    45Ca electron microscopic autoradiography was used to examine the effects of buffer composition and temperature on the distribution of calcium in rabbit renal artery smooth muscle cells. The results show that the relative distribution of calcium is dependent on both the buffer used (Tris or Krebs) and the temperature of the bathing solution (25 degrees C or 34 degrees C). Krebs buffer at 34 degrees C gave the highest relative activity in the plasma membrane, sarcoplasmic reticulum, and mitochondria. Buffer and temperature had little effect on the relative activity of the nucleus or cytoplasm. Next, we identified the cellular sites of calcium accumulation after 5, 15, 30, or 60 min exposure to 45 Ca in Krebs buffer at 34 degrees C. The results show that sarcoplasmic reticulum and plasma membrane are the primary sites of calcium accumulation during influx into these cells. Although the amount of 45 Ca in the cell continues to increase with longer exposure, the relative distribution of calcium is essentially the same after 5 or 60 min. The data also indicate that the relative activity of plasma membrane + sarcoplasmic reticulum (a combination site that includes sarcoplasmic reticulum within a mean distance of 275 nm of the plasma membrane) is similar to the membrane alone and is lower than the sarcoplasmic reticulum alone

  10. A direct gateway into the extracellular space: Unconventional secretion of FGF2 through self-sustained plasma membrane pores. (United States)

    Steringer, Julia P; Nickel, Walter


    As illustrated by a diverse set of examples in this special issue, multiple mechanisms of protein secretion have been identified in eukaryotes that do not involve the endoplasmic reticulum (ER) and the Golgi apparatus. Here we focus on the type I pathway with Fibroblast Growth Factor 2 (FGF2) being the most prominent example. Unconventional secretion of FGF2 from cells is mediated by direct protein translocation across the plasma membrane. A unique feature of this process is the ability of FGF2 to form its own membrane translocation intermediate through oligomerization and membrane insertion. This process depends on the phosphoinositide PI(4,5)P 2 at the inner leaflet and results in the formation of lipidic membrane pores in the plasma membrane. Various lines of evidence suggest that these pores are characterized by a toroidal architecture with FGF2 oligomers being accommodated in the center of these structures. At the outer leaflet of the plasma membrane, membrane proximal heparan sulfate proteoglycans are required for the final step of FGF2 translocation into the extracellular space. Based upon mutually exclusive interactions of FGF2 with PI(4,5)P 2 versus heparan sulfates, an assembly/disassembly pathway has been proposed to be the underlying principle of directional transport of FGF2 across the plasma membrane. Thus, the core mechanism of unconventional secretion of FGF2 is based upon three discrete steps with (i) PI(4,5)P 2 dependent oligomerization of FGF2 at the inner leaflet, (ii) insertion of membrane spanning FGF2 oligomers into the plasma membrane and (iii) disassembly at the outer leaflet mediated by heparan sulfates that subsequently retain FGF2 on cell surfaces. This process has recently been reconstituted with an inside-out membrane model system using giant unilamellar vesicles providing a compelling explanation of how FGF2 reaches the extracellular space in an ER/Golgi independent manner. This review is part of a Special Issue of SCDB on

  11. Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment

    Directory of Open Access Journals (Sweden)

    Sano Kouichi


    Full Text Available Abstract Background It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV. Results We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-α stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1 and U1 cells (latently infected U937 cells with HIV-1 displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-α, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane. Conclusion Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute

  12. Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation. (United States)

    Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan


    Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

  13. An Adaptable Spectrin/Ankyrin-Based Mechanism for Long-Range Organization of Plasma Membranes in Vertebrate Tissues. (United States)

    Bennett, Vann; Lorenzo, Damaris N


    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

  14. Biomechanics and Thermodynamics of Nanoparticle Interactions with Plasma and Endosomal Membrane Lipids in Cellular Uptake and Endosomal Escape (United States)


    To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(d,l-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In

  15. Hemocompatibility and oxygenation performance of polysulfone membranes grafted with polyethylene glycol and heparin by plasma-induced surface modification. (United States)

    Wang, Weiping; Zheng, Zhi; Huang, Xin; Fan, Wenling; Yu, Wenkui; Zhang, Zhibing; Li, Lei; Mao, Chun


    Polyethylene glycol (PEG) and heparin (Hep) were grafted onto polysulfone (PSF) membrane by plasma-induced surface modification to prepare PSF-PEG-Hep membranes used for artificial lung. The effects of plasma treatment parameters, including power, gas type, gas flow rate, and treatment time, were investigated, and different PEG chains were bonded covalently onto the surface in the postplasma grafting process. Membrane surfaces were characterized by water contact angle, PEG grafting degree, attenuated total reflectance-Fourier transform infrared spectroscopy, ultraviolet-visible spectrophotometry, X-ray photoelectron spectroscopy, critical water permeability pressure, and scanning electron microscopy. Protein adsorption, platelet adhesion, and coagulation tests showed significant improvement in the hemocompatibility of PSF-PEG-Hep membranes compared to pristine PSF membrane. Gas exchange tests through PSF-PEG6000-Hep membrane showed that when the flow rate of porcine blood reached 5.0 L/min, the permeation fluxes of O 2 and CO 2 reached 192.6 and 166.9 mL/min, respectively, which were close to the gas exchange capacity of a commercial membrane oxygenator. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1737-1746, 2017. © 2016 Wiley Periodicals, Inc.

  16. Evidence for a role of plasma membrane calcium pumps in neurodegenerative disease: Recent developments. (United States)

    Strehler, Emanuel E; Thayer, Stanley A


    Plasma membrane Ca 2+ ATPases (PMCAs) are a major system for calcium extrusion from all cells. Different PMCA isoforms and splice variants are involved in the precise temporal and spatial handling of Ca 2+ signals and the re-establishment of resting Ca 2+ levels in the nervous system. Lack or inappropriate expression of specific PMCAs leads to characteristic neuronal phenotypes, which may be reciprocally exacerbated by genetic predisposition through alleles in other genes that modify PMCA interactions, regulation, and function. PMCA dysfunction is often poorly compensated in neurons and may lead to changes in synaptic transmission, altered excitability and, with long-term calcium overload, eventual cell death. Decrease and functional decline of PMCAs are hallmarks of neurodegeneration during aging, and mutations in specific PMCAs are responsible for neuronal dysfunction and accelerated neurodegeneration in many sensory and cognitive diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Plasma membrane Ca2+-ATPases in the nervous system during development and ageing (United States)

    Mata, Ana M; Sepulveda, M Rosario


    Calcium signaling is used by neurons to control a variety of functions, including cellular differentiation, synaptic maturation, neurotransmitter release, intracellular signaling and cell death. This review focuses on one of the most important Ca2+ regulators in the cell, the plasma membrane Ca2+-ATPase (PMCA), which has a high affinity for Ca2+ and is widely expressed in brain. The ontogeny of PMCA isoforms, linked to specific requirements of Ca2+ during development of different brain areas, is addressed, as well as their function in the adult tissue. This is based on the high diversity of variants in the PMCA family in brain, which show particular kinetic differences possibly related to specific localizations and functions of the cell. Conversely, alterations in the activity of PMCAs could lead to changes in Ca2+ homeostasis and, consequently, to neural dysfunction. The involvement of PMCA isoforms in certain neuropathologies and in brain ageing is also discussed. PMID:21537478

  18. Influence of quantum dot labels on single molecule movement in the plasma membrane

    DEFF Research Database (Denmark)

    Clausen, Mathias P.; Lagerholm, B. Christoffer


    a biotin ligase acceptor peptide (BLAP) or an acyl carrier protein (ACP) tag, respectively. Trajectories of the differently labeled GPI-anchored molecules were recorded simultaneously in dual-color experiments at rates of ~25 -~1500 Hz. Knowing the effect of different labels is of utmost importance......Single particle tracking results are very dependent on the probe that is used. In this study we have investigated the influence that functionalized quantum dots (QDs) have on the recorded movement in single molecule tracking experiments of plasma membrane species in live cells. Potential issues...... functionalization tag (e.g. streptavidin (sAv)) or the presence of multiple mono- or multivalent functionalization tags per QD. In this work, we have compared commercially available sAv-QDs of different sizes with custom prepared Co enzyme A (CoA)-QDs both targeting a GPI-anchored protein modified with either...

