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Sample records for activity cell membrane

  1. Clustering of T cell ligands on artificial APC membranes influences T cell activation and protein kinase C theta translocation to the T cell plasma membrane.

    PubMed

    Giannoni, Francesca; Barnett, Joellen; Bi, Kun; Samodal, Rodrigo; Lanza, Paola; Marchese, Patrizia; Billetta, Rosario; Vita, Randi; Klein, Mark R; Prakken, Berent; Kwok, William W; Sercarz, Eli; Altman, Amnon; Albani, Salvatore

    2005-03-15

    T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase theta, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.

  2. Phosphatidic acid phosphatase and phospholipdase A activities in plasma membranes from fusing muscle cells.

    PubMed

    Kent, C; Vagelos, P R

    1976-06-17

    Plasma membrane from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distributions of the plasma membrane markers, (Na+ + k+)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.

  3. Thylakoid Membrane Maturation and PSII Activation Are Linked in Greening Synechocystis sp. PCC 6803 Cells1

    PubMed Central

    Barthel, Sandra; Bernát, Gábor; Seidel, Tobias; Rupprecht, Eva; Kahmann, Uwe; Schneider, Dirk

    2013-01-01

    Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. While they are crucial for phototrophic growth of cyanobacterial cells, biogenesis of thylakoid membranes is not well understood yet. Dark-grown Synechocystis sp. PCC 6803 cells contain only rudimentary thylakoid membranes but still a relatively high amount of phycobilisomes, inactive photosystem II and active photosystem I centers. After shifting dark-grown Synechocystis sp. PCC 6803 cells into the light, “greening” of Synechocystis sp. PCC 6803 cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions, was monitored. Complete restoration of a typical thylakoid membrane system was observed within 24 hours after an initial lag phase of 6 to 8 hours. Furthermore, activation of photosystem II complexes and restoration of a functional photosynthetic electron transport chain appears to be linked to the biogenesis of organized thylakoid membrane pairs. PMID:23922268

  4. Glucocorticoid hormones increase the activity of plasma membrane alkaline phosphodiesterase I in rat hepatoma cells.

    PubMed Central

    Rousseau, G G; Amar-Costesec, A; Verhaegen, M; Granner, D K

    1980-01-01

    In rat hepatoma cells the synthetic glucocorticoid dexamethasone causes a 3-fold increase in the activity of the plasma membrane enzyme alkaline phosphodiesterase I (oligonucleat 5'-nucleotidohydrolase, EC 3.1.4.1). The data are consistent with an induction phenomenon mediated by the glucocorticoid receptor involved in tyrosine aminotransferase induction. The effect on alkaline phosphodiesterase I is not a reflection of a general membrane effect of dexamethasone, because the activity of three other enzymes of the plasma membrane is unaffected. On the other hand, nucleoside diphosphatase (nucleoside diphosphate phosphohydrolase acting on ADP) activity is inhibited. Thus, two more enzymes sensitive to glucocorticoids have been identified in a cell line in which these hormones influence only very few gene products. This paper describes enzymatic changes in the plasma membrane of rat hepatoma cells in which glucocorticoids normalize a number of membrane-associated processes that are considered to be characteristic of transformed cells. PMID:6102383

  5. Electrochemiluminescence imaging for parallel single-cell analysis of active membrane cholesterol.

    PubMed

    Zhou, Junyu; Ma, Guangzhong; Chen, Yun; Fang, Danjun; Jiang, Dechen; Chen, Hong-Yuan

    2015-08-18

    Luminol electrochemiluminescence (ECL) imaging was developed for the parallel measurement of active membrane cholesterol at single living cells, thus establishing a novel electrochemical detection technique for single cells with high analysis throughput and low detection limit. In our strategy, the luminescence generated from luminol and hydrogen peroxide upon the potential was recorded in one image so that hydrogen peroxide at the surface of multiple cells could be simultaneously analyzed. Compared with the classic microelectrode array for the parallel single-cell analysis, the plat electrode only was needed in our ECL imaging, avoiding the complexity of electrode fabrication. The optimized ECL imaging system showed that hydrogen peroxide as low as 10 μM was visible and the efflux of hydrogen peroxide from cells could be determined. Coupled with the reaction between active membrane cholesterol and cholesterol oxidase to generate hydrogen peroxide, active membrane cholesterol at cells on the electrode was analyzed at single-cell level. The luminescence intensity was correlated with the amount of active membrane cholesterol, validating our system for single-cell cholesterol analysis. The relative high standard deviation on the luminescence suggested high cellular heterogeneities on hydrogen peroxide efflux and active membrane cholesterol, which exhibited the significance of single-cell analysis. This success in ECL imaging for single-cell analysis opens a new field in the parallel measurement of surface molecules at single cells.

