Science.gov

Sample records for increased plasma membrane

  1. Adipocyte cell size enlargement involves plasma membrane area increase.

    PubMed

    Chowdhury, H H; Zorec, R

    2012-07-01

    The adipocyte enlargement is associated with an increase in the cytoplasmic lipid content, but how the plasma membrane area follows this increase is poorly understood. We monitored single-cell membrane surface area fluctuations, which mirror the dynamics of exocytosis and endocytosis. We employed the patch-clamp technique to measure membrane capacitance (C(m)), a parameter linearly related to the plasma membrane area. Specifically, we studied whether insulin affects membrane area dynamics in adipocytes. A five-minute cell exposure to insulin increased resting C(m) by 12 ± 4%; in controls the change in C(m) was not different from zero. We measured cell diameter of isolated rat adipocytes microscopically. Twenty-four hour exposure of cells to insulin resulted in a significant increase in cell diameter by 5.1 ± 0.6%. We conclude that insulin induces membrane area increase, which may in chronic hyperinsulinemia promote the enlargement of plasma membrane area, acting in concert with other insulin-mediated metabolic effects on adipocytes. PMID:22540353

  2. Reducing Plasma Membrane Sphingomyelin Increases Insulin Sensitivity ▿

    PubMed Central

    Li, Zhiqiang; Zhang, Hongqi; Liu, Jing; Liang, Chien-Ping; Li, Yan; Li, Yue; Teitelman, Gladys; Beyer, Thomas; Bui, Hai H.; Peake, David A.; Zhang, Youyan; Sanders, Phillip E.; Kuo, Ming-Shang; Park, Tae-Sik; Cao, Guoqing; Jiang, Xian-Cheng

    2011-01-01

    It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency- or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity. PMID:21844222

  3. Elevated cAMP increases aquaporin-3 plasma membrane diffusion.

    PubMed

    Marlar, Saw; Arnspang, Eva C; Koffman, Jennifer S; Løcke, Else-Merete; Christensen, Birgitte M; Nejsum, Lene N

    2014-03-15

    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 exits via basolateral AQP3 and AQP4. Upon long-term stimulation with AVP or during thirst, expression levels of both AQP2 and AQP3 are increased; however, there is so far no evidence for short-term AVP regulation of AQP3 or AQP4. To facilitate the increase in transepithelial water transport, AQP3 may 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 if AQP3 could be short-term regulated by AVP. k-Space image correlation spectroscopy (kICS) analysis of time-lapse image sequences of basolateral enhanced green fluorescent protein-tagged AQP3 (AQP3-EGFP) revealed that the forskolin-mediated elevation of cAMP increased the diffusion coefficient by 58% from 0.0147 ± 0.0082 μm(2)/s (control) to 0.0232 ± 0.0085 μm(2)/s (forskolin, P < 0.05). Quantum dot-conjugated antibody labeling also revealed a significant increase in AQP3 diffusion upon forskolin treatment by 44% [0.0104 ± 0.0040 μm(2)/s (control) vs. 0.0150 ± 0.0016 μm(2)/s (forskolin, P < 0.05)]. Immunoelectron microscopy showed no obvious difference in AQP3-EGFP expression levels or localization in the plasma membrane upon forskolin stimulation. Thus AQP3-EGFP diffusion is altered upon increased cAMP, which may correspond to basolateral adaptations in response to the increased apical water readsorption. PMID:24452376

  4. Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate.

    PubMed

    Tang, Xiaoyun; Zhao, Yuan Y; Dewald, Jay; Curtis, Jonathan M; Brindley, David N

    2016-04-01

    Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 μg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs. PMID:26884614

  5. Hypoxia directly increases serotonin transport by porcine pulmonary artery endothelial cell (PAEC) plasma membrane vesicles

    SciTech Connect

    Bhat, G.B.; Block, E.R. )

    1990-02-26

    Alterations in the physical state and composition of membrane lipids have been shown to interfere with a number of critical cellular and membrane functions including transmembrane transport. The authors have reported that hypoxia has profound effects upon the physical state and lipid composition of the PAEC plasma membrane bilayer and have suggested that this is responsible for increased serotonin uptake by these cells. In order to determine whether hypoxia has a direct effect on the plasma membrane transport of serotonin, they measured serotonin transport activity (1) in plasma membrane vesicles isolated from normoxic (20% O{sub 2}-5% CO{sub 2}) and hypoxic (0% O{sub 2}-5% CO{sub 2}) PAEC and (2) in PAEC plasma membrane vesicles that were exposed directly to normoxia or hypoxia. A 24-h exposure of PAEC to hypoxia resulted in a 40% increase in specific serotonin transport by plasma membrane vesicles derived from these cells. When plasma membrane vesicles were isolated and then directly exposed to normoxia or hypoxia for 1 h at 37C, a 31% increase in specific 5-HT transport was observed in hypoxic vesicles. Hypoxia did not alter the Km of serotonin transport (normoxia = 3.47 {mu}M versus hypoxia = 3.76 {mu}M) but markedly increased the maximal rate of transport (V{sup max}) (normoxia = 202.4 pmol/min/mg protein versus hypoxia = 317.9 pmol/min/mg protein). These results indicate that hypoxia increases serotonin transport in PAEC by a direct effect on the plasma membrane leading to an increase in the effective number of transporter molecules without alteration in transporter affinity for serotonin.

  6. Filamin-A Increases the Stability and Plasma Membrane Expression of Polycystin-2

    PubMed Central

    Wang, Qian; Zheng, Wang; Wang, Zuocheng; Yang, JungWoo; Hussein, Shaimaa; Tang, Jingfeng; Chen, Xing-Zhen

    2015-01-01

    Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in ~15% of autosomal dominant polycystic kidney disease. Filamins are actin-binding proteins implicated in scaffolding and membrane stabilization. Here we studied the effects of filamin on PC2 stability using filamin-deficient human melanoma M2, filamin-A (FLNA)-replete A7, HEK293 and IMCD cells together with FLNA siRNA/shRNA knockdown (KD). We found that the presence of FLNA is associated with higher total and plasma membrane PC2 protein expression. Western blotting analysis in combination with FLNA KD showed that FLNA in A7 cells represses PC2 degradation, prolonging the half-life from 2.3 to 4.4 hours. By co-immunoprecipitation and Far Western blotting we found that the FLNA C-terminus (FLNAC) reduces the FLNA-PC2 binding and PC2 expression, presumably through competing with FLNA for binding PC2. We further found that FLNA mediates PC2 binding with actin through forming complex PC2-FLNA-actin. FLNAC acted as a blocking peptide and disrupted the link of PC2 with actin through disrupting the PC2-FLNA-actin complex. Finally, we demonstrated that the physical interaction of PC2-FLNA is Ca-dependent. Taken together, our current study indicates that FLNA anchors PC2 to the actin cytoskeleton through complex PC2-FLNA-actin to reduce degradation and increase stability, and possibly regulate PC2 function in a Ca-dependent manner. PMID:25861040

  7. Filamin-a increases the stability and plasma membrane expression of polycystin-2.

    PubMed

    Wang, Qian; Zheng, Wang; Wang, Zuocheng; Yang, JungWoo; Hussein, Shaimaa; Tang, Jingfeng; Chen, Xing-Zhen

    2015-01-01

    Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in ~15% of autosomal dominant polycystic kidney disease. Filamins are actin-binding proteins implicated in scaffolding and membrane stabilization. Here we studied the effects of filamin on PC2 stability using filamin-deficient human melanoma M2, filamin-A (FLNA)-replete A7, HEK293 and IMCD cells together with FLNA siRNA/shRNA knockdown (KD). We found that the presence of FLNA is associated with higher total and plasma membrane PC2 protein expression. Western blotting analysis in combination with FLNA KD showed that FLNA in A7 cells represses PC2 degradation, prolonging the half-life from 2.3 to 4.4 hours. By co-immunoprecipitation and Far Western blotting we found that the FLNA C-terminus (FLNAC) reduces the FLNA-PC2 binding and PC2 expression, presumably through competing with FLNA for binding PC2. We further found that FLNA mediates PC2 binding with actin through forming complex PC2-FLNA-actin. FLNAC acted as a blocking peptide and disrupted the link of PC2 with actin through disrupting the PC2-FLNA-actin complex. Finally, we demonstrated that the physical interaction of PC2-FLNA is Ca-dependent. Taken together, our current study indicates that FLNA anchors PC2 to the actin cytoskeleton through complex PC2-FLNA-actin to reduce degradation and increase stability, and possibly regulate PC2 function in a Ca-dependent manner. PMID:25861040

  8. Painful nerve injury increases plasma membrane Ca2+-ATPase activity in axotomized sensory neurons

    PubMed Central

    2012-01-01

    Background The plasma membrane Ca2+-ATPase (PMCA) is the principal means by which sensory neurons expel Ca2+ and thereby regulate the concentration of cytoplasmic Ca2+ and the processes controlled by this critical second messenger. We have previously found that painful nerve injury decreases resting cytoplasmic Ca2+ levels and activity-induced cytoplasmic Ca2+ accumulation in axotomized sensory neurons. Here we examine the contribution of PMCA after nerve injury in a rat model of neuropathic pain. Results PMCA function was isolated in dissociated sensory neurons by blocking intracellular Ca2+ sequestration with thapsigargin, and cytoplasmic Ca2+ concentration was recorded with Fura-2 fluorometry. Compared to control neurons, the rate at which depolarization-induced Ca2+ transients resolved was increased in axotomized neurons after spinal nerve ligation, indicating accelerated PMCA function. Electrophysiological recordings showed that blockade of PMCA by vanadate prolonged the action potential afterhyperpolarization, and also decreased the rate at which neurons could fire repetitively. Conclusion We found that PMCA function is elevated in axotomized sensory neurons, which contributes to neuronal hyperexcitability. Accelerated PMCA function in the primary sensory neuron may contribute to the generation of neuropathic pain, and thus its modulation could provide a new pathway for peripheral treatment of post-traumatic neuropathic pain. PMID:22713297

  9. Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing.

    PubMed

    Torres, M A; Ravagnani, G M; Leal, D F; Martins, S M M K; Muro, B B D; Meirelles, F V; Papa, F O; Dell'aqua, J A; Alvarenga, M A; Moretti, A S; De Andrade, A F C

    2016-05-01

    Boar spermatozoa arising from the sperm-rich ejaculate fraction are reported to have a more stable plasma membrane and are more resistant to cold shock and premature acrosome reaction than spermatozoa from the whole ejaculate. Furthermore, seminal plasma (SP) can increase the cryotolerance of boar spermatozoa, and in other domestic species, it has the ability to reverse cryopreservation damage. This study aimed to evaluate the effects of boar SP arising from the whole sperm-rich ejaculate fraction (SP-SRF) on the integrity, stability, and peroxidation of sperm membranes after thawing. Each ejaculate ( = 24) was divided among 4 treatments: control (CT), centrifuged and suspended in autologous SP-SRF (CS), centrifuged with withdrawn SP-SRF (CW), and post-thawed SP arising from the whole sperm-rich fraction addition to CW (CWSP). After thawing, all treatments were incubated for 5, 60, and 120 min and were analyzed for membrane integrity, fluidity, and peroxidation by flow cytometer. The absence of SP-SRF increased the lipid disorder ( < 0.05) but had no effect on lipid peroxidation ( > 0.05) or membrane integrity ( > 0.05). However, the increase in lipid disorder by withdrawal of SP-SRF was reversed by SP-SRF addition ( < 0.05) to the post-thawing medium, whereas plasma and acrosomal membrane integrity ( > 0.05) and lipid peroxidation ( > 0.05) were unchanged. In conclusion, despite the centrifugation effects, the addition of SP arising from the whole sperm-rich fraction to post-thawed boar semen decreased sperm lipid disorder without an influence of the sperm membrane integrity and peroxidation. PMID:27285688

  10. Terpenes Increase the Lipid Dynamics in the Leishmania Plasma Membrane at Concentrations Similar to Their IC50 Values

    PubMed Central

    Camargos, Heverton Silva; Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Fernandes, Kelly Souza; Dorta, Miriam Leandro; Alonso, Antonio

    2014-01-01

    Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×106 parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC50 values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC50 values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC50 values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4–9%) at their respective IC50 values. For assays with high cell concentrations (2×109 parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC50 values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis. PMID:25101672

  11. Whey Protein Hydrolysate Increases Translocation of GLUT-4 to the Plasma Membrane Independent of Insulin in Wistar Rats

    PubMed Central

    Morato, Priscila Neder; Lollo, Pablo Christiano Barboza; Moura, Carolina Soares; Batista, Thiago Martins; Camargo, Rafael Ludemann; Carneiro, Everardo Magalhães; Amaya-Farfan, Jaime

    2013-01-01

    Whey protein (WP) and whey protein hydrolysate (WPH) have the recognized capacity to increase glycogen stores. The objective of this study was to verify if consuming WP and WPH could also increase the concentration of the glucose transporters GLUT-1 and GLUT-4 in the plasma membrane (PM) of the muscle cells of sedentary and exercised animals. Forty-eight Wistar rats were divided into 6 groups (n = 8 per group), were treated and fed with experimental diets for 9 days as follows: a) control casein (CAS); b) WP; c) WPH; d) CAS exercised; e) WP exercised; and f) WPH exercised. After the experimental period, the animals were sacrificed, muscle GLUT-1 and GLUT-4, p85, Akt and phosphorylated Akt were analyzed by western blotting, and the glycogen, blood amino acids, insulin levels and biochemical health indicators were analyzed using standard methods. Consumption of WPH significantly increased the concentrations of GLUT-4 in the PM and glycogen, whereas the GLUT-1 and insulin levels and the health indicators showed no alterations. The physical exercise associated with consumption of WPH had favorable effects on glucose transport into muscle. These results should encourage new studies dealing with the potential of both WP and WPH for the treatment or prevention of type II diabetes, a disease in which there is reduced translocation of GLUT-4 to the plasma membrane. PMID:24023607

  12. Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR.

    PubMed

    Chen, Yi-Yung; Rosario, Fredrick J; Shehab, Majida Abu; Powell, Theresa L; Gupta, Madhulika B; Jansson, Thomas

    2015-12-01

    Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (-72%, P<0.0001) and SNAT-1 (-42%, P<0.05) and SNAT-2 (-31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR. PMID:26374858

  13. Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway.

    PubMed

    Caraballo, Juan Carlos; Yshii, Cecilia; Butti, Maria L; Westphal, Whitney; Borcherding, Jennifer A; Allamargot, Chantal; Comellas, Alejandro P

    2011-04-01

    During pulmonary edema, the alveolar space is exposed to a hypoxic environment. The integrity of the alveolar epithelial barrier is required for the reabsorption of alveolar fluid. Tight junctions (TJ) maintain the integrity of this barrier. We set out to determine whether hypoxia creates a dysfunctional alveolar epithelial barrier, evidenced by an increase in transepithelial electrical conductance (G(t)), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithelial cells (AEC) exposed to mild hypoxia (Po(2) = 50 mmHg) for 30 and 60 min decreased occludin abundance at the plasma membrane and significantly increased G(t). Other cell adhesion molecules such as E-cadherin and claudins were not affected by hypoxia. AEC exposed to hypoxia increased superoxide, but not hydrogen peroxide (H(2)O(2)). Overexpression of superoxide dismutase 1 (SOD1) but not SOD2 prevented the hypoxia-induced G(t) increase and occludin reduction in AEC. Also, overexpression of catalase had a similar effect as SOD1, despite not detecting any increase in H(2)O(2) during hypoxia. Blocking PKC-ζ and protein phosphatase 2A (PP2A) prevented the hypoxia-induced occludin reduction at the plasma membrane and increase in G(t). In summary, we show that superoxide, PKC-ζ, and PP2A are involved in the hypoxia-induced increase in G(t) and occludin reduction at the plasma membrane in AEC. PMID:21257729

  14. Benzoyl peroxide increases UVA-induced plasma membrane damage and lipid oxidation in murine leukemia L1210 cells.

    PubMed

    Ibbotson, S H; Lambert, C R; Moran, M N; Lynch, M C; Kochevar, I E

    1998-01-01

    Ultraviolet A radiation induces oxidative stress and cell damage. The purpose of this investigation was to examine whether ultraviolet A-induced cell injury was amplified by the presence of a non-ultraviolet A absorbing molecule capable of generating free radicals. Benzoyl peroxide was used as a lipid soluble potential radical-generating agent. Plasma membrane permeability assessed by trypan blue uptake was used to measure cell damage in murine leukemia L1210 cells. Cells were irradiated with a pulsed Nd/YAG laser at 355 nm using 0-160 J per cm2. The ratio of the fluence-response slope in the presence of 40 microM benzoyl peroxide to that of irradiated controls was 4.3 +/- 2.6. Benzoyl peroxide alone or benzoyl peroxide added after irradiation did not cause increased trypan blue uptake. The ratio of the fluence-response slopes in the presence of 40 microM benzoyl peroxide to that of irradiated controls was 4.7 +/- 1.4 when cells were irradiated (0-43 J per cm2) with a xenon lamp, filtered to remove wavelengths <320 nm. The increased trypan blue uptake in 355 nm-irradiated cells in the presence of benzoyl peroxide was inhibited in a concentration-dependent manner by butylated hydroxytoluene, vitamin E, and trolox, a water-soluble vitamin E derivative. Lipid oxidation, assessed as thiobarbituric acid reactive substances, was significantly increased in samples irradiated with ultraviolet A in the presence of benzoyl peroxide at fluences >34 J per cm2. The increased trypan blue uptake and thiobarbituric acid reactive substances were inhibited by butylated hydroxytoluene. These results suggest that agents not absorbing ultraviolet A radiation may enhance ultraviolet A-initiated oxidative stress in cells. PMID:9424093

  15. Layered plasma polymer composite membranes

    DOEpatents

    Babcock, W.C.

    1994-10-11

    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 [>=]2 and is the number of selective layers. 2 figs.

  16. Challenges in plasma membrane phosphoproteomics

    PubMed Central

    Orsburn, Benjamin C; Stockwin, Luke H; Newton, Dianne L

    2011-01-01

    The response to extracellular stimuli often alters the phosphorylation state of plasma membrane-associated proteins. In this regard, generation of a comprehensive membrane phosphoproteome can significantly enhance signal transduction and drug mechanism studies. However, analysis of this subproteome is regarded as technically challenging, given the low abundance and insolubility of integral membrane proteins, combined with difficulties in isolating, ionizing and fragmenting phosphopeptides. In this article, we highlight recent advances in membrane and phosphoprotein enrichment techniques resulting in improved identification of these elusive peptides. We also describe the use of alternative fragmentation techniques, and assess their current and future value to the field of membrane phosphoproteomics. PMID:21819303

  17. Lysosomes and the plasma membrane

    PubMed Central

    Andrews, Norma W.

    2002-01-01

    Studies of the cell invasion mechanism of the parasite Trypanosoma cruzi led to a series of novel findings, which revealed a previously unsuspected ability of conventional lysosomes to fuse with the plasma membrane. This regulated exocytic process, previously regarded mostly as a specialization of certain cell types, was recently shown to play an important role in the mechanism by which cells reseal their plasma membrane after injury. PMID:12147679

  18. All-trans retinoic acid increases expression of aquaporin-5 and plasma membrane water permeability via transactivation of Sp1 in mouse lung epithelial cells.

    PubMed

    Nomura, Johji; Horie, Ichiro; Seto, Mayumi; Nagai, Kazufumi; Hisatsune, Akinori; Miyata, Takeshi; Isohama, Yoichiro

    2006-12-29

    Aquaporin-5 (AQP5) is a water-selective channel protein that is expressed in lacrimal glands, salivary glands, and distal lung. Several studies using AQP5 knockout mice have revealed that AQP5 plays an important role in maintaining water homeostasis in the lung. We report here that all-trans retinoic acid (atRA) increases plasma membrane water permeability, AQP5 mRNA and protein expression, and AQP5 promoter activity in MLE-12 cells. The promoter activation induced by atRA was diminished by mutation at the Sp1/Sp3 binding element (SBE), suggesting that the SBE mediates the effects of atRA. In addition, atRA increased the binding of Sp1 to the SBE without changing the levels of Sp1 in the nucleus. Taken together, our data indicate that atRA increases AQP5 expression through transactivation of Sp1, leading to an increase in plasma membrane water permeability. PMID:17097063

  19. Cellular membrane collapse by atmospheric-pressure plasma jet

    SciTech Connect

    Kim, Kangil; Sik Yang, Sang E-mail: ssyang@ajou.ac.kr; Jun Ahn, Hak; Lee, Jong-Soo E-mail: ssyang@ajou.ac.kr; Lee, Jae-Hyeok; Kim, Jae-Ho

    2014-01-06

    Cellular membrane dysfunction caused by air plasma in cancer cells has been studied to exploit atmospheric-pressure plasma jets for cancer therapy. Here, we report that plasma jet treatment of cervical cancer HeLa cells increased electrical conductivity across the cellular lipid membrane and caused simultaneous lipid oxidation and cellular membrane collapse. We made this finding by employing a self-manufactured microelectrode chip. Furthermore, increased roughness of the cellular lipid membrane and sequential collapse of the membrane were observed by atomic force microscopy following plasma jet treatment. These results suggest that the cellular membrane catastrophe occurs via coincident altered electrical conductivity, lipid oxidation, and membrane roughening caused by an atmospheric-pressure plasma jet, possibly resulting in cellular vulnerability to reactive species generated from the plasma as well as cytotoxicity to cancer cells.

  20. A novel synaptobrevin/VAMP homologous protein (VAMP5) is increased during in vitro myogenesis and present in the plasma membrane.

    PubMed

    Zeng, Q; Subramaniam, V N; Wong, S H; Tang, B L; Parton, R G; Rea, S; James, D E; Hong, W

    1998-09-01

    cDNA clones encoding a novel protein (VAMP5) homologous to synaptobrevins/VAMPs are detected during database searches. The predicted 102-amino acid VAMP5 harbors a 23-residue hydrophobic region near the carboxyl terminus and exhibits an overall amino acid identity of 33% with synaptobrevin/VAMP1 and 2 and cellubrevin. Northern blot analysis reveals that the mRNA for VAMP5 is preferentially expressed in the skeletal muscle and heart, whereas significantly lower levels are detected in several other tissues but not in the brain. During in vitro differentiation (myogenesis) of C2C12 myoblasts into myotubes, the mRNA level for VAMP5 is increased approximately 8- to 10-fold. Immunoblot analysis using antibodies specific for VAMP5 shows that the protein levels are also elevated approximately 6-fold during in vitro myogenesis of C2C12 cells. Indirect immunofluorescence microscopy and immunoelectron microscopy reveal that VAMP5 is associated with the plasma membrane as well as intracellular perinuclear and peripheral vesicular structures of myotubes. Epitope-tagged versions of VAMP5 are similarly targeted to the plasma membrane. PMID:9725904

  1. The Plasma Membrane Calcium Pump

    NASA Technical Reports Server (NTRS)

    Rasmussen, H.

    1983-01-01

    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.

  2. Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

    PubMed

    Cerecedo, D; Martínez-Vieyra, Ivette; Sosa-Peinado, Alejandro; Cornejo-Garrido, Jorge; Ordaz-Pichardo, Cynthia; Benítez-Cardoza, Claudia

    2016-08-01

    Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of β-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension. PMID:27137675

  3. CD63 Interacts with the Carboxy-Terminus of the Colonic H+,K+-ATPase to Increase Plasma Membrane Localization and Rb+-Uptake

    PubMed Central

    Codina, Juan; Li, Jian; DuBose, Thomas D.

    2005-01-01

    The carboxy-terminus of the colonic H+,K+-ATPase is required for stable assembly with the β-subunit, translocation to the plasma membrane and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKα2, we selected 84 amino acids in the carboxy-terminus of the α-subunit of mouse colonic H+,K+-ATPase (CT-HKα2) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize interaction of CT-HKα2 with CD63, recombinant CT-HKα2 and CD63 were synthesized in vitro, incubated, and complexes immunoprecipitated. CT-HKα 2 protein (but not CT-HKα1) co-precipitated with CD63, confirming stable assembly of HKα2 with CD63. In HEK-293 transfected with HKα2 plus β1-Na+,K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKα2/NKβ1 and 86Rb+-uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKα2/NKβ1 complex in the cell membrane. PMID:15647390

  4. The loss of plasma membrane lysopip and an increase of PIP sub 2 result from treatment of carrot cells with fungal enzymes

    SciTech Connect

    Chen, Q.; Boss, W.F. )

    1989-04-01

    The plasma membranes of carrot cells grown in suspension culture are enriched with PIP, lysoPIP, and PIP{sub 2}. To determine whether or not these lipids are involved in signal transduction, we have challenged the cells with a mixture of fungal cellulases, Driselase, and monitored the changes in the phosphoinositides and in the phosphoinositide kinase activity. With cell prelabeled with ({sup 3}H)inositol, two major changes are observed: (1) lysoPIP decreases 30% compared to the sorbitol control and (2) PIP{sub 2} doubles. There is no increase in IP, IP{sub 2}, or IP{sub 3}. In vitro phosphorylation studies using ({gamma}-{sup 32}P)ATP indicate that the increase in PIP{sub 2} is due in part to activation of the PIP kinase. These data suggest that the role of the polyphosphoinositides in signal transduction in plants may involve activation of the PIP kinase and/or activation of A type phospholipases rather than C type phospholipases.

  5. Characterization of α-Crystallin-Plasma Membrane Binding*

    PubMed Central

    Cobb, Brian A.; Petrash, J. Mark

    2010-01-01

    α-Crystallin, a large lenticular protein complex made up of two related subunits (αA- and αB-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract. To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and recombinant human αA- and αB-crystallins conjugated to a small fluorescent tag (Alexa350®). Both αA and αB homopolymer complexes, as well as a reconstituted 3:1 heteromeric complex, bind to lens membranes in a specific, saturable, and partially irreversible manner that is sensitive to both time and temperature. The amount of α-crystallin that binds to the membrane increases under acidic pH conditions and upon removal of exposed intrinsic membrane protein domains but is not affected at high ionic strength, suggesting that α-crystallin binds to the fiber cell plasma membranes mainly through hydrophobic interactions. The binding capacity and affinity for the reconstituted 3:1 heteromeric complex were measured to be 3.45 ± 0.11 ng/μg of membrane and 4.57 ± 0.50 × 10−4 μg−1 of membrane, respectively. The present membrane binding data support the hypothesis that the physical properties of a mixed α-crystallin complex may hold particular relevance for the function of α-crystallin within the lens. PMID:10692476

  6. Reverse-osmosis membranes by plasma polymerization

    NASA Technical Reports Server (NTRS)

    Hollahan, J. R.; Wydeven, T.

    1972-01-01

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

  7. Transport proteins of the plant plasma membrane

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  8. Protein Homeostasis at the Plasma Membrane

    PubMed Central

    2014-01-01

    The plasma membrane (PM) and endocytic protein quality control (QC) in conjunction with the endosomal sorting machinery either repairs or targets conformationally damaged membrane proteins for lysosomal/vacuolar degradation. Here, we provide an overview of emerging aspects of the underlying mechanisms of PM QC that fulfill a critical role in preserving cellular protein homeostasis in health and diseases. PMID:24985330

  9. Glycan Moieties as Bait to Fish Plasma Membrane Proteins.

    PubMed

    Fang, Fei; Zhao, Qun; Sui, Zhigang; Liang, Yu; Jiang, Hao; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-05-17

    Plasma membrane proteome analysis is of significance for screening candidate biomarkers and drug targets. However, due to their low abundance and lack of specific groups that can enable their capture, the plasma membrane proteins (PMPs) are under-represented. On the basis of the fact that PMPs are embedded in or anchored to the phospholipid bilayer of the plasma membrane and the glycan moieties of proteins and lipids located on the plasma membrane are exposed outside of the cell surface, we proposed a strategy to capture PMPs, termed as glycan moieties-directed PMPs enrichment (GMDPE). With the glycan moieties exposed outside of the cells as bait to ensure the selectivity and the phospholipid bilayer as raft to provide the sensitivity, we applied this strategy into the plasma membrane proteome analysis of HeLa cells, and in total, 772 PMPs were identified, increased by 4.5 times compared to those identified by the reported cell surface biotinylation method. Notably, among them, 86 CD antigens and 16 ion channel proteins were confidently identified. All these results demonstrated that our proposed approach has great potential in the large scale plasma membrane proteome profiling. PMID:27088673

  10. Plasma membrane disruption: repair, prevention, adaptation

    NASA Technical Reports Server (NTRS)

    McNeil, Paul L.; Steinhardt, Richard A.