  19. The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy (United States)

    Mohamed, Tamer M. A.; Abou-Leisa, Riham; Stafford, Nicholas; Maqsood, Arfa; Zi, Min; Prehar, Sukhpal; Baudoin-Stanley, Florence; Wang, Xin; Neyses, Ludwig; Cartwright, Elizabeth J.; Oceandy, Delvac


    The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition. PMID:27020607

  20. Influence of quantum dot labels on single molecule movement in the plasma membrane

    DEFF Research Database (Denmark)

    Clausen, Mathias P.; Lagerholm, B. Christoffer


    Single particle tracking results are very dependent on the probe that is used. In this study we have investigated the influence that functionalized quantum dots (QDs) have on the recorded movement in single molecule tracking experiments of plasma membrane species in live cells. Potential issues...... in labeling single molecules with QDs (and other particles e.g. gold particles) are induction of cross-linking of the target molecules, which can cause activation of signaling pathways or reduced mobility, and steric hindrance as a result of the probe size. Cross-linking can be a result of the multivalent...... functionalization tag (e.g. streptavidin (sAv)) or the presence of multiple mono- or multivalent functionalization tags per QD. In this work, we have compared commercially available sAv-QDs of different sizes with custom prepared Co enzyme A (CoA)-QDs both targeting a GPI-anchored protein modified with either...

  1. Effect of passive transport of water through plasma membrane in production of extracellular enzyme. (United States)

    Mahmoodi, M; Najafpour, G D; Mohammadi, M


    In this article, availability and control of water in solid-state fermentation (SSF) were investigated. Based on passive transport of water through plasma membranes, a new model was proposed for calculation and control of water activities in the mixture of solids. The validity of theoretical model and accuracy of the proposed model were proved by experimental data. This model was used for production of pectinases via mixed-SSF with the aid of a rotary drum bioreactor. It was found that in case of extracellular enzyme production, the new model is in good agreement with experimental data for the control of water activities in the mixed-SSF. Exact control of water activity in SFF, the production of endo- and exo-pectinases was relatively enhanced. Based on theoretical view point, the prominence of this new model in control of water activity was also proved.

  2. Quantitative analysis of plasma membrane proteome using two-dimensional difference gel electrophoresis. (United States)

    Tang, Wenqiang


    The plasma membrane (PM) controls cell's exchange of both material and information with the outside environment, and PM-associated proteins play key roles in cellular regulation. Numerous cell surface receptors allow cells to perceive and respond to various signals from neighbor cells, pathogens, or the environment; large numbers of transporter and channel proteins control material uptake or release. Quantitative proteomic analysis of PM-associated proteins can identify key proteins involved in signal transduction and cellular regulation. Here, we describe a protocol for quantitative proteomic analysis of PM proteins using two-dimensional difference gel electrophoresis. The protocol has been successfully employed to identify new components of the brassinosteroid signaling pathway, and should also be applicable to the studies of other plant signal transduction pathways and regulatory mechanisms.

  3. Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S.cerevisiae

    DEFF Research Database (Denmark)

    L. Rudashevskaya, Elena; Ye, Juanying; Jensen, Ole Nørregaard

    +-ATPases are app. 60 amino acid residues longer than their yeast homologous. Yeast is found to phosphorylate at least one residue within the plant C-terminus. At the same time a wide range of investigations on structure, function, regulation and interaction of H+-ATPase is carried out with implication...... functioning of the residues and suggests, that plant H+-ATPase could be regulated by phosphorylation at several sites being in yeast cells. Plant H+-ATPase purified from yeast cells by his-tag affinity chromatography was subjected to IMAC and TiO2 for enrichment of phosphopeptides. The phosphopeptides were...... It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H...

  4. Garbage on, garbage off: new insights into plasma membrane protein quality control. (United States)

    MacGurn, Jason A


    Maintenance of cellular protein quality - by restoring misfolded proteins to their native state and by targeting terminally misfolded or damaged proteins for degradation - is a critical function of all cells. To ensure protein quality, cells have evolved various organelle-specific quality control mechanisms responsible for recognizing and responding to misfolded proteins at different subcellular locations of the cell. Recently, several publications have begun to elucidate mechanisms of quality control that operate at the plasma membrane (PM), recognizing misfolded PM proteins and targeting their endocytic trafficking and lysosomal degradation. Here, I discuss these recent developments in our understanding of PM quality control mechanisms and how they relate to global protein quality control strategies in the cell. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, P.; Custódio, M.R. [Instituto de Biociências, Departamento de Fisiologia, Universidade de São Paulo, Rua do Matão, Travessa 14, #101, São Paulo 05508-900, SP (Brazil); Zanotto, F.P., E-mail: [Instituto de Biociências, Departamento de Fisiologia, Universidade de São Paulo, Rua do Matão, Travessa 14, #101, São Paulo 05508-900, SP (Brazil); Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, São Paulo 04044-020 (Brazil)


    gill cell plasma membrane of crabs are suggested.

  6. Maternal Plasma Procalcitonin Concentrations in Pregnancy Complicated by Preterm Premature Rupture of Membranes

    Directory of Open Access Journals (Sweden)

    Andrzej Torbé


    Full Text Available Objectives. Our objective is to compare maternal plasma procalcitonin concentrations in preterm premature rupture of membranes (pPROM and premature rupture of membranes (PROM at term with their levels in uncomplicated pregnancy, and to determine whether these concentrations are useful in the diagnosis of pPROM cases suspected of infection and in the prediction of pPROM-to-delivery interval. Study design. Forty eight patients with pPROM, 30 with PROM at term, 31 healthy women at preterm gestation, and 33 healthy women at term were included. In pPROM group, analysis of procalcitonin concentrations with reference to leucocytosis, serum C-reactive protein, vaginal fluid culture, neonatal infection, histological chorioamnionitis and pPROM-to-delivery interval was carried out. Results. Procalcitonin concentrations in pPROM and PROM at term cases were comparable. However, in both groups procalcitonin values were significantly higher than in healthy controls in approximate gestational age. In pPROM group, procalcitonin concentrations between the patients with and without laboratory indices of infection were comparable, as well as between patients who gave birth to newborns with and without congenital infection, and between patients with and without histological chorioamnionitis. The predictive values of procalcitonin determinations were poor. Conclusion. The value of maternal plasma procalcitonin determinations in the diagnostics of pPROM cases suspected of intraamniotic infection, as well as for the prediction of pPROM-to-delivery interval, newborn's infection or histological chorioamnionitis is unsatisfactory. However, procalcitonin concentrations are elevated, both in patients with preterm and term PROMs in comparison to healthy pregnants, and therefore further evaluations are necessary to establish the role of procalcitonin in the pathophysiology of pregnancy.

  7. Glycosylation stabilizes hERG channels on the plasma membrane by decreasing proteolytic susceptibility. (United States)

    Lamothe, Shawn M; Hulbert, Maggie; Guo, Jun; Li, Wentao; Yang, Tonghua; Zhang, Shetuan


    The human ether-a-go-go related gene ( hERG)-encoded channel hERG undergoes N-linked glycosylation at position 598, which is located in the unusually long S5-pore linker of the channel. In other work we have demonstrated that hERG is uniquely susceptible to proteolytic cleavage at the S5-pore linker by proteinase K (PK) and calpain (CAPN). The scorpion toxin BeKm-1, which binds to the S5-pore linker of hERG, protects hERG from such cleavage. In the present study, our data revealed that, compared with normal glycosylated hERG channels, nonglycosylated hERG channels were significantly more susceptible to cleavage by extracellular PK. Furthermore, the protective effect of BeKm-1 on hERG from PK-cleavage was lost when glycosylation of hERG was inhibited. The inactivation-deficient mutant hERG channels S620T and S631A were resistant to PK cleavage, and inhibition of glycosylation rendered both mutants susceptible to PK cleavage. Compared with normal glycosylated channels, nonglycosylated hERG channels were also more susceptible to cleavage mediated by CAPN, which was present in the medium of human embryonic kidney cells under normal culture conditions. Inhibition of CAPN resulted in an increase of nonglycosylated hERG current. In summary, our results revealed that N-linked glycosylation protects hERG against protease-mediated degradation and thus contributes to hERG channel stability on the plasma membrane.-Lamothe, S. M., Hulbert, M., Guo, J., Li, W., Yang, T., Zhang, S. Glycosylation stabilizes hERG channels on the plasma membrane by decreasing proteolytic susceptibility.