  6. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    NASA Astrophysics Data System (ADS)

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-09-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

  7. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane.

    PubMed

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  8. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    PubMed Central

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  9. Phosphatidylinositol kinase is activated in membranes derived from cells treated with epidermal growth factor.

    PubMed Central

    Walker, D H; Pike, L J

    1987-01-01

    The ability of epidermal growth factor (EGF) to stimulate phosphatidylinositol (PtdIns) kinase activity in A431 cells was examined. The incorporation of 32P from [gamma-32P]ATP into PtdIns by A431 membranes was increased in membranes prepared from cells that had been pretreated with EGF. Demonstration of a stimulation of the PtdIns kinase activity by EGF required the use of subconfluent cultures and was dependent on the inclusion of protease inhibitors in the buffers used to prepare the membranes. Stimulation of the PtdIns kinase activity was rapid. The activation peaked 2 min after the addition of EGF and declined slowly thereafter. Half-maximal stimulation of the PtdIns kinase occurred at 7 nM EGF. Kinetic analyses of the reaction indicated that treatment of the cells with EGF resulted in a decrease in the Km for PtdIns with no change in the Vmax. The kinetic parameters for the utilization of ATP were unchanged in the EGF-treated membranes compared to the control membranes. Pretreatment of the cells with the phorbol ester phorbol 12-myristate 13-acetate blocked the ability of EGF to stimulate PtdIns kinase activity. These findings demonstrate that a PtdIns kinase activity in A431 cells is regulated by EGF and provide a good system for examining the mechanism by which EGF stimulates the activity of this intracellular enzyme. PMID:2823265

  10. Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells.

    PubMed

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R; Grewal, Thomas; Gaus, Katharina

    2016-01-01

    Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

  11. Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells.

    PubMed

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R; Grewal, Thomas; Gaus, Katharina

    2016-01-01

    Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells. PMID:26977592

  12. Estimating the magnitude of near-membrane PDE4 activity in living cells.

    PubMed

    Xin, Wenkuan; Feinstein, Wei P; Britain, Andrea L; Ochoa, Cristhiaan D; Zhu, Bing; Richter, Wito; Leavesley, Silas J; Rich, Thomas C

    2015-09-15

    Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments.

  13. Vaccinia extracellular virions enter cells by macropinocytosis and acid-activated membrane rupture

    PubMed Central

    Schmidt, Florian Ingo; Bleck, Christopher Karl Ernst; Helenius, Ari; Mercer, Jason

    2011-01-01

    Vaccinia virus (VACV), the model poxvirus, produces two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). EV particles possess two membranes and therefore require an unusual cellular entry mechanism. By a combination of fluorescence and electron microscopy as well as flow cytometry, we investigated the cellular processes that EVs required to infect HeLa cells. We found that EV particles were endocytosed, and that internalization and infection depended on actin rearrangements, activity of Na+/H+ exchangers, and signalling events typical for the macropinocytic mechanism of endocytosis. To promote their internalization, EVs were capable of actively triggering macropinocytosis. EV infection also required vacuolar acidification, and acid exposure in endocytic vacuoles was needed to disrupt the outer EV membrane. Once exposed, the underlying MV-like particle presumably fused its single membrane with the limiting vacuolar membrane. Release of the viral core into the host cell cytosol allowed for productive infection. PMID:21792173

  14. Vaccinia extracellular virions enter cells by macropinocytosis and acid-activated membrane rupture.

    PubMed

    Schmidt, Florian Ingo; Bleck, Christopher Karl Ernst; Helenius, Ari; Mercer, Jason