    2003-01-01

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

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

    SciTech Connect

    Haylett, T.; Thilo, L.

    1986-10-01

    Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D/sub 1/, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from <0.1% to a steady-state level of approx.2.5% of the total label. As analyzed by NaDodSO/sub 4/ PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only approx.1% of internalized membrane is recycled via a membrane pool of secondary lysosomes.

  12. Homeostasis of plasma membrane viscosity in fluctuating temperatures.

    PubMed

    Martinière, Alexandre; Shvedunova, Maria; Thomson, Adrian J W; Evans, Nicola H; Penfield, Steven; Runions, John; McWatters, Harriet G

    2011-10-01

    Temperature has a direct effect at the cellular level on an organism. For instance, in the case of biomembranes, cooling causes lipids to lose entropy and pack closely together. Reducing temperature should, in the absence of other factors, increase the viscosity of a lipid membrane. We have investigated the effect of temperature variation on plasma membrane (PM) viscosity. We used dispersion tracking of photoactivated green fluorescent protein (GFP) and fluorescence recovery after photobleaching in wild-type and desaturase mutant Arabidopsis thaliana plants along with membrane lipid saturation analysis to monitor the effect of temperature and membrane lipid composition on PM viscosity. Plasma membrane viscosity in A. thaliana is negatively correlated with ambient temperature only under constant-temperature conditions. In the more natural environment of temperature cycles, plants actively manage PM viscosity to counteract the direct effects of temperature. Plasma membrane viscosity is regulated by altering the proportion of desaturated fatty acids. In cold conditions, cell membranes accumulate desaturated fatty acids, which decreases membrane viscosity and vice versa. Moreover, we show that control of fatty acid desaturase 2 (FAD2)-dependent lipid desaturation is essential for this homeostasis of membrane viscosity. Finally, a lack of FAD2 function results in aberrant temperature responses. PMID:21762166

  13. Yeast Ist2 Recruits the Endoplasmic Reticulum to the Plasma Membrane and Creates a Ribosome-Free Membrane Microcompartment

    PubMed Central

    Lorenz, Holger; Schwappach, Blanche; Seedorf, Matthias

    2012-01-01

    The endoplasmic reticulum (ER) forms contacts with the plasma membrane. These contacts are known to function in non-vesicular lipid transport and signaling. Ist2 resides in specific domains of the ER in Saccharomyces cerevisiae where it binds phosphoinositide lipids at the cytosolic face of the plasma membrane. Here, we report that Ist2 recruits domains of the yeast ER to the plasma membrane. Ist2 determines the amount of cortical ER present and the distance between the ER and the plasma membrane. Deletion of IST2 resulted in an increased distance between ER and plasma membrane and allowed access of ribosomes to the space between the two membranes. Cells that overexpress Ist2 showed an association of the nucleus with the plasma membrane. The morphology of the ER and yeast growth were sensitive to the abundance of Ist2. Moreover, Ist2-dependent effects on cytosolic pH and genetic interactions link Ist2 to the activity of the H+ pump Pma1 in the plasma membrane during cellular adaptation to the growth phase of the culture. Consistently we found a partial colocalization of Ist2-containing cortical ER and Pma1-containing domains of the plasma membrane. Hence Ist2 may be critically positioned in domains that couple functions of the ER and the plasma membrane. PMID:22808051

  14. Fluidity of pea root plasma membranes under altered gravity

    NASA Astrophysics Data System (ADS)

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

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

  15. Modification of polyethylene terephthalate track membrane properties by ammonia plasma

    NASA Astrophysics Data System (ADS)

    Kravets, Lyubov; Dmitriev, Serguei; Dinescu, George; Lazea, Andrada; Raiciu, Eric

    2004-09-01

    The properties of polyethylene terephthalate track membranes (PET TM) exposed to ammonia are investigated. The influence of the conditions of plasma treatment on the basic characteristics of the membranes, namely pore size and shape, wettability, water permeability, is studied. PET TM of the thickness of 10 μ m with the effective pore diameter of 0.215 μ m (pore density 2\\cdot 10^8 cm-2) were under study. The plasma treatment was performed on a plasma-chemical installation realizing a RF-discharge on the frequency 13.56 MHz. The process was conducted in a dynamic mode. Before delivering vapours of the plasma forming gas, the chamber was beforehand vacuumed down to residual pressure of 10-2 Torr. One side of the membranes was subjected to plasma. The discharge parameters (gas pressure in the vacuum chamber, discharge power) and the duration of plasma action were varied. It has been figured out that when treating the membranes in plasma of the explored gas there are two competing processes: etching of a polymeric matrix and deposition of a polymeric layer on their surface. It has been shown that at a short time of plasma action and low values of the discharge parameters, an etching process is mainly observed. Decrease in the thickness of the membranes and increase in the effective pore diameter testifies it. A result of the gas-discharge etching is also a hydrophilization of the TM surface stipulated by formation of polar function groups in the points of breaking chemical bonds. Here the value of the water contact angle of surface decreases down to 45-50 degrees in some cases. It has been shown that at a longer action of the plasma and increase of the discharge parameters, as accumulation in the chamber of etch products takes place, a process of deposition of a polymeric film becomes dominating, and it is proved by increasing the width of the membranes and changing their color. The value of the water contact angle of surface in this case is grown and, depending

  16. Radiofrequency plasma polymerized perfluoroionomer membrane materials

    SciTech Connect

    Danilich, M.J.; Gervasio, D.F.; Marchant, R.E.

    1993-12-31

    Ion exchange membranes have received considerable attention in recent years. Applications of ion exchange membranes have included such electrochemical systems as water and organic electrolyzers, redox-flow batteries, and sensors. This work is a study of radiofrequency plasma polymerization of perfluorinated acid-containing monomers and a perfluorinated {open_quotes}backbone{close_quotes} comonomer as a method for synthesizing novel polyionomer film coatings for use as membranes on electrodes and biomedical sensors. The results indicate that, by altering the deposition conditions, some control can be exercised over the retention of acid functional groups by plasma polymers. Using AC impedance measurements, the ionic conductivity of these films was found to be two to four orders of magnitude higher than their aqueous environments. In addition, several of the acid-containing plasma polymerized films were hydrophilic, having an advancing water contact angle of less than fifteen degrees. The initial results of this study have demonstrated the feasibility of using acid-containing plasma polymers as crosslinked membrane materials suitable for use with electrochemical sensors and biosensors.

  17. Cholesterol Asymmetry in Synaptic Plasma Membranes

    PubMed Central

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

    2010-01-01

    Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: 1) chronic ethanol consumption; 2) statins; 3) aging; and 4) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density-lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, p-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function. PMID:21214553

  18. Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells.

    PubMed

    Cooper, R A

    1978-01-01

    Cholesterol and phospholipid are the two major lipids of the red cell membrane. Cholesterol is insoluble in water but is solubilized by phospholipids both in membranes and in plasma lipoproteins. Morever, cholesterol exchanges between membranes and lipoproteins. An equilibrium partition is established based on the amount of cholesterol relative to phospholipid (C/PL) in these two compartments. Increases in the C/PL of red cell membranes have been studied under three conditions: First, spontaneous increases in vivo have been observed in the spur red cells of patients with severe liver disease; second, similar red cell changes in vivo have been induced by the administration of cholesterol-enriched diets to rodents and dogs; third, increases in membrane cholesterol have been induced in vitro by enriching the C/PL of the lipoprotein environment with cholesterol-phospholipid dispersions (liposomes) having a C/PL of greater than 1.0. In each case, there is a close relationship between the C/PL of the plasma environment and the C/PL of the red cell membrane. In vivo, the C/PL mole ratio of red cell membranes ranges from a normal value of 0.09--1.0 to values which approach but do not reach 2.0. In vitro, this ratio approaches 3.0. Cholesterol enrichment of red cell membranes directly influences membrane lipid fluidity, as assessed by the rotational diffusion of hydrophobic fluorescent probes such as diphenyl hexatriene (DPH). A close correlation exists between increases in red cell membrane C/PL and decreases in membrane fluidity over the range of membrane C/PL from 1.0 to 2.0; however, little further change in fluidity occurs when membrane C/PL is increased to 2.0--3.0. Cholesterol enrichment of red cell membranes is associated with the transformation of cell contour to one which is redundant and folded, and this is associated with a decrease in red cell filterability in vitro. Circulation in vivo in the presence of the spleen further modifies cell shape to a spiny

  19. Rupture of plasma membrane under tension.

    PubMed

    Tan, Samuel Chun Wei; Yang, Tianyi; Gong, Yingxue; Liao, Kin

    2011-04-29

    We present a study on the rupture behavior of single NIH 3T3 mouse fibroblasts under tension using micropipette aspiration. Membrane rupture was characterized by breaking and formation of an enclosed membrane linked to a tether at the cell apex. Three different rupture modes, namely: single break, initial multiple breaks, and continuous multiple breaks, were observed under similar loading condition. The measured mean tensile strengths of plasma membrane were 3.83 ± 1.94 and 3.98 ± 1.54mN/m for control cells and cells labeled with TubulinTracker, respectively. The tensile strength data was described by Weibull distribution. For the control cells, the Weibull modulus and characteristic strength were 1.86 and 4.40 mN/m, respectively; for cells labeled with TubulinTracker, the Weibull modulus and characteristic strength were 2.68 and 4.48 mN/m, respectively. Based on the experimental data, the estimated average transmembrane proteins-lipid cleavage strength was 2.64 ± 0.64 mN/m. From the random sampling of volume ratio of transmembrane proteins in cell membrane, we concluded that the Weibull characteristic of plasma membrane strength was likely to be originated from the variation in transmembrane proteins-lipid interactions. PMID:21288526

  20. Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses

    PubMed Central

    Corrotte, M.; Castro-Gomes, T.; Koushik, A.B.; Andrews, N.W.

    2016-01-01

    Rapid plasma membrane repair is essential to restore cellular homeostasis and improve cell survival after injury. Several mechanisms for plasma membrane repair have been proposed, including formation of an intracellular vesicle patch, reduction of plasma membrane tension, lesion removal by endocytosis, and/or shedding of the wounded membrane. Under all conditions studied to date, plasma membrane repair is strictly dependent on the entry of calcium into cells, from the extracellular medium. Calcium-dependent exocytosis of lysosomes is an important early step in the plasma membrane repair process, and defects in plasma membrane repair have been observed in cells carrying mutations responsible for serious lysosomal diseases, such as Chediak–Higashi (Huynh, Roth, Ward, Kaplan, & Andrews, 2004) and Niemann–Pick Disease type A (Tam et al., 2010). A functional role for release of the lysosomal enzyme acid sphingomyelinase, which generates ceramide on the cell surface and triggers endocytosis, has been described (Corrotte et al., 2013; Tam et al., 2010). Therefore, procedures for measuring the extent of lysosomal fusion with the plasma membrane of wounded cells are important indicators of the cellular repair response. The importance of carefully selecting the methodology for experimental plasma membrane injury, in order not to adversely impact the membrane repair machinery, is becoming increasingly apparent. Here, we describe physiologically relevant methods to induce different types of cellular wounds, and sensitive assays to measure the ability of cells to secrete lysosomes and reseal their plasma membrane. PMID:25665445

  1. Mechanical properties of the plasma membrane of isolated plant protoplasts

    SciTech Connect

    Wolfe, J.; Steponkus, P.L.

    1983-01-01

    The volume of isolated protoplasts of rye (Secale cereale L. cv Puma) in a suspending solution at constant concentration is shown to be negligibly changed by tensions in the plasma membrane which approach that tension necessary to lyse them. This allows a detailed investigation of the plasma membrane stress-strain relation by micropipette aspiration. Over periods less than a second, the membrane behaves as an elastic two-dimensional fluid with an area modulus of elasticity of 230 millinewtons per meter. Over longer periods, the stress-strain relation approaches a surface energy law--the resting tension is independent of area and has a value of the order 100 micronewtons per meter. Over longer periods the untensioned area, which is defined as the area that would be occupied by the molecules in the membrane at any given time if the tension were zero, increases with time under large imposed tensions and decreases under sufficiently small tension. It is proposed that these long term responses are the result of exchange of material between the plane of the membrane and a reservoir of membrane material. The irreversibility of large contractions in area is demonstrated directly, and the behavior of protoplasts during osmotically induced cycles of contraction and expansion is explained in terms of the membrane stress-strain relation.

  2. Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

    PubMed Central

    Morsomme, P; de Kerchove d'Exaerde, A; De Meester, S; Thinès, D; Goffeau, A; Boutry, M

    1996-01-01

    In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway. Images PMID:8896445

  3. Plasma surface modification of nanofiltration (NF) thin-film composite (TFC) membranes to improve anti organic fouling

    NASA Astrophysics Data System (ADS)

    Kim, Eun-Sik; Yu, Qingsong; Deng, Baolin

    2011-09-01

    Commercial nanofiltration (NF) thin-film composite (TFC) membranes were treated by low-pressure NH3 plasma, and the effects of the plasma treatment were investigated in terms of the membrane hydrophilicity, pure water flux, salt rejection, protein adsorption, and humic acid fouling. Experimental results indicated that the membrane surface hydrophilicity was increased by the plasma treatment, and changes in the hydrophilicity as well as membrane performance including permeate flux and fouling varied with the original membrane characteristics (e.g., roughness and hydrophilicity). Water flux of plasma treated membranes was the highest with 10 min and 90 W of plasma treatment, and salt rejection was mainly affected by the intensity of the plasma power. Results of bovine serum albumin (BSA) adsorption demonstrated that the protein adsorption decreased with increasing plasma treatment time. The plasma treatment that resulted in more negatively charged surfaces could also better prevent Aldrich humic acid (AHA) attachment on the membrane surface.

  4. Membrane raft association is a determinant of plasma membrane localization

    PubMed Central

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

    2014-01-01

    The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting. PMID:24912166

  5. Plasma Membrane Transporters in Modern Liver Pharmacology

    PubMed Central

    Marin, Jose J. G.

    2012-01-01

    The liver plays a crucial role in the detoxification of drugs used in the treatment of many diseases. The liver itself is the target for drugs aimed to modify its function or to treat infections and tumours affecting this organ. Both detoxification and pharmacological processes occurring in the liver require the uptake of the drug by hepatic cells and, in some cases, the elimination into bile. These steps have been classified as detoxification phase 0 and phase III, respectively. Since most drugs cannot cross the plasma membrane by simple diffusion, the involvement of transporters is mandatory. Several members of the superfamilies of solute carriers (SLC) and ATP-binding cassette (ABC) proteins, with a minor participation of other families of transporters, account for the uptake and efflux, respectively, of endobiotic and xenobiotic compounds across the basolateral and apical membranes of hepatocytes and cholangiocytes. These transporters are also involved in the sensitivity and refractoriness to the pharmacological treatment of liver tumours. An additional interesting aspect of the role of plasma membrane transporters in liver pharmacology regards the promiscuity of many of these carriers, which accounts for a variety of drug-drug, endogenous substances-drug and food components-drug interactions with clinical relevance. PMID:24278693

  6. Regulation of Plasma Membrane Recycling by CFTR

    NASA Astrophysics Data System (ADS)

    Bradbury, Neil A.; Jilling, Tamas; Berta, Gabor; Sorscher, Eric J.; Bridges, Robert J.; Kirk, Kevin L.

    1992-04-01

    The gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) is defective in patients with cystic fibrosis. Although the protein product of the CFTR gene has been proposed to function as a chloride ion channel, certain aspects of its function remain unclear. The role of CFTR in the adenosine 3',5'-monophosphate (cAMP)-dependent regulation of plasma membrane recycling was examined. Adenosine 3',5'-monophosphate is known to regulate endocytosis and exocytosis in chloride-secreting epithelial cells that express CFTR. However, mutant epithelial cells derived from a patient with cystic fibrosis exhibited no cAMP-dependent regulation of endocytosis and exocytosis until they were transfected with complementary DNA encoding wild-type CFTR. Thus, CFTR is critical for cAMP-dependent regulation of membrane recycling in epithelial tissues, and this function of CFTR could explain in part the pleiotropic nature of cystic fibrosis.

  7. Electron microscopy methods for studying plasma membranes.

    PubMed

    Beckett, Alison J; Prior, Ian A

    2015-01-01

    Electron microscopy allows direct visualization of the underlying organization of cell surface components on a nano-scale. Immuno-gold labelling of isolated plasma membranes generates point patterns that enable mapping of protein and lipid distributions. 2D spatial statistics reveals the extent to which these distributions are clustered or dispersed and allows the extent of co-localization between different cell surface components to be precisely determined. This approach has been successfully applied to the study of signalling network organization and the consequences of physiological changes in modulating cell surface function. PMID:25331134

  8. Chronic and acute ethanol treatment modifies fluidity and composition in plasma membranes of a human hepatic cell line (WRL-68).

    PubMed

    Gutiérrez-Ruiz, M C; Gómez, J L; Souza, V; Bucio, L

    1995-04-01

    The aim of this study was to compare the effects of chronic (0.1 mol/L ethanol exposure during 30 days) and acute (0.5 mol/L ethanol exposure during 24 h) ethanol treatment on the physical properties and the lipid composition of plasma membranes of the WRL-68 cells (fetal human hepatic cell line). Using fluorescence polarization we found that ethanol treatment reduced membrane anisotropy due to disorganization of acyl chains in plasma membranes and consequently increased fluidity, as measured with the diphenylhexatriene probe. Addition of ethanol in vitro reduced anisotropy in control plasma membranes, whereas chronically ethanol-treated plasma membranes were relatively tolerant to the in vitro addition of ethanol. Acutely ethanol-treated plasma membranes exhibited a smaller anisotropy parameter value than control plasma membranes. We found a decrease in total phospholipid content in acute ethanol WRL-68 plasma membranes. Cholesterol content was increased in both ethanol treatments, and we also found a significant decrease in phosphatidylinositol and phosphatidylcholine and an increase in phosphatidylethanolamine content in ethanol-treated plasma membranes. Our data showed that ethanol treatment decreased the anisotropy parameter consistently with increased fluidity, while increasing the cholesterol/phospholipid ratio of plasma membranes of WRL-68 cells, but only chronically ethanol-treated plasma membranes exhibited tolerance to the in vitro addition of ethanol. It is important to note that some changes that were interpreted as a result of chronic ethanol treatment were also present in short-period ethanol treatments. PMID:7583873

  9. Inside job: ligand-receptor pharmacology beneath the plasma membrane

    PubMed Central

    Babcock, Joseph J; Li, Min

    2013-01-01

    Most drugs acting on the cell surface receptors are membrane permeable and thus able to engage their target proteins in different subcellular compartments. However, these drugs' effects on cell surface receptors have historically been studied on the plasma membrane alone. Increasing evidence suggests that small molecules may also modulate their targeted receptors through membrane trafficking or organelle-localized signaling inside the cell. These additional modes of interaction have been reported for functionally diverse ligands of GPCRs, ion channels, and transporters. Such intracellular drug-target engagements affect cell surface expression. Concurrent intracellular and cell surface signaling may also increase the complexity and therapeutic opportunities of small molecule modulation. Here we discuss examples of ligand-receptor interactions that are present in both intra- and extracellular sites, and the potential therapeutic opportunities presented by this phenomenon. PMID:23685953

  10. Whey cheese: membrane technology to increase yields.

    PubMed

    Riera, Francisco; González, Pablo; Muro, Claudia

    2016-02-01

    Sweet cheese whey has been used to obtain whey cheese without the addition of milk. Pre-treated whey was concentrated by nanofiltration (NF) at different concentration ratios (2, 2.5 and 2.8) or by reverse osmosis (RO) (2-3 times). After the concentration, whey was acidified with lactic acid until a final pH of 4.6-4.8, and heated to temperatures between 85 and 90 °C. The coagulated fraction (supernatant) was collected and freely drained over 4 h. The cheese-whey yield and protein, fat, lactose and ash recoveries in the final product were calculated. The membrane pre-concentration step caused an increase in the whey-cheese yield. The final composition of products was compared with traditional cheese-whey manufacture products (without membrane concentration). Final cheese yields found were to be between 5 and 19.6%, which are higher than those achieved using the traditional 'Requesón' process. PMID:26869115

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

    PubMed

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

    2015-01-01

    Understanding the adaptation of plants to acid rain is important to find feasible approaches to alleviate such damage to plants. We studied effects of acid rain on plasma membrane H(+)-ATPase activity and transcription, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate during stress and recovery periods. Simulated acid rain at pH 5.5 did not affect plasma membrane H(+)-ATPase activity, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate. Plasma membrane H(+)-ATPase activity and transcription in leaves treated with acid rain at pH 3.5 was increased to maintain ion homeostasis by transporting excessive H(+) out of cells. Then intracellular H(+) was close to the control after a 5-day recovery, alleviating damage on membrane and sustaining photosynthetic efficiency and growth. Simulated acid rain at pH 2.5 inhibited plasma membrane H(+)-ATPase activity by decreasing the expression of H(+)-ATPase at transcription level, resulting in membrane damage and abnormal intracellular H(+), and reduction in photosynthetic efficiency and relative growth rate. After a 5-day recovery, all parameters in leaves treated with pH 2.5 acid rain show alleviated damage, implying that the increased plasma membrane H(+)-ATPase activity and its high expression were involved in repairing process in acid rain-stressed plants. Our study suggests that plasma membrane H(+)-ATPase can play a role in adaptation to acid rain for rice seedlings. PMID:25087500

  12. Binding contribution between synaptic vesicle membrane and plasma membrane proteins in neurons: an AFM study.

    PubMed

    Sritharan, K C; Quinn, A S; Taatjes, D J; Jena, B P

    1998-01-01

    The final step in the exocytotic process is the docking and fusion of membrane-bound secretory vesicles at the cell plasma membrane. This docking and fusion is brought about by several participating vesicle membrane, plasma membrane and soluble cytosolic proteins. A clear understanding of the interactions between these participating proteins giving rise to vesicle docking and fusion is essential. In this study, the binding force profiles between synaptic vesicle membrane and plasma membrane proteins have been examined for the first time using the atomic force microscope. Binding force contributions of a synaptic vesicle membrane protein VAMP1, and the plasma membrane proteins SNAP-25 and syntaxin, are also implicated from these studies. Our study suggests that these three proteins are the major, if not the only contributors to the interactive binding force that exist between the two membranes. PMID:10452835

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

    PubMed Central

    Singh, Prabhakar; Kesharwani, Rajesh Kumar; Misra, Krishna; Rizvi, Syed Ibrahim

    2016-01-01

    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. PMID:26904287

  14. Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration.

    PubMed Central

    Deliconstantinos, G; Ramantanis, G

    1983-01-01

    A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5'-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5'-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration. PMID:6309144

  15. Surface Modification of Polypropylene Membrane by RF Methane/Oxygen Mixture Plasma Treatment

    NASA Astrophysics Data System (ADS)

    Tsai, Ching-Yuan; Juang, Ruey-Shin; Huang, Chun

    2011-08-01

    The hydrophilic surface modification of micro-porous polypropylene (PP) membranes is achieved by low-pressure 13.56 MHz RF methane (CH4)/oxygen (O2) gas mixture plasma treatment. The changes in surface wettability and surface free energy were examined by static contact angle analysis. The static water contact angle of the plasma modified membrane notably decreased with increases in treatment time and plasma power. The obvious increase in the surface energy of polypropylene membranes due to CH4/O2 mixture gas plasma treatments was also observed. Optical emission spectroscopy (OES) was used to analyze the chemical species of CH4/O2 mixture gas plasma treatment. The variations in the surface morphology and chemical structure of the micro-porous PP membranes were confirmed by confocal laser scanning microscopy (CLSM), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) measurements. XPS analysis showed significantly higher surface concentrations of oxygen functional groups for CH4/O2 mixture gas plasma-modified polypropylene membrane surfaces than for the originally unmodified polypropylene membrane surface. The experimental results show the important role of chemical species in the interaction between a CH4/O2 mixture gas plasma and a membrane surface, which can be controlled by surface modification to tailor the hydrophilicity of the membrane to the requirements of various applications.

  16. Selective photosensitizer delivery into plasma membrane for effective photodynamic therapy.

    PubMed

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

    2014-10-10

    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. PMID:24892975

  17. Order of lipid phases in model and plasma membranes

    PubMed Central

    Kaiser, Hermann-Josef; Lingwood, Daniel; Levental, Ilya; Sampaio, Julio L.; Kalvodova, Lucie; Rajendran, Lawrence; Simons, Kai

    2009-01-01

    Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165–3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective. PMID:19805351

  18. Channelopathies linked to plasma membrane phosphoinositides

    PubMed Central

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

    2014-01-01

    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

  19. Dynamics of photoinduced cell plasma membrane injury.

    PubMed Central

    Thorpe, W P; Toner, M; Ezzell, R M; Tompkins, R G; Yarmush, M L

    1995-01-01

    We have developed a video microscopy system designed for real-time measurement of single cell damage during photolysis under well defined physicochemical and photophysical conditions. Melanoma cells cultured in vitro were treated with the photosensitizer (PS), tin chlorin e6 (SnCe6) or immunoconjugate (SnCe6 conjugated to a anti-ICAM monoclonal antibody), and illuminated with a 10 mW He/Ne laser at a 630 nm wavelength. Cell membrane integrity was assessed using the vital dye calcein-AM. In experiments in which the laser power density and PS concentration were varied, it was determined that the time lag before cell rupture was inversely proportional to the estimated singlet oxygen flux to the cell surface. Microscopic examination of the lytic event indicated that photo-induced lysis was caused by a point rupture of the plasma membrane. The on-line nature of this microscopy system offers an opportunity to monitor the dynamics of the cell damage process and to gain insights into the mechanism governing photolytic cell injury processes. Images FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 7 PMID:7612864

  20. Phosphorylation and Interaction with the 14-3-3 Protein of the Plasma Membrane H+-ATPase are Involved in the Regulation of Magnesium-Mediated Increases in Aluminum-Induced Citrate Exudation in Broad Bean (Vicia faba. L).