  8. Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane. (United States)

    Salanenka, Yuliya; Verstraeten, Inge; Löfke, Christian; Tabata, Kaori; Naramoto, Satoshi; Glanc, Matouš; Friml, Jiří


    The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses. Copyright © 2018 the Author(s). Published by PNAS.

  9. Enrichment and proteomic analysis of plasma membrane from rat dorsal root ganglions

    Directory of Open Access Journals (Sweden)

    Lin Yong


    Full Text Available Abstract Background Dorsal root ganglion (DRG neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. Plasma membrane (PM of DRG cells plays important roles in their functions. PM proteins are main performers of the functions. However, mainly due to the very low amount of DRG that leads to the difficulties in PM sample collection, few proteomic analyses on the PM have been reported and it is a subject that demands further investigation. Results By using aqueous polymer two-phase partition in combination with high salt and high pH washing, PMs were efficiently enriched, demonstrated by western blot analysis. A total of 954 non-redundant proteins were identified from the plasma membrane-enriched preparation with CapLC-MS/MS analysis subsequent to protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE or shotgun digestion. 205 (21.5% of the identified proteins were unambiguously assigned as PM proteins, including a large number of signal proteins, receptors, ion channel and transporters. Conclusion The aqueous polymer two-phase partition is a simple, rapid and relatively inexpensive method. It is well suitable for the purification of PMs from small amount of tissues. Therefore, it is reasonable for the DRG PM to be enriched by using aqueous two-phase partition as a preferred method. Proteomic analysis showed that DRG PM was rich in proteins involved in the fundamental biological processes including material exchange, energy transformation and information transmission, etc. These data would help to our further understanding of the fundamental DRG functions.

  10. Free-flow electrophoresis of plasma membrane vesicles enriched by two-phase partitioning enhances the quality of the proteome from Arabidopsis seedlings

    DEFF Research Database (Denmark)

    de Michele, Roberto; McFarlane, Heather E; Parsons, Harriet Tempé


    using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane...

  11. Spermine modulates fungal morphogenesis and activates plasma membrane H+-ATPase during yeast to hyphae transition

    Directory of Open Access Journals (Sweden)

    Antônio Jesus Dorighetto Cogo


    Full Text Available Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H+-ATPase. Morphological transition was followed microscopically, and the H+-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H+-ATPase activity and H+ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

  12. Antifungal activity of alkanols against Zygosaccharomyces bailii and their effects on fungal plasma membrane. (United States)

    Fujita, Ken-Ichi; Fujita, Tomoko; Kubo, Isao


    A series of aliphatic primary alkanols from C(6) to C(13) were tested for antifungal activity against a food spoilage fungus Zygosaccharomyces bailii using a broth dilution method and were compared for their effects against Saccharomyces cerevisiae and Z. rouxii. Decanol (C(10)) was found to be the most potent fungicide against Z. bailii at a minimum fungicidal concentration of 50 microg/ml (0.31 mM), whereas undecanol (C(11)) was found to be the most potent fungistatic at a minimum inhibitory concentration of 25 microg/ml (0.14 mM). The time-kill curve study showed that decanol was fungicidal against Z. bailii at any growth stage. Octanol (C(8)) increased plasma membrane fluidity in the spheroplast cells of S. cerevisiae. The primary antifungal action of alkanols comes from their ability to disrupt the native membrane-associated function of integral proteins nonspecifically as nonionic surface-active agents (surfactants). The antifungal activity of decanol against Z. bailii was slightly enhanced in combination with anethole. (c) 2008 John Wiley & Sons, Ltd.

  13. Potassium transport at the plasma membrane of the food spoilage yeast Zygosaccharomyces bailii. (United States)

    Demidchik, Vadim; Macpherson, Neil; Davies, Julia M


    Zygosaccharomyces bailii is a commercially important spoilage yeast capable of growth at low pH in the presence of weak organic acid preservatives, such as benzoic acid. A patch-clamp electrophysiological analysis of plasma membrane K+ transport revealed a high conductance pathway for low-affinity K+ uptake. In contrast to the equivalent K+ transporter in Saccharomyces cerevisiae, this system remained operative at low extracellular pH and may therefore facilitate K+ uptake in K(+)-rich and acidic beverages. Benzoate inhibited growth, increased intracellular K+ content, yet decreased the magnitude of the K+ uptake conductance; specifically, the hyperpolarization-activated inwardly-rectifying component was reduced. It is proposed that this adaptation helps maintain a hyperpolarized membrane voltage to effect continued ATPase-mediated H+ extrusion and so combat preservative-induced cytosolic acidosis. Again in contrast to S. cerevisiae, the K+ conductance was relatively insensitive to increased extracellular Ca2+. Paradoxically (and unlike S. cerevisiae) increasing extracellular Ca2+ inhibited growth, suggesting a simple expedient to limit spoilage by Z. bailii.

  14. Purification and characterization of an NADH-hexacyanoferrate(III) reductase from spinach leaf plasma membrane. (United States)

    Bérczi, A; Fredlund, K M; Møller, I M


    Plasma membranes were purified from spinach (Spinacea oleracea L.) leaves by aqueous two-phase partitioning. The NADH-hexacyanoferrate(III) reductase was released from the membrane by Chaps solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size-exclusion chromatography on FPLC. A major band of 45 kDa and a minor contaminant of 66 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The band at 45 kDa cross-reacted with antibodies raised against an NADH-hexacyanoferrate(III) reductase from potato tuber microsomes. The native size of the enzyme was 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Two-dimensional gel electrophoresis, isoelectric focusing, followed by SDS-PAGE revealed three main bands of identical molecular weight with pI of 5.3-5.6. The enzyme contained about one flavin adenine dinucleotide (FAD) per 45-kDa subunit as determined by fluorescence spectroscopy, was specific for the beta-hydrogen of NADH, preferred NADH over NADPH as electron donor, and preferred hexacyanoferrate(III) as electron acceptor, e.g., it reduced Fe3+-EDTA, cytochrome c, oxygen, and duroquinone at 70% whereas FAD, flavin mononucleotide, duroquinone, and ubiquinone0 did not affect the activity.

  15. Exploring the Underlying Biophysics of Eukaryotic Plasma Membrane Asymmetry by Sum-Frequency Vibrational Spectroscopy (United States)

    Conboy, John


    A central issue in molecular biology is the movement of lipids across the cellular membrane. The translocation of lipids is involved in cell apoptosis, the viral infection of living cells, the functioning of antibiotics, antiseptics and drugs, and the regulation and growth of cells. There have been a number of studies attempting to find the putative proteins responsive for lipid transbilayer movement in eukaryotic cells. This has led to a large number of theories about the mechanism of transbilayer movement of lipids in cellular systems and the physical process by which lipid compositional asymmetry in the plasma membrane of eukaryotic cells is maintained. Using methods of classical surface chemistry coupled with nonlinear optical methods, we have developed a novel analytical approach, using sum-frequency vibrational spectroscopy (SFVS), to selectively probe lipid compositional asymmetry in a planar supported lipid bilayer. This new method allows for the detection of lipid flip-flop kinetics and compositional asymmetry without the need for a fluorescent or spin-labeled lipid species. The effect of lipid composition, headgroup and fatty acid chemical structure, on the rate and thermodynamics of lipid transbilayer migration and the electrostatic induction of lipid asymmetry will be discussed.

  16. Insulin Protects Pancreatic Acinar Cells from Cytosolic Calcium Overload and Inhibition of Plasma Membrane Calcium Pump* (United States)

    Mankad, Parini; James, Andrew; Siriwardena, Ajith K.; Elliott, Austin C.; Bruce, Jason I. E.