    2011-08-31

    Vaccinia virus (VACV), the model poxvirus, produces two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). EV particles possess two membranes and therefore require an unusual cellular entry mechanism. By a combination of fluorescence and electron microscopy as well as flow cytometry, we investigated the cellular processes that EVs required to infect HeLa cells. We found that EV particles were endocytosed, and that internalization and infection depended on actin rearrangements, activity of Na(+)/H(+) exchangers, and signalling events typical for the macropinocytic mechanism of endocytosis. To promote their internalization, EVs were capable of actively triggering macropinocytosis. EV infection also required vacuolar acidification, and acid exposure in endocytic vacuoles was needed to disrupt the outer EV membrane. Once exposed, the underlying MV-like particle presumably fused its single membrane with the limiting vacuolar membrane. Release of the viral core into the host cell cytosol allowed for productive infection. PMID:21792173

  15. Sirtuin Activation: A Role for Plasma Membrane in the Cell Growth Puzzle

    PubMed Central

    2013-01-01

    For more than 20 years, the observation that impermeable oxidants can stimulate cell growth has not been satisfactorily explained. The discovery of sirtuins provides a logical answer to the puzzle. The NADH-dependent transplasma membrane electron transport system, which is stimulated by growth factors and interventions such as calorie restriction, can transfer electrons to external acceptors and protect against stress-induced apoptosis. We hypothesize that the activation of plasma membrane electron transport contributes to the cytosolic NAD+ pool required for sirtuin to activate transcription factors necessary for cell growth and survival. PMID:23033342

  16. Ionizing radiations increase the activity of the cell surface glycohydrolases and the plasma membrane ceramide content.

    PubMed

    Aureli, Massimo; Bassi, Rosaria; Prinetti, Alessandro; Chiricozzi, Elena; Pappalardi, Brigida; Chigorno, Vanna; Di Muzio, Nadia; Loberto, Nicoletta; Sonnino, Sandro

    2012-12-01

    We detected significant levels of β-glucosidase, β-galactosidase, sialidase Neu3 and sphingomyelinase activities associated with the plasma membrane of fibroblasts from normal and Niemann-Pick subjects and of cells from breast, ovary, colon and neuroblastoma tumors in culture. All of the cells subjected to ionizing radiations showed an increase of the activity of plasma membrane β-glucosidase, β-galactosidase and sialidase Neu3, in addition of the well known increase of activity of plasma membrane sphingomyelinase, under similar conditions. Human breast cancer cell line T47D was studied in detail. In these cells the increase of activity of β-glucosidase and β-galactosidase was parallel to the increase of irradiation dose up to 60 Gy and continued with time, at least up to 72 h from irradiation. β-glucosidase increased up to 17 times and β-galactosidase up to 40 times with respect to control. Sialidase Neu3 and sphingomyelinase increased about 2 times at a dose of 20 Gy but no further significant differences were observed with increase of radiation dose and time. After irradiation, we observed a reduction of cell proliferation, an increase of apoptotic cell death and an increase of plasma membrane ceramide up to 3 times, with respect to control cells. Tritiated GM3 ganglioside has been administered to T47D cells under conditions that prevented the lysosomal catabolism. GM3 became component of the plasma membranes and was transformed into LacCer, GlcCer and ceramide. The quantity of ceramide produced in irradiated cells was about two times that of control cells.

  17. Beyond apoptosis: The mechanism and function of phosphatidylserine asymmetry in the membrane of activating mast cells

    PubMed Central

    Rysavy, Noel M.; Shimoda, Lori M. N.; Dixon, Alyssa M.; Speck, Mark; Stokes, Alexander J.; Turner, Helen; Umemoto, Eric Y.

    2014-01-01

    Loss of plasma membrane asymmetry is a hallmark of apoptosis, but lipid bilayer asymmetry and loss of asymmetry can contribute to numerous cellular functions and responses that are independent of programmed cell death. Exofacial exposure of phosphatidylserine occurs in lymphocytes and mast cells after antigenic stimulation and in the absence of apoptosis, suggesting that there is a functional requirement for phosphatidylserine exposure in immunocytes. In this review we examine current ideas as to the nature of this functional role in mast cell activation. Mechanistically, there is controversy as to the candidate proteins responsible for phosphatidylserine translocation from the internal to external leaflet, and here we review the candidacies of mast cell PLSCR1 and TMEM16F. Finally we examine the potential relationship between functionally important mast cell membrane perturbations and phosphatidylserine exposure during activation. PMID:25759911

  18. Trans-activity of Plasma Membrane-associated Ganglioside Sialyltransferase in Mammalian Cells*

    PubMed Central

    Vilcaes, Aldo A.; Demichelis, Vanina Torres; Daniotti, Jose L.