    PubMed

    Chen, Qi; Kan, Qi; Wang, Ping; Yu, Wenqian; Yu, Yuzhen; Zhao, Yan; Yu, Yongxiong; Li, Kunzhi; Chen, Limei

    2015-06-01

    Several studies have shown that external application of micromolar magnesium (Mg) can increase the resistance of legumes to aluminum (Al) stress by enhancing Al-induced citrate exudation. However, the exact mechanism underlying this regulation remains unknown. In this study, the physiological and molecular mechanisms by which Mg enhances Al-induced citrate exudation to alleviate Al toxicity were investigated in broad bean. Micromolar concentrations of Mg that alleviated Al toxicity paralleled the stimulation of Al-induced citrate exudation and increased the activity of the plasma membrane (PM) H(+)-ATPase. Northern blot analysis shows that a putative MATE-like gene (multidrug and toxic compound extrusion) was induced after treatment with Al for 4, 8 and 12 h, whereas the mRNA abundance of the MATE-like gene showed no significant difference between Al plus Mg and Al-only treatments during the entire treatment period. Real-time reverse transcription-PCR (RT-PCR) and Western blot analyses suggest that the transcription and translation of the PM H(+)-ATPase were induced by Al but not by Mg. In contrast, immunoprecipitation suggests that Mg enhanced the phosphorylation levels of VHA2 and its interaction with the vf14-3-3b protein under Al stress. Taken together, our results suggest that micromolar concentrations of Mg can alleviate the Al rhizotoxicity by increasing PM H(+)-ATPase activity and Al-induced citrate exudation in YD roots. This enhancement is likely to be attributable to Al-induced increases in the expression of the MATE-like gene and vha2 and Mg-induced changes in the phosphorylation levels of VHA2, thus changing its interaction with the vf14-3-3b protein. PMID:25745032

  1. The Liver Connexin32 Interactome Is a Novel Plasma Membrane-Mitochondrial Signaling Nexus

    PubMed Central

    2013-01-01

    Connexins are the structural subunits of gap junctions and act as protein platforms for signaling complexes. Little is known about tissue-specific connexin signaling nexuses, given significant challenges associated with affinity-purifying endogenous channel complexes to the level required for interaction analyses. Here, we used multiple subcellular fractionation techniques to isolate connexin32-enriched membrane microdomains from murine liver. We show, for the first time, that connexin32 localizes to both the plasma membrane and inner mitochondrial membrane of hepatocytes. Using a combination of immunoprecipitation-high throughput mass spectrometry, reciprocal co-IP, and subcellular fractionation methodologies, we report a novel interactome validated using null mutant controls. Eighteen connexin32 interacting proteins were identified. The majority represent resident mitochondrial proteins, a minority represent plasma membrane, endoplasmic reticulum, or cytoplasmic partners. In particular, connexin32 interacts with connexin26 and the mitochondrial protein, sideroflexin-1, at the plasma membrane. Connexin32 interaction enhances connexin26 stability. Converging bioinformatic, biochemical, and confocal analyses support a role for connexin32 in transiently tethering mitochondria to connexin32-enriched plasma membrane microdomains through interaction with proteins in the outer mitochondrial membrane, including sideroflexin-1. Complex formation increases the pool of sideroflexin-1 that is present at the plasma membrane. Together, these data identify a novel plasma membrane/mitochondrial signaling nexus in the connexin32 interactome. PMID:23590695

  2. Properties of Plasma Membrane from Pea Root Seedlings under Altered Gravity

    NASA Astrophysics Data System (ADS)

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

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

  3. Membrane Compartment Occupied by Can1 (MCC) and Eisosome Subdomains of the Fungal Plasma Membrane

    PubMed Central

    Douglas, Lois M.; Wang, Hong X.; Li, Lifang; Konopka, James B.

    2011-01-01

    Studies on the budding yeast Saccharomyces cerevisiae have revealed that fungal plasma membranes are organized into different subdomains. One new domain termed MCC/eisosomes consists of stable punctate patches that are distinct from lipid rafts. The MCC/eisosome domains correspond to furrows in the plasma membrane that are about 300 nm long and 50 nm deep. The MCC portion includes integral membrane proteins, such as the tetraspanners Sur7 and Nce102. The adjacent eisosome includes proteins that are peripherally associated with the membrane, including the BAR domains proteins Pil1 and Lsp1 that are thought to promote membrane curvature. Genetic analysis of the MCC/eisosome components indicates these domains broadly affect overall plasma membrane organization. The mechanisms regulating the formation of MCC/eisosomes in model organisms will be reviewed as well as the role of these plasma membrane domains in fungal pathogenesis and response to antifungal drugs. PMID:22368779

  4. Effect of plasma membrane fluidity on serotonin transport by endothelial cells

    SciTech Connect

    Block, E.R.; Edwards, D. )

    1987-11-01

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

  5. Increased plasma homocysteine in liver cirrhosis.

    PubMed

    Bosy-Westphal, A; Petersen, S; Hinrichsen, H; Czech, N; J Müller, M

    2001-05-01

    Background: Homocysteine (Hcy), is an atherogenic and thrombogenic risk factor which has also been proposed to be involved in hepatic fibrinogenesis. Hcy metabolism, depends on the cofactors folate, vit. B12, and the vit. B6 vitamer pyridoxalphosphate (PLP). Metabolism of these vitamins is frequently disturbed in cirrhotics, but little is known about plasma Hcy levels in these patients. Methods: Plasma levels of Hcy, methionine, serine, cysteine, PLP, vit. B12 and folate, and standard clinical/biochemical parameters of liver disease were measured in 43 postabsorptive patients with biopsy proven cirrhosis of different origin. Results: 74% of the patients had elevated plasma Hcy levels defined as >13.4 µmol/l (mean+2SD of healthy age matched controls). Increased plasma Hcy concentrations were seen in alcoholic as well as in non-alcoholic cirrhosis. Excluding patients with impaired renal function (n=7), Hcy concentrations remained elevated in 69% of the patients. We found a high prevalence of pathological plasma vitamin concentrations of 33% for increased vit. B12 levels and 5% and 80% for decreased folate and vit. B6 levels, respectively. Mean plasma vitamin B12 concentrations increased, folate remained unchanged and PLP concentrations decreased with deteriorating liver function. Hcy concentrations were correlated with levels of creatinine (r=0.44, P<0.01), serine (r=-0.46, P<0.01), and cysteine (r=0.38, P<0.05), but showed no association with parameters of liver function and with plasma levels of folate, vit. B12 und vit. B6. This was contrary to data obtained in healthy individuals. In a stepwise multiple regression serine and cysteine best explained the variance in Hcy levels. Conclusions: Elevated basal Hcy-plasma levels are frequently seen cirrhotic patients. Variations of Hcy concentration in liver cirrhosis are not explained by plasma levels of cofactors of Hcy metabolism. PMID:11282484

  6. Surface monofunctionalized polymethyl pentene hollow fiber membranes by plasma treatment and hemocompatibility modification for membrane oxygenators

    NASA Astrophysics Data System (ADS)

    Huang, Xin; Wang, Weiping; Zheng, Zhi; Fan, Wenling; Mao, Chun; Shi, Jialiang; Li, Lei

    2016-01-01

    The hemocompatibility of polymethyl pentene (PMP) hollow fiber membranes (HFMs) was improved through surface modification for membrane oxygenator applications. The modification was performed stepwise with the following: (1) oxygen plasma treatment, (2) functionalization of monosort hydroxyl groups through NaBH4 reduction, and (3) grafting 2-methacryloyloxyethyl phosphorylcholine (MPC) or heparin. SEM, ATR-FTIR, and XPS analyses were conducted to confirm successful grafting during the modification. The hemocompatibility of PMP HFMs was analyzed and compared through protein adsorption, platelet adhesion, and coagulation tests. Pure CO2 and O2 permeation rates, as well as in vitro gas exchange rates, were determined to evaluate the mass transfer properties of PMP HFMs. SEM results showed that different nanofibril topographies were introduced on the HFM surface. ATR-FTIR and XPS spectra indicated the presence of functionalization of monosort hydroxyl group and the grafting of MPC and heparin. Hemocompatibility evaluation results showed that the modified PMP HFMs presented optimal hemocompatibility compared with pristine HFMs. Gas permeation results revealed that gas permeation flux increased in the modified HFMs because of dense surface etching during the plasma treatment. The results of in vitro gas exchange rates showed that all modified PMP HFMs presented decreased gas exchange rates because of potential surface fluid wetting. The proposed strategy exhibits a potential for fabricating membrane oxygenators for biomedical applications to prevent coagulation formation and alter plasma-induced surface topology and composition.

  7. Plasma Membrane Lesions In Anthracycline-Resistant Tumor Cells Probed Using A Fluorescent Dye

    NASA Astrophysics Data System (ADS)

    Burke, Thomas G.; Doroshow, James H.

    1989-06-01

    Human cancer cells selected for resistance to several structurally unrelated cytotoxic drugs are known to display plasma membrane alterations such as amplified levels of a variety of glycoproteins, modifications in lipid composition, alterations in membrane fluidity and increased cellular fragility to osmotic shock. We have studied the plasma membrane fluidity of HL60 human leukemia cells and MCF-7 human breast cancer cells that have been selected for acquired resistance against the cytocidal effects of the anthracycline anticancer drug Adriamycin. Fluidity measurements were accomplished by evaluating the fluorescence anisotropy of the plasma membrane specific probe trimethylamino-1,6-dipihenylhexatriene (TMA.DPH) bound to whole, living cells. TMA.DPH anisotropy values for MCF-7 sensitive and 12-fold resistant cells were 0.306 and 0.285, respectively, while anisotropy values for HL-60 sensitive and 80-fold resistant cells lines were 0.310 and 0.295, respectively. In all cases, cell viability exceeded 97% and anisotropy values were subject to a day-to-day uncertainty of +/-2%. Our results demonstrate that increased plasma membrane fluidity apparently accompanies the development of resistance in both cell lines. Because it is known that increased membrane fluidity results in significantly decreased Adriamycin binding in artificial membrane systems, we propose here that decreased drug associations with fluidized, plasma membrane lipid bilayer regions may be a mechanism which contributes, in part, to the reduced rates of drug accumulation observed in HL60 and MCF-7 cells resistant to Adriamycin.

  8. Dysferlinopathy Fibroblasts Are Defective in Plasma Membrane Repair

    PubMed Central

    Matsuda, Chie; Kiyosue, Kazuyuki; Nishino, Ichizo; Goto, Yuichi; Hayashi, Yukiko K.

    2015-01-01

    Background: Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, and is involved in sarcolemmal repair. Primary cultured myoblasts and myotubes established from patient muscle biopsies have been widely utilized to explore the molecular mechanism of dysferlinopathy. Objectives: The purpose of this study was to explore the possible utility of dermal fibroblasts from dysferlin-deficient patients and SJL mice as a tool for studying dysferlinopathy. Methods: Dysferlin protein expression in fibroblasts from dysferlin-deficient patients and SJL mice was analyzed by immunoblotting and immunocytochemistry. The membrane wound-repair assay was performed on the fibroblasts using a confocal microscope equipped with a UV-laser. The membrane blebbing assay using hypotonic shock, in which normal membrane blebbing is detected only in the presence of dysferlin, was also performed using human and mouse fibroblasts. Results: Mis-sense mutated dysferlin was expressed at a very low level in fibroblasts from a dysferlinopathy patient, and lower expression level of truncated dysferlin was observed in SJL mouse fibroblast. Fibroblasts from patients with dysferlinopathy and SJL mice showed attenuated membrane repair and did not form membrane blebs in response to hypoosmotic shock. Proteosomal inhibitior increased mis-sense mutated or truncated dysferlin levels, and restored membrane blebbing, however, proteosomal inhibition failed to improve levels of dysferlin with non-sense or frame-shift mutation. Conclusion: Fibroblasts from dysferlinopathy patients and SJL mice showed attenuated plasma membrane repair, and could be a tool for studying dysferlinopathy. PMID:26579332

  9. Effect of ion bombardment on plasma-driven superpermeation of hydrogen isotopes through a niobium membrane

    NASA Astrophysics Data System (ADS)

    Notkin, M. E.; Livshits, A. I.; Bruneteau, A. M.; Bacal, M.

    2001-08-01

    Hydrogen plasma-driven permeation through the superpermeable niobium membrane was investigated under bombardment of the input membrane surface with hydrogen, deuterium and helium ions with energy 0-250 eV over the range of membrane temperature 910-1420 K. The membrane surface was covered with a nonmetal monolayer generating a potential barrier responsible for the superpermeability to suprathermal hydrogen particles. Both an increase of ion energy and an increase of mass of sputtering ions result in a significant decrease of permeability due to destruction of the nonmetal monolayer, when the ion energy is higher than the threshold energy of surface film sputtering. On the contrary, the increase of the membrane temperature results in the decrease of the ion bombardment effect and in the increase of the membrane permeability due to recovery of the surface barrier through segregation of impurities dissolved in the membrane bulk onto the membrane surface. To increase the membrane ability to recover the potential barrier, oxygen was dissolved in the membrane bulk up to a concentration of 2.5 at.%. This resulted in a significant decrease of the damaging effect of ion bombardment and in the extension of the range of the membrane temperature and ion energy over which plasma-driven superpermeability was observed.

  10. The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

    PubMed

    van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N

    1995-06-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes

  11. The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

    PubMed Central

    van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N

    1995-01-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes

  12. Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury 1

    PubMed Central

    Iswari, S.; Palta, Jiwan P.

    1989-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  15. Lysosomal involvement in cellular turnover of plasma membrane sphingomyelin.

    PubMed

    Sutrina, S L; Chen, W W

    1984-04-18

    At least two isoenzymes of sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal acid sphingomyelinase and nonlysosomal magnesium-dependent neutral sphingomyelinase, catalyse the degradation of sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of sphingomyelin metabolism, type A Niemann-Pick disease, is characterized by a deficiency of lysosomal acid sphingomyelinase. To investigate the involvement of lysosomes in the degradation of cellular membrane sphingomyelin, we have undertaken studies to compare the turnover of plasma membrane sphingomyelin in fibroblasts from a patient with type A Niemann-Pick disease, which completely lack acid sphingomyelinase activity but retain nonlysosomal neutral sphingomyelinase activity, with turnover in fibroblasts from normal individuals. Plasma membrane sphingomyelin was labeled by incubating cells at low temperature with phosphatidylcholine vesicles containing radioactive sphingomyelin. A fluorescent analog of sphingomyelin, N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine (NBD-sphingomyelin) is seen to be readily transferred at low temperature from phosphatidylcholine liposomes to the plasma membranes of cultured human fibroblasts. Moreover, when kinetic studies were done in parallel, a constant ratio of [14C]oleoylsphingosylphosphorylcholine ( [14C]sphingomyelin) to NBD-sphingomyelin was taken up at low temperature by the fibroblast cells, suggesting that [14C]sphingomyelin undergoes a similar transfer. The comparison of sphingomyelin turnover at 37 degrees C in normal fibroblasts compared to Niemann-Pick diseased fibroblasts shows that a rapid turnover of plasma membrane-associated sphingomyelin within the first 30 min appears to be similar in both normal and Niemann-Pick diseased cells. This rapid turnover appears to be primarily due to rapid removal of the [14C]sphingomyelin from the cell surface into the incubation medium. During

  16. No primexine and plasma membrane undulation is essential for primexine deposition and plasma membrane undulation during microsporogenesis in Arabidopsis.

    PubMed

    Chang, Hai-Shuang; Zhang, Cheng; Chang, Yu-Hua; Zhu, Jun; Xu, Xiao-Feng; Shi, Zhi-Hao; Zhang, Xiao-Lei; Xu, Ling; Huang, Hai; Zhang, Sen; Yang, Zhong-Nan

    2012-01-01

    Primexine deposition and plasma membrane undulation are the initial steps of pollen wall formation. However, little is known about the genes involved in this important biological process. Here, we report a novel gene, NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION (NPU), which functions in the early stage of pollen wall development in Arabidopsis (Arabidopsis thaliana). Loss of NPU function causes male sterility due to a defect in callose synthesis and sporopollenin deposition, resulting in disrupted pollen in npu mutants. Transmission electronic microscopy observation demonstrated that primexine deposition and plasma membrane undulation are completely absent in the npu mutants. NPU encodes a membrane protein with two transmembrane domains and one intracellular domain. In situ hybridization analysis revealed that NPU is strongly expressed in microspores and the tapetum during the tetrad stage. All these results together indicate that NPU plays a vital role in primexine deposition and plasma membrane undulation during early pollen wall development. PMID:22100644

  17. Composite plasma polymerized sulfonated polystyrene membrane for PEMFC

    SciTech Connect

    Nath, Bhabesh Kumar; Khan, Aziz; Chutia, Joyanti

    2015-10-15

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

  18. Membrane-based Therapeutic Plasma Exchange: A New Frontier for Nephrologists.

    PubMed

    Gashti, Casey N

    2016-09-01

    Therapeutic plasma exchange has long been utilized to manage a variety of immune-mediated diseases. The underlying principle is the removal of a circulating pathogenic substance from the plasma and substitution with a replacement fluid. Different methodologies of plasma separation include the use of centrifuge, which relies on the variation in the specific gravity of blood components, and membrane-based separation, which relies on particle size. With advancements in technology and clinical insight into disease pathophysiology, membrane technology has become more biocompatible, safer, and more adaptable to conventional hemodialysis and hemofiltration machines. As such, nephrologists, who are familiar with management of extracorporeal blood purification systems, are increasingly involved with membrane-based plasma separation. This review aims to highlight the technical aspects of membrane-based separation, review the prescription for therapy, and draw comparisons with the centrifuge-based technique when applicable. PMID:27062015

  19. Gravity Responsive NADH Oxidase of the Plasma Membrane

    NASA Technical Reports Server (NTRS)

    Morre, D. James (Inventor)

    2002-01-01

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

  20. Sixteen-Day Bedrest Significantly Increases Plasma Colloid Osmotic Pressure

    NASA Technical Reports Server (NTRS)

    Hargens, Alan R.; Hsieh, S. T.; Murthy, G.; Ballard, R. E.; Convertino, V. A.; Wade, Charles E. (Technical Monitor)

    1994-01-01

    Upon exposure to microgravity, astronauts lose up to 10% of their total plasma volume, which may contribute to orthostatic intolerance after space flight. Because plasma colloid osmotic pressure (COP) is a primary factor maintaining plasma volume, our objective was to measure time course changes in COP during microgravity simulated by 6 deg. head-down tilt (HDT). Seven healthy male subjects (30-55 years of age) were placed in HDT for 16 days. For the purpose of another study, three of the seven subjects were chosen to exercise on a cycle ergometer on day 16. Blood samples were drawn immediately before bedrest on day 14 of bedrest, 18-24 hours following exercise while all subjects were still in HDT and 1 hour following bedrest termination. Plasma COP was measured in all 20 microliter EDTA-treated samples using an osmometer fitted with a PM 30 membrane. Data were analyzed with paired and unpaired t-tests. Plasma COP on day 14 of bedrest (29.9 +/- 0.69 mmHg) was significantly higher (p less than 0.005) than the control, pre-bedrest value (23.1 +/- 0.76 mmHg). At one hour of upright recovery after HDT, plasma COP remained significantly elevated (exercise: 26.9 +/- 0.87 mmHg; no exercise: 26.3 +/- 0.85 mmHg). Additionally, exercise had no significant effect on plasma COP 18-24 hours following exercise (exercise: 27.8 +/- 1.09 mmHg; no exercise: 27.1 +/- 0.78 mmHg). Our results demonstrate that plasma COP increases significantly with microgravity simulated by HDT. However, preliminary results indicate exercise during HDT does not significantly affect plasma COP.

  1. Oxytocin regulates the plasma membrane Ca2+ transport in rat myometrium.

    PubMed Central

    Enyedi, A; Brandt, J; Minami, J; Penniston, J T

    1989-01-01

    Development of myometrium in young female rats was stimulated by administration of diethylstilboestrol. Plasma membrane and sarcoplasmic reticulum from rat myometrium were separated by a new and rapid method using a Percoll gradient. Calcium uptake was inhibited in plasma membrane vesicles isolated from oxytocin-treated myometrium, while no consistent effect of oxytocin was found on the Ca2+ uptake in the sarcoplasmic reticulum. Oxytocin regulated the plasma membrane Ca2+ pump by decreasing its apparent affinity for Ca2+ without affecting its maximal velocity. The K1/2 for Ca2+ in the absence of calmodulin was 0.41 +/- 0.04 microM in normal membranes; this was increased to 0.93 +/- 0.12 microM in oxytocin-treated membranes. Calmodulin decreased the K1/2 for Ca2+ to 0.27 +/- 0.027 microM and oxytocin also increased this, to 0.46 +/- 0.061 microM. The effect of oxytocin on the plasma membrane Ca2+ pump was highly dependent on the hormonal status of the animals. When the diethylstilboestrol was administered together with progesterone, the inhibitory action of oxytocin was totally suppressed, consistent with the expected action of this agent. The results suggest that regulation of the plasma membrane Ca2+ pump may be important in the prolonged elevation of intracellular Ca2+ caused by oxytocin. PMID:2775210

  2. Facilitative plasma membrane transporters function during ER transit

    PubMed Central

    Takanaga, Hitomi; Frommer, Wolf B.

    2010-01-01

    Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na+-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.—Takanaga, H., Frommer, W. B. Facilitative plasma membrane transporters function during ER transit. PMID:20354141

  3. Paracrine signaling through plasma membrane hemichannels☆

    PubMed Central

    Wang, Nan; De Bock, Marijke; Decrock, Elke; Bol, Mélissa; Gadicherla, Ashish; Vinken, Mathieu; Rogiers, Vera; Bukauskas, Feliksas F.; Bultynck, Geert; Leybaert, Luc

    2013-01-01

    Plasma membrane hemichannels composed of connexin (Cx) proteins are essential components of gap junction channels but accumulating evidence suggests functions of hemichannels beyond the communication provided by junctional channels. Hemichannels not incorporated into gap junctions, called unapposed hemichannels, can open in response to a variety of signals, electrical and chemical, thereby forming a conduit between the cell’s interior and the extracellular milieu. Open hemichannels allow the bidirectional passage of ions and small metabolic or signaling molecules of below 1–2 kDa molecular weight. In addition to connexins, hemichannels can also be formed by pannexin (Panx) proteins and current evidence suggests that Cx26, Cx32, Cx36, Cx43 and Panx1, form hemichannels that allow the diffusive release of paracrine messengers. In particular, the case is strong for ATP but substantial evidence is also available for other messengers like glutamate and prostaglandins or metabolic substances like NAD+ or glutathione. While this field is clearly in expansion, evidence is still lacking at essential points of the paracrine signaling cascade that includes not only messenger release, but also downstream receptor signaling and consequent functional effects. The data available at this moment largely derives from in vitro experiments and still suffers from the difficulty of separating the functions of connexin-based hemichannels from gap junctions and from pannexin hemichannels. However, messengers like ATP or glutamate have universal roles in the body and further defining the contribution of hemichannels as a possible release pathway is expected to open novel avenues for better understanding their contribution to a variety of physiological and pathological processes. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions. PMID:22796188

  4. Neomycin inhibits the phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate stimulation of plasma membrane ATPase activity

    SciTech Connect

    Chen, Qiuyun; Boss, W.F. )

    1991-05-01

    The inositol phospholipids, phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP{sub 2}), have been shown to increase the vanadate-sensitive ATPase activity of plant plasma membranes. In this paper, the authors show the effect of various concentrations of phosphatidyinositol, PIP, and PIP{sub 2} on the plasma membrane vanadate-sensitive ATPase activity. PIP and PIP{sub 2} at concentrations at 10 nanomoles per 30 microgram membrane protein per milliliter of reaction mixture caused a twofold and 1.8-fold increase in the ATPase activity, respectively. The effect of these negatively charged phospholipids on the ATPase activity was inhibited by adding the positively charged aminoglycoside, neomycin. Neomycin did not affect the endogenous plasma membrane ATPase activity in the absence of exogenous lipids.

  5. Influence of decavanadate on rat synaptic plasma membrane ATPases activity.

    PubMed

    Krstić, Danijela; Colović, Mirjana; Bosnjaković-Pavlović, Nada; Spasojević-De Bire, Anne; Vasić, Vesna

    2009-09-01

    The in vitro influence of decameric vanadate species on Na+/K+-ATPase, plasma membrane Ca2+-ATPase (PMCA)-calcium pump and ecto-ATPase activity, using rat synaptic plasma membrane (SPM) as model system was investigated, whereas the commercial porcine cerebral cortex Na+/K+-ATPase served as a reference. The thermal behaviour of the synthesized decavanadate (V10) has been studied by differential scanning calorimetry and thermogravimetric analysis, while the type of polyvanadate anion was identified using the IR spectroscopy. The concentration-dependent responses to V10 of all enzymes were obtained. The half-maximum inhibitory concentration (IC50) of the enzyme activity was achieved at (4.74 +/- 1.15) x 10(-7) mol/l for SPM Na+/K+-ATPase, (1.30 +/- 0.10) x 10(-6) mol/l for commercial Na+/K+-ATPase and (3.13 +/- 1.70) x 10(-8) mol/l for Ca2+-ATPase, while ecto-ATPase is significantly less sensitive toward V10 (IC50 = (1.05 +/- 0.10) x 10(-4) mol/l) than investigated P-type ATPases. Kinetic analysis showed that V10 inhibited Na+/K+-ATPase by reducing the maximum enzymatic velocity and apparent affinity for ATP (increasing K(m) value), implying a mixed mode of interaction between V10 and P-type ATPases. PMID:20037196

  6. Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3.

    PubMed

    Li, Caiyue; Ma, Wenbo; Yin, Shikui; Liang, Xin; Shu, Xiaodong; Pei, Duanqing; Egan, Terrance M; Huang, Jufang; Pan, Aihua; Li, Zhiyuan

    2016-05-01

    The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3-transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists-activated Ca(2+) influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein-protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes. PMID:26818531

  7. Plasma membrane phosphoinositide balance regulates cell shape during Drosophila embryo morphogenesis

    PubMed Central

    Reversi, Alessandra; Loeser, Eva; Subramanian, Devaraj; Schultz, Carsten

    2014-01-01

    Remodeling of cell shape during morphogenesis is driven by the coordinated expansion and contraction of specific plasma membrane domains. Loss of this coordination results in abnormal cell shape and embryonic lethality. Here, we show that plasma membrane lipid composition plays a key role in coordinating plasma membrane contraction during expansion. We found that an increase in PI(4,5)P2 levels caused premature actomyosin contraction, resulting in the formation of shortened cells. Conversely, acute depletion of PI(4,5)P2 blocked plasma membrane expansion and led to premature actomyosin disassembly. PI(4,5)P2-mediated contractility is counteracted by PI(3,4,5)P3 and the zygotic gene bottleneck, which acts by limiting myosin recruitment during plasma membrane expansion. Collectively, these data support a model in which the ratio of PI(4,5)P2/PI(3,4,5)P3 coordinates actomyosin contractility and plasma membrane expansion during tissue morphogenesis, thus ensuring proper cell shape. PMID:24798734

  8. The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane.

    PubMed

    Chen, P; Hwang, T C; Gillis, K D

    2001-08-01

    The mechanism whereby cAMP stimulates Cl(-) flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1-43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl(-) current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1-43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur. PMID:11479341

  9. The Relationship between Camp, Ca2+, and Transport of Cftr to the Plasma Membrane

    PubMed Central

    Chen, Peng; Hwang, Tzyh-Chang; Gillis, Kevin D.

    2001-01-01

    The mechanism whereby cAMP stimulates Cl− flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1–43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl− current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1–43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca2+ in Calu-3 cells. However, no increase in Cl− current accompanies Ca2+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca2+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur. PMID:11479341

  10. [Isolation and characteristics of the plasma membrane fraction from the swine myometrium].