    Acute pancreatitis is a serious and sometimes fatal inflammatory disease of the pancreas without any reliable treatment or imminent cure. In recent years, impaired metabolism and cytosolic Ca2+ ([Ca2+]i) overload in pancreatic acinar cells have been implicated as the cardinal pathological events common to most forms of pancreatitis, regardless of the precise causative factor. Therefore, restoration of metabolism and protection against cytosolic Ca2+ overload likely represent key therapeutic untapped strategies for the treatment of this disease. The plasma membrane Ca2+-ATPase (PMCA) provides a final common path for cells to “defend” [Ca2+]i during cellular injury. In this paper, we use fluorescence imaging to show for the first time that insulin treatment, which is protective in animal models and clinical studies of human pancreatitis, directly protects pancreatic acinar cells from oxidant-induced cytosolic Ca2+ overload and inhibition of the PMCA. This protection was independent of oxidative stress or mitochondrial membrane potential but appeared to involve the activation of Akt and an acute metabolic switch from mitochondrial to predominantly glycolytic metabolism. This switch to glycolysis appeared to be sufficient to maintain cellular ATP and thus PMCA activity, thereby preventing Ca2+ overload, even in the face of impaired mitochondrial function. PMID:22128146

  17. Latent nitrate reductase activity is associated with the plasma membrane of corn roots (United States)

    Ward, M. R.; Grimes, H. D.; Huffaker, R. C.


    Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

  18. Mechanical loading disrupts osteocyte plasma membranes which initiates mechanosensation events in bone. (United States)

    Yu, Kanglun; Sellman, David P; Bahraini, Anoosh; Hagan, Mackenzie L; Elsherbini, Ahmed; Vanpelt, Kayce T; Marshall, Peyton L; Hamrick, Mark W; McNeil, Anna; McNeil, Paul L; McGee-Lawrence, Meghan E


    Osteocytes sense loading in bone, but their mechanosensation mechanisms remain poorly understood. Plasma membrane disruptions (PMD) develop with loading under physiological conditions in many cell types (e.g., myocytes, endothelial cells). These PMD foster molecular flux across cell membranes that promotes tissue adaptation, but this mechanosensation mechanism had not been explored in osteocytes. Our goal was to investigate whether PMD occur and initiate consequent mechanotransduction in osteocytes during physiological loading. We found that osteocytes experience PMD during in vitro (fluid flow) and in vivo (treadmill exercise) mechanical loading, in proportion to the level of stress experienced. In fluid flow studies, osteocyte PMD preferentially formed with rapid as compared to gradual application of loading. In treadmill studies, osteocyte PMD increased with loading in weight bearing locations (tibia), but this trend was not seen in non-weight bearing locations (skull). PMD initiated osteocyte mechanotransduction including calcium signaling and expression of c-fos, and repair rates of these PMD could be enhanced or inhibited pharmacologically to alter downstream mechanotransduction and osteocyte survival. PMD may represent a novel mechanosensation pathway in bone and a target for modifying skeletal adaptation signaling in osteocytes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:653-662, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  19. 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.


    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

  20. Glucocorticoid receptor localizes to adherens junctions at the plasma membrane of keratinocytes.

    Directory of Open Access Journals (Sweden)

    Olivera Stojadinovic

    Full Text Available Glucocorticoids are important regulators of epidermal tissue homeostasis. As such, their clinical applications are widespread, ranging from inflammatory skin disorders to keloids and cancer. Glucocorticoids exert their effect by binding to glucocorticoid receptor (GR which translocates to the nucleus and regulates gene expression (genomic effect. In addition, GR has rapid non- genomic effects that are mediated by cell signaling proteins and do not involve gene transcription. Although genomic effects of GR in the epidermis are well documented, the non-genomic effects are not completely understood. Therefore, we utilized immunostaining and immunoprecipitations to determine specific localization of the GR in human keratinocytes that may contribute to non-genomic effects of glucocorticoid action. Here we describe a novel finding of GR localization to the plasma membrane of keratinocytes. Immunocytochemistry showed co-localization of GR with α-catenin. Immunoprecipitation of the membranous fraction revealed an association of GR with α-catenin, confirming its localization to adherens junctions. We conclude that GR localization to adherens junctions of keratinocytes provides a new mechanism of non-genomic signaling by glucocorticoids which may have significant biological and clinical impact.

  1. Preferential transfer of certain plasma membrane proteins onto T and B cells by trogocytosis.

    Directory of Open Access Journals (Sweden)

    Sandrine Daubeuf

    Full Text Available T and B cells capture antigens via membrane fragments of antigen presenting cells (APC in a process termed trogocytosis. Whether (and how a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM. Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.

  2. Immobilization of Na,K-ATPase isolated from rat brain synaptic plasma membranes

    Directory of Open Access Journals (Sweden)



    Full Text Available Rat brain Na,K-ATPase partially purified by SDS from synaptic plasma membranes (SPM was immobilized by adsorption on nitrocellulose (NC, polyvinylidene fluoride (PVDF and glass fiber (GF membranes. Partial SDS solubilization increased the enzyme activity by 40 %. With regard to the preservation of the enzyme activity, nitrocellulose was shown to be the optimal support for the immobilization. The enzyme showed the highest percentage activity (14 % after 30 min of SPM adsorption, at 20°C under the vaccum, with 25 mg of proteins per NC disc filter. In addition, adsorption on NC stabilizes the Na,K-ATPase, since the activity was substantial 72 h after adsorption at 20°C. After adsorption, the sensitivity of the enzyme to HgCl2and CdCll2 inhibition was higher. The results show that immobilized Na,K-ATPase SPM can be used as a practical model for the detection of metal ions in different samples.

  3. Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry (United States)

    Roelants, Françoise M.; Su, Brooke M.; von Wulffen, Joachim; Ramachandran, Subramaniam; Sartorel, Elodie; Trott, Amy E.


    Plasma membrane function requires distinct leaflet lipid compositions. Two of the P-type ATPases (flippases) in yeast, Dnf1 and Dnf2, translocate aminoglycerophospholipids from the outer to the inner leaflet, stimulated via phosphorylation by cortically localized protein kinase Fpk1. By monitoring Fpk1 activity in vivo, we found that Fpk1 was hyperactive in cells lacking Gin4, a protein kinase previously implicated in septin collar assembly. Gin4 colocalized with Fpk1 at the cortical site of future bud emergence and phosphorylated Fpk1 at multiple sites, which we mapped. As judged by biochemical and phenotypic criteria, a mutant (Fpk111A), in which 11 sites were mutated to Ala, was hyperactive, causing increased inward transport of phosphatidylethanolamine. Thus, Gin4 is a negative regulator of Fpk1 and therefore an indirect negative regulator of flippase function. Moreover, we found that decreasing flippase function rescued the growth deficiency of four different cytokinesis mutants, which suggests that the primary function of Gin4 is highly localized control of membrane lipid asymmetry and is necessary for optimal cytokinesis. PMID:25646086

  4. The plasma membrane proteome of maize roots grown under low and high iron conditions. (United States)

    Hopff, David; Wienkoop, Stefanie; Lüthje, Sabine


    Iron (Fe) homeostasis is essential for life and has been intensively investigated for dicots, while our knowledge for species in the Poaceae is fragmentary. This study presents the first proteome analysis (LC-MS/MS) of plasma membranes isolated from roots of 18-day old maize (Zea mays L.). Plants were grown under low and high Fe conditions in hydroponic culture. In total, 227 proteins were identified in control plants, whereas 204 proteins were identified in Fe deficient plants and 251 proteins in plants grown under high Fe conditions. Proteins were sorted by functional classes, and most of the identified proteins were classified as signaling proteins. A significant number of PM-bound redox proteins could be identified including quinone reductases, heme and copper-containing proteins. Most of these components were constitutive, and others could hint at an involvement of redox signaling and redox homeostasis by change in abundance. Energy metabolism and translation seem to be crucial in Fe homeostasis. The response to Fe deficiency includes proteins involved in development, whereas membrane remodeling and assembly and/or repair of Fe-S clusters is discussed for Fe toxicity. The general stress response appears to involve proteins related to oxidative stress, growth regulation, an increased rigidity and synthesis of cell walls and adaption of nutrient uptake and/or translocation. This article is part of a Special Issue entitled: Plant Proteomics in Europe. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis (United States)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.