    2011-01-01

    Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (cis-catalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition. PMID:21768099

  19. Receptor-independent, direct membrane binding leads to cell surface lipid sorting and Syk kinase activation in dendritic cells

    PubMed Central

    Ng, Gilbert; Sharma, Karan; Ward, Sandra M.; Desrosiers, Melanie D.; Stephens, Leslie A.; Schoel, W. Michael; Li, Tonglei; Lowell, Clifford A.; Ling, Chang-Chun; Amrein, Matthias W.; Shi, Yan

    2008-01-01

    Summary Binding of particulate antigens by antigen presenting cells (APC) is a critical step in immune activation. Previously, we demonstrated that uric acid crystals are potent adjuvants, initiating a robust adaptive immune response. However, the mechanisms of activation are unknown. Using atomic force microscopy as a tool for real time single cell activation analysis, we report that uric acid crystals can directly engage cellular membranes, particularly the cholesterol components, with a force substantially stronger than protein based cellular contacts. Binding of particulate substances activates Syk kinase-dependent signaling in dendritic cells (DCs). These observations suggest a mechanism whereby immune cell activation can be triggered by solid structures via membrane lipid alteration without the requirement for specific cell surface receptors, and a testable hypothesis for crystal-associated arthropathies, inflammation and adjuvanticity. PMID:18993083

  20. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    NASA Astrophysics Data System (ADS)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  1. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells.

    PubMed

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway. PMID:27657873

  2. Disruption of cell membranes via laser-activated, acoustically active, carbon nanoparticles

    NASA Astrophysics Data System (ADS)

    Holguin, Stefany; Prausnitz, Mark; Thadhani, Naresh

    2015-06-01

    Physical drug delivery methods provide an avenue to overcome the selectivity of the cell membrane via physical forces that disrupt cell membranes and drive drug molecules into the cytosol. When carbon black nanoparticles in suspension with cells and drug molecules are exposed to nanosecond-pulsed laser light, high uptake and cell viability are observed. This laser-carbon nanoparticle interaction causes thermal expansion and local vaporization that results in the release of acoustic waves into the surrounding medium. These combined energy transduction mechanisms, phenomena called transient nanoparticle energy transduction (TNET), are responsible for disruption of the cell membrane and subsequent efficient intracellular drug uptake while maintaining high cell viability. The overall objective of this work is to investigate TNET and the bioeffects associated with physical disruption of cell membranes for drug delivery via laser-carbon nanoparticle interactions. For example, varying and quantifying energy input to carbon nanoparticles by way of laser beam manipulation, assists in the understanding and assessment of subsequent bioeffects. Results of work performed to date will be presented. National Science Foundation Graduate Research Fellowship under Grant No. 0946809, Georgia Tech University Center of Exemplary Mentoring (UCEM) & the Alfred P. Sloan Foundation.

  3. Membrane potential and endocytic activity control disintegration of cell-cell adhesion and cell fusion in vinculin-injected MDBK cells.

    PubMed

    Palovuori, Riitta; Myrsky, Essi; Eskelinen, Sinikka

    2004-09-01

    Cell fusion occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. We have developed an experimental model for epithelial cell fusion which permits analysis of the processes during junction disintegration and formation of polykaryons (Palovuori and Eskelinen [2000] Eur. J. Cell. Biol. 79: 961-974). In the present work, we analyzed the process in detail. Cell fusion was achieved by microinjecting into the cytoplasm of kidney epithelial Madin-Darby bovine kidney (MDBK) cells TAMRA-tagged vinculin, which incorporated into lateral membranes, focal adhesions and nucleus, and, prior fusion, induced internalization of actin, cadherin and plakoglobin to small clusters in cytoplasm. Injected vinculin was still visible at lateral membranes after removal of junctional proteins indicating that it was tightly associated and perturbed the cell-cell contact sites resulting in membrane fragmentation. Injection of active Rac together with vinculin induced accumulation of cadherin to the membranes, but did not affect vinculin-membrane association. However, it hampered cell fusion probably by supporting adherens junctions. In order to stop endocytosis, we lowered intracellular pH of vinculin-injected cells to 5.5 with the aid of nigericin in KCl buffer. In acidified cells, injected vinculin delineated lateral membranes as thick layers, cadherin remained in situ, and cell fusion was completely inhibited. Since this treatment also leads to cell depolarization, we checked the vinculin incorporation in a KCl solution containing nigericin at neutral pH. In these circumstances, both endogenous and injected vinculin delineated lateral membranes as very thin discontinuous layers, but still fusion was hampered most likely due to perturbation in the initial vinculin-membrane association. We suggest that vinculin might function as a sensor of the environment triggering cell fusion during development in circumstances where membrane potential and local