    PubMed

    Kondratiuk, T P; Bychenok, S F; Prishchepa, L A; Babich, L G; Kurskiĭ, M D

    1986-01-01

    An accelerated method is developed for isolating a fraction of plasma membranes of pig myometrium using ultracentrifugation within the sucrose density gradient (15% and 30%). The membranes possessed the high activity of 5'-nucleotidase and Na+, K+-ATPase and the low activity of rhotenon-insensitive NADH-cytochrome c reductase. The vesicularized preparations of plasma membranes are able of ATP-dependent accumulation of Ca2+ (7.5 +/- 0.3 nmol. 45Ca2+ per 1 mg of protein for 15 min). Phosphate increases the calcium accumulation in the presence of ATP and Mg2+. Ionophore A 23187 promotes a complete and rapid release of the previously active-accumulated calcium. The release of 45Ca2+ accumulated by the membrane fraction may be reached by introduction of 1 mM EGTA or DS-Na into the incubation medium, that evidences for the cation accumulation inside closed structures. Using concanavalin-A-sepharose 4B it is shown that 60% of membrane vesicles are turned inside out. The low saponine concentrations (0.0005%) which inhibit Ca2+-accumulation by plasma membranes but not by the endoplasmic reticulum inhibit this process by 60-70% in preparations of the isolated membrane fraction. The method has certain advantages over the previously applied methods used for isolating of plasma membrane fragments from smooth muscles. PMID:3016962

  11. Fluconazole treatment hyperpolarizes the plasma membrane of Candida cells.

    PubMed

    Elicharova, Hana; Sychrova, Hana

    2013-11-01

    Five pathogenic Candida species were compared in terms of their osmotolerance, tolerance to toxic sodium and lithium cations, and resistance to fluconazole. The species not only differed, in general, in their tolerance to high osmotic pressure (C. albicans and C. parapsilosis being the most osmotolerant) but exhibited distinct sensitivities to toxic sodium and lithium cations, with C. parapsilosis and C. tropicalis being very tolerant but C. krusei and C. dubliniensis sensitive to LiCl. The treatment of both fluconazole-susceptible (C. albicans and C. parapsilosis) and fluconazole-resistant (C. dubliniensis, C. krusei and C. tropicalis) growing cells with subinhibitory concentrations of fluconazole resulted in substantially elevated intracellular Na(+) levels. Using a diS-C3(3) assay, for the first time, to monitor the relative membrane potential (ΔΨ) of Candida cells, we show that the fluconazole treatment of growing cells of all five species results in a substantial hyperpolarization of their plasma membranes, which is responsible for an increased non-specific transport of toxic alkali metal cations and other cationic drugs (e.g., hygromycin B). Thus, the combination of relatively low doses of fluconazole and drugs, whose import into the tested Candida strains is driven by the cell membrane potential, might be especially potent in terms of its ability to inhibit the growth of or even kill various Candida species. PMID:23547882

  12. Plasma jet accelerator optimization with supple membrane model

    NASA Astrophysics Data System (ADS)

    Galkin, S. A.; Bogatu, I. N.; Kim, J. S.

    2006-10-01

    High density (>=3x10^17cm-3) and high Mach number (M>10) plasma jets have important applications such as plasma rotation, refueling and disruption mitigation in tokamaks. The most deleterious blow-by instability occurs in coaxial plasma accelerators; hence electrode shape optimization is required to accelerate plasmas to ˜200 km/s [1]. A full 3D particle simulation takes a huge computational time. We have developed a membrane model to provide a good starting point and further physical insight for a full 3D optimization. Our model approximates the axisymmetrical plasma by a thin supple conducting membrane with a distributed mass, located between the electrodes, and connects them to model dynamics of the blow-by instability and to conduct the optimization. The supple membrane is allowed to slip along the conductors freely or with some friction as affected by Lorenz force, generated by magnetic field inside the chamber and current on membrane. The total mass and the density distribution represent the initial plasma. The density is redistributed adiabatically during the acceleration. An external electrical circuit with capacitance, inductance and resistivity is a part of the model. The membrane model simulation results will be compared to the 2D fluid MACH2 results and then will be used to guide a full 3D optimization by the LSP code. 1. http://hyperv.com/projects/pic/

  13. Roles of charged particles and reactive species on cell membrane permeabilization induced by atmospheric-pressure plasma irradiation

    NASA Astrophysics Data System (ADS)

    Sasaki, Shota; Kanzaki, Makoto; Hokari, Yutaro; Tominami, Kanako; Mokudai, Takayuki; Kanetaka, Hiroyasu; Kaneko, Toshiro

    2016-07-01

    As factors that influence cell membrane permeabilization during direct and indirect atmospheric-pressure plasma irradiation, charged particle influx, superoxide anion radicals (O2 ‑•), and hydrogen peroxide (H2O2) in plasma-irradiated solution were evaluated. These are the three strong candidate factors and might multiply contribute to cell membrane permeabilization. In particular, a shorter plasma diffusion distance leads to the enhancement of the direct effects such as charged particle influx and further increase cell membrane permeability. In addition, O2 ‑• dissipates over time (a life span of the order of minutes) in plasma-irradiated water, and the deactivation of a plasma-irradiated solution in term of cell membrane permeabilization occurs in a life span of the same order. These results could promote the understanding of the mechanism of plasma-induced cell membrane permeabilization.

  14. A locally-induced increase in intracellular Ca2+ propagates cell-to-cell in the presence of plasma membrane Ca2+ ATPase inhibitors in non-excitable cells.

    PubMed

    Nakano, Tadashi; Koujin, Takako; Suda, Tatsuya; Hiraoka, Yasushi; Haraguchi, Tokuko

    2009-11-19

    Intercellular Ca(2+) waves are commonly observed in many cell types. In non-excitable cells, intercellular Ca(2+) waves are mediated by gap junctional diffusion of a Ca(2+) mobilizing messenger such as IP(3). Since Ca(2+) is heavily buffered in the cytosolic environment, it has been hypothesized that the contribution of the diffusion of Ca(2+) to intercellular Ca(2+) waves is limited. Here, we report that in the presence of plasma membrane Ca(2+) ATPase inhibitors, locally-released Ca(2+) from the flash-photolysis of caged-Ca(2+) appeared to induce further Ca(2+) release and were propagated from one cell to another, indicating that Ca(2+) was self-amplified to mediate intercellular Ca(2+) waves. Our findings support the notion that non-excitable cells can establish a highly excitable medium to communicate local responses with distant cells. PMID:19840794

  15. Membrane stress increases cation permeability in red cells.

    PubMed

    Johnson, R M

    1994-11-01

    The human red cell is known to increase its cation permeability when deformed by mechanical forces. Light-scattering measurements were used to quantitate the cell deformation, as ellipticity under shear. Permeability to sodium and potassium was not proportional to the cell deformation. An ellipticity of 0.75 was required to increase the permeability of the membrane to cations, and flux thereafter increased rapidly as the limits of cell extension were reached. Induction of membrane curvature by chemical agents also did not increase cation permeability. These results indicate that membrane deformation per se does not increase permeability, and that membrane tension is the effector for increased cation permeability. This may be relevant to some cation permeabilities observed by patch clamping. PMID:7858123

  16. Essentially All Excess Fibroblast Cholesterol Moves from Plasma Membranes to Intracellular Compartments

    PubMed Central

    Lange, Yvonne; Ye, Jin; Steck, Theodore L.

    2014-01-01

    It has been shown that modestly increasing plasma membrane cholesterol beyond its physiological set point greatly increases the endoplasmic reticulum and mitochondrial pools, thereby eliciting manifold feedback responses that return cell cholesterol to its resting state. The question arises whether this homeostatic mechanism reflects the targeting of cell surface cholesterol to specific intracellular sites or its general equilibration among the organelles. We now show that human fibroblast cholesterol can be increased as much as two-fold from 2-hydroxypropyl-β-cyclodextrin without changing the size of the cell surface pool. Rather, essentially all of the added cholesterol disperses rapidly among cytoplasmic membranes, increasing their overall cholesterol content by as much as five-fold. We conclude that the level of plasma membrane cholesterol is normally at capacity and that even small increments above this physiological set point redistribute essentially entirely to intracellular membranes, perhaps down their chemical activity gradients. PMID:25014655

  17. Reversal of carbon tetrachloride induced changes in microviscosity and lipid composition of liver plasma membrane by colchicine in rats.

    PubMed Central

    Solis-Herruzo, J A; De Gando, M; Ferrer, M P; Hernandez Muñoz, I; Fernandez-Boya, B; De la Torre, M P; Muñoz-Yague, M T

    1993-01-01

    Colchicine is beneficial in the treatment of cirrhotic patients, it prevents changes in plasma membrane bound enzymes induced by CCl4 intoxication. In this study, lipid composition and microviscosity were measured in liver plasma membranes isolated from rats given CCl4. Microviscosity values increased in rats given CCl4 for six weeks but fell considerably in those given CCl4 for 10 weeks. Both these changes were absent when colchicine was given with CCl4. The cholesterol/phospholipid molar ratios and lipid peroxide values increased but plasma membrane phospholipids, the length of fatty acyl chains, and the unsaturation index fell significantly after CCl4 intoxication. Colchicine treatment also prevented these changes. Changes in the lipid composition of liver plasma membranes were significantly correlated with lipid peroxidation. Colchicine prevents changes in the physicochemical properties of liver plasma membranes induced by longterm CCl4 treatment, probably by blocking peroxidation of unsaturated fatty acids. PMID:8244117

  18. Strong Static Magnetic Fields Increase the Gel Signal in Partially Hydrated DPPC/DMPC Membranes.

    PubMed

    Tang, Jennifer; Alsop, Richard J; Schmalzl, Karin; Epand, Richard M; Rheinstädter, Maikel C

    2015-01-01

    NIt was recently reported that static magnetic fields increase lipid order in the hydrophobic membrane core of dehydrated native plant plasma membranes [Poinapen, Soft Matter 9:6804-6813, 2013]. As plasma membranes are multicomponent, highly complex structures, in order to elucidate the origin of this effect, we prepared model membranes consisting of a lipid species with low and high melting temperature. By controlling the temperature, bilayers coexisting of small gel and fluid domains were prepared as a basic model for the plasma membrane core. We studied molecular order in mixed lipid membranes made of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) using neutron diffraction in the presence of strong static magnetic fields up to 3.5 T. The contribution of the hydrophobic membrane core was highlighted through deuterium labeling the lipid acyl chains. There was no observable effect on lipid organization in fluid or gel domains at high hydration of the membranes. However, lipid order was found to be enhanced at a reduced relative humidity of 43%: a magnetic field of 3.5 T led to an increase of the gel signal in the diffraction patterns of 5%. While all biological materials have weak diamagnetic properties, the corresponding energy is too small to compete against thermal disorder or viscous effects in the case of lipid molecules. We tentatively propose that the interaction between the fatty acid chains' electric moment and the external magnetic field is driving the lipid tails in the hydrophobic membrane core into a better ordered state. PMID:26426063

  19. Strong Static Magnetic Fields Increase the Gel Signal in Partially Hydrated DPPC/DMPC Membranes

    PubMed Central

    Tang, Jennifer; Alsop, Richard J.; Schmalzl, Karin; Epand, Richard M.; Rheinstädter, Maikel C.

    2015-01-01

    It was recently reported that static magnetic fields increase lipid order in the hydrophobic membrane core of dehydrated native plant plasma membranes [Poinapen, Soft Matter 9:6804-6813, 2013]. As plasma membranes are multicomponent, highly complex structures, in order to elucidate the origin of this effect, we prepared model membranes consisting of a lipid species with low and high melting temperature. By controlling the temperature, bilayers coexisting of small gel and fluid domains were prepared as a basic model for the plasma membrane core. We studied molecular order in mixed lipid membranes made of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) using neutron diffraction in the presence of strong static magnetic fields up to 3.5 T. The contribution of the hydrophobic membrane core was highlighted through deuterium labeling the lipid acyl chains. There was no observable effect on lipid organization in fluid or gel domains at high hydration of the membranes. However, lipid order was found to be enhanced at a reduced relative humidity of 43%: a magnetic field of 3.5 T led to an increase of the gel signal in the diffraction patterns of 5%. While all biological materials have weak diamagnetic properties, the corresponding energy is too small to compete against thermal disorder or viscous effects in the case of lipid molecules. We tentatively propose that the interaction between the fatty acid chains’ electric moment and the external magnetic field is driving the lipid tails in the hydrophobic membrane core into a better ordered state. PMID:26426063

  20. Surface modification of nanoporous alumina membranes by plasma polymerization

    NASA Astrophysics Data System (ADS)

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

    2008-06-01

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

  1. Deposition of polymeric perfluored thin films in proton ionic membranes by plasma processes

    NASA Astrophysics Data System (ADS)

    Polak, Peter Lubomir; Mousinho, Ana Paula; Ordonez, Nelson; da Silva Zambom, Luis; Mansano, Ronaldo Domingues

    2007-10-01

    In this work the surfaces of polymeric membranes based on Nafion (proton conducting material), used in proton exchange membranes fuel cells (PEMFC) had been modified by plasma deposition of perfluored polymers, in order to improve its functioning in systems of energy generation (fuel cells). The deposition increases the chemical resistance of the proton ionic polymers without losing the electrical properties. The processing of the membranes also reduces the permeability of the membranes to the alcohols (methanol and ethanol), thus preventing poisoning of the fuel cell. The processing of the membranes of Nafion was carried through in a system of plasma deposition using a mixture of CF 4 and H 2 gases. The plasma processing was made mainly to increase the chemical resistance and result in hydrophobic surfaces. The Fourier transformed infrared (FTIR) technique supplies a spectrum with information about the CF n bond formation. Through the Rutherford back scattering (RBS) technique it was possible to verify the deposition rate of the polymeric layer. The plasma process with composition of 60% of CF 4 and 40% of H 2 presented the best deposition rate. By the spectrum analysis for the optimized configuration, it was possible to verify that the film deposition occurred with a thickness of 90 nm, and fluorine concentration was nearly 30%. Voltammetry made possible to verify that the fluorination increases the membranes chemical resistance, improving the stability of Nafion, becoming an attractive process for construction of fuel cells.

  2. Effects of non-thermal plasma on the electrical properties of an erythrocyte membrane

    NASA Astrophysics Data System (ADS)

    Lee, Jin Young; Baik, Ku Youn; Kim, Tae Soo; Lim, Jaekwan; Uhm, Han S.; Choi, Eun Ha

    2015-09-01

    Non-thermal plasma is used here for membrane oxidation and permeabilization in which the electrical properties of an erythrocyte membrane are investigated after treatments. The zeta potential as measured by electrophoresis shows the increased negativity of the membrane surface potential (Ψs). The secondary electron emission coefficient ( γ) measured by a focused ion beam shows a decrease in the dipole potential (Ψd) of lipid molecules. The voltage-sensitive fluorescent intensity as measured by flow cytometry shows a decrease in the trans-membrane potential (ΔΨ) through the lipid bilayer membrane. These results allow us to take a step forward to unveil the complex events occurring in plasma-treated cells.

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  6. Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43

    PubMed Central

    Gauthier-Kemper, Anne; Igaev, Maxim; Sündermann, Frederik; Janning, Dennis; Brühmann, Jörg; Moschner, Katharina; Reyher, Hans-Jürgen; Junge, Wolfgang; Glebov, Konstantin; Walter, Jochen; Bakota, Lidia; Brandt, Roland

    2014-01-01

    Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43. PMID:25165142

  7. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies

    PubMed Central

    Zidovetzki, Raphael

    2007-01-01

    The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol. PMID:17493580

  8. Detection of glycoproteins in the Acanthamoeba plasma membrane

    SciTech Connect

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

    1988-11-01

    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.

  9. Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase

    PubMed Central

    De Michelis, Maria Ida; Pugliarello, Maria Chiara; Rasi-Caldogno, Franca

    1989-01-01

    The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a Kd of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (Kd 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H+-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H+-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H+-ATPase is mediated by fusicoccin binding to its plasma membrane receptor. PMID:16666723

  10. Palmitoylation of POTE family proteins for plasma membrane targeting

    SciTech Connect

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

    2007-11-23

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

  11. Increased Membrane Cholesterol in Lymphocytes Diverts T-Cells toward an Inflammatory Response

    PubMed Central

    Surls, Jacqueline; Nazarov-Stoica, Cristina; Kehl, Margaret; Olsen, Cara; Casares, Sofia; Brumeanu, Teodor-D.

    2012-01-01

    Cell signaling for T-cell growth, differentiation, and apoptosis is initiated in the cholesterol-rich microdomains of the plasma membrane known as lipid rafts. Herein, we investigated whether enrichment of membrane cholesterol in lipid rafts affects antigen-specific CD4 T-helper cell functions. Enrichment of membrane cholesterol by 40–50% following squalene administration in mice was paralleled by an increased number of resting CD4 T helper cells in periphery. We also observed sensitization of the Th1 differentiation machinery through co-localization of IL-2Rα, IL-4Rα, and IL-12Rβ2 subunits with GM1 positive lipid rafts, and increased STAT-4 and STAT-5 phosphorylation following membrane cholesterol enrichment. Antigen stimulation or CD3/CD28 polyclonal stimulation of membrane cholesterol-enriched, resting CD4 T-cells followed a path of Th1 differentiation, which was more vigorous in the presence of increased IL-12 secretion by APCs enriched in membrane cholesterol. Enrichment of membrane cholesterol in antigen-specific, autoimmune Th1 cells fostered their organ-specific reactivity, as confirmed in an autoimmune mouse model for diabetes. However, membrane cholesterol enrichment in CD4+ Foxp3+ T-reg cells did not alter their suppressogenic function. These findings revealed a differential regulatory effect of membrane cholesterol on the function of CD4 T-cell subsets. This first suggests that membrane cholesterol could be a new therapeutic target to modulate the immune functions, and second that increased membrane cholesterol in various physiopathological conditions may bias the immune system toward an inflammatory Th1 type response. PMID:22723880

  12. Decreasing Outer Hair Cell Membrane Cholesterol Increases Cochlear Electromechanics

    NASA Astrophysics Data System (ADS)

    Brownell, William E.; Jacob, Stefan; Hakizimana, Pierre; Ulfendahl, Mats; Fridberger, Anders

    2011-11-01

    The effect of decreasing membrane cholesterol on the mechanical response of the cochlea to acoustic and/or electrical stimulation was monitored using laser interferometry. In contrast to pharmacological interventions that typically decrease cochlear electromechanics, reducing membrane cholesterol increased the response. The electromechanical response in untreated preparations was asymmetric with greater displacements in response to positive currents and cholesterol depletion increased the asymmetry. The results confirm that outer hair cell electromotility is enhanced by low membrane cholesterol. The asymmetry of the response indicates the outer hair cell resting membrane potential is hyperpolarized relative to the voltage of maximum gain for the outer hair cell voltage-displacement function. The magnitude of the response increase suggests a non-uniform distribution of cholesterol along the lateral wall of normal adult outer hair cells.

  13. Comparative Analysis of Techniques to Purify Plasma Membrane Proteins

    PubMed Central

    Weekes, Michael P.; Antrobus, Robin; Lill, Jennie R.; Duncan, Lidia M.; Hör, Simon; Lehner, Paul J.

    2010-01-01

    The aim of this project was to identify the best method for the enrichment of plasma membrane (PM) proteins for proteomics experiments. Following tryptic digestion and extended liquid chromatography-tandem mass spectrometry acquisitions, data were processed using MaxQuant and Gene Ontology (GO) terms used to determine protein subcellular localization. The following techniques were examined for the total number and percentage purity of PM proteins identified: (a) whole cell lysate (total number, 84–112; percentage purity, 9–13%); (b) crude membrane preparation (104–111; 17–20%); (c) biotinylation of surface proteins with N-hydroxysulfosuccinimydyl-S,S-biotin and streptavidin pulldown (78–115; 27–31%); (d) biotinylation of surface glycoproteins with biocytin hydrazide and streptavidin pulldown (41–54; 59–85%); or (e) biotinylation of surface glycoproteins with amino-oxy-biotin (which labels the sialylated fraction of PM glycoproteins) and streptavidin pulldown (120; 65%). A two- to threefold increase in the overall number of proteins identified was achieved by using stop and go extraction tip (StageTip)-based anion exchange (SAX) fractionation. Combining technique (e) with SAX fractionation increased the number of proteins identified to 281 (54%). Analysis of GO terms describing these proteins identified a large subset of proteins integral to the membrane with no subcellular assignment. These are likely to be of PM location and bring the total PM protein identifications to 364 (68%). This study suggests that selective biotinylation of the cell surface using amino-oxy-biotin in combination with SAX fractionation is a useful method for identification of sialylated PM proteins. PMID:20808639

  14. Modulation of Plasma Membrane Ca2+-ATPase by Neutral Phospholipids

    PubMed Central

    Pignataro, María Florencia; Dodes-Traian, Martín M.; González-Flecha, F. Luis; Sica, Mauricio; Mangialavori, Irene C.; Rossi, Juan Pablo F. C.

    2015-01-01

    The effects of lipids on membrane proteins are likely to be complex and unique for each membrane protein. Here we studied different detergent/phosphatidylcholine reconstitution media and tested their effects on plasma membrane Ca2+ pump (PMCA). We found that Ca2+-ATPase activity shows a biphasic behavior with respect to the detergent/phosphatidylcholine ratio. Moreover, the maximal Ca2+-ATPase activity largely depends on the length and the unsaturation degree of the hydrocarbon chain. Using static light scattering and fluorescence correlation spectroscopy, we monitored the changes in hydrodynamic radius of detergent/phosphatidylcholine particles during the micelle-vesicle transition. We found that, when PMCA is reconstituted in mixed micelles, neutral phospholipids increase the enzyme turnover. The biophysical changes associated with the transition from mixed micelles to bicelles increase the time of residence of the phosphorylated intermediate (EP), decreasing the enzyme turnover. Molecular dynamics simulations analysis of the interactions between PMCA and the phospholipid bilayer in which it is embedded show that in the 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer, charged residues of the protein are trapped in the hydrophobic core. Conversely, in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer, the overall hydrophobic-hydrophilic requirements of the protein surface are fulfilled the best, reducing the thermodynamic cost of exposing charged residues to the hydrophobic core. The apparent mismatch produced by a 1,2-dioleoyl-sn-glycero-3-phosphocholine thicker bilayer could be a structural foundation to explain its functional effect on PMCA. PMID:25605721

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  16. Bovine plasma proteins increase virulence of Haemophilus somnus in mice.

    PubMed

    Geertsema, Roger S; Kimball, Richard A; Corbeil, Lynette B

    2007-01-01

    The role of bovine serum or plasma proteins in Haemophilus somnus virulence was investigated in a mouse model of septicemia. An increase in virulence was detected when the organism was pre-incubated for 5 min and inoculated with fetal calf serum. When purified bovine serum or plasma proteins were pre-incubated with H. somnus before inoculating into mice, transferrin was found to increase virulence. Bovine lactoferrin was also noted to increase virulence, but to a lesser extent and had a delayed time course when compared with transferrin. Using an ELISA assay, an increased amount of H. somnus whole cells and culture supernatant bound to bovine transferrin when the organism was grown in iron-restricted media. Lactoferrin also bound to H. somnus, but binding was not affected by growth in iron-restricted media and it was eliminated with 2M NaCl, which reversed charge mediated binding. Transferrin, but not lactoferrin, supported growth of H. somnus on iron-depleted agar based media using a disk assay. Therefore, lactoferrin increased virulence by an undetermined mechanism whereas transferrin increased virulence of H. somnus by binding to iron-regulated outer-membrane proteins (IROMPs) and providing iron to the pathogen. PMID:17125964

  17. Crystal structure of the plasma membrane proton pump.

    PubMed

    Pedersen, Bjørn P; Buch-Pedersen, Morten J; Morth, J Preben; Palmgren, Michael G; Nissen, Poul

    2007-12-13

    A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi, and Na+,K+-ATPase (the sodium-potassium pump) in animals. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis. The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na+,K+-ATPase and Ca2+-ATPase are type II. Electron microscopy has revealed the overall shape of proton pumps, 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 where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement. PMID:18075595

  18. Cocaine induction of dopamine transporter trafficking to the plasma membrane.

    PubMed

    Little, Karley Y; Elmer, Lawrence W; Zhong, Huailing; Scheys, Joshua O; Zhang, Lian

    2002-02-01

    Several previous human postmortem experiments have detected an increase in striatal [(3)H]WIN 35428 binding to the dopamine transporter (DAT) in chronic cocaine users. However, animal experiments have found considerable variability in DAT radioligand binding levels in brain after cocaine administration, perhaps caused by length and dose of treatment and type of radioligand used. The present experiments tested the hypothesis that [(3)H]WIN 35428 binding and [(3)H]dopamine uptake would be increased by exposure to cocaine through alterations in DAT cellular trafficking, rather than increased protein synthesis. Experiments were conducted in stably hDAT-transfected N2A cells and assessed the dose response and time course of cocaine effects on [(3)H]WIN 35428 binding to the DAT, [(3)H]dopamine uptake, measures of DAT protein and mRNA, as well as DAT subcellular location. Cocaine doses of 10(-6) M caused statistically significant increases in [(3)H]WIN 35428 binding and [(3)H]dopamine uptake after 12 and 3 h, respectively. Despite these increases in DAT function, there was no change in DAT total protein or mRNA. Immunofluorescence and biotinylation experiments indicated that cocaine treatment induced increases in plasma membrane DAT immunoreactivity and intracellular decreases. The present model system may further our understanding of regulatory alterations in DAT radioligand binding and function caused by cocaine exposure. PMID:11809869

  19. Modification of plasma membrane organization in tobacco cells elicited by cryptogein.

    PubMed

    Gerbeau-Pissot, Patricia; Der, Christophe; Thomas, Dominique; Anca, Iulia-Andra; Grosjean, Kevin; Roche, Yann; Perrier-Cornet, Jean-Marie; Mongrand, Sébastien; Simon-Plas, Françoise

    2014-01-01

    Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the "membrane raft" hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment. PMID:24235133

  20. Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein1[W

    PubMed Central

    Gerbeau-Pissot, Patricia; Der, Christophe; Thomas, Dominique; Anca, Iulia-Andra; Grosjean, Kevin; Roche, Yann; Perrier-Cornet, Jean-Marie; Mongrand, Sébastien; Simon-Plas, Françoise

    2014-01-01

    Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the “membrane raft” hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment. PMID:24235133

  1. 25-Hydroxycholesterol Increases the Availability of Cholesterol in Phospholipid Membranes

    SciTech Connect

    Olsen, Brett N.; Schlesinger, Paul H.; Ory, Daniel S.; Baker, Nathan A.

    2011-02-01

    Side-chain oxysterols are enzymatically generated oxidation products of cholesterol that serve a central role in mediating cholesterol homeostasis. Recent work has shown that side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), alter membrane structure in very different ways from cholesterol, suggesting a possible mechanism for how these oxysterols regulate cholesterol homeostasis. Here we extend our previous work, using molecular dynamics simulations of 25-HC and cholesterol mixtures in 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers to examine interactions between 25-HC and cholesterol in the same bilayer. When added to cholesterol-containing membranes, 25-HC causes larger changes in membrane structure than when added to cholesterol-free membranes, demonstrating interactions between the two sterols. We also find that the presence of 25-HC changes the position, orientation, and solvent accessibility of cholesterol, shifting it into the water interface and therefore its availability to external acceptors. This is consistent with experimental results showing that oxysterols can trigger cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. These interactions provide a potential mechanism for 25-HC-mediated regulation of cholesterol trafficking and homeostasis through direct modulation of cholesterol availability.