    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  6. Membrane potential of cells and its regulation during aging. 2. Report: the effect of hormones on the level of the cellular plasma membrane polarization. (United States)

    Frolkis, V V; Tanin, S A; Gorban, E N; Bogatskaya, L N; Sabko, V E


    Age-dependent changes in the polarization of plasma membranes (PM) of various cell types and the mechanisms responsible for its regulation were studied in the experiments on the adult (6-8 and old (28-32 months) Wistar male rats. It was found that the effect of the hormones on the PM polarization level is altered during aging. This being related to shifts in the number and affinity of the hormonal receptors, energetic processes and protein synthesis in the cell.

  7. Single-molecule imaging technique to study the dynamic regulation of GPCR function at the plasma membrane

    NARCIS (Netherlands)

    Snaar-Jagalska, B.E.; Cambi, A.; Schmidt, T.; de Keijzer, S.


    The lateral diffusion of a G-protein-coupled receptor (GPCR) in the plasma membrane determines its interaction capabilities with downstream signaling molecules and critically modulates its function. Mechanisms that control GPCR mobility, like compartmentalization, enable a cell to fine-tune its

  8. Protein 90 Recognized as an Iron-Binding Protein Associated with the Plasma Membrane of HeLa Cells

    Czech Academy of Sciences Publication Activity Database

    Kovář, Jan; Štýbrová, Hana; Novák, J.; Ehrlichová, Marie; Truksa, Jaroslav; Koc, Michal; Kriegerbecková, Karin; Scheiber-Mojdehkar, B.; Goldenberg, H.

    1-2, č. 14 (2004), s. 41-46 ISSN 1015-8987 R&D Projects: GA AV ČR IAA5052702; GA ČR GA301/01/0041 Keywords : heat shock protein 90 * iron -binding protein * plasma membrane Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.093, year: 2004

  9. Plasma membrane calcium ATPases are important components of receptor-mediated signaling in plant immune responses and development.

    NARCIS (Netherlands)

    Frei dit Frey, N.; Mbengue, M.; Kwaaitaal, M.A.C.J.; Nitsch, L.M.C.; Altenbach, D.; Häweker, H.; Lozano-Duran, R.; Njo, M.F.; Beeckman, T.; Huettel, B.; Borst, J.W.; Panstruga, R.; Robatzek, S.


    Plasma membrane-resident receptor kinases (RKs) initiate signaling pathways important for plant immunity and development. In Arabidopsis (Arabidopsis thaliana), the receptor for the elicitor-active peptide epitope of bacterial flagellin, flg22, is encoded by FLAGELLIN SENSING2 (FLS2), which promotes

  10. CHX14 is a plasma membrane K-efflux transporter that regulates K+ redistribution in "Arabidopsis thaliana" (United States)

    Potassium (K(+)) is essential for plant growth and development, yet the molecular identity of many K(+) transporters remains elusive. Here we characterized cation/H(+) exchanger (CHX) 14 as a plasma membrane K(+) transporter. "CHX14" expression was induced by elevated K(+) and histochemical analysis...

  11. Bipolar Plasma Membrane Distribution of Phosphoinositides and Their Requirement for Auxin-Mediated Cell Polarity and Patterning in Arabidopsis

    NARCIS (Netherlands)

    Tejos, R.; Sauer, M.; Vanneste, S.; Palacios-Gomez, M.; Li, H.; Heilmann, M.; van Wijk, R.; Vermeer, J.E.M.; Heilmann, I.; Munnik, T.; Friml, J.


    Cell polarity manifested by asymmetric distribution of cargoes, such as receptors and transporters, within the plasma membrane (PM) is crucial for essential functions in multicellular organisms. In plants, cell polarity (re)establishment is intimately linked to patterning processes. Despite the

  12. Super-resolution microscopy reveals the insulin-resistance-regulated reorganization of GLUT4 on plasma membranes. (United States)

    Gao, Lan; Chen, Junling; Gao, Jing; Wang, Hongda; Xiong, Wenyong


    GLUT4 (also known as SLC2A4) is essential for glucose uptake in skeletal muscles and adipocytes, which play central roles in whole-body glucose metabolism. Here, using direct stochastic optical reconstruction microscopy (dSTORM) to investigate the characteristics of plasma-membrane-fused GLUT4 at the single-molecule level, we have demonstrated that insulin and insulin resistance regulate the spatial organization of GLUT4 in adipocytes. Stimulation with insulin shifted the balance of GLUT4 on the plasma membrane toward a more dispersed configuration. In contrast, insulin resistance induced a more clustered distribution of GLUT4 and increased the mean number of molecules per cluster. Furthermore, our data demonstrate that the F 5 QQI motif and lipid rafts mediate the maintenance of GLUT4 clusters on the plasma membrane. Mutation of F 5 QQI (F 5 QQA-GLUT4) induced a more clustered distribution of GLUT4; moreover, destruction of lipid rafts in adipocytes expressing F 5 QQA-GLUT4 dramatically decreased the percentage of large clusters and the mean number of molecules per cluster. In conclusion, our data clarify the effects of insulin stimulation or insulin resistance on GLUT4 reorganization on the plasma membrane and reveal new pathogenic mechanisms of insulin resistance. © 2017. Published by The Company of Biologists Ltd.

  13. Plasma Membrane Proteomics of Arabidopsis Suspension-Cultured Cells Associated with Growth Phase Using Nano-LC-MS/MS. (United States)

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


    Arabidopsis thaliana suspension-cultured cells (T87 line) are important model system for studies of responses to biotic and abiotic stresses at the cellular level in vitro since the cells have certain advantages compared with the whole plant system. However, the physiological and morphological characteristics of the cells are influenced by the progress of the growth phase of cells, which may result in different stress tolerance. To obtain comprehensive proteome profiles of the plasma membrane of Arabidopsis thaliana T87 suspension-cultured cells at the lag, log, or stationary growth phase, a shotgun proteomics method using nano-LC-MS/MS is used. The results obtained indicate that proteome profiles of the plasma membrane with the progress of the growth phase of cells dynamically changed, which may be associated with the physiological and morphological characteristics of the plasma membrane of the suspension-cultured cells. The proteomics results are further applied to explain different responsive patterns in the plasma membrane to cold acclimation and ABA treatment, which lead to understanding of different freezing tolerance associated with the growth phase of the cells.

  14. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    Energy Technology Data Exchange (ETDEWEB)

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S. (Mount Sinai School of Medicine, New York, NY (USA)); Wada, H.; Horio, Y. (Univ. of Osaka (Japan))


    The hepatic plasma membrane fatty acid-binding protein (h-FABP{sub PM}) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABP{sub PM} have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP{sub PM} reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of ({sup 3}H)oleate but not that of ({sup 35}S)sulfobromophthalein or ({sup 14}C)taurocholate. The inhibition of oleate uptake produced by anti-h-FABP{sub PM} can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABP{sub PM} and mGOT are closely related.

  15. Cellular processes and pathways that protect Saccharomyces cerevisiae cells against the plasma membrane-perturbing compound chitosan.

    NARCIS (Netherlands)

    Zakrzewska, A.M.; Boorsma, A.; Delneri, D.; Brul, S.; Oliver, S.G.; Klis, F.M.


    Global fitness analysis makes use of a genomic library of tagged deletion strains. We used this approach to study the effect of chitosan, which causes plasma membrane stress. The data were analyzed using T-profiler, which was based on determining the sensitivities of groups of deletion strains to

  16. Cloning of a cDNA encoding a developmentally regulated 22 kDa polypeptide from tobacco leaf plasma membrane

    NARCIS (Netherlands)

    Gantet, P; Masson, F; Domergue, O; MarquisMention, M; Bauw, G; Inze, D; Rossignol, M; delaServe, BT


    A polypeptide doublet (P18-P19, ca 22 kDa, pI 4.5) has been shown to accumulate in tobacco leaf plasma membrane in a development-dependent way, under constant environmental conditions. P18 and P19 were purified by 2D-PAGE and microsequenced. Microsequences revealed only small differences between the

  17. Regulation of mitogen-activated protein kinase pathways by the plasma membrane Na+/H+ exchanger, NHE1

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Darborg, Barbara Vasek; Rentsch, Maria Louise


    activity is regulated by a three-tiered phosphorelay system, which is in turn regulated by a complex network of signaling events and scaffolding proteins. The ubiquitous plasma membrane Na(+)/H(+) exchanger NHE1 is activated by, and implicated in, the physiological/pathophysiological responses to many...