  4. Membrane-bound complement regulatory activity is decreased on vaccinia virus-infected cells.

    PubMed Central

    Baranyi, L; Okada, N; Baranji, K; Takizawa, H; Okada, H

    1994-01-01

    Decay accelerating factor (DAF), membrane cofactor protein (MCP), complement receptor 1 and mouse Crry are cell surface-bound complement regulatory proteins capable of inhibiting C3 convertase activity on cell membranes, and therefore provide a substantial protection from attack by homologous complement activated either by the classical or by the alternative pathway. Decrease in complement regulatory activity might lead to spontaneous complement deposition and subsequent cell injury. MoAb 5I2 can inhibit the complement regulatory activity of molecules on rat cells, resulting in deposition of homologous complement. The antigen recognized by 5I2 MoAb in rats is homologous to mouse Crry. Fifteen to 20 h after infection with vaccinia virus, in vitro cultured KDH-8 rat hepatoma cells show a strong decrease in expression of Crry-like antigen, and proved to be sensitive to complement deposition when 1:5 diluted normal rat serum was added to the culture medium as a source of complement. Addition of complement to the cultured KDH-8 cells infected with a very low dose of vaccinia virus (1 plaque-forming unit (PFU)/1000 cells) substantially reduced spreading of virus infection in the cell culture, while inactivation of complement by heat or zymosan treatment abrogated the protective effect. PMID:7923872

  5. Sweetness-induced activation of membrane dipole potential in STC-1 taste cells.

    PubMed

    Chen, Li-Chun; Xie, Ning-Ning; Deng, Shao-Ping

    2016-12-01

    The biological functions of cell membranes strongly influence the binding and transport of molecular species. We developed STC-1 cell line stably expressing the sweet taste receptor (T1R2/T1R3), and explored the possible correlation between sweeteners and membrane dipole potential of STC-1 cells. In this study, sweetener-induced dipole potential activation was elucidated using a fluorescence-based measurement technique, by monitoring the voltage sensitive probe Di-8-ANEPPS using a dual wavelength ratiometric approach. It indicated that the presence of sweeteners resulted in cell membrane dipole potential change, and interaction of artificial sweeteners with taste cells resulted in a greater reduction in potential compared with natural sweeteners. Our work presents a newly developed approach using a fluorescence-based measurement technique to study sweetener-induced dipole potential activation of STC-1 cells. This new approach could be used as a complementary tool to study the function of sweet taste receptors or other GPCRs and helps to understand the basis sweetness mechanism. PMID:27374594

  6. Antibacterial Activity of Shikimic Acid from Pine Needles of Cedrus deodara against Staphylococcus aureus through Damage to Cell Membrane

    PubMed Central

    Bai, Jinrong; Wu, Yanping; Liu, Xiaoyan; Zhong, Kai; Huang, Yina; Gao, Hong

    2015-01-01

    Shikimic acid (SA) has been reported to possess antibacterial activity against Staphylococcus aureus, whereas the mode of action of SA is still elusive. In this study, the antibacterial activity and mechanism of SA toward S. aureus by cell membrane damage was investigated. After SA treatment, massive K+ and nucleotide leakage from S. aureus, and a significant change in the membrane potential was observed, suggesting SA may act on the membrane by destroying the cell membrane permeability. Through transmission electron microscopic observations we further confirmed that SA can disrupt the cell membrane and membrane integrity. Meanwhile, SA was found to be capable of reducing the membrane fluidity of the S. aureus cell. Moreover, the fluorescence experiments indicated that SA could quench fluorescence of Phe residues of the membrane proteins, thus demonstrating that SA can bind to S. aureus membrane proteins. Therefore, these results showed the antibacterial activity of SA against S. aureus could be caused by the interactions of SA with S. aureus membrane proteins and lipids, resulting in causing cell membrane dysfunction and bacterial damage or even death. This study reveals the potential use of SA as an antibacterial agent. PMID:26580596

  7. AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

    PubMed Central

    Thavarajah, Thanusi; Medvedev, Sergei; Bowden, Peter; Marshall, John G.; Antonescu, Costin N.