  2. Magnetic apatite for structural insights on the plasma membrane

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  3. Functional Implications of Plasma Membrane Condensation for T Cell Activation

    PubMed Central

    Quinn, Carmel M.; Engelhardt, Karin; Williamson, David; Grewal, Thomas; Jessup, Wendy; Harder, Thomas; Gaus, Katharina

    2008-01-01

    The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process. PMID:18509459

  4. Estradiol's interesting life at the cell's plasma membrane.

    PubMed

    Caldwell, J D; Gebhart, V M; Jirikowski, G F

    2016-07-01

    Clearly, we have presented here evidence of a very complex set of mechanisms and proteins involved with various and intricate actions of steroids at the plasma membrane. Steroids do MUCH more at the plasma membrane than simply passing passively through it. They may sit in the membrane; they are bound by numerous proteins in the membrane, including ERs, SHBG, steroid-binding globulin receptors, and perhaps elements of cellular architecture such as tubulin. It also seems likely that the membrane itself responds graphically to the presence of steroids by actually changing its shape as well, perhaps, as accumulating steroids. Clara Szego suggested in the 1980s that actions of E2 at one level would act synergistically with its actions at another level (e.g. membrane actions would complement nuclear actions). Given the sheer number of proteins involved in steroid actions, just at the membrane level, it seems unlikely that every action of a steroid on every potential protein effector will act to the same end. It seems more likely that these multiple effects and sites of effect of steroids contribute to the confusion that exists as to what actions steroids always have. For example, there is confusion with regard to synthetic agents (SERMs etc.) that have different and often opposite actions depending on which organ they act upon. A better understanding of the basic actions of steroids should aid in understanding the variability of their clinical effects. PMID:27018128

  5. An Endosome-to-Plasma Membrane Pathway Involved in Trafficking of a Mutant Plasma Membrane ATPase in Yeast

    PubMed Central

    Luo, Wen-jie; Chang, Amy

    2000-01-01

    The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37°C. Several vps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane. In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, and vps8 mutants. Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36 cells. After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane. In both vps1 and vps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface. Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1. Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (and vps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64. Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane. These data support a model in which mutant Pma1 is diverted from the Golgi to the surface in vps1 cells. We hypothesize that in vps8 and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway. PMID:10679016

  6. Red wine activates plasma membrane redox system in human erythrocytes.

    PubMed

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

    2016-05-01

    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. PMID:26866566

  7. Intrinsic stability of Brassicaceae plasma membrane in relation to changes in proteins and lipids as a response to salinity.

    PubMed

    Chalbi, Najla; Martínez-Ballesta, Ma Carmen; Youssef, Nabil Ben; Carvajal, Micaela

    2015-03-01

    Changes in plasma membrane lipids, such as sterols and fatty acids, have been observed as a result of salt stress. These alterations, together with modification of the plasma membrane protein profile, confer changes in the physical properties of the membrane to be taken into account for biotechnological uses. In our experiments, the relationship between lipids and proteins in three different Brassicaceae species differing in salinity tolerance (Brassica oleracea, B. napus and Cakile maritima) and the final plasma membrane stability were studied. The observed changes in the sterol (mainly an increase in sitosterol) and fatty acid composition (increase in RUFA) in each species led to physical adaptation of the plasma membrane to salt stress. The in vitro vesicles stability was higher in the less tolerant (B. oleracea) plants together with low lipoxygenase activity. These results indicate that the proteins/lipids ratio and lipid composition is an important aspect to take into account for the use of natural vesicles in plant biotechnology. PMID:25544590

  8. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas

    NASA Astrophysics Data System (ADS)

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-01

    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.

  9. Exclusive photorelease of signalling lipids at the plasma membrane

    PubMed Central

    Nadler, André; Yushchenko, Dmytro A.; Müller, Rainer; Stein, Frank; Feng, Suihan; Mulle, Christophe; Carta, Mario; Schultz, Carsten

    2015-01-01

    Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems. PMID:26686736

  10. Exclusive photorelease of signalling lipids at the plasma membrane.

    PubMed

    Nadler, André; Yushchenko, Dmytro A; Müller, Rainer; Stein, Frank; Feng, Suihan; Mulle, Christophe; Carta, Mario; Schultz, Carsten

    2015-01-01

    Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems. PMID:26686736

  11. Mammalian gamete plasma membranes re-assessments and reproductive implications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. On the puzzling distribution of cholesterol in the plasma membrane.

    PubMed

    Giang, H; Schick, M

    2016-09-01

    The distribution of cholesterol between the two leaves of the plasma membrane in mammalian cells presents a conundrum; given cholesterol's known affinity for sphingomyelin, which resides predominantly in the exoplasmic leaf, why is it that experiment finds a majority of the cholesterol in the cytoplasmic leaf? This article reviews a recently proposed solution to this puzzle. PMID:26724709

  13. Granuphilin exclusively mediates functional granule docking to the plasma membrane

    PubMed Central

    Mizuno, Kouichi; Fujita, Takuji; Gomi, Hiroshi; Izumi, Tetsuro

    2016-01-01

    In regulated exocytosis, it is generally assumed that vesicles must stably “dock” at the plasma membrane before they are primed to become fusion-competent. However, recent biophysical analyses in living cells that visualize fluorescent secretory granules have revealed that exocytic behaviors are not necessarily uniform: some granules beneath the plasma membrane are resistant to Ca2+ -triggered release, while others are accelerated to fuse without a pause for stable docking. These findings suggest that stable docking is unnecessary, and can even be inhibitory or nonfunctional, for fusion. Consistently, pancreatic β cells deficient in the Rab27 effector, granuphilin, lack insulin granules directly attached to the plasma membrane in electron micrographs but nevertheless exhibit augmented exocytosis. Here we directly compare the exocytic behaviors between granuphilin-positive and -negative insulin granules. Although granuphilin makes granules immobile and fusion-reluctant beneath the plasma membrane, those granuphilin-positive, docked granules release a portion of granuphilin upon fusion, and fuse at a frequency and time course similar to those of granuphilin-negative undocked granules. Furthermore, granuphilin forms a 180-nm cluster at the site of each docked granule, along with granuphilin-interacting Rab27a and Munc18-1 clusters. These findings indicate that granuphilin is an exclusive component of the functional and fusion-inhibitory docking machinery of secretory granules. PMID:27032672

  14. A TRiP to the plasma membrane

    PubMed Central

    Ghosh, Debapriya; Voets, Thomas

    2015-01-01

    TRP ion channels are ubiquitously present in the mammalian body and take part in numerous key physiological functions, including temperature sensing, taste perception, osmo-regulation, cardiac function, renal function, development, and glucose homeostasis. The mechanisms whereby TRP channels are transported to the plasma membrane, where most of them exert their physiological actions, remains a poorly understood aspect of TRP channel biology.

  15. Expression of a plasma membrane proteolipid during differentiation of neuronal and glial cells in primary culture.

    PubMed

    Shea, T B; Fischer, I; Sapirstein, V

    1986-09-01

    Plasma membrane proteolipid protein (PM-PLP) synthesis was examined in embryonic rat neurons and neonatal rat glial cells during differentiation in culture. Glial cultures were treated with 1 mM N6, O2, dibutyryl cyclic adenosine monophosphate (dbcAMP) following confluency to induce differentiation, which resulted in the elaboration of long cellular processes. However, no changes in the biosynthetic level of PM-PLP was observed during the differentiation of these cells. Neurons differentiated spontaneously in culture, forming cellular aggregates immediately following plating and elaborating a network of neurites over 7 days. The differentiation of neurons was accompanied by a seven-fold increase in PM-PLP synthesis with increases in biosynthetic increase in PM-PLP synthesis with increases in biosynthetic rate observed between days 1 and 3 and between days 3 and 7 in culture. Ultrastructural examination of neurons indicated that the Golgi apparatus was also developing during this period of time, with an increase in both the number of lamellae and generation of vesicles. The transport of PM-PLP to the plasma membrane was therefore examined in neurons at day 7 in culture by pulse labeling experiments with monensin and colchicine. Monensin (1 microM) was found to inhibit the appearance of radiolabeled PM-PLP in the plasma membrane by 63%, indicating that a functional Golgi apparatus is required for transport of PM-PLP to its target membrane. Colchicine (125 microM) also inhibited the appearance of newly synthesized PM-PLP in the plasma membrane by greater than 40%, suggesting that microtubules may also be required for PM-PLP transport to the plasma membrane. PMID:3016181

  16. Inhibition of microbial growth on chitosan membranes by plasma treatment.

    PubMed

    de Oliveira Cardoso Macêdo, Marina; de Macêdo, Haroldo Reis Alves; Gomes, Dayanne Lopes; de Freitas Daudt, Natália; Rocha, Hugo Alexandre Oliveira; Alves, Clodomiro

    2013-11-01

    The use of polymeric medical devices has stimulated the development of new sterilization methods. The traditional techniques rely on ethylene oxide, but there are many questions concerning the carcinogenic properties of the ethylene oxide residues adsorbed on the materials after processing. Another common technique is the gamma irradiation process, but it is costly, its safe operation requires an isolated site, and it also affects the bulk properties of the polymers. The use of gas plasma is an elegant alternative sterilization technique. The plasma promotes efficient inactivation of the microorganisms, minimizes damage to the materials, and presents very little danger for personnel and the environment. In this study we used plasma for microbial inhibition of chitosan membranes. The membranes were treated with oxygen, methane, or argon plasma for different time periods (15, 30, 45, or 60 min). For inhibition of microbial growth with oxygen plasma, the time needed was 60 min. For the methane plasma, samples were successfully treated after 30, 45, and 60 min. For argon plasma, all treatment periods were effective. PMID:24251774

  17. S-Acylation of the cellulose synthase complex is essential for its plasma membrane localization.

    PubMed

    Kumar, Manoj; Wightman, Raymond; Atanassov, Ivan; Gupta, Anjali; Hurst, Charlotte H; Hemsley, Piers A; Turner, Simon

    2016-07-01

    Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. How interactions between membranes and the CSC are regulated is currently unknown. Here, we demonstrate that all catalytic subunits of the CSC, known as cellulose synthase A (CESA) proteins, are S-acylated. Analysis of Arabidopsis CESA7 reveals four cysteines in variable region 2 (VR2) and two cysteines at the carboxy terminus (CT) as S-acylation sites. Mutating both the VR2 and CT cysteines permits CSC assembly and trafficking to the Golgi but prevents localization to the plasma membrane. Estimates suggest that a single CSC contains more than 100 S-acyl groups, which greatly increase the hydrophobic nature of the CSC and likely influence its immediate membrane environment. PMID:27387950

  18. Control of Plasma Membrane Permeability by ABC Transporters.

    PubMed

    Khakhina, Svetlana; Johnson, Soraya S; Manoharlal, Raman; Russo, Sarah B; Blugeon, Corinne; Lemoine, Sophie; Sunshine, Anna B; Dunham, Maitreya J; Cowart, L Ashley; Devaux, Frédéric; Moye-Rowley, W Scott

    2015-05-01

    ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5Δ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3Δ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5Δ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5Δ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2. PMID:25724885

  19. Superhydrophilic poly(L-lactic acid) electrospun membranes for biomedical applications obtained by argon and oxygen plasma treatment

    NASA Astrophysics Data System (ADS)

    Correia, D. M.; Ribeiro, C.; Botelho, G.; Borges, J.; Lopes, C.; Vaz, F.; Carabineiro, S. A. C.; Machado, A. V.; Lanceros-Méndez, S.

    2016-05-01

    Poly(L-lactic acid), PLLA, electrospun membranes and films were plasma treated at different times and power with argon (Ar) and oxygen (O2), independently, in order to modify the hydrophobic nature of the PLLA membranes. Both Ar and O2 plasma treatments promote an increase in fiber average size of the electrospun membranes from 830 ± 282 nm to 866 ± 361 and 1179 ± 397 nm, respectively, for the maximum exposure time (970 s) and power (100 W). No influence of plasma treatment was detected in the physical-chemical characteristics of PLLA, such as chemical structure, polymer phase or degree of crystallinity. On the other hand, an increase in the roughness of the films was obtained both with argon and oxygen plasma treatments. Surface wettability studies revealed a decrease in the contact angle with increasing plasma treatment time for a given power and with increasing power for a given time in membranes and films and superhydrophilic electrospun fiber membranes were obtained. Results showed that the argon and oxygen plasma treatments can be used to tailor hydrophilicity of PLLA membranes for biomedical applications. MTT assay results indicated that plasma treatments under Ar and O2 do not influence the metabolic activity of MC3T3-E1 pre-osteoblast cells.

  20. Impact of ionic liquids in aqueous solution on bacterial plasma membranes studied with molecular dynamics simulations.

    PubMed

    Lim, Geraldine S; Zidar, Jernej; Cheong, Daniel W; Jaenicke, Stephan; Klähn, Marco

    2014-09-01

    The impact of five different imidazolium-based ionic liquids (ILs) diluted in water on the properties of a bacterial plasma membrane is investigated using molecular dynamics (MD) simulations. Cations considered are 1-octyl-3-methylimidazolium (OMIM), 1-octyloxymethyl-3-methylimidazolium (OXMIM), and 1-tetradecyl-3-methylimidazolium (TDMIM), as well as the anions chloride and lactate. The atomistic model of the membrane bilayer is designed to reproduce the lipid composition of the plasma membrane of Gram-negative Escherichia coli. Spontaneous insertion of cations into the membrane is observed in all ILs. Substantially more insertions of OMIM than of OXMIM occur and the presence of chloride reduces cation insertions compared to lactate. In contrast, anions do not adsorb onto the membrane surface nor diffuse into the bilayer. Once inserted, cations are oriented in parallel to membrane lipids with cation alkyl tails embedded into the hydrophobic membrane core, while the imidazolium-ring remains mostly exposed to the solvent. Such inserted cations are strongly associated with one to two phospholipids in the membrane. The overall order of lipids decreased after OMIM and OXMIM insertions, while on the contrary the order of lipids in the vicinity of TDMIM increased. The short alkyl tails of OMIM and OXMIM generate voids in the bilayer that are filled by curling lipids. This cation induced lipid disorder also reduces the average membrane thickness. This effect is not observed after TDMIM insertions due to the similar length of cation alkyl chain and the fatty acids of the lipids. This lipid-mimicking behavior of inserted TDMIM indicates a high membrane affinity of this cation that could lead to an enhanced accumulation of cations in the membrane over time. Overall, the simulations reveal how cations are inserted into the bacterial membrane and how such insertions change its properties. Moreover, the different roles of cations and anions are highlighted and the fundamental

  1. Plant cell plasma membrane structure and properties under clinostatting

    NASA Astrophysics Data System (ADS)

    Polulakh, Yu. A.; Zhadko, S. I.; Klimchuk, D. A.; Baraboy, V. A.; Alpatov, A. N.; Sytnik, K. M.

    Structural-functional organization of plasma membrane of pea roots seedling was investigated by methods of chemiluminescence, fluorescence probes, chromatography and freeze-fracture studies under normal conditions and clinostatting. Phase character of lipid peroxidation intensity was fixed. The initial phase of this process is characterized by lipid peroxidation decreasing with its next induction. The primary changes depending on free-radical mechanisms of lipid peroxidation were excellently revealed by chemiluminescence. Plasmalemma microviscosity increased on the average of 15-20 % under microgravity at the initial stages of its phenomenon. There were major changes of phosphatidilcholine and phosphatidilethanolamine contents. The total quantity of phospholipids remained rather stable. Changes of phosphatide acid concentration point to degradation and phospholipids biosynthesis. There were increases of unsaturated fatty acids mainly at the expense of linoleic and linolenic acids and also a decrease of saturated fatty acid content at the expense of palmitic and stearic acids. Unsaturation index of fatty acids increased as well. On the whole fatty acid composition was variable in comparison with phospholipids. Probably it is one of mechanisms of maintaining of microviscosity within definite limits. Considerable structural changes in organization of plasmalemma protein-lipid complex were not revealed by the freeze-fracture studies.

  2. Surface modification of PTMSP membranes by plasma treatment: Asymmetry of transport in organic solvent nanofiltration.

    PubMed

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

    2015-08-01

    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. PMID:25482845

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

    PubMed

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

    2015-01-01

    It may be possible to regulate the cell colonization of biodegradable polymer nanofibrous membranes by plasma treatment and by the density of the fibers. To test this hypothesis, nanofibrous membranes of different fiber densities were treated by oxygen plasma with a range of plasma power and exposure times. Scanning electron microscopy and mechanical tests showed significant modification of nanofibers after plasma treatment. The intensity of the fiber modification increased with plasma power and exposure time. The exposure time seemed to have a stronger effect on modifying the fiber. The mechanical behavior of the membranes was influenced by the plasma treatment, the fiber density, and their dry or wet state. Plasma treatment increased the membrane stiffness; however, the membranes became more brittle. Wet membranes displayed significantly lower stiffness than dry membranes. X-ray photoelectron spectroscopy (XPS) analysis showed a slight increase in oxygen-containing groups on the membrane surface after plasma treatment. Plasma treatment enhanced the adhesion and growth of HaCaT keratinocytes on nanofibrous membranes. The cells adhered and grew preferentially on membranes of lower fiber densities, probably due to the larger area of void spaces between the fibers. PMID:25085812

  4. Membrane damage and active but nonculturable state in liquid cultures of Escherichia coli treated with an atmospheric pressure plasma jet.

    PubMed

    Dolezalova, Eva; Lukes, Petr

    2015-06-01

    Electrical discharge plasmas can efficiently inactivate various microorganisms. Inactivation mechanisms caused by plasma, however, are not fully understood because of the complexity of both the plasma and biological systems. We investigated plasma-induced inactivation of Escherichia coli in water and mechanisms by which plasma affects bacterial cell membrane integrity. Atmospheric pressure argon plasma jet generated at ambient air in direct contact with bacterial suspension was used as a plasma source. We determined significantly lower counts of E. coli after treatment by plasma when they were assayed using a conventional cultivation technique than using a fluorescence-based LIVE/DEAD staining method, which indicated that bacteria may have entered the viable-but-nonculturable state (VBNC). We did not achieve resuscitation of these non-culturable cells, however, we detected their metabolic activity through the analysis of cellular mRNA, which suggests that cells may have been rather in the active-but-nonculturable state (ABNC). We hypothesize that peroxidation of cell membrane lipids by the reactive species produced by plasma was an important pathway of bacterial inactivation. Amount of malondialdehyde and membrane permeability of E. coli to propidium iodide increased with increasing bacterial inactivation by plasma. Membrane damage was also demonstrated by detection of free DNA in plasma-treated water. PMID:25212700

  5. Osmotic swelling of hepatocytes increases membrane conductance but not membrane capacitance.

    PubMed Central

    Graf, J; Rupnik, M; Zupancic, G; Zorec, R

    1995-01-01

    We have used the whole-cell patch-clamp technique to study changes in membrane conductance and membrane capacitance after osmotic swelling in rat hepatocytes. Hypoosmotic solutions induced an instantaneous increase in the volume of patch-clamped cells that was followed by a slow decline reminiscent of regulatory volume decrease as seen in intact cells. These morphological changes were associated with a transient increase in membrane conductance. The rise in conductance was not correlated with changes in capacitance, neither in time after the initiation of cell swelling nor in magnitude. Therefore we conclude that an osmotically induced increase in conductance is probably a result of the activation of existent channels in the plasmalemma and not a result of the fusion of vesicle membrane containing ionic channels. Images FIGURE 1 PMID:7540428

  6. Effect of clofibrate on the enzyme activity of rat liver plasma membranes.

    PubMed

    Renaud, G; Foliot, A; Marais, J; Infante, R

    1980-03-15

    The activity of 3 plasma membranes marker enzymes (5'-nucleotidase, Mg++-ATPase and alkaline phosphodiesterase-I) was determined in plasma membranes isolated from liver of control and of clofibrate-treated rats. A complete indentity of plasma membranes enzyme activity in the 2 groups of experimental animals was observed for the 3 enzymes studied. PMID:6102923

  7. Cross-tolerance of human placental plasma membranes of smokers to fluidizing effects of alcohol

    SciTech Connect

    Sastry, B.V.R.; Horst, M.A.; Naukam, R.J. )

    1991-03-11

    There is cross-tolerance between ethanol and several centrally acting drugs at the membrane level. In order to evaluate cross-tolerance between maternal smoking during pregnancy and alcohol, the authors have prepared plasma membranes of human term placentas from nonsmokers (NS, n=5) and smokers (S, 24 {plus minus} 8 cigarettes/day, n=5) and studied their microviscosities by steady state fluorescence polarization using trans-1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe. These experiments gave the following results: (a) microviscosity was increased by maternal smoking; (b) alcohol decreased microviscosity of the membranes of smokers; (c) exogenous nicotine did not exert any significant effect on the membranes of smokers and nonsmokers. Therefore, the increase in the rigidity of placental plasma membranes is due to chronic smoking, and these membranes are tolerant to the fluidizing effects of alcohol. Cross-tolerance between smoking and ethanol suggests a common hydrophobic locus of the apparent adaptation at the membrane level.

  8. Imaging plasma membrane deformations with pTIRFM.

    PubMed

    Passmore, Daniel R; Rao, Tejeshwar C; Peleman, Andrew R; Anantharam, Arun

    2014-01-01

    To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress. Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or diD. DiD intercalates in the membrane plane with a "fixed" orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561 nm laser (p-pol, s-pol). A 488 nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation. PMID:24747638

  9. Proteome Analysis of the Plasma Membrane of Mycobacterium Tuberculosis

    PubMed Central

    Arora, Shalini; Kosalai, K.; Namane, Abdelkader; Pym, Alex S.; Cole, Stewart T.

    2002-01-01

    The plasma membrane of Mycobacterium tuberculosis is likely to contain proteins that could serve as novel drug targets, diagnostic probes or even components of a vaccine against tuberculosis. With this in mind, we have undertaken proteome analysis of the membrane of M. tuberculosis H37Rv. Isolated membrane vesicles were extracted with either a detergent (Triton X114) or an alkaline buffer (carbonate) following two of the protocols recommended for membrane protein enrichment. Proteins were resolved by 2D-GE using immobilized pH gradient (IPG) strips, and identified by peptide mass mapping utilizing the M. tuberculosis genome database. The two extraction procedures yielded patterns with minimal overlap. Only two proteins, both HSPs, showed a common presence. MALDI–MS analysis of 61 spots led to the identification of 32 proteins, 17 of which were new to the M. tuberculosis proteome database. We classified 19 of the identified proteins as ‘membrane-associated’; 14 of these were further classified as ‘membrane-bound’, three of which were lipoproteins. The remaining proteins included four heat-shock proteins and several enzymes involved in energy or lipid metabolism. Extraction with Triton X114 was found to be more effective than carbonate for detecting ‘putative’ M. tuberculosis membrane proteins. The protocol was also found to be suitable for comparing BCG and M. tuberculosis membranes, identifying ESAT-6 as being expressed selectively in M. tuberculosis. While this study demonstrates for the first time some of the membrane proteins of M. tuberculosis, it also underscores the problems associated with proteomic analysis of a complex membrane such as that of a mycobacterium. PMID:18629250

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

    PubMed Central

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

    2003-01-01

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

  11. Amine Enrichment of Thin-Film Composite Membranes via Low Pressure Plasma Polymerization for Antimicrobial Adhesion.

    PubMed

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

    2015-07-15

    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. PMID:26083007

  12. Fractionation of liver plasma membranes prepared by zonal centrifugation

    PubMed Central

    Evans, W. H.

    1970-01-01

    1. Plasma membranes were isolated from crude nuclear sediments from mouse and rat liver by a rate-dependent centrifugation through a sucrose density gradient contained in the `A' type zonal rotor. 2. The membranes were further purified by isopycnic centrifugation, and characterized enzymically, chemically and morphologically. 3. When the plasma-membrane fraction of sucrose density 1.17g/cm3 was dispersed in a tight-fitting homogenizer, two subfractions of densities 1.12 and 1.18 were obtained by isopycnic centrifugation. 4. The light subfraction contained 5′-nucleotidase, nucleoside diphosphatase, leucine naphthylamidase and Mg2+-stimulated adenosine triphosphatase activities at higher specific activities than unfractionated membranes. The heavy subfraction was deficient in the above enzymes but contained higher Na++K+-stimulated adenosine triphosphatase activity. 5. The light subfraction contained twice as much phospholipid and cholesterol, and three times as much N-acetylneuraminic acid relative to unit protein weight as the heavy subfraction. Polyacrylamide-gel electrophoresis indicated differences in protein composition. 6. Electron microscopy showed the light subfraction to be vesicular. The heavy subfraction contained membrane strips with junctional complexes in addition to vesicles. ImagesPLATE 2PLATE 3PLATE 1 PMID:4315049

  13. The microviscosity of liver plasma membranes of rats fed with oleoylanilide.

    PubMed Central

    Pagani, R; Portoles, M T; Gavilanes, F G; Garcia-Barreno, P; Municio, A M

    1984-01-01

    Oleoylanilide was administered orally to groups of rats according to different patterns. Oleoylanilide was perfused at different concentrations through rat liver. Oleoylanilide was added to isolated hepatocytes. Oleoylanilide was added to plasma-membrane preparations. Membrane preparations were obtained after experiments performed in vivo and perfusion experiments and, by using 1,6-diphenylhexa-1,3,5-triene as fluorescence probe, the fluorescence polarization parameter was measured, from which the microviscosity (eta) was calculated. In all cases the microviscosity decreased markedly. Addition of oleoylanilide to hepatocyte preparations and to isolated membranes produced the same effect, increasing the fluidity of the membranes. These data suggest that oleoylanilide partitions into the membrane, disordering some lipid interactions. PMID:6712608

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

    SciTech Connect

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

    1988-11-01

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

  15. Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L. ) storage tissue

    SciTech Connect

    Giannini, J.L.; Gildensoph, L.H.; Briskin, D.P.

    1987-05-01

    Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.

  16. Homeostasis of the apical plasma membrane during regulated exocytosis in the salivary glands of live rodents

    PubMed Central

    Masedunskas, Andrius; Sramkova, Monika; Weigert, Roberto

    2011-01-01

    In exocrine organs such as the salivary glands, fluids and proteins are secreted into ductal structures by distinct mechanisms that are tightly coupled. In the acinar cells, the major secretory units of the salivary glands, fluids are secreted into the acinar canaliculi through paracellular and intracellular transport, whereas proteins are stored in large granules that undergo exocytosis and fuse with the apical plasma membranes releasing their content into the canaliculi. Both secretory processes elicit a remodeling of the apical plasma membrane that has not been fully addressed in in vitro or ex vivo models. Recently, we have studied regulated exocytosis in the salivary glands of live rodents, focusing on the role that actin and myosin plays in this process. We observed that during exocytosis both secretory granules and canaliculi are subjected to the hydrostatic pressure generated by fluid secretion. Furthermore, the absorption of the membranes of the secretory granules contributes to the expansion and deformation of the canaliculi. Here we suggest that the homeostasis of the apical plasma membranes during exocytosis is maintained by various strategies that include: (1) membrane retrieval via compensatory endocytosis, (2) increase of the surface area via membrane folds and (3) recruitment of a functional actomyosin complex. Our observations underscore the important relationship between tissue architecture and cellular response, and highlight the potential of investigating biological processes in vivo by using intravital microscopy. PMID:22754613

  17. A study of the Interaction Between Cetirizine and Plasma Membrane of Eosinophils, Neutrophils, Platelets and Lymphocytes using A fluorescence Technique

    PubMed Central

    Oggiano, N.; Giorgi, P. L.; Rihoux, J-P.

    1994-01-01

    The effect of cetirizine on plasma membrane fluidity and heterogeneity of human eosinophils, neutrophils, platelets and lymphocytes was investigated using a fluorescence technique. Membrane fluidity and heterogeneity were studied by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4- trimethylammonium-phenyl)-6-phenyl-1, 3, 5-hexatriene (TMA-DPH) incorporated in the membrane. The results demonstrate that cetirizine (1 μg/ml) induced a significant increase in the Hpid order in the exterior part of the membrane and a decrease in membrane heterogeneity in eosinophils, neutrophils and platelets. Moreover, cetirizine blocked the PAF induced changes in membrane fluidity in these cells. Cetirizine did not influence significantly the plasma membrane of lymphocytes. These data may partially explain the effect ofcetirizine on inflammatory cell activities. PMID:18472948

  18. Caveolae protect endothelial cells from membrane rupture during increased cardiac output

    PubMed Central

    Cheng, Jade P.X.; Mendoza-Topaz, Carolina; Howard, Gillian; Chadwick, Jessica; Shvets, Elena; Cowburn, Andrew S.; Dunmore, Benjamin J.; Crosby, Alexi; Morrell, Nicholas W.