  18. Lateral mobility of plasma membrane lipids in a molluscan egg: Evidence for an animal/vegetal polarity

    NARCIS (Netherlands)

    Laat, S.W. de; Speksnijder, J.E.; Dohmen, M.R.; Zoelen, E. van; Tertoolen, L.G.J.; Bluemink, J.G.


    The lateral diffusion of the lipid analog C₁₄-diI (3', 3'-dihexadecylindocarbocyanine iodide) was measured in the plasma membrane of early embryos of the mollusc Nassarius reticulatus using the FPR-(Fluorescence Photobleaching Recovery) method. At almost all stages measured (from

  19. Estimation of the Asymmetrical Arrangement of Plasma Membrane Aminophospholipids. An Experimental Assay for Students of Biochemistry and Molecular Biology. (United States)

    Sanchez-Yague, J.; And Others


    Describes an experiment to discover the topology of plasma membrane aminophospholipids (phosphatidylethanolamine and phosphatidylserine) using whole platelets and trinitrobenzene sulfonate (TNBS) as a probe. Reports changes in phospholipid distribution during platelet activation with simultaneous action of thrombia and collagen. Details the…

  20. Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.

    NARCIS (Netherlands)

    Gutierrez, R.; Lindeboom, J.J.; Paredez, A.R.; Emons, A.M.C.; Ehrhardt, D.W.


    Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. Studies using in vivo markers confirmed that one function of the cortical microtubule array is

  1. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe


    Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary tran......(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps....... transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na(+),K......Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary...

  2. ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Y.C.; Liu, C.


    Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.

  3. Two subunits of the exocyst, Sec3p and Exo70p, can function exclusively on the plasma membrane. (United States)

    Liu, Dongmei; Li, Xia; Shen, David; Novick, Peter


    The exocyst is an octameric complex that tethers secretory vesicles to the plasma membrane in preparation for fusion. We anchored each subunit with a transmembrane (TM) domain at its N- or C-terminus. Only N-terminally anchored TM-Sec3p and C-terminally anchored Exo70p-TM proved functional. These findings orient the complex with respect to the membrane and establish that Sec3p and Exo70p can function exclusively on the membrane. The functions of TM-Sec3p and Exo70p-TM were largely unaffected by blocks in endocytic recycling suggesting that they act on the plasma membrane, rather than on secretory vesicles. Cytosolic pools of the other exocyst subunits were unaffected in TM-sec3 cells, while they were partially depleted in exo70-TM cells. Blocking actin-dependent delivery of secretory vesicles in act1-3 cells results in loss of Sec3p from the purified complex. Our results are consistent with a model in which Sec3p and Exo70p can function exclusively on the plasma membrane while the other subunits are brought to them on secretory vesicles. © 2018 by The American Society for Cell Biology.

  4. Membrane-based therapeutic plasma exchange (mTPE): Technical and clinical experience. (United States)

    Gashti, Casey N; Andreoli, Daniel C; Patel, Dipal


    Therapeutic plasma exchange (TPE) has long been utilized to manage a variety of immune-mediated diseases. The basic principle relies on removal of circulating pathogenic substances from the bloodstream. Methods of plasma separation include centrifuge (cTPE) and membrane (mTPE). Although mTPE has existed for a few decades, recent advances in developing highly permeable filters that are compatible with currently existing dialysis machines has opened a new frontier. Published data in the area of technical and clinical experience with mTPE is lacking. We report our single center experience of 998 inpatient mTPE treatments performed in 237 patients at a large tertiary care academic center. The most common treatment indication was neurologic. We found a very low incidence of patient-reported complications. Filter clotting without the use of anticoagulation occurred in 7.7% of treatments. Laboratory parameters that significantly changed during the course of therapy included serum potassium, platelet count, and partial thromboplastin time. We found that mTPE can be safely and efficiently performed as an alternative to cTPE, and suggest an individualized approach when prescribing this therapy. © 2017 Wiley Periodicals, Inc.

  5. Effects of wheat germ agglutinin on tunicate egg activation and fertilization: is there a plasma membrane sperm receptor system on Ascidia ceratodes eggs? (United States)

    Flannery, B; Epel, D


    Little work has been carried out on the sperm recognition systems present on the egg plasma membrane. Here it is shown that wheat germ agglutinin (WGA) interferes with the sperm-interacting system on the plasma membrane of eggs of the ascidian, Ascidia ceratodes. The WGA activates the dechorionated egg, indicating that a plasma membrane sugar residue can be directly tied to egg activation. Low concentrations of this lectin do not activate the eggs, but reduce fertilizability. This observation suggests that the WGA binding site might be part of a sperm reception-activation complex in the plasma membrane. While WGA also affects sperm binding to the chorion, the mechanisms of sperm interaction at the plasma membrane and chorion show different sensitivities to lectins, sugars and enzymes.

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

    Raza Shaikh, Saame; Brown, David A


    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.

  7. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe


    transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na(+),K(+)-ATPase...... maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps.......Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary...

  8. Enhancement in biological response of Ag-nano composite polymer membranes using plasma treatment for fabrication of efficient bio materials (United States)

    Agrawal, Narendra Kumar; Sharma, Tamanna Kumari; Chauhan, Manish; Agarwal, Ravi; Vijay, Y. K.; Swami, K. C.


    Biomaterials are nonviable material used in medical devices, intended to interact with biological systems, which are becoming necessary for the development of artificial material for biological systems such as artificial skin diaphragm, valves for heart and kidney, lenses for eye etc. Polymers having novel properties like antibacterial, antimicrobial, high adhesion, blood compatibility and wettability are most suitable for synthesis of biomaterial, but all of these properties does not exist in any natural or artificial polymeric material. Nano particles and plasma treatment can offer these properties to the polymers. Hence a new nano-biomaterial has been developed by modifying the surface and chemical properties of Ag nanocomposite polymer membranes (NCPM) by Argon ion plasma treatment. These membranes were characterized using different techniques for surface and chemical modifications occurred. Bacterial adhesion and wettability were also tested for these membranes, to show direct use of this new class of nano-biomaterial for biomedical applications.

  9. Parallel artificial liquid membrane extraction of new psychoactive substances in plasma and whole blood. (United States)

    Vårdal, Linda; Askildsen, Hilde-Merete; Gjelstad, Astrid; Øiestad, Elisabeth Leere; Edvardsen, Hilde Marie Erøy; Pedersen-Bjergaard, Stig


    Parallel artificial liquid membrane extraction (PALME) was combined with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the potential for screening of new psychoactive substances (NPS) was investigated for the first time. PALME was performed in 96-well format comprising a donor plate, a supported liquid membrane (SLM), and an acceptor plate. Uncharged NPS were extracted from plasma or whole blood, across an organic SLM, and into an aqueous acceptor solution, facilitated by a pH gradient. MDAI (5,6-methylenedioxy-2-aminoindane), methylone, PFA (para-fluoroamphetamine), mCPP (meta-chlorophenylpiperazine), pentedrone, methoxetamine, MDPV (methylenedioxypyrovalerone), ethylphenidate, 2C-E (2,5-dimethoxy-4-ethylphenethylamine), bromo-dragonfly, and AH-7921 (3,4-dichloro-N-{[1-(dimethylamino)cyclohexyl]methyl}benzamide) were selected as representative NPS. Optimization of operational parameters was necessary as the NPS were novel to PALME, and because PALME was performed from whole blood for the very first time. In the PALME method developed for plasma, NPS were extracted from a 250μL alkalized donor solution consisting of 125μL plasma sample, 115μL 40mM NaOH, and 10μL internal standard. In the PALME method from whole blood, the 250μL alkalized donor solution consisted of 100μL whole blood, 50μL deionized water, 75μL 80mM NaOH, and 25μL internal standard. In both methods, extraction was accomplished across an SLM of 5μL dodecyl acetate with 1% trioctylamine (w/w), and further into an acidic acceptor solution of 50μL 20mM formic acid. The extraction was promoted by agitation at 900rpm and was carried out for 120min. Method validation was performed and the following parameters were considered: linearity, limits of quantification (LOQ), intra- and inter-day precision, accuracy, extraction recoveries, carry-over, and matrix effects. The validation results were in accordance with FDA guidelines. Copyright © 2017 Elsevier B.V. All

  10. Do 14-3-3 proteins and plasma membrane H+-ATPases interact in the barley epidermis in response to the barley powdery mildew fungus?