    2015-01-01

    The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of β1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute

  8. Effect of various concentration of sulfuric acid for Nafion membrane activation on the performance of fuel cell

    NASA Astrophysics Data System (ADS)

    Pujiastuti, Sri; Onggo, Holia

    2016-02-01

    This work proposes an activation treatment to Nafion 117 membrane with sulfuric acid in various concentrations. The main goal of this study is to increase the Nafion 117 membrane performance, which is determined by proton number in the membrane and membrane performance in Polymer Electrolyte Membrane Fuel Cell (PEMFC). This work was developed using sulfuric acids in four different concentrations: 1, 2, 3, and 4 M. The surface morphology and functional groups of activated membranes were studied using Scanning Electron Microscope and Fourier Transform Infrared, respectively. The proton number absorbed in membranes was observed by gravimetric measurements. The performances of activated membranes in PEMFC were studied by single cell measurements with H2/O2 operation. The experimental results showed that activation of Nafion membrane did not change its surface morphology and functional groups. The proton number increased when the concentration of sulfuric acid is increased from 1 to 3 M and from 1 to 4 M. On the other hand, there is no significant increase when the concentration of sulfuric acid was increased from 1 to 2 M. Similar trends were observed when testing activated membrane performance in PEMFC, especially for current density at 0.6 V and maximum power. It is assumed that there is a correlation between the increase of sulfuric acid concentration in activation process with the increase of proton number in the membrane that are available for facilitating of transfer protons from the anode to the cathode.

  9. The Immunosuppressive Activity of Amniotic Membrane Mesenchymal Stem Cells on T Lymphocytes

    PubMed Central

    Alikarami, Fatemeh; Yari, Fatemeh; Amirizadeh, Naser; Nikougoftar, Mahin; Jalili, Mohammad Ali

    2015-01-01

    Background: Mesenchymal Stem Cells (MSCs) are isolated from different sources like placenta. The placenta and its membranes like Amniotic Membrane (AM) are readily available and easy to work with. There is only limited knowledge on the immunomodulatory properties of human Amniotic Membrane-derived Mesenchymal Stem Cells (hAM-MSCs). The aim of this study was to survey the suppressive activity of hAM-MSCs on T lymphocytes in vitro. Methods: Human AMs were obtained after caesarean section births from healthy women. After enzymatic digestion, cells were cultured and hAM-MSCs were obtained. In addition, human T lymphocytes were isolated and co-cultured with hAM-MSCs for 72 hr in the presence or absence of phytohemagglutinin (PHA). Subsequently, proliferation of T cells was analyzed using BrdU and subsequently flow cytometry technique. Besides, the production of IL-4 and IFN-γ was examined by ELISA method. Additionally, the expression of activation markers (CD38, HLA-DR) was studied on T lymphocytes by flow cytometry technique. Results: It was revealed that hAM-MSCs could significantly suppress the proliferation of T lymphocytes (p≤0.01) and significantly decrease the production of IFN-γ by T cells (p<0.05). hAM-MSCs also down regulated the expression of activation markers on the surface of T lymphocytes, CD38 and HLA-DR. The difference was significant between the case and control samples (p<0.05). All the comparisons were carried out between the case (Tcell+PHA+hAM-MSCs) and control (Tcell+PHA) groups. Conclusion: In conclusion, hAM-MSCs could inhibit the (mitogen-activated) T cells even in the absence of blood monocytes. Besides, hAM-MSCs-mediated inhibition of T lymphocytes was combined with down regulation of activation markers. PMID:26306147

  10. Comparative Transport Activity of Intact Cells, Membrane Vesicles, and Mesosomes of Bacillus licheniformis

    PubMed Central

    MacLeod, Robert A.; Thurman, Paul; Rogers, H. J.