    2015-01-01

    Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1−/− mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo. PMID:26459598

  19. G-1-activated membrane estrogen receptors mediate increased contractility of the human myometrium.

    PubMed

    Maiti, K; Paul, J W; Read, M; Chan, E C; Riley, S C; Nahar, P; Smith, R

    2011-06-01

    Estrogens are key mediators of increased uterine contractility at labor. We sought to determine whether membrane-associated estrogen receptors, such as the recently described seven-transmembrane receptor G protein-coupled receptor 30 (GPR30), mediated some of this effect. Using human myometrium obtained at term cesarean section before or after the onset of labor, we demonstrated the presence of GPR30 mRNA and protein using quantitative RT-PCR and Western blotting. GPR30 receptor was localized to the cell membrane and often colocalized with calveolin-1. Using the specific estrogen membrane receptor agonist G-1 and myometrial explants, we showed that membrane receptor activation led to phosphorylation of MAPK and the actin-modifying small heat shock protein 27. Using myometrial strips incubated with G-1 or vehicle we demonstrated that estrogen membrane receptor activation increased the myometrial contractile response to oxytocin. These data suggest that activation of the plasma membrane estrogen receptor GPR30 likely participates in the physiology of the human myometrium during pregnancy and identifies it as a potential target to modify uterine activity. PMID:21427217

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

    PubMed

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

    2015-10-14

    Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement. PMID:26248320

  1. Mechanical Properties of the Plasma Membrane of Isolated Plant Protoplasts 1

    PubMed Central

    Wolfe, Joe; Steponkus, Peter L.

    1983-01-01

    The volume of isolated protoplasts of rye (Secale cereale L. cv Puma) in a suspending solution at constant concentration is shown to be negligibly changed by tensions in the plasma membrane which approach that tension necessary to lyse them. This allows a detailed investigation of the plasma membrane stress-strain relation by micropipette aspiration. Over periods less than a second, the membrane behaves as an elastic two-dimensional fluid with an area modulus of elasticity of 230 millinewtons per meter. Over longer periods, the stress-strain relation approaches a surface energy law—the resting tension is independent of area and has a value of the order 100 micronewtons per meter. Over longer periods the untensioned area, which is defined as the area that would be occupied by the molecules in the membrane at any given time if the tension were zero, increases with time under large imposed tensions and decreases under sufficiently small tension. It is proposed that these long term responses are the result of exchange of material between the plane of the membrane and a reservoir of membrane material. The irreversibility of large contractions in area is demonstrated directly, and the behavior of protoplasts during osmotically induced cycles of contraction and expansion is explained in terms of the membrane stress-strain relation. Images Fig. 2 PMID:16662817

  2. In Situ Quantification of Protein Binding to the Plasma Membrane

    PubMed Central

    Smith, Elizabeth M.; Hennen, Jared; Chen, Yan; Mueller, Joachim D.

    2015-01-01

    This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of protein expression levels, and a fit of the binding curve determines the number of binding sites and the dissociation coefficient. We experimentally verified the technique, using myosin-1C-EGFP as a model system and fit its binding curve. Furthermore, we studied the protein-lipid interactions of the membrane binding domains from lactadherin and phospholipase C-δ1 to evaluate the feasibility of using competition binding experiments to identify specific lipid-protein interactions in living cells. Finally, we applied the technique to determine the lipid specificity, the number of binding sites, and the dissociation coefficient of membrane binding for the Gag matrix domain of human T-lymphotropic virus type 1, which provides insight into early assembly steps of the retrovirus. PMID:26039166

  3. b-Type Cytochromes in Higher Plant Plasma Membranes 1

    PubMed Central

    Asard, Han; Venken, Mireille; Caubergs, Roland; Reijnders, Willem; Oltmann, Fred L.; De Greef, Jan A.

    1989-01-01

    The composition and characteristics of b-type cytochromes from higher plant plasma membranes, purified using aqueous two-phase partitioning, were investigated. At least three different cytochromes were identified by their wavelength maxima and redox midpoint potentials (E0′). Cytochrome b-560.7 (E0′ from + 110 to + 160 millivolts) was present in zucchini (Cucurbita pepo) hypocotyls and bean (Phaseolus vulgaris L.) hooks, although in different concentrations. The main component in cauliflower (Brassica oleracea L.) inflorescences (cytochrome b-558.8) is probably functionally similar to this cytochrome. The plasma membrane generally contains two to three cytochrome species. However, the occurrence and concentrations were species dependent. The high potential cytochrome can be reduced by ascorbate but not NADH, and may be involved in blue light perception. PMID:16666854

  4. Neobiosynthesis of glycosphingolipids by plasma membrane-associated glycosyltransferases.

    PubMed

    Crespo, Pilar M; Demichelis, Vanina Torres; Daniotti, José L

    2010-09-17

    Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the endoplasmic reticulum and in the Golgi complex. These neobiosynthesized gangliosides move via vesicular transport to the plasma membrane, becoming components of the external leaflet. Gangliosides can undergo endocytosis followed by recycling to the cell surface or sorting to the Golgi complex or lysosomes for remodeling and catabolism. Recently, glycosphingolipid catabolic enzymes (glycohydrolases) have been found to be associated with the plasma membrane, where they display activity on the membrane components. In this work, we demonstrated that ecto-ganglioside glycosyltransferases may catalyze ganglioside synthesis outside the Golgi compartment, particularly at the cell surface. Specifically, we report the first direct evidence of expression and activity of CMP-NeuAc:GM3 sialyltransferase (Sial-T2) at the cell surface of epithelial and melanoma cells, with membrane-integrated ecto-Sial-T2 being able to sialylate endogenously synthesized GM3 ganglioside as well as exogenously incorporated substrate. Interestingly, we also showed that ecto-Sial-T2 was able to synthesize GD3 ganglioside at the cell surface using the endogenously synthesized cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) available at the extracellular milieu. In addition, the expression of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase (GalNAc-T) was also detected at the cell surface of epithelial cells, whose catalytic activity was only observed after feeding the cells with exogenous GM3 substrate. Thus, the relative interplay between the plasma membrane-associated glycosyltransferase and glycohydrolase activities, even when acting on a common substrate, emerges as a potential level of regulation of the local glycosphingolipid composition in response to different external and internal stimuli. PMID:20639193

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

    NASA Astrophysics Data System (ADS)

    Han, Yu; Song, Shuijun; Lu, Yin; Zhu, Dongfa

    2016-08-01

    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/m2 h to 2812.7 L/m2 h and the equilibrium flux of BSA solution increased from 31 L/m2 h to 53 L/m2 h.

  6. Transformable DNA nanocarriers for plasma membrane targeted delivery of cytokine.

    PubMed

    Sun, Wujin; Ji, Wenyan; Hu, Quanyin; Yu, Jicheng; Wang, Chao; Qian, Chenggen; Hochu, Gabrielle; Gu, Zhen

    2016-07-01

    Direct delivery of cytokines using nanocarriers holds great promise for cancer therapy. However, the nanometric scale of the vehicles made them susceptible to size-dependent endocytosis, reducing the plasma membrane-associated apoptosis signaling. Herein, we report a tumor microenvironment-responsive and transformable nanocarrier for cell membrane targeted delivery of cytokine. This formulation is comprised of a phospholipase A2 (PLA2) degradable liposome as a shell, and complementary DNA nanostructures (designated as nanoclews) decorated with cytokines as the cores. Utilizing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a model cytokine, we demonstrate that the TRAIL loaded DNA nanoclews are capable of transforming into nanofibers after PLA2 activation. The nanofibers with micro-scaled lengths efficiently present the loaded TRAIL to death receptors on the cancer cell membrane and amplified the apoptotic signaling with reduced TRAIL internalization. PMID:27131597

  7. Detecting protein association at the T cell plasma membrane.

    PubMed

    Baumgart, Florian; Schütz, Gerhard J

    2015-04-01

    At the moment, many models on T cell signaling rely on results obtained via rather indirect methodologies, which makes direct comparison and conclusions to the in vivo situation difficult. Recently, a variety of new imaging methods were developed, which have the potential to directly shed light onto the mysteries of protein association at the T cell membrane. While the new modalities are extremely promising, for a broad readership it may be difficult to judge the results, since technological shortcomings are not always obvious. In this review article, we put key questions on the mechanism of protein interactions in the T cell plasma membrane into relation with techniques that allow to address such questions. We discuss applicability of the techniques, their strengths and weaknesses. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling. PMID:25300585

  8. Role of plasma membrane transporters in muscle metabolism.

    PubMed Central

    Zorzano, A; Fandos, C; Palacín, M

    2000-01-01

    Muscle plays a major role in metabolism. Thus it is a major glucose-utilizing tissue in the absorptive state, and changes in muscle insulin-stimulated glucose uptake alter whole-body glucose disposal. In some conditions, muscle preferentially uses lipid substrates, such as fatty acids or ketone bodies. Furthermore, muscle is the main reservoir of amino acids and protein. The activity of many different plasma membrane transporters, such as glucose carriers and transporters of carnitine, creatine and amino acids, play a crucial role in muscle metabolism by catalysing the influx or the efflux of substrates across the cell surface. In some cases, the membrane transport process is subjected to intense regulatory control and may become a potential pharmacological target, as is the case with the glucose transporter GLUT4. The goal of this review is the molecular characterization of muscle membrane transporter proteins, as well as the analysis of their possible regulatory role. PMID:10903126

  9. Analysis of plasma membrane phosphoinositides from fusogenic carrot cells

    SciTech Connect

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

    1987-04-01

    Phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP/sub 2/) were found to be associated with the plasma membrane-rich fractions isolated by aqueous polymer two-phase partitioning from fusogenic cells. They represented at least 5% and 0.7% of the total inositol-labeled lipids in the plasma membrane-rich fractions, respectively, and were present in a ratio of about 7:1 (PIP:PIP/sub 2/). In addition, two unidentified inositol-labeled compounds, which together were approximately 3% of the inositol-labeled lipids, were found predominantly in the plasma membrane-rich fractions and migrated between PIP/sub 2/ and PIP. The R/sub f/s of these compounds were approximately 0.31 and 0.34 in the solvent system CHCl/sub 3/:MeOH:15N NH/sub 4/OH:H/sub 2/O (90:90:7:22) using LK5 plates presoaked in 1% potassium oxalate. These compounds incorporated /sup 32/P/sub i/, (/sup 3/H)inositol and were hydrolyzed in mild base. These data suggested that they were glycero-phospholipids. Although the compounds did not comigrate with lysoPIP obtained from bovine brain (R/sub f/ approx. 0.35), when endogenous PIP was hydrolyzed to lysoPIP, the breakdown product migrated in the region of the unidentified inositol lipids.

  10. Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition.

    PubMed Central

    Avery, S V; Howlett, N G; Radice, S

    1996-01-01

    One major mechanism of copper toxicity towards microorganisms is disruption of plasma membrane integrity. In this study, the influence of plasma membrane fatty acid composition on the susceptibility of Saccharomyces cerevisiae to Cu2+ toxicity was investigated. Microbial fatty acid composition is highly variable, depending on both intrinsic and environmental factors. Manipulation was achieved in this study by growth in fatty acid-supplemented medium. Whereas cells grown under standard conditions contained only saturated and monounsaturated fatty acids, considerable incorporation of the diunsaturated fatty acid linoleate (18:2) (to more than 65% of the total fatty acids) was observed in both whole-cell homogenates and plasma membrane-enriched fractions from cells grown in linoleate-supplemented medium. Linoleate enrichment had no discernible effect on the growth of S. cerevisiae. However, linoleate-enriched cells were markedly more susceptible to copper-induced plasma membrane permeabilization. Thus, after addition of Cu(NO3)2, rates of cellular K+ release (loss of membrane integrity) were at least twofold higher from linoleate-supplemented cells than from unsupplemented cells; this difference increased with reductions in the Cu2+ concentration supplied. Levels of cellular Cu accumulation were also higher in linoleate-supplemented cells. These results were correlated with a very marked dependence of whole-cell Cu2+ toxicity on cellular fatty acid unsaturation. For example, within 10 min of exposure to 5 microM Cu2+, only 3% of linoleate-enriched cells remained viable (capable of colony formation). In contrast, 100% viability was maintained in cells previously grown in the absence of a fatty acid supplement. Cells displaying intermediate levels of linoleate incorporation showed intermediate Cu2+ sensitivity, while cells enriched with the triunsaturated fatty acid linolenate (18:3) were most sensitive to Cu2+. These results demonstrate for the first time that changes

  11. Differences in Organizational Structure of Insulin Receptor on Rat Adipocyte and Liver Plasma Membranes: Role of Disulfide Bonds

    NASA Astrophysics Data System (ADS)

    Schweitzer, John B.; Smith, Robert M.; Jarett, Leonard

    1980-08-01

    Binding of 125I-labeled insulin to rat liver and adipocyte plasma membranes has been investigated after treatment of the membranes with agents that modify disulfide bonds or sulfhydryl groups. Dithiothreitol, a disulfide-reducing agent, produced a bimodal response in adipocyte plasma membranes with dose-dependent increases in binding occurring over the range of 0-1 mM dithiothreitol; 5 mM dithiothreitol produced decreased binding. Insulin binding reached its maximal increase at 1 mM and was 3 times control values. Scatchard analysis of the 1 mM dithiothreitol effect revealed a straight line plot indicative of one class of sites with a Ka of 1.0× 108 M-1 which is intermediate between the two Kas obtained from the curvilinear Scatchard plot of control membranes. There was a 20-fold increase in the number of intermediate-affinity receptors compared to high-affinity receptors. The increased 125I-labeled insulin binding after dithiothreitol treatment was reversed by oxidized glutathione in a dose-dependent manner. Interposition of treatment with N-ethylmaleimide, an alkylating agent, prevented oxidized glutathione from reversing the dithiothreitol effect. Reduced glutathione produced the same effect as dithiothreitol. Liver plasma membranes treated with up to 1 mM dithiothreitol exhibited a maximum increase in insulin binding of 20% compared to control. Dithiothreitol at 5 mM decreased insulin binding below that of control membranes. The results indicate that the dithiothreitol effect on insulin binding to adipocyte plasma membranes is due to disruption of disulfide bonds, and that the structural organization of the insulin receptor on the plasma membranes is different for liver and for adipose tissue. The data imply that the insulin receptors on the plasma membrane of adipocytes possess at least two functionally distinct subclasses of disulfide bond but liver insulin receptors do not.

  12. The Structure of the Yeast Plasma Membrane SNARE Complex Reveals Destabilizing Water Filled Cavities

    SciTech Connect

    Strop, P.; Kaiser, S.E.; Vrljic, M.; Brunger, A.T.

    2009-05-26

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7{angstrom} resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.

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

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (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.

  14. Agonist-activated Ca2+ influx occurs at stable plasma membrane and endoplasmic reticulum junctions

    PubMed Central

    Treves, Susan; Vukcevic, Mirko; Griesser, Johanna; Armstrong, Clara-Franzini; Zhu, Michael X.; Zorzato, Fancesco

    2010-01-01

    Junctate is a 33 kDa integral protein of sarco(endo)plasmic reticulum membranes that forms a macromolecular complex with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and TRPC3 channels. TIRF microscopy shows that junctate enhances the number of fluorescent puncta on the plasma membrane. The size and distribution of these puncta are not affected by the addition of agonists that mobilize Ca2+ from Ins(1,4,5)P3-sensitive stores. Puncta are associated with a significantly larger number of peripheral junctions between endoplasmic reticulum and plasma membrane, which are further enhanced upon stable co-expression of junctate and TRPC3. The gap between the membranes of peripheral junctions is bridged by regularly spaced electron-dense structures of 10 nm. Ins(1,4,5)P3 inhibits the interaction of the cytoplasmic N-terminus of junctate with the ligand-binding domain of the Ins(1,4,5)P3 receptor. Furthermore, Ca2+ influx evoked by activation of Ins(1,4,5)P3 receptors is increased where puncta are located. We conclude that stable peripheral junctions between the plasma membrane and endoplasmic reticulum are the anatomical sites of agonist-activated Ca2+ entry. PMID:21062895

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

    PubMed

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

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  16. Water permeability of polyethylene terephthalate track membranes modified in plasma of dimethylaniline

    NASA Astrophysics Data System (ADS)

    Kravets, Lyubov; Dmitriev, Serguei; Gilman, Alla; Drachev, Alexander

    2004-09-01

    The surface properties and hydrodynamic characteristics of composite membranes consisting of a porous substrate, on which a polymer layer from a direct current discharge in a mixture of air and vapours of dimethylaniline was deposited, have been investigated. As a substrate, we used poly(ethylene) terephthalate track membrane (PET TM) of the thickness of 10 μ m and the effective pore diameter of 0.215 μ m (pore density is 2\\cdot 10^8 cm-2). The performed researches show that when treating the membranes in plasma, two competing processes are observed: deposition of the polymer layer on a membrane surface, that testifies increase of the mass of sample, and etching of a polymeric matrix which causes growth of effective pore diameter. The last process is stipulated by presence of oxygen in the gas mixture. Decreasing the degree of overweight of the sample at increasing the treatment time leads us to a supposition that a dominating process in this case becomes the process of gas-discharge etching. In all cases, if treating PET TM, a drop of the water contact angle occurs, i.e. hydrophilization of the membrane surface takes place that is connected first of all with a grafting of polymer layer containing polar functional groups. The research in the hydrodynamic characteristics of the initial PET TM and the membranes modified in plasma at neutral and subacid pH value of filtrate leads to a linear dependence of their permeability upon the quantity of applied pressure. It is connected with a viscous character of the flow, that is, when the diameter of the pores of the membrane is much more than the size of the water molecules. This fact shows that the macromolecules of the deposited polymer layer in this case have a compact conformation, which does not hinder the water molecules infiltration. At a lower pH value of the filtrate, the picture cardinally changes. For modified in plasma membranes a diversion from the linear relation is observed. This means that in this case

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

    PubMed

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

    2015-12-01

    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. PMID:26658212

  18. A specific gastrin receptor on plasma membranes of antral smooth muscle.

    PubMed

    Baur, S; Bacon, V C

    1976-12-20

    Plasma membranes with a 17 fold enrichment in 5'-nucleotidase over homogenate were prepared from antral smooth muscle. A specific gastrin receptor on the plasma membranes has been demonstrated. By Scatchard analysis receptor has a Kaff of 2x10(9)M(-1) and a binding capacity of 5x10(-14) moles/mg of membrane protein. PMID:15625862

  19. Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium

    PubMed Central

    Milenkovic, Ljiljana

    2009-01-01

    The function of primary cilia depends critically on the localization of specific proteins in the ciliary membrane. A major challenge in the field is to understand protein trafficking to cilia. The Hedgehog (Hh) pathway protein Smoothened (Smo), a 7-pass transmembrane protein, moves to cilia when a ligand is received. Using microscopy-based pulse-chase analysis, we find that Smo moves through a lateral transport pathway from the plasma membrane to the ciliary membrane. Lateral movement, either via diffusion or active transport, is quite distinct from currently studied pathways of ciliary protein transport in mammals, which emphasize directed trafficking of Golgi-derived vesicles to the base of the cilium. We anticipate that this alternative route will be used by other signaling proteins that function at cilia. The path taken by Smo may allow novel strategies for modulation of Hh signaling in cancer and regeneration. PMID:19948480

  20. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts, progress report

    SciTech Connect

    Steponkus, P L

    1993-01-01

    Our goal is to provide a mechanistic understanding of the cellular and molecular aspects of freezing injury and cold acclimation from a perspective of the structural and functional integrity of the plasma membrane -- the primary site of freezing injury in winter cereals. We have utilized protoplasts isolated from leaves of winter rye (Secale cereale L. cv Puma) to study the cryobehavior of the plasma membrane during a freeze/thaw cycle. The focus of our current studies is on lesions in the plasma membrane that result from severe freeze-induced dehydration and result in the alteration of the semipermeable characteristics of the plasma membrane so that the protoplasts are osmotically unresponsive. In protoplasts isolated from non-acclimated rye leaves (NA protoplasts), injury is associated with the formation of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal II phase transitions in the plasma membrane and the subtending lamellae. However, lamellar-to-hexagonal II phase transitions are not observed following severe dehydration of protoplasts isolated from cold-acclimated rye leaves (ACC protoplasts). Rather, injury is associated with the fracture-jump lesion,'' which, in freeze-fracture electron microscopy studies, is manifested as localized deviations in the fracture face of the plasma membrane. The fracture plane jumps'' from the plasma membrane to either subtending aparticulate lamellae or aparticulate regions of various endomembranes (predominantly chloroplast envelopes) that are in close apposition with the plasma membrane.

  1. Reconstitution of a plasma-membrane H(+)-ATPase into bilayer lipid membrane.

    PubMed

    Ziegler, W; Slayman, C L; Cartwright, C P

    1993-10-01

    The plasma membrane H(+)-ATPase of Neurospora has been reconstituted into planar lipid bilayer membranes by means of the vesicle-fusion technique described by Finkelstein and his collaborators (Zimmerberg et al., 1980; Cohen et al., 1980, 1984; Akabas et al., 1984). Enzyme was first transferred from isolated plasma membrane fragments into asolectin vesicles by a detergent-dialysis procedure (Perlin et al., 1984). After H(+)-pumping activity had been checked by quenching of acridine orange fluorescence, the vesicles were fused into performed bilayers. Critical features of the fusion process include (i) attachment of the vesicles to the bilayer in the presence of divalent cations (Mg++), and (ii) rapid osmotic swelling, which was enhanced by prior sonication or freeze-thawing of the vesicles, and/or by inclusions of physiologic channels. Enough proton pumps could be thus incorporated into bilayers to achieve ATP-driven, vanadate-sensitive currents of 0.04-0.4 pA. Aqueous solutions of low ionic strength were used to suppress conductance fluctuations due to the channels, and when that precaution was taken, we could demonstrate the proton pump the work against membrane potentials of at least 50 mV. PMID:8181690

  2. Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae.

    PubMed

    Garver, William S; Krishnan, Kumar; Gallagos, Jayme R; Michikawa, Makoto; Francis, Gordon A; Heidenreich, Randall A

    2002-04-01

    The Niemann-Pick C1 (NPC1) protein regulates cholesterol transport from late endosomes-lysosomes to other intracellular compartments. In this article, cholesterol transport to caveolin-1 and caveolin-2 containing compartments, such as the trans-Golgi network (TGN) and plasma membrane caveolae, was examined in normal (NPC+/+), NPC heterozygous (NPC+/-), and NPC homozygous (NPC-/-) human fibroblasts. The expression and distribution of NPC1 in each cell type were similar, and characterized by a finely dispersed, granular staining pattern. The expression of caveolin-1 and caveolin-2 was increased in NPC+/- and NPC-/- fibroblasts, although the distribution in each cell type was similar and characterized by predominant staining of the TGN and plasma membrane. The TGN in NPC+/+ fibroblasts was relatively cholesterol-enriched, whereas the TGN in NPC+/- and NPC-/- fibroblasts was partially or completely cholesterol-deficient, respectively. Consistent with studies demonstrating the transport of cholesterol from the TGN to plasma membrane caveolae, the concentration of cholesterol in plasma membrane caveolae isolated from NPC+/- and NPC-/- fibroblasts was significantly decreased, even though the total concentration of plasma membrane cholesterol in each cell type was similar. These studies demonstrate that NPC1 regulates cholesterol transport to caveolin-1 and caveolin-2 containing compartments such as the TGN and plasma membrane caveolae. PMID:11907140

  3. Solid polymer electrolyte composite membrane comprising plasma etched porous support

    DOEpatents

    Liu, Han; LaConti, Anthony B.

    2010-10-05

    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.

  4. Increasing Plasma Science Interest among Underprivileged Students

    NASA Astrophysics Data System (ADS)

    Merali, Aliya; Wissel, Stephanie; Quinn, Maureen; Hamlet, Nicole; Zwicker, Andrew

    2012-10-01

    For the past three years, PPPL has partnered with NASA to study dusty plasmas, convective flows, and other related topics in μ-gravity. During the last year, we worked with a group of highly-motivated but underprivileged high school students to develop an experiment looking at the Rayleigh-Taylor instability in varying gravitational environments. The students were challenged to design, build and test the experiment to be flown aboard NASA's ``Weightless Wonder.'' Students met weekly with PPPL mentors over the course of the 9-month school year. The program provided students with long-term exposure to a scientific experiment, a sense of experimental ownership, and an opportunity to experience the scientific method outside of a classroom setting. Participants used inquiry-based learning techniques to design and build the experiment. We report on impact of the program and the outcome of the students' continued work.

  5. Switchable hydrophobic/hydrophilic surface of electrospun poly (l-lactide) membranes obtained by CF₄microwave plasma treatment

    SciTech Connect

    Yue, Mengyao; Zhou, Baoming; Jiao, Kunyan; Qian, Xiaoming; Xu, Zhiwei; Teng, Kunyue; Zhao, Lihuan; Wang, Jiajun; Jiao, Yanan

    2014-11-29

    A switchable surface that promotes either hydrophobic or hydrophilic wettability of poly (L-lactide) (PLLA) microfibrous membranes is obtained by CF₄ microwave plasma treatment in this paper. The results indicated that both etching and grafting process occurred during the CF₄ plasma treatment and these two factors synergistically affected the final surface wettability of PLLA membranes. When plasma treatment was taken under a relatively low power, the surface wettability of PLLA membranes turned from hydrophobic to hydrophilic. Especially when CF₄ plasma treatment was taken under 100 W for 10 min and 150 W for 5 min, the water contact angle sharply decreased from 116 ± 3.0° to ~0°. According to Field-emission scanning electron microscopy (FESEM) results, the PLLA fibers were notably etched by CF₄ plasma treatment. Combined with the X-ray photoelectron spectroscopy (XPS) measurements, only a few fluorine-containing groups were grafted onto the surface, so the etching effect directly affected the surface wettability of PLLA membranes in low plasma power condition. However, with the plasma power increasing to 200 W, the PLLA membrane surface turned to hydrophobic again. In contrast, the morphology changes of PLLA fiber surfaces were not obvious while a large number of fluorine-containing groups grafted onto the surface. So the grafting effect gradually became the major factor for the final surface wettability.

  6. Switchable hydrophobic/hydrophilic surface of electrospun poly (l-lactide) membranes obtained by CF₄microwave plasma treatment

    DOE PAGESBeta

    Yue, Mengyao; Zhou, Baoming; Jiao, Kunyan; Qian, Xiaoming; Xu, Zhiwei; Teng, Kunyue; Zhao, Lihuan; Wang, Jiajun; Jiao, Yanan

    2014-11-29

    A switchable surface that promotes either hydrophobic or hydrophilic wettability of poly (L-lactide) (PLLA) microfibrous membranes is obtained by CF₄ microwave plasma treatment in this paper. The results indicated that both etching and grafting process occurred during the CF₄ plasma treatment and these two factors synergistically affected the final surface wettability of PLLA membranes. When plasma treatment was taken under a relatively low power, the surface wettability of PLLA membranes turned from hydrophobic to hydrophilic. Especially when CF₄ plasma treatment was taken under 100 W for 10 min and 150 W for 5 min, the water contact angle sharply decreasedmore » from 116 ± 3.0° to ~0°. According to Field-emission scanning electron microscopy (FESEM) results, the PLLA fibers were notably etched by CF₄ plasma treatment. Combined with the X-ray photoelectron spectroscopy (XPS) measurements, only a few fluorine-containing groups were grafted onto the surface, so the etching effect directly affected the surface wettability of PLLA membranes in low plasma power condition. However, with the plasma power increasing to 200 W, the PLLA membrane surface turned to hydrophobic again. In contrast, the morphology changes of PLLA fiber surfaces were not obvious while a large number of fluorine-containing groups grafted onto the surface. So the grafting effect gradually became the major factor for the final surface wettability.« less

  7. Plasma membrane calcium channels in cancer: Alterations and consequences for cell proliferation and migration.

    PubMed

    Déliot, Nadine; Constantin, Bruno

    2015-10-01

    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

  8. G-protein-coupled receptor participates in 20-hydroxyecdysone signaling on the plasma membrane

    PubMed Central

    2014-01-01

    Background Animal steroid hormones are conventionally known to initiate signaling via a genomic pathway by binding to the nuclear receptors. The mechanism by which 20E initiates signaling via a nongenomic pathway is unclear. Results We illustrate that 20E triggered the nongenomic pathway through a plasma membrane G-protein-coupled receptor (named ErGPCR) in the lepidopteran insect Helicoverpa armigera. The transcript of ErGPCR was increased at the larval molting stage and metamorphic molting stage by 20E regulation. Knockdown of ErGPCR via RNA interference in vivo blocked larval–pupal transition and suppressed 20E-induced gene expression. ErGPCR overexpression in the H. armigera epidermal cell line increased the 20E-induced gene expression. Through ErGPCR, 20E modulated Calponin nuclear translocation and phosphorylation, and induced a rapid increase in cytosolic Ca2+ levels. The inhibitors of T-type voltage-gated calcium channels and canonical transient receptor potential calcium channels repressed the 20E-induced Ca2+ increase. Truncation of the N-terminal extracellular region of ErGPCR inhibited its localization on the plasma membrane and 20E-induced gene expression. ErGPCR was not detected to bind with the steroid hormone analog [3H]Pon A. Conclusion These results suggest that ErGPCR participates in 20E signaling on the plasma membrane. PMID:24507557

  9. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    NASA Technical Reports Server (NTRS)

    Caldwell, C.