    DEFF Research Database (Denmark)

    Finnie, C.; Andersen, C.H.; Borch, J.


    , or treatment with fusicoccin, results in an increase in fusicoccin binding ability of barley leaf membranes. Overlay assays show a fungus-induced increase in binding of digoxygenin-labelled 14-3-3 protein to several proteins including a 100 kDa membrane protein, probably the plasma membrane H...

  11. Poly(hydroxyethyl methacrylate) based affinity membranes for in vitro removal of anti-dsDNA antibodies from SLE plasma. (United States)

    Uzun, Lokman; Yavuz, Handan; Osman, Bilgen; Celik, Hamdi; Denizli, Adil


    The preparation of polymeric membrane using affinity technology for application in blood filtration devices is described here. DNA attached poly(hydroxyethyl methacrylate) (PHEMA) based microporous affinity membrane was prepared for selective removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma in in vitro. In order to further increase blood-compatibility of affinity membrane, aminoacid based comonomer N-methacryloyl-L-alanine (MAAL) was included in the polymerization recipe. PHEMAAL membrane was produced by a photopolymerization technique and then characterized by swelling tests and scanning electron microscope (SEM) studies. Blood-compatibility tests were also performed. The water swelling ratio of PHEMAAL membrane increased significantly (133.2%) compared with PHEMA (58%). PHEMAAL membrane has large pores around in the range of 5-10 microm. All the clotting times increased when compared with PHEMA membrane. Loss of platelets and leukocytes was very low. DNA loading was 7.8 mg/g. There was a very low anti-dsDNA-antibody adsorption onto the plain PHEMAAL membrane, about 78 IU/g. The PHEMAAL-DNA membrane adsorbed anti-dsDNA-antibody in the range of 10-68 x 10(3)IU/g from SLE plasma. Anti-dsDNA-antibody concentration decreased significantly from 875 to 144 IU/ml with the time. Anti-dsDNA-antibodies could be repeatedly adsorbed and eluted without noticeable loss in the anti-dsDNA-antibody adsorption amount. (c) 2010 Elsevier B.V. All rights reserved.

  12. Key steps in type III secretion system (T3SS) towards translocon assembly with potential sensor at plant plasma membrane. (United States)

    Ji, Hongtao; Dong, Hansong


    Many plant- and animal-pathogenic Gram-negative bacteria employ the type III secretion system (T3SS) to translocate effector proteins from bacterial cells into the cytosol of eukaryotic host cells. The effector translocation occurs through an integral component of T3SS, the channel-like translocon, assembled by hydrophilic and hydrophobic proteinaceous translocators in a two-step process. In the first, hydrophilic translocators localize to the tip of a proteinaceous needle in animal pathogens, or a proteinaceous pilus in plant pathogens, and associate with hydrophobic translocators, which insert into host plasma membranes in the second step. However, the pilus needs to penetrate plant cell walls in advance. All hydrophilic translocators so far identified in plant pathogens are characteristic of harpins: T3SS accessory proteins containing a unitary hydrophilic domain or an additional enzymatic domain. Two-domain harpins carrying a pectate lyase domain potentially target plant cell walls and facilitate the penetration of the pectin-rich middle lamella by the bacterial pilus. One-domain harpins target plant plasma membranes and may play a crucial role in translocon assembly, which may also involve contrapuntal associations of hydrophobic translocators. In all cases, sensory components in the target plasma membrane are indispensable for the membrane recognition of translocators and the functionality of the translocon. The conjectural sensors point to membrane lipids and proteins, and a phosphatidic acid and an aquaporin are able to interact with selected harpin-type translocators. Interactions between translocators and their sensors at the target plasma membrane are assumed to be critical for translocon assembly. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  13. Modification of plasma membrane electron transport in cultured rose cells by UV-C radiation and fungal elicitor

    International Nuclear Information System (INIS)

    Murphy, T.M.; Auh, C.K.; Schorr, R.; Grobe, C.


    Previous experiments have shown that treatments of suspension-cultured cells of Rosa damascena Mill. with UV radiation or with fungal elicitors stimulates the synthesis of H 2 O 2 by the cells. To test the hypothesis that this synthesis involves reduction of O 2 at the plasma membrane and to identify the mechanism of the reduction, we have determined the effects of UV and elicitor on redox reactions associated with the plasma membrane. Elicitor prepared from cell walls of Phytophthora sp. (14 μg solids/ml) inhibited the reduction of ferricyanide by intact cells by 98%; UV-C (primarily 254 nm, up to 19,500 J/m 2 ) inhibited this reduction by 40%. Neither treatment inhibited the reduction of Fe(III)-EDTA by intact cells. Intact cells oxidized NADH in the absence of external oxidizing agent, and the rate of oxidation was increased by UV and elicitor. Cells that were poisoned with arsenite and CCCP catalyzed the reduction of Fe(III)-EDTA in the presence of external NADH, and this ability was slightly stimulated by UV and elicitor. UV irradiation (6,480 J/m 2 ) of cells resulted in a 27% inhibition of the specific activity of NADH-ferricyanide oxidoreductase in plasma membrane isolated from those cells. Elicitor treatment of cells for at least 90 min resulted in a 50% inhibition of the enzyme's specific activity in isolated plasma membrane; this inhibition was reversed by addition of Triton-X100 in the assay mixture. The results suggest that UV and elicitor alter the flow of electrons in the plasma membrane, reversibly inhibiting NADH-cytochrome b reductase, the putative key enzyme in the pathway of ferricyanide reduction, and stimulating (or at least not inhibiting) the pathway of Fe(III)-EDTA reduction

  14. Isobaric tags for relative and absolute quantification- based comparative proteomics reveals the features of plasma membrane-associated proteomes of pollen grains and pollen tubes from Lilium davidii. (United States)

    Han, Bing; Chen, Sixue; Dai, Shaojun; Yang, Ning; Wang, Tai


    Mature pollen grains (PGs) from most plant species are metabolically quiescent. However, once pollinated onto stigma, they quickly hydrate and germinate. A PG can give rise to a vegetative cell-derived polarized pollen tube (PT), which represents a specialized polar cell. The polarized PT grows by the tip and requires interaction of different signaling molecules localized in the apical plasma membrane and active membrane trafficking. The mechanisms underlying the interaction and membrane trafficking are not well understood. In this work, we purified PG and PT plasma-membrane vesicles from Lilium davidii Duch. using the aqueous two-phase partition technique, then enriched plasma membrane proteins by using Brij58 and KCl to remove loosely bound contaminants. We identified 223 integral and membrane-associated proteins in the plasma membrane of PGs and PTs by using isobaric tags for relative and absolute quantification (iTRAQ) and 2-D high-performance liquid chromatography-tandem mass spectrometry. More than 68% of the proteins have putative transmembrane domains and/or lipid-modified motifs. Proteins involved in signal transduction, membrane trafficking and transport are predominant in the plasma-membrane proteome. We revealed most components of the clathrin-dependent endocytosis pathway. Statistical analysis revealed 14 proteins differentially expressed in the two development stages: in PTs, six upregulated and eight downregulated are mainly involved in signaling, transport and membrane trafficking. These results provide novel insights into polarized PT growth. © 2010 Institute of Botany, Chinese Academy of Sciences.