    1973-01-01

    Sodium ion was shown to stimulate strongly the transport of l-glutamic acid into cells of Bacillus licheniformis 6346 His−. Lithium ion had a slight capacity to replace Na+ in this capacity, but K+ was without effect. Three of five amino acids tested. l-glutamic acid, l-aspartic acid, and l-alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l-histidine and l-lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l- or d-lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K2(5) or K2(10). Sodium ion stimulated transport of glutamic acid by membrane vesicles. PMID:4347247

  11. Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations.

    PubMed

    Jansen, J; De Napoli, I E; Fedecostante, M; Schophuizen, C M S; Chevtchik, N V; Wilmer, M J; van Asbeck, A H; Croes, H J; Pertijs, J C; Wetzels, J F M; Hilbrands, L B; van den Heuvel, L P; Hoenderop, J G; Stamatialis, D; Masereeuw, R

    2015-11-16

    The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

  12. Crosslinking of membrane immunoglobulins and B-cell activation: a simple model based on percolation theory.

    PubMed

    Faro, J; Velasco, S

    1993-11-22

    In immune network models it is assumed that membrane immunoglobulin (mIg) crosslinking leads to B-cell activation. To analyse further the implications of this idea, a model of B-cell activation by ligand-induced mIg crosslinking in the absence of cell-to-cell interactions is proposed. The present model, based on a simple crosslinking mechanism previously proposed by other authors, assumes that activation of B-cells is possible once crosslinks of mIgs percolate and that percolation of crosslinks can only happen within a relatively short time tau. Given a lattice (regular or not), a molecular cluster is said to percolate or to become a percolating cluster if it spans the whole lattice (this is the case, for instance, of a polymer in a gel phase). From this model of B-cell activation we define the activation function fa (LK) as the fraction of B-cells activated after tau minutes of interaction with a ligand at concentration L and with affinity K. Numerical calculations show that, for current estimates of kinetic constants involved in the interaction of a given ligand with a B-cell clone, the activation function fa shifts when k-, the dissociation rate constant, is varied below 10(-3) s-1, this shift being linearly proportional to the variation of k-. This result contradicts and, therefore, challenges the assumption in immune network models that the activation function is identical for all ligands. This is important because the behaviour of at least some of those immune network models is quite sensitive to the relative values of the activation function thresholds.

  13. Composite fuel cell membranes

    DOEpatents

    Plowman, Keith R.; Rehg, Timothy J.; Davis, Larry W.; Carl, William P.; Cisar, Alan J.; Eastland, Charles S.

    1997-01-01

    A bilayer or trilayer composite ion exchange membrane suitable for use in a fuel cell. The composite membrane has a high equivalent weight thick layer in order to provide sufficient strength and low equivalent weight surface layers for improved electrical performance in a fuel cell. In use, the composite membrane is provided with electrode surface layers. The composite membrane can be composed of a sulfonic fluoropolymer in both core and surface layers.

  14. Composite fuel cell membranes

    DOEpatents

    Plowman, K.R.; Rehg, T.J.; Davis, L.W.; Carl, W.P.; Cisar, A.J.; Eastland, C.S.

    1997-08-05

    A bilayer or trilayer composite ion exchange membrane is described suitable for use in a fuel cell. The composite membrane has a high equivalent weight thick layer in order to provide sufficient strength and low equivalent weight surface layers for improved electrical performance in a fuel cell. In use, the composite membrane is provided with electrode surface layers. The composite membrane can be composed of a sulfonic fluoropolymer in both core and surface layers.

  15. Role of membrane oxidation in controlling the activity of human group IIa secretory phospholipase A(2) toward apoptotic lymphoma cells.