    1983-01-01

    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.

  10. ATP11C Facilitates Phospholipid Translocation across the Plasma Membrane of All Leukocytes

    PubMed Central

    Yabas, Mehmet; Jing, Weidong; Shafik, Sarah

    2016-01-01

    Organization of the plasma membrane into specialized substructures in different blood lineages facilitates important biological functions including proper localization of receptors at the plasma membrane as well as the initiation of crucial intracellular signaling cascades. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. This asymmetry is actively maintained by membrane-embedded lipid transporters, but there is only limited data available about the molecular identity of the predominantly active transporters and their substrate specificity in different leukocyte subsets. We demonstrate here that the P4-type ATPase ATP11C mediates significant flippase activity in all murine leukocyte subsets. Loss of ATP11C resulted in a defective internalization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in comparison to control cells. The diminished flippase activity caused increased PS exposure on 7-aminoactinomycin D− (7-AAD−) viable pro-B cells freshly isolated from the bone marrow of ATP11C-deficient mice, which was corrected upon a 2-hour resting period in vitro. Despite the impaired flippase activity in all immune cell subsets, the only other blood cell type with an accumulation of PS on the surface were viable 7-AAD− developing T cells but this did not result in any discernable effect on their development in the thymus. These findings show that all leukocyte lineages exhibit flippase activity, and identify ATP11C as an aminophospholipid translocase in immune cells. PMID:26799398

  11. The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump.

    PubMed Central

    Zaman, G J; Flens, M J; van Leusden, M R; de Haas, M; Mülder, H S; Lankelma, J; Pinedo, H M; Scheper, R J; Baas, F; Broxterman, H J

    1994-01-01

    The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an expression vector containing MRP cDNA. MRP-overexpressing SW-1573 cells are resistant to doxorubicin, daunorubicin, vincristine, VP-16, colchicine, and rhodamine 123, but not to 4'-(9-acridinylamino)methanesulfon-m-anisidide or taxol. The intracellular accumulation of drug (daunorubicin, vincristine, and VP-16) is decreased and the efflux of drug (daunorubicin) is increased in the transfectant. The decreased accumulation of daunorubicin is abolished by permeabilization of the plasma membrane with digitonin, showing that MRP can lower the intracellular daunorubicin level against a concentration gradient. Anti-MRP antisera predominantly stain the plasma membrane of MRP-overexpressing cells. We conclude that MRP is a plasma membrane drug-efflux pump. Images PMID:7916458

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

    SciTech Connect

    Steponkus, P.L.

    1991-01-01

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

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

    PubMed

    Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

    2014-01-01

    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. PMID:25302608

  14. Measuring Local Viscosities near Plasma Membranes of Living Cells with Photonic Force Microscopy.

    PubMed

    Jünger, Felix; Kohler, Felix; Meinel, Andreas; Meyer, Tim; Nitschke, Roland; Erhard, Birgit; Rohrbach, Alexander

    2015-09-01

    The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle's hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 μm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 μs timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions. PMID:26331245

  15. Measuring Local Viscosities near Plasma Membranes of Living Cells with Photonic Force Microscopy

    PubMed Central

    Jünger, Felix; Kohler, Felix; Meinel, Andreas; Meyer, Tim; Nitschke, Roland; Erhard, Birgit; Rohrbach, Alexander

    2015-01-01

    The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle’s hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 μm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 μs timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions. PMID:26331245

  16. A mechanistic study to increase understanding of titanium dioxide nanoparticles-increased plasma glucose in mice.

    PubMed

    Hu, Hailong; Li, Li; Guo, Qian; Jin, Sanli; Zhou, Ying; Oh, Yuri; Feng, Yujie; Wu, Qiong; Gu, Ning

    2016-09-01

    Titanium dioxide nanoparticle (TiO2 NP) is an authorized food additive. Previous studies determined oral administration of TiO2 NPs increases plasma glucose in mice via inducing insulin resistance. An increase in reactive oxygen species (ROS) has been considered the possible mechanism of increasing plasma glucose. However, persistently high plasma glucose is also a mechanism of increasing ROS. This study aims to explore whether TiO2 NPs increase plasma glucose via ROS. We found after oral administration of TiO2 NPs, an increase in ROS preceded an increase in plasma glucose. Subsequently, mice were treated with two antioxidants (resveratrol and vitamin E) at the same time as oral administration of TiO2 NPs. Results showed resveratrol and vitamin E reduced TiO2 NPs-increased ROS. An increase in plasma glucose was also inhibited. Further research showed resveratrol and vitamin E inhibited the secretion of TNF-α and IL-6, and the phosphorylation of JNK and p38 MAPK, resulting in improved insulin resistance. These results suggest TiO2 NPs increased ROS levels, and then ROS activated inflammatory cytokines and phosphokinases, and thus induced insulin resistance, resulting in an increase in plasma glucose. Resveratrol and vitamin E can reduce TiO2 NPs-increased ROS and thereby inhibit an increase in plasma glucose in mice. PMID:27430421

  17. Myelin Basic Protein Induces Neuron-Specific Toxicity by Directly Damaging the Neuronal Plasma Membrane

    PubMed Central

    Zheng, Sixin; Liu, Xiao; Jin, Jinghua; Ren, Yi; Luo, Jianhong

    2014-01-01

    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. PMID:25255088

  18. Simultaneous Measurements of Cytoplasmic K+ Concentration and the Plasma Membrane Electrical Parameters in Single Membrane Samples of Chara corallina

    PubMed Central

    Beilby, Mary J.; Blatt, Michael R.

    1986-01-01

    The electrophysiological properties of cytoplasm-rich fragments (single membrane samples) prepared from internodal cells of Chara corallina were explored in conjunction with K+-sensitive microelectrode and current-voltage (I-V) measurements. This system eliminated the problem of the inaccessible cytoplasmic layer, while preserving many of the electrical characteristics of the intact cells. In 0.1 millimolar external K concentration (Ko+), the resting conductance (membrane conductance Gm, 0.85 ± 0.25 Siemens per square meter (±standard error)) of the single membrane samples, was dominated by the proton pump, as suggested by the response of the near-linear I-V characteristic to changes in external pH. Initial cytoplasmic K+ activities (aK+), judged most reliable, gave values of 117 ± 67 millimolar; stable aK+ values were 77 ± 31 millimolar. Equilibrium potentials for K+ (Nernst equilibrium potential) (EK) calculated, using either of these data sets, were near the mean membrane potential (Vm). On a cell-to-cell basis, however, EK was generally negative of the Vm, despite an electrogenic contribution from the Chara proton pump. When Ko+ was increased to 1.0 millimolar or above, Gm rose (by 8- to 10-fold in 10 millimolar Ko+), the steady state I-V characteristics showed a region of negative slope conductance, and Vm followed EK. These results confirm previous studies which implicated a Ko+-induced and voltage-dependent permeability to K+ at the Chara plasma membrane. They provide an explanation for transitions between apparent Ko+-insensitive and Ko+-sensitive (`K+ electrode') behavior displayed by the membrane potential, as recorded in many algae and higher plant cells. PMID:16665044

  19. Subproteomics: identification of plasma membrane proteins from the yeast Saccharomyces cerevisiae.

    PubMed

    Navarre, Catherine; Degand, Hervé; Bennett, Keiryn L; Crawford, Janne S; Mørtz, Ejvind; Boutry, Marc

    2002-12-01

    As a consequence of their poor solubility during isoelectric focusing, integral membrane proteins are generally absent from two-dimensional gel proteome maps. In order to analyze the yeast plasma membrane proteome, a plasma membrane purification protocol was optimized in order to reduce contaminating membranes and cytosolic proteins. Specifically, the new fractionation scheme largely depleted the plasma membrane fraction of cytosolic proteins by deoxycholate stripping and ribosomal proteins by sucrose gradient flotation. The plasma membrane complement was resolved by two-dimensional electrophoresis using the cationic detergent cetyl trimethyl ammonium bromide in the first, and sodium dodecyl sulfate in the second dimension, and fifty spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectometry. In spite of the presence of still contaminating ribosomal proteins, major proteins corresponded to known plasma membrane residents, the ABC transporters Pdr5p and Snq2p, the P-type H(+)-ATPase Pma1p, the glucose transporter Hxt7p, the seven transmembrane-span Mrh1p, the low affinity Fe(++) transporter Fet4p, the twelve-span Ptr2p, and the plasma membrane anchored casein kinase Yck2p. The four transmembrane-span proteins Sur7p and Nce102p were also present in the isolated plasma membranes, as well as the unknown protein Ygr266wp that probably contains a single transmembrane span. Thus, combining subcellular fractionation with adapted two-dimensional electrophoresis resulted in the identification of intrinsic plasma membrane proteins. PMID:12469340

  20. Induction of stable ER–plasma-membrane junctions by Kv2.1 potassium channels

    PubMed Central

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

    2015-01-01

    ABSTRACT 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 Ca2+ signaling. PMID:25908859

  1. Induction of stable ER-plasma-membrane junctions by Kv2.1 potassium channels.

    PubMed

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

    2015-06-01

    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. PMID:25908859

  2. A plasma-membrane linker for the phosphoinositide-specific phospholipase C in tobacco plants.

    PubMed

    Nakamura, Kimiyo; Sano, Hiroshi

    2009-01-01

    We previously screened genes that were transcriptionally activated during the early stage of wound response in tobacco plants (Nicotiana tabacum), and isolated a particular clone, which encoded a membrane-located protein, designated as NtC7. Upon overexpression in tobacco plants, NtC7 conferred a marked tolerance to osmotic stress, suggesting it to be involved in maintenance of osmotic adjustments. In this study, we searched for proteins which interact with NtC7 by the yeast two-hybrid screening, and isolated a clone encoding phosphoinositide-specific phospholipase C, designated as NtPI-PLC. Physical interaction between NtC7 and C2 domain of NtPI-PLC was confirmed by the pull-down assay. Expression of fused protein to green-fluorescence protein in onion epidermal cell layers indicated both proteins to predominantly localize to the plasma membrane. Their interaction in planta was shown by the bimolecular fluorescence complementation, which exhibited a clear fluorescence of reconstituted yellow fluorescence protein. Transcripts of NtC7 and NtPI-PLC were markedly increased 30 to 60 min after wounding. PI-PLC is one of key enzymes in metabolism of inositol phospholipids, which function in signal transduction and also in response to stresses including osmotic changes. It was shown to localize to plasma-membrane and, to a lesser extent, to cytosol. However, molecular mechanism of membrane localization has remained to be determined, because of the apparent lack of domains for membrane association. The present results suggest that one of such mechanisms is tethering NtPI-PLC to the plasma membrane through interaction with NtC7, which possesses a transmembrane domain at the C-terminus. PMID:19704699

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

    PubMed

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

    2014-12-01

    Membrane pathway for intracellular cadmium (Cd(2+)) accumulation is not fully elucidated in many organisms and has not been studied in crab gill cells. To characterize membrane Cd(2+) transport of anterior and posterior gill cells of Ucides cordatus, a hypo-hyper-regulating crab, a change in intracellular Cd(2+) concentration under various experimental conditions was examined by using FluoZin, a fluorescent probe. The membrane Cd(2+) transport was estimated by the augmentation of FluoZin fluorescence induced by extracellular application of CdCl2 and different inhibitors. Addition of extracellular calcium (Ca(2+)) to the cells affected little the fluorescence of FluoZin, confirming that Cd(2+) was the main ion increasing intracellular fluorescence. Ca(2+) channels blockers (nimodipine and verapamil) decreased Cd(2+) influx as well as vanadate, a Ca(2+)-ATPase blocker. Chelating intracellular Ca(2+) (BAPTA) decreased Cd(2+) influx in gill cells, while increasing intracellular Ca(2+) (caffeine) augmented Cd influx. Cd(2+) and ATP added at different temporal conditions were not effective at increasing intracellular Cd(2+) accumulation. Ouabain (Na(+)/K(+)-ATPase inhibitor) increased Cd(2+) influx probably through a change in intracellular Na and/or a change in cell membrane potential. Routes of Cd(2+) influx, a non-essential metal, through the gill cell plasma membrane of crabs are suggested. PMID:25456216

  4. Polyamine Binding to Plasma Membrane Vesicles Isolated from Zucchini Hypocotyls.

    PubMed Central

    Tassoni, A.; Antognoni, F.; Bagni, N.

    1996-01-01

    The general features of [14C]spermidine binding to plasmalemma vesicles isolated from zucchini (Cucurbita pepo L.) etiolated hypocotyls are reported in the present paper. The specific interaction of the polyamine with the plasma membranes was reversible and thermolabile, since it decreased by about 50% in the assay performed at 40[deg]C compared to that carried out on ice. On the contrary, nonspecific binding was unaffected by temperature. Specific spermidine binding showed a pH dependence with a maximum at pH 8.0 and it reached saturation between 0.75 and 1 mM external spermidine concentration. The value of the dissociation constant calculated from Scatchard analysis was 4.4 x 10-5 M. Specific spermidine interaction appeared to be sensitive to detergents and was markedly reduced by the presence of divalent cations, such as Mg2+ and Ca2+, whereas it was stimulated by monovalent cations. Polyamine binding sites were highly sensitive to pronase treatment. Competition experiments, performed using a series of compounds structurally related to spermidine, may provide some indication of the characteristics of spermidine binding sites. The results presented here suggest that specific spermidine binding occurs mainly with the protein component of the plasma membrane. PMID:12226221

  5. A Plasma Membrane Association Module in Yeast Amino Acid Transporters.

    PubMed

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

    2016-07-29

    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 silico analyses and mutational studies we found that the C-terminal sequences of Gap1, Bap2, Hip1, Tat1, Tat2, Mmp1, Sam3, Agp1, and Gnp1 are about 50 residues long, associate with the PM, and have features that discriminate them from the termini of organellar amino acid transporters. We show that this sequence (named PMasseq) contains an amphipathic α-helix and the FWC signature, which is palmitoylated by palmitoyltransferase Pfa4. Variations of PMasseq, found in different AAPs, lead to different mobilities and localization patterns, whereas the disruption of the sequence has an adverse effect on cell viability. We propose that PMasseq modulates the function and localization of AAPs along the PM. PMasseq is one of the most complex protein signals for plasma membrane association across species and can be used as a delivery vehicle for the PM. PMID:27226538

  6. Structure and Function of Thyroid Hormone Plasma Membrane Transporters

    PubMed Central

    Schweizer, Ulrich; Johannes, Jörg; Bayer, Dorothea; Braun, Doreen

    2014-01-01

    Thyroid hormones (TH) cross the plasma membrane with the help of transporter proteins. As charged amino acid derivatives, TH cannot simply diffuse across a lipid bilayer membrane, despite their notorious hydrophobicity. The identification of monocarboxylate transporter 8 (MCT8, SLC16A2) as a specific and very active TH transporter paved the way to the finding that mutations in the MCT8 gene cause a syndrome of psychomotor retardation in humans. The purpose of this review is to introduce the current model of transmembrane transport and highlight the diversity of TH transmembrane transporters. The interactions of TH with plasma transfer proteins, T3 receptors, and deiodinase are summarized. It is shown that proteins may bind TH owing to their hydrophobic character in hydrophobic cavities and/or by specific polar interaction with the phenolic hydroxyl, the aminopropionic acid moiety, and by weak polar interactions with the iodine atoms. These findings are compared with our understanding of how TH transporters interact with substrate. The presumed effects of mutations in MCT8 on protein folding and transport function are explained in light of the available homology model. PMID:25538896

  7. Carboxylic Acids Plasma Membrane Transporters in Saccharomyces cerevisiae.

    PubMed

    Casal, Margarida; Queirós, Odília; Talaia, Gabriel; Ribas, David; Paiva, Sandra

    2016-01-01

    This chapter covers the functionally characterized plasma membrane carboxylic acids transporters Jen1, Ady2, Fps1 and Pdr12 in the yeast Saccharomyces cerevisiae, addressing also their homologues in other microorganisms, as filamentous fungi and bacteria. Carboxylic acids can either be transported into the cells, to be used as nutrients, or extruded in response to acid stress conditions. The secondary active transporters Jen1 and Ady2 can mediate the uptake of the anionic form of these substrates by a H(+)-symport mechanism. The undissociated form of carboxylic acids is lipid-soluble, crossing the plasma membrane by simple diffusion. Furthermore, acetic acid can also be transported by facilitated diffusion via Fps1 channel. At the cytoplasmic physiological pH, the anionic form of the acid prevails and it can be exported by the Pdr12 pump. This review will highlight the mechanisms involving carboxylic acids transporters, and the way they operate according to the yeast cell response to environmental changes, as carbon source availability, extracellular pH and acid stress conditions. PMID:26721276

  8. Hybrid lipids increase nanoscale fluctuation lifetimes in mixed membranes

    NASA Astrophysics Data System (ADS)

    Palmieri, Benoit; Safran, Samuel A.

    2013-09-01

    A recently proposed ternary mixture model is used to predict fluctuation domain lifetimes in the one phase region. The membrane is made of saturated, unsaturated, and hybrid lipids that have one saturated and one unsaturated hydrocarbon chain. The hybrid lipid is a natural linactant which can reduce the packing incompatibility between saturated and unsaturated lipids. The fluctuation lifetimes are predicted as a function of the hybrid lipid fraction and the fluctuation domain size. These lifetimes can be increased by up to three orders of magnitude compared to the case of no hybrids. With hybrid, small length scale fluctuations have sizable amplitudes even close to the critical temperature and, hence, benefit from enhanced critical slowing down. The increase in lifetime is particularly important for nanometer scale fluctuation domains where the hybrid orientation and the other lipids composition are highly coupled.

  9. High-protein-PUFA supplementation, red blood cell membranes, and plasma antioxidant activity in volleyball athletes.

    PubMed

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

    2008-06-01

    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. PMID:18562771

  10. Supramolecular architecture of endoplasmic reticulum-plasma membrane contact sites.

    PubMed

    Fernández-Busnadiego, Rubén

    2016-04-15

    The endoplasmic reticulum (ER) forms membrane contact sites (MCS) with most other cellular organelles and the plasma membrane (PM). These ER-PM MCS, where the membranes of the ER and PM are closely apposed, were discovered in the early days of electron microscopy (EM), but only recently are we starting to understand their functional and structural diversity. ER-PM MCS are nowadays known to mediate excitation-contraction coupling (ECC) in striated muscle cells and to play crucial roles in Ca(2+)and lipid homoeostasis in all metazoan cells. A common feature across ER-PM MCS specialized in different functions is the preponderance of cooperative phenomena that result in the formation of large supramolecular assemblies. Therefore, characterizing the supramolecular architecture of ER-PM MCS is critical to understand their mechanisms of function. Cryo-electron tomography (cryo-ET) is a powerful EM technique uniquely positioned to address this issue, as it allows 3D imaging of fully hydrated, unstained cellular structures at molecular resolution. In this review I summarize our current structural knowledge on the molecular organization of ER-PM MCS and its functional implications, with special emphasis on the emerging contributions of cryo-ET. PMID:27068966

  11. The 82-plex plasma protein signature that predicts increasing inflammation

    PubMed Central

    Tepel, Martin; Beck, Hans C.; Tan, Qihua; Borst, Christoffer; Rasmussen, Lars M.

    2015-01-01

    The objective of the study was to define the specific plasma protein signature that predicts the increase of the inflammation marker C-reactive protein from index day to next-day using proteome analysis and novel bioinformatics tools. We performed a prospective study of 91 incident kidney transplant recipients and quantified 359 plasma proteins simultaneously using nano-Liquid-Chromatography-Tandem Mass-Spectrometry in individual samples and plasma C-reactive protein on the index day and the next day. Next-day C-reactive protein increased in 59 patients whereas it decreased in 32 patients. The prediction model selected and validated 82 plasma proteins which determined increased next-day C-reactive protein (area under receiver-operator-characteristics curve, 0.772; 95% confidence interval, 0.669 to 0.876; P < 0.0001). Multivariable logistic regression showed that 82-plex protein signature (P < 0.001) was associated with observed increased next-day C-reactive protein. The 82-plex protein signature outperformed routine clinical procedures. The category-free net reclassification index improved with 82-plex plasma protein signature (total net reclassification index, 88.3%). Using the 82-plex plasma protein signature increased net reclassification index with a clinical meaningful 10% increase of risk mainly by the improvement of reclassification of subjects in the event group. An 82-plex plasma protein signature predicts an increase of the inflammatory marker C-reactive protein. PMID:26445912

  12. Cobalt oxide nanoparticles can enter inside the cells by crossing plasma membranes

    PubMed Central

    Bossi, Elena; Zanella, Daniele; Gornati, Rosalba; Bernardini, Giovanni

    2016-01-01

    The ability of nanoparticles (NPs) to be promptly uptaken by the cells makes them both dangerous and useful to human health. It was recently postulated that some NPs might cross the plasma membrane also by a non-endocytotic pathway gaining access to the cytoplasm. To this aim, after having filled mature Xenopus oocytes with Calcein, whose fluorescence is strongly quenched by divalent metal ions, we have exposed them to different cobalt NPs quantifying quenching as evidence of the increase of the concentration of Co2+ released by the NPs that entered into the cytoplasm. We demonstrated that cobalt oxide NPs, but not cobalt nor cobalt oxide NPs that were surrounded by a protein corona, can indeed cross plasma membranes. PMID:26924527

  13. An efficient organic solvent based extraction method for the proteomic analysis of Arabidopsis plasma membranes.

    PubMed

    Mitra, Srijeet K; Walters, Benjamin T; Clouse, Steven D; Goshe, Michael B

    2009-06-01

    Membrane proteins are involved in diverse cellular processes and are an integral component of many signaling cascades, but due to their highly hydrophobic nature and the complexities associated with studying these proteins in planta, alternative methods are being developed to better characterize these proteins on a proteome-wide scale. In our previous work ( Mitra , S. K. et al. J. Proteome Res. 2007 , 6 , ( 5 ), 1933 - 50 ), methanol-assisted solubilization was determined to facilitate the identification of both hydrophobic and hydrophilic membrane proteins compared to Brij-58 solubilization and was particularly effective for leucine-rich repeat receptor-like kinases (LRR RLKs). To improve peptide identification and to overcome sample losses after tryptic digestion, we have developed an effective chloroform extraction method to promote plasma membrane protein identification. The use of chloroform extraction over traditional solid-phase extraction (SPE) prior to off-line strong cation exchange liquid chromatography (SCXC) and reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis facilitated the removal of chlorophylls, major contaminants of plant tissue preparations that can affect downstream analysis, in addition to the effective removal of trypsin used in the digestion. On the basis of a statistically derived 5% false discovery rate, the chloroform extraction procedure increased the identification of unique peptides for plasma membrane proteins over SPE by 70% which produced nearly a 2-fold increase in detection of membrane transporters and LRR RLKs without increased identification of contaminating Rubisco and ribosomal peptides. Overall, the combined use of methanol and chloroform provides an effective method to study membrane proteins and can be readily applied to other tissues and cells types for proteomic analysis. PMID:19334764

  14. Toxin Pores Endocytosed During Plasma Membrane Repair Traffic into the Lumen of MVBs for Degradation

    PubMed Central

    Corrotte, Matthias; Fernandes, Maria Cecilia; Tam, Christina; Andrews, Norma W.