  15. Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes. (United States)

    Stone, Matthew B; Shelby, Sarah A; Núñez, Marcos F; Wisser, Kathleen; Veatch, Sarah L


    Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

  16. Detergent-dependent separation of postsynaptic density, membrane rafts and other subsynaptic structures from the synaptic plasma membrane of rat forebrain. (United States)

    Zhao, LiYing; Sakagami, Hiroyuki; Suzuki, Tatsuo


    We systematically investigated the purification process of post-synaptic density (PSD) and post-synaptic membrane rafts (PSRs) from the rat forebrain synaptic plasma membranes by examining the components and the structures of the materials obtained after the treatment of synaptic plasma membranes with TX-100, n-octyl β-d-glucoside (OG) or 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO). These three detergents exhibited distinct separation profiles for the synaptic subdomains. Type I and type II PSD proteins displayed mutually exclusive distribution. After TX-100 treatment, type I PSD was recovered in two fractions: a pellet and an insoluble fraction 8, which contained partially broken PSD-PSR complexes. Conventional PSD was suggested to be a mixture of these two PSD pools and did not contain type II PSD. An association of type I PSD with PSRs was identified in the TX-100 treatment, and those with type II PSD in the OG and CHAPSO treatments. An association of GABA receptors with gephyrin was easily dissociated. OG at a high concentration solubilized the type I PSD proteins. CHAPSO treatment resulted in a variety of distinct fractions, which contained certain novel structures. Two different pools of GluA, either PSD or possibly raft-associated, were identified in the OG and CHAPSO treatments. These results are useful in advancing our understanding of the structural organization of synapses at the molecular level. We systematically investigated the purification process of post-synaptic density (PSD) and synaptic membrane rafts by examining the structures obtained after treatment of the SPMs with TX-100, n-octyl β-d-glucoside or CHAPSO. Differential distribution of type I and type II PSD, synaptic membrane rafts, and other novel subdomains in the SPM give clues to understand the structural organization of synapses at the molecular level. © 2014 International Society for Neurochemistry.

  17. Mini-Scale Isolation and Preparation of Plasma Membrane Proteins from Potato Roots for LC/MS Analysis. (United States)

    Jozefowicz, Anna M; Matros, Andrea; Witzel, Katja; Mock, Hans-Peter


    Plasma membrane (PM) proteins are of special interest due to their function in exchanging material and information with the external environment as well as their role in cellular regulation. In quantitative proteomic studies PM proteins are underrepresented mostly because they constitute only small percent of all membrane proteins. Strong demand is placed on plasma membrane enrichment methods. For decades two-phase partitioning Dextran T500/PEG 3350 isolation protocols were applied for many different animal and plant species and also a variety of tissue types. The typical quantity of material used in the enrichment protocols is 10-30 g of fresh weight. The main difficulty of working with in vitro cultivated plants is the low amount of material, especially when roots are examined. In addition, roots are frequently characterized by low protein concentrations. Our protocol established for roots of in vitro cultivated potato plants is adjusted to amounts of fresh weight not exceeding 7.5 g and allows studying the plasma membrane proteome by LC-MS.

  18. Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.

    LENUS (Irish Health Repository)

    Knodler, Leigh A


    The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K(6)R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt\\/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K(6)R mutant. At later times, fewer SCV were decorated with SopB-K(6)R compared with SopB. Instead SopB-K(6)R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.

  19. Live cell plasma membranes do not exhibit a miscibility phase transition over a wide range of temperatures. (United States)

    Lee, Il-Hyung; Saha, Suvrajit; Polley, Anirban; Huang, Hector; Mayor, Satyajit; Rao, Madan; Groves, Jay T


    Lipid/cholesterol mixtures derived from cell membranes as well as their synthetic reconstitutions exhibit well-defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid/cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid-phase separation is not generally observed in cell membranes, and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown. A fundamental property of phase transitions is that the variation of tagged particle diffusion with temperature exhibits an abrupt change as the system passes through the transition, even when the two phases are distributed in a nanometer-scale emulsion. We support this using a variety of Monte Carlo and atomistic simulations on model lipid membrane systems. However, temperature-dependent fluorescence correlation spectroscopy of labeled lipids and membrane-anchored proteins in live cell membranes shows a consistently smooth increase in the diffusion coefficient as a function of temperature. We find no evidence of a discrete miscibility phase transition throughout a wide range of temperatures: 14-37 °C. This contrasts the behavior of giant plasma membrane vesicles (GPMVs) blebbed from the same cells, which do exhibit phase transitions and macroscopic phase separation. Fluorescence lifetime analysis of a DiI probe in both cases reveals a significant environmental difference between the live cell and the GPMV. Taken together, these data suggest the live cell membrane may avoid the miscibility phase transition inherent to its lipid constituents by actively regulating physical parameters, such as tension, in the membrane.

  20. Calcium-regulated anion channels in the plasma membrane of Lilium longiflorum pollen protoplasts. (United States)

    Tavares, Bárbara; Dias, Pedro Nuno; Domingos, Patrícia; Moura, Teresa Fonseca; Feijó, José Alberto; Bicho, Ana


    • Currents through anion channels in the plasma membrane of Lilium longiflorum pollen grain protoplasts were studied under conditions of symmetrical anionic concentrations by means of patch-clamp whole-cell configuration. • With Cl(-) -based intra- and extracellular solutions, three outward-rectifying anion conductances, I(Cl1) , I(Cl2) and I(Cl3) , were identified. These three activities were discriminated by differential rundown behaviour and sensitivity to 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), which could not be attributed to one or more channel types. All shared strong outward rectification, activated instantaneously and displayed a slow time-dependent activation for positive potentials. All showed modulation by intracellular calcium ([Ca(2+) ](in) ), increasing intensity from 6.04 nM up to 0.5 mM (I(Cl1) ), or reaching a maximum value with 8.50 μM (I(Cl2) and I(Cl3) ). • After rundown, the anionic currents measured using NO(3) (-) -based solutions were indistinguishable, indicating that the permeabilities of the channels for Cl(-) and NO(3) (-) are similar. Additionally, unitary anionic currents were measured from outside-out excised patches, confirming the presence of individual anionic channels. • This study shows for the first time the presence of a large anionic conductance across the membrane of pollen protoplasts, resulting from the presence of Ca(2+) -regulated channels. A similar conductance was also found in germinated pollen. We hypothesize that these putative channels may be responsible for the large anionic fluxes previously detected by means of self-referencing vibrating probes. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  1. Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane. (United States)

    Solanko, Lukasz M; Sullivan, David P; Sere, Yves Y; Szomek, Maria; Lunding, Anita; Solanko, Katarzyna A; Pizovic, Azra; Stanchev, Lyubomir D; Pomorski, Thomas Günther; Menon, Anant K; Wüstner, Daniel


    Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that membrane-impermeant collisional quenchers (spin-labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass-bead lysis or repeated freeze-thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis. (United States)

    Aloui, Achref; Recorbet, Ghislaine; Lemaître-Guillier, Christelle; Mounier, Arnaud; Balliau, Thierry; Zivy, Michel; Wipf, Daniel; Dumas-Gaudot, Eliane


    In arbuscular mycorrhizal (AM) roots, the plasma membrane (PM) of the host plant is involved in all developmental stages of the symbiotic interaction, from initial recognition to intracellular accommodation of intra-radical hyphae and arbuscules. Although the role of the PM as the agent for cellular morphogenesis and nutrient exchange is especially accentuated in endosymbiosis, very little is known regarding the PM protein composition of mycorrhizal roots. To obtain a global overview at the proteome level of the host PM proteins as modified by symbiosis, we performed a comparative protein profiling of PM fractions from Medicago truncatula roots either inoculated or not with the AM fungus Rhizophagus irregularis. PM proteins were isolated from root microsomes using an optimized discontinuous sucrose gradient; their subsequent analysis by liquid chromatography followed by mass spectrometry (MS) identified 674 proteins. Cross-species sequence homology searches combined with MS-based quantification clearly confirmed enrichment in PM-associated proteins and depletion of major microsomal contaminants. Changes in protein amounts between the PM proteomes of mycorrhizal and non-mycorrhizal roots were monitored further by spectral counting. This workflow identified a set of 82