    2012-01-01

    Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca2+-dependent manner. Resealing involves Ca2+-dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes. PMID:22212686

  15. Plasma Modified Polypropylene Membranes as the Lithium-Ion Battery Separators

    NASA Astrophysics Data System (ADS)

    Wang, Zhengduo; Zhu, Huiqin; Yang, Lizhen; Wang, Xinwei; Liu, Zhongwei; Chen, Qiang

    2016-04-01

    To reduce the thermal shrinkage of the polymeric separators and improve the safety of the Li-ion batteries, plasma treatment and plasma enhanced vapor chemical deposition (PECVD) of SiOx-like are carried out on polypropylene (PP) separators, respectively. Critical parameters for separator properties, such as the thermal shrinkage rate, porosity, wettability, and mechanical strength, are evaluated on the plasma treated PP membranes. O2 plasma treatment is found to remarkably improve the wettability, porosity and electrolyte uptake. PECVD SiOx-like coatings are found to be able to effectively reduce the thermal shrinkage rate of the membranes and increase the ionic conductivity. The electrolyte-philicity of the SiOx-like coating surface can be tuned by the varying O2 content in the gas mixture during the deposition. Though still acceptable, the mechanical strength is reduced after PECVD, which is due to the plasma etching. supported by National Natural Science Foundation of China (Nos. 11175024, 11375031), the Beijing Institute of Graphic and Communication Key Project of China (No. 23190113051), the Shenzhen Science and Technology Innovation Committee of China (No. JCYJ20130329181509637), BJNSFC (No. KZ201510015014), and the State Key Laboratory of Electrical Insulation and Power Equipment of China (No. EIPE15208)

  16. Cationic nanoparticles induce nanoscale disruption in living cell plasma membranes.

    PubMed

    Chen, Jiumei; Hessler, Jessica A; Putchakayala, Krishna; Panama, Brian K; Khan, Damian P; Hong, Seungpyo; Mullen, Douglas G; Dimaggio, Stassi C; Som, Abhigyan; Tew, Gregory N; Lopatin, Anatoli N; Baker, James R; Holl, Mark M Banaszak; Orr, Bradford G

    2009-08-13

    It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes. PMID:19606833

  17. Plasma membrane electron transport in pancreatic β-cells is mediated in part by NQO1.

    PubMed

    Gray, Joshua P; Eisen, Timothy; Cline, Gary W; Smith, Peter J S; Heart, Emma

    2011-07-01

    Plasma membrane electron transport (PMET), a cytosolic/plasma membrane analog of mitochondrial electron transport, is a ubiquitous system of cytosolic and plasma membrane oxidoreductases that oxidizes cytosolic NADH and NADPH and passes electrons to extracellular targets. While PMET has been shown to play an important role in a variety of cell types, no studies exist to evaluate its function in insulin-secreting cells. Here we demonstrate the presence of robust PMET activity in primary islets and clonal β-cells, as assessed by the reduction of the plasma membrane-impermeable dyes WST-1 and ferricyanide. Because the degree of metabolic function of β-cells (reflected by the level of insulin output) increases in a glucose-dependent manner between 4 and 10 mM glucose, PMET was evaluated under these conditions. PMET activity was present at 4 mM glucose and was further stimulated at 10 mM glucose. PMET activity at 10 mM glucose was inhibited by the application of the flavoprotein inhibitor diphenylene iodonium and various antioxidants. Overexpression of cytosolic NAD(P)H-quinone oxidoreductase (NQO1) increased PMET activity in the presence of 10 mM glucose while inhibition of NQO1 by its inhibitor dicoumarol abolished this activity. Mitochondrial inhibitors rotenone, antimycin A, and potassium cyanide elevated PMET activity. Regardless of glucose levels, PMET activity was greatly enhanced by the application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle. We propose a model for the role of PMET as a regulator of glycolytic flux and an important component of the metabolic machinery in β-cells. PMID:21505151

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

    PubMed Central

    Martinière, Alexandre; Runions, John

    2013-01-01

    Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment. PMID:24381579

  19. Assembly and Comparison of Plasma Membrane SNARE Acceptor Complexes.

    PubMed

    Kreutzberger, Alex J B; Liang, Binyong; Kiessling, Volker; Tamm, Lukas K

    2016-05-24

    Neuronal exocytotic membrane fusion occurs on a fast timescale and is dependent on interactions between the vesicle SNARE synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1a and SNAP-25 with a 1:1:1 stoichiometry. Reproducing fast fusion rates as observed in cells by reconstitution in vitro has been hindered by the spontaneous assembly of a 2:1 syntaxin-1a:SNAP-25 complex on target membranes that kinetically alters the binding of synaptobrevin-2. Previously, an artificial SNARE acceptor complex consisting of 1:1:1 syntaxin-1a(residues 183-288):SNAP-25:syb(residues 49-96) was found to greatly accelerate the rates of lipid mixing of reconstituted target and vesicle SNARE proteoliposomes. Here we present two (to our knowledge) new procedures to assemble membrane-bound 1:1 SNARE acceptor complexes that produce fast and efficient fusion without the need of the syb(49-96) peptide. In the first procedure, syntaxin-1a is purified in a strictly monomeric form and subsequently assembled with SNAP-25 in detergent with the correct 1:1 stoichiometry. In the second procedure, monomeric syntaxin-1a and dodecylated (d-)SNAP-25 are separately reconstituted into proteoliposomes and subsequently assembled in the plane of merged target lipid bilayers. Examining single particle fusion between synaptobrevin-2 proteoliposomes and planar-supported bilayers containing the two different SNARE acceptor complexes revealed similar fast rates of fusion. Changing the stoichiometry of syntaxin-1a and d-SNAP-25 in the target bilayer had significant effects on docking, but little effect on the rates of synaptobrevin-2 proteoliposome fusion. PMID:27178662

  20. The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes

    PubMed Central

    Zak, Elena; Norling, Birgitta; Maitra, Radhashree; Huang, Fang; Andersson, Bertil; Pakrasi, Himadri B.

    2001-01-01

    During oxygenic photosynthesis in cyanobacteria and chloroplasts of plants and eukaryotic algae, conversion of light energy to biologically useful chemical energy occurs in the specialized thylakoid membranes. Light-induced charge separation at the reaction centers of photosystems I and II, two multisubunit pigment-protein complexes in the thylakoid membranes, energetically drive sequential photosynthetic electron transfer reactions in this membrane system. In general, in the prokaryotic cyanobacterial cells, the thylakoid membrane is distinctly different from the plasma membrane. We have recently developed a two-dimensional separation procedure to purify thylakoid and plasma membranes from the genetically widely studied cyanobacterium Synechocystis sp. PCC 6803. Immunoblotting analysis demonstrated that the purified plasma membrane contained a number of protein components closely associated with the reaction centers of both photosystems. Moreover, these proteins were assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, as evidenced from nondenaturing green gel and low-temperature fluorescence spectroscopy data. Furthermore, electron paramagnetic resonance spectroscopic analysis showed that in the partially assembled photosystem I core complex in the plasma membrane, the P700 reaction center was capable of undergoing light-induced charge separation. Based on these data, we propose that the plasma membrane, and not the thylakoid membrane, is the site for a number of the early steps of biogenesis of the photosynthetic reaction center complexes in these cyanobacterial cells. PMID:11687660

  1. Asymmetric distribution of a fluorescent sterol in synaptic plasma membranes: effects of chronic ethanol consumption.

    PubMed

    Wood, W G; Schroeder, F; Hogy, L; Rao, A M; Nemecz, G

    1990-06-27

    Ethanol-induced structural changes in membranes have in some studies been attributed to an increase in total membrane cholesterol. Consistent changes in cholesterol content, however, have not been observed in membranes of ethanol consuming animals and alcoholic patients. This study examined the hypotheses that cholesterol was asymmetrically distributed in synaptic plasma membranes (SPM) and that chronic ethanol consumption alters the transbilayer distribution of cholesterol. Dehydroergosterol, a fluorescent cholesterol analogue was used to examine sterol distribution and exchange in chronic ethanol-treated and pair-fed control groups. The cytofacial leaflet was found to have significantly more dehydroergosterol as compared to the exofacial leaflet. This asymmetric distribution was significantly reduced by chronic ethanol consumption as was sterol transport. Total cholesterol content did not differ between the two groups. Chronic ethanol consumption appeared to alter transbilayer sterol distribution as determined by the incorporation and distribution of dehydroergosterol in SPM. The changes in transbilayer sterol distribution are consistent with recent reports on the asymmetric effects of ethanol in vitro ((1988) Biochim. Biophys. Acta 946, 85-94) and in vivo ((1989) J. Neurochem. 52, 1925-1930) on membrane leaflet structure. The results of this study also underscore the importance of examining membrane lipid domains in addition to the total content of different lipids. PMID:2364080

  2. The Plasma Membrane Ca2+ ATPase and the Plasma Membrane Sodium Calcium Exchanger Cooperate in the Regulation of Cell Calcium

    PubMed Central

    Brini, Marisa; Carafoli, Ernesto

    2011-01-01

    Calcium is an ambivalent signal: it is essential for the correct functioning of cell life, but may also become dangerous to it. The plasma membrane Ca2+ ATPase (PMCA) and the plasma membrane Na+/Ca2+ exchanger (NCX) are the two mechanisms responsible for Ca2+ extrusion. The NCX has low Ca2+ affinity but high capacity for Ca2+ transport, whereas the PMCA has a high Ca2+ affinity but low transport capacity for it. Thus, traditionally, the PMCA pump has been attributed a housekeeping role in maintaining cytosolic Ca2+, and the NCX the dynamic role of counteracting large cytosolic Ca2+ variations (especially in excitable cells). This view of the roles of the two Ca2+ extrusion systems has been recently revised, as the specific functional properties of the numerous PMCA isoforms and splicing variants suggests that they may have evolved to cover both the basal Ca2+ regulation (in the 100 nM range) and the Ca2+ transients generated by cell stimulation (in the μM range). PMID:21421919

  3. Plasma Membrane Permeabilization by Trains of Ultrashort Electric Pulses

    PubMed Central

    Ibey, Bennett L.; Mixon, Dustin G.; Payne, Jason A.; Bowman, Angela; Sickendick, Karl; Wilmink, Gerald J.; Roach, W. Patrick; Pakhomov, Andrei G.

    2010-01-01

    Ultrashort electric pulses (USEP) cause long-lasting increase of cell membrane electrical conductance, and that a single USEP increased cell membrane electrical conductance proportionally to the absorbed dose (AD) with a threshold of about 10 mJ/g. The present study extends quantification of the membrane permeabilization effect to multiple USEP and employed a more accurate protocol that identified USEP effect as the difference between post- and pre-exposure conductance values (Δg) in individual cells. We showed that Δg can be increased by either increasing the number of pulses at a constant E-field, or by increasing the E-field at a constant number of pulses. For 60-ns pulses, an E-field threshold of 6 kV/cm for a single pulse was lowered to less than 1.7 kV/cm by applying 100-pulse or longer trains. However, the reduction of the E-field threshold was only achieved at the expense of a higher AD compared to a single pulse exposure. Furthermore, the effect of multiple pulses was not fully determined by AD, suggesting that cells permeabilized by the first pulse(s) in the train become less vulnerable to subsequent pulses. This explanation was corroborated by a model that treated multiple-pulse exposures as a series of single-pulse exposures and assumed an exponential decline of cell susceptibility to USEP as Δg increased after each pulse during the course of the train. PMID:20171148

  4. Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

    PubMed

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2015-08-18

    Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic

  5. Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry.

    PubMed

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

    2016-08-01

    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. PMID:26369275

  6. Surface Modification of Polypropylene Microporous Membrane by Atmospheric-Pressure Plasma Immobilization of N,N-dimethylamino Ethyl Methacrylate

    NASA Astrophysics Data System (ADS)

    Zhong, Shaofeng

    2010-10-01

    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.

  7. NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes.

    PubMed

    Cirillo, Federica; Ghiroldi, Andrea; Fania, Chiara; Piccoli, Marco; Torretta, Enrica; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

    2016-05-13

    NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. PMID:26987901

  8. The plasma membrane shuttling of CAPRI is related to regulation of mast cell activation

    SciTech Connect

    Nakamura, Rika; Furuno, Tadahide; Nakanishi, Mamoru . E-mail: mamoru@dpc.agu.ac.jp

    2006-08-18

    The Ca{sup 2+}-promoted Ras inactivator (CAPRI), a Ras GTPase-activating protein, is involved in the inactivation of mitogen-activated protein kinase pathway. However, a precise role of CAPRI in immune responses is still unknown. Here we showed that overexpression of CAPRI suppresses antigen-induced degranulation and cytokine production in mast cells (RBL cells). Antigen elicited the translocation of CAPRI to the plasma membrane from the cytoplasm, which was concomitant with the increase in the intracellular Ca{sup 2+} concentration. The nuclear import of extracellular signal-regulated kinase 2 (ERK2) occurred after the re-localization of CAPRI to the cytoplasm in the mast cells, suggesting that the early phase of ERK2 activation is eliminated. A mutant of GAP-related domain, CAPRI(R472S), showed a feeble translocation to the plasma membrane but did not affect the degranulation, ERK2 activation, and cytokine production. The results suggested that the translocation of CAPRI to the plasma membranes regulates crucially cellular responses in mast cells.

  9. HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein.

    PubMed

    Chen, Jianbo; Rahman, Sheikh Abdul; Nikolaitchik, Olga A; Grunwald, David; Sardo, Luca; Burdick, Ryan C; Plisov, Sergey; Liang, Edward; Tai, Sheldon; Pathak, Vinay K; Hu, Wei-Shau

    2016-01-12

    Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms. PMID:26712001

  10. Ethylene activates a plasma membrane Ca(2+)-permeable channel in tobacco suspension cells.

    PubMed

    Zhao, Min-Gui; Tian, Qiu-Ying; Zhang, Wen-Hao

    2007-01-01

    Here, the effects of the ethylene-releasing compound, ethephon, and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), on ionic currents across plasma membranes and on the cytosolic Ca(2+) activity ([Ca(2+)](c)) of tobacco (Nicotiana tabacum) suspension cells were characterized using a patch-clamp technique and confocal laser scanning microscopy. Exposure of tobacco protoplasts to ethephon and ACC led to activation of a plasma membrane cation channel that was permeable to Ba(2+), Mg(2+) and Ca(2+), and inhibited by La(3+), Gd(3+) and Al(3+). The ethephon- and ACC-induced Ca(2+)-permeable channel was abolished by the antagonist of ethylene perception (1-metycyclopropene) and by the inhibitor of ACC synthase (aminovinylglycin), indicating that activation of the Ca(2+)-permeable channels results from ethylene. Ethephon elicited an increase in the [Ca(2+)](c) of tobacco suspension cells, as visualized by the Ca(2+)-sensitive probe Fluo-3 and confocal microscopy. The ethephon-induced elevation of [Ca(2+)](c) was markedly inhibited by Gd(3+) and BAPTA, suggesting that an influx of Ca(2+) underlies the elevation of [Ca(2+)](c). These results indicate that an elevation of [Ca(2+)](c), resulting from activation of the plasma membrane Ca(2+)-permeable channels by ethylene, is an essential component in ethylene signaling in plants. PMID:17447907

  11. Fatty acid composition of plasma lipids and erythrocyte membranes during simulated extravehicular activity

    NASA Astrophysics Data System (ADS)

    Skedina, M. A.; Katuntsev, V. P.; Buravkova, L. B.; Naidina, V. P.

    Ten subjects (from 27 to 41 years) have been participated in 32 experiments. They were decompressed from ground level to 40-35 kPa in altitude chamber when breathed 100% oxygen by mask and performed repeated cycles of exercises (3.0 Kcal/min). The intervals between decompressions were 3-5 days. Plasma lipid and erythrocyte membrane fatty acid composition was evaluated in the fasting venous blood before and immediately after hypobaric exposure. There were 7 cases decompression sickness (DCS). Venous gas bubbles (GB) were detected in 27 cases (84.4%). Any significant changes in the fatty acid composition of erythrocyte membranes and plasma didn't practically induce after the first decompression. However, by the beginning of the second decompression the total lipid level in erythrocyte membranes decreased from 54.6 mg% to 40.4 mg% in group with DCS symptoms and from 51.2 mg% to 35.2 mg% (p < 0.05) without DCS symptoms. In group with DCS symptoms a tendency to increased level of saturated fatty acids in erythrocyte membranes (16:0, 18:0), the level of the polyunsaturated linoleic fatty acid (18:2) and arachidonic acid (20:4) tended to be decreased by the beginning of the second decompression. Insignificant changes in blood plasma fatty acid composition was observed in both groups. The obtained biochemical data that indicated the simulated extravehicular activity (EVA) condition is accompanied by the certain changes in the blood lipid metabolism, structural and functional state of erythrocyte membranes, which are reversible. The most pronounced changes are found in subjects with DCS symptoms.

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

    PubMed Central

    Eskandari, Farzaneh; Momeni, Hamid Reza

    2016-01-01

    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. PMID:27141548

  13. Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

    PubMed

    Wen, Peter J; Grenklo, Staffan; Arpino, Gianvito; Tan, Xinyu; Liao, Hsien-Shun; Heureaux, Johanna; Peng, Shi-Yong; Chiang, Hsueh-Cheng; Hamid, Edaeni; Zhao, Wei-Dong; Shin, Wonchul; Näreoja, Tuomas; Evergren, Emma; Jin, Yinghui; Karlsson, Roger; Ebert, Steven N; Jin, Albert; Liu, Allen P; Shupliakov, Oleg; Wu, Ling-Gang

    2016-01-01

    Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30-300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging. PMID:27576662

  14. Complement-mediated production of plasma-membrane vesicles from rat fat-cells.

    PubMed

    Richardson, P J; Luzio, J P

    1980-03-15

    1. Rat isolated fat-cells were coated with rabbit anti-(rat erythrocyte) antibody and incubated with fresh guinea-pig serum for 25 min at 37 degrees C, which resulted in a more than 95% release of the cytosolic enzyme lactate dehydrogenase. 2. Under these conditions fragmentation of the plasma membrane was examined by following the plasma-membrane markers 5'-nucleotidase, adrenaline-sensitive adenylate cyclase and membrane-bound rabbit immunoglobulin G through a differential-centrifugation fractionation procedure. 3. Approx. 50% of the plasma-membrane markers remained associated with triacylglycerol. Of the remainder more than half was pelleted by centrifugation at 10 000 g for 30 min. 4. The 10 000 g supernatant was fractionated by centrifugation on a sucrose density gradient (15-50%, w/w). This procedure resulted in the production of two visible white bands on the density gradient. The bands consisted of vesicles derived from the plasma membrane, since they coincided with peaks of 5'-nucleotidase activity, contained membrane-bound immunoglobulin G and the denser one had adenylate cyclase activity. The phospholipid and protein contents of the vesicles were determined and compared with those in purified plasma membrane. 5. It is suggested that complement-mediated lysis of rat fat-cells caused the production of plasma-membrane vesicles that differ in composition from the whole plasma membrane. PMID:6249263

  15. TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

    SciTech Connect

    Magini, Alessandro; Polchi, Alice; Urbanelli, Lorena; Cesselli, Daniela; Beltrami, Antonio; Tancini, Brunella; Emiliani, Carla

    2013-10-18

    Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane.

  16. The isolation and partial characterization of the plasma membrane from Trypanosoma brucei.

    PubMed

    Voorheis, H P; Gale, J S; Owen, M J; Edwards, W

    1979-04-15

    Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase. PMID:486094

  17. Detection of boar sperm plasma membrane protein using Rhodamine 640; implications for cryobiology and physiology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhodamine 640 (R640) was used to detect changes in boar sperm plasma membrane protein (PMP) during cryopreservation; a poorly understood phenomenon. The protocol was adapted for boar sperm so that semen samples (n = 17) could be analyzed for PMP (R640 positive) and plasma membrane integrity (PMI; Y...

  18. Thymocyte plasma membrane: the location of specific glucocorticoid binding sites

    SciTech Connect

    Sergeev, P.V.; Kalinin, G.V.; Dukhanin, A.S.

    1987-01-01

    In modern molecular endocrinology it is now possible to determine the localization of receptors for biologically active substances with the aid of ligands, with high affinity for the receptor, immobilized on polymers. The purpose of this paper is to study the ability of hydrocortisone (HC), immobilized on polyvinylpyrrolidone (PVP-HC), to reduce binding of tritium-HC by thymocytes of adrenalectomized rats. It is determined that specific binding sites for HC on rat thymocytes are also accessible for PVP-HC, which, due to the fact that this immobilized version of HC does not penetrate into the cell, leads to the conclusion that the binding sites for HC itself are located in the plasma membrane.

  19. Elevated plasma corticosterone increases metabolic rate in a terrestrial salamander.

    PubMed

    Wack, Corina L; DuRant, Sarah E; Hopkins, William A; Lovern, Matthew B; Feldhoff, Richard C; Woodley, Sarah K

    2012-02-01

    Plasma glucocorticoid hormones (GCs) increase intermediary metabolism, which may be reflected in whole-animal metabolic rate. Studies in fish, birds, and reptiles have shown that GCs may alter whole-animal energy expenditure, but results are conflicting and often involve GC levels that are not physiologically relevant. A previous study in red-legged salamanders found that male courtship pheromone increased plasma corticosterone (CORT; the primary GC in amphibians) concentrations in males, which could elevate metabolic processes to sustain courtship behaviors. To understand the possible metabolic effect of elevated plasma CORT, we measured the effects of male courtship pheromone and exogenous application of CORT on oxygen consumption in male red-legged salamanders (Plethodon shermani). Exogenous application of CORT elevated plasma CORT to physiologically relevant levels. Compared to treatment with male courtship pheromone and vehicle, treatment with CORT increased oxygen consumption rates for several hours after treatment, resulting in 12% more oxygen consumed (equivalent to 0.33 J) during our first 2h sampling period. Contrary to our previous work, treatment with pheromone did not increase plasma CORT, perhaps because subjects used in this study were not in breeding condition. Pheromone application did not affect respiration rates. Our study is one of the few to evaluate the influence of physiologically relevant elevations in CORT on whole-animal metabolism in vertebrates, and the first to show that elevated plasma CORT increases metabolism in an amphibian. PMID:22047668

  20. Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells.

    PubMed

    Abu-Arish, Asmahan; Pandzic, Elvis; Goepp, Julie; Matthes, Elizabeth; Hanrahan, John W; Wiseman, Paul W

    2015-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function. PMID:26153705

  1. Increased Uptake of Chelated Copper Ions by Lolium perenne Attributed to Amplified Membrane and Endodermal Damage

    PubMed Central

    Johnson, Anthea; Singhal, Naresh

    2015-01-01

    The contributions of mechanisms by which chelators influence metal translocation to plant shoot tissues are analyzed using a combination of numerical modelling and physical experiments. The model distinguishes between apoplastic and symplastic pathways of water and solute movement. It also includes the barrier effects of the endodermis and plasma membrane. Simulations are used to assess transport pathways for free and chelated metals, identifying mechanisms involved in chelate-enhanced phytoextraction. Hypothesized transport mechanisms and parameters specific to amendment treatments are estimated, with simulated results compared to experimental data. Parameter values for each amendment treatment are estimated based on literature and experimental values, and used for model calibration and simulation of amendment influences on solute transport pathways and mechanisms. Modeling indicates that chelation alters the pathways for Cu transport. For free ions, Cu transport to leaf tissue can be described using purely apoplastic or transcellular pathways. For strong chelators (ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA)), transport by the purely apoplastic pathway is insufficient to represent measured Cu transport to leaf tissue. Consistent with experimental observations, increased membrane permeability is required for simulating translocation in EDTA and DTPA treatments. Increasing the membrane permeability is key to enhancing phytoextraction efficiency. PMID:26512647

  2. STIM Proteins and the Endoplasmic Reticulum-Plasma Membrane Junctions

    PubMed Central

    Carrasco, Silvia; Meyer, Tobias

    2013-01-01

    Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca2+ levels and directly activate Orai PM Ca2+ channels across the junction space. In an inverse process, a voltage-gated PM Ca2+ channel can directly open ER ryanodine-receptor Ca2+ channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca2+ signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes. PMID:21548779

  3. A membrane-separator interface for mass-spectrometric analysis of blood plasma

    NASA Astrophysics Data System (ADS)

    Elizarov, A. Yu.; Gerasimov, D. G.

    2014-09-01

    We demonstrate the possibility of rapid mass-spectrometric determination of the content of anesthetic agents in blood plasma with the aid of a membrane-separator interface. The interface employs a hydrophobic selective membrane that is capable of separating various anesthetic drugs (including inhalation anesthetic sevofluran, noninhalation anesthetic thiopental, hypnotic propofol, and opioid analgesic fentanyl) from the blood plasma and introducing samples into a mass spectrometer. Analysis of the blood plasma was not accompanied by the memory effect and did not lead to membrane degradation. Results of clinical investigation of the concentration of anesthetics in the blood plasma of patients are presented.

  4. Effect of serum lipoproteins on the adenylate cyclase activity of rat liver plasma membranes.

    PubMed Central

    Ghiselli, G; Sirtori, C R; Nicosia, S

    1981-01-01

    Four rat lipoprotein classes [lymph chylomicrons, VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins] were tested for their ability to affect basal adenylate cyclase (EC 4.6.1.1) activity of rat liver plasma membranes. All the lipoproteins, with the exception of lymph chylomicrons, effectively increase the enzyme activity. VLD lipoproteins are the most active class (67% maximal increase), followed by HD lipoproteins (33%) and LD lipoproteins (23%). The effect of VLD lipoproteins is additive to that elicited by GTP or GTP plus glucagon (at least within a certain concentration range). VLD lipoproteins affect only the Vmax. of the enzyme, not the Km. PMID:7317023

  5. VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals.

    PubMed

    Taubenblatt, P; Dedieu, J C; Gulik-Krzywicki, T; Morel, N

    1999-10-01

    Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin. After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoichiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process

  6. Multi-walled carbon nanotubes injure the plasma membrane of macrophages

    SciTech Connect

    Hirano, Seishiro Kanno, Sanae; Furuyama, Akiko

    2008-10-15

    Carbon nanotubes (CNTs) are emerging nanotechnology materials which are likely to be mass-produced in the near future. However, prior to mass-production, certain health-related concerns should first be addressed. For example, when inhaled, the thin-fibrous shape and the biopersistent characteristics of CNTs may cause pulmonary diseases, in a manner similar to asbestos. In the present study, mouse macrophages (J774.1) were exposed to highly-purified multi-walled CNTs (MWCNTs, 67 nm) or to UICC crocidolite in order to evaluate the toxicity of these nano-size fibers. The cytotoxicity of MWCNTs was found to be higher than that of crocidolite. The toxic effect of MWCNTs was not affected by N-acetylcysteine, an antioxidant, or buthionine sulfoximine, a glutathione synthesis inhibitor. cDNA microarray analyses suggested that the cytotoxicity of MWCNTs could not be explained satisfactorily by either an increase or decrease of gene expression, although mRNA levels of some cytokines were slightly increased by MWCNTs. Moreover, MWCNTs did not significantly activate either MAP kinases such as ERK, JNK and p38, nor common apoptosis pathways such as caspase 3 and PARP. Electron microscopic studies indicated that MWCNTs associate with the plasma membrane of macrophages and disrupt the integrity of the membrane. Several proteins were found to adsorb onto MWCNTs when MWCNT-exposed macrophages were gently lysed. One of these proteins was macrophage receptor with collagenous structure (MARCO). MARCO-transfected CHO-K1 cells associated with MWCNTs more rapidly than mock-transfected cells. These results indicate that MWCNTs probably trigger cytotoxic effects in phagocytotic cells by reacting with MARCO on the plasma membrane and rupturing the plasma membrane.

  7. Aspirin Increases the Solubility of Cholesterol in Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Alsop, Richard; Barrett, Matthew; Zheng, Sonbo; Dies, Hannah; Rheinstadter, Maikel

    2014-03-01

    Aspirin (ASA) is often prescribed for patients with high levels of cholesterol for the secondary prevention of myocardial events, a regimen known as the Low-Dose Aspirin Therapy. We have recently shown that Aspirin partitions in lipid bilayers. However, a direct interplay between ASA and cholesterol has not been investigated. Cholesterol is known to insert itself into the membrane in a dispersed state at moderate concentrations (under ~37.5%) and decrease fluidity of membranes. We prepared model lipid membranes containing varying amounts of both ASA and cholesterol molecules. The structure of the bilayers as a function of ASA and cholesterol concentration was determined using high-resolution X-ray diffraction. At cholesterol levels of more than 40mol%, immiscible cholesterol plaques formed. Adding ASA to the membranes was found to dissolve the cholesterol plaques, leading to a fluid lipid bilayer structure. We present first direct evidence for an interaction between ASA and cholesterol on the level of the cell membrane.

  8. Reduction in lateral lipid mobility of lipid bilayer membrane by atmospheric pressure plasma irradiation

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

    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.

  9. Removal of toxic substances by a selective membrane plasma separator.

    PubMed

    Nakae, Hajime; Hattori, Tomoko; Igarashi, Toshiko; Okuyama, Manabu; Tajimi, Kimitaka

    2014-06-01

    We devised a method of plasma exchange with dialysis (PED), in which selective plasma exchange (sPE) is performed using a selective membrane plasma separator (EC-2A) with an albumin-sieving coefficient of 0.3 while the dialysate flows outside the hollow fibers, and reported the usefulness of the system for treating acute liver failure. Thereafter, EC-4A with an albumin-sieving coefficient of 0.6 was developed, which was expected to be even more effective for removing protein-bound substances. In order to examine whether or not EC-4A might be applicable to blood purification therapy against drug poisoning, we compared the efficacies of sPE, PED, and direct hemoperfusion (DHP) using an activated carbon column for the removal of phenobarbital and lithium. Subjects undergoing the extracorporeal circulation study were assigned to the sPE group, PED group, or DHP group, and the changes in the blood concentrations of phenobarbital and lithium were measured over 180 min. A significant decrease of the phenobarbital concentration over time was seen in the PED group, as compared to that in the sPE group (P < 0.0001), while no significant difference in the concentration was observed between the PED and DHP groups. The PED group showed a significant decrease of the lithium concentration over time, as compared to the DHP group (P < 0.0001), while no significant difference in the concentration was observed between the PED and sPE groups. Thus, PED was as effective as DHP for removing phenobarbital and was as effective as sPE for removing lithium. These results suggest that PED therapy using EC-4A may be a feasible modality for the treatment of drug poisoning. PMID:24965293

  10. Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability.

    PubMed

    Schapire, Arnaldo L; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A

    2008-12-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness. PMID:19088329