    PubMed

    Gibbons, Elizabeth; Nelson, Jennifer; Anderson, Lynn; Brewer, Kelly; Melchor, Stephanie; Judd, Allan M; Bell, John D

    2013-02-01

    The membranes of healthy lymphocytes normally resist hydrolysis by secretory phospholipase A(2). However, they become susceptible during the process of apoptosis. Previous experiments have demonstrated the importance of certain physical changes to the membrane during cell death such as a reduction in membrane lipid order and exposure of phosphatidylserine on the membrane surface. Nevertheless, those investigations also showed that at least one additional factor was required for rapid hydrolysis by the human group IIa phospholipase isozyme. This study was designed to test the possibility that oxidation of membrane lipids is the additional factor. Flow cytometry and confocal microscopy with a fluorescent probe of oxidative potential suggested that oxidation of the plasma membrane occurs during apoptosis stimulated by thapsigargin. When oxidative potential was high, the activity of human group IIa secretory phospholipase A(2) was enhanced 30- to 100-fold compared to that observed with conditions sufficient for maximal hydrolysis by other secretory phospholipase A(2) isoforms. Direct oxidation of cell membranes with either of two oxidizing agents also stimulated hydrolysis by secretory phospholipase A(2). Both oxidizers caused externalization of phosphatidylserine, but a change in lipid order did not always occur. These results demonstrated that membrane oxidation strongly stimulates human group IIa secretory phospholipase A(2) activity toward apoptotic cells. Interestingly, the change in membrane order, previously thought to be imperative for high rates of hydrolysis, was not required when membrane lipids were oxidized. Whether phosphatidylserine exposure is still necessary with oxidation remains unresolved since the two events could not be deconvoluted.

  16. Recruitment of Slp-76 to the Membrane and Glycolipid-Enriched Membrane Microdomains Replaces the Requirement for Linker for Activation of T Cells in T Cell Receptor Signaling

    PubMed Central

    Boerth, Nancy J.; Sadler, Jeffrey J.; Bauer, Daniel E.; Clements, James L.; Gheith, Shereen M.; Koretzky, Gary A.

    2000-01-01

    Two hematopoietic-specific adapters, src homology 2 domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224–244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cγ1 phosphorylation, extracellular signal–regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224–244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane. PMID:11015445

  17. Anti-Candida activity of geraniol involves disruption of cell membrane integrity and function.

    PubMed

    Sharma, Y; Khan, L A; Manzoor, N

    2016-09-01

    Candidiasis is a major problem in immunocompromised patients. Candida, an opportunistic fungal pathogen, is a major health concern today as conventional drugs are highly toxic with undesirable side effects. Their fungistatic nature is responsible for drug resistance in continuously evolving strains. Geraniol, an acyclic monoterpene alcohol, is a component of several plant essential oils. In the present study, an attempt has been made to understand the antifungal activity of geraniol at the cell membrane level in three Candida species. With an MIC of 30-130μg/mL, this natural compound was fungicidal at concentrations 2×MIC. There was complete suppression of fungal growth at MIC values (growth curves) and encouragingly geraniol is non-toxic even at the concentrations approaching 5×MIC (hemolysis assay). Exposed cells showed altered morphology, wherein the cells appeared either broken or shrivelled up (SEM studies). Significant reduction was seen in ergosterol levels at sub-MIC and glucose-induced H(+) efflux at concentrations>MIC values. Our results suggest that geraniol disrupts cell membrane integrity by interfering with ergosterol biosynthesis and inhibiting the very crucial PM-ATPase. It may hence be used in the management and treatment of both superficial and invasive candidiasis but further studies are required to elaborate its mode of action. PMID:27554866

  18. Resistance of spheroplasts and whole cells of Pseudomonas aeruginosa to bactericidal activity of various biocides: evidence of the membrane implication.

    PubMed

    Guérin-Méchin, Laurence; Leveau, Jean-Yves; Dubois-Brissonnet, Florence

    2004-01-01

    To emphasise the role of outer and inner membranes in the resistance of Pseudomonas aeruginosa to bactericidal activity of various disinfectants, spheroplasts and whole cells were compared. Spheroplasts are more sensitive than whole cells to quaternary ammonium compounds such as didecyl dimethyl ammonium bromide (DDAB) and C16-benzalkonium chloride. The outer membrane acts as a barrier to prevent these disinfectants from entering the cell. It seems to have no influence on activities of smaller molecules such as C12, C14-benzalkonium chlorides and sodium dichloroisocyanurate. For tri-sodium phosphate, the presence of outer membrane emphasized the action of the molecule. Moreover, resistance of DDAB-adapted spheroplasts to bactericidal activity of DDAB is higher than the resistance of non-adapted spheroplasts. This suggests that the inner membrane could also play a role in resistance to DDAB. PMID:15160607

  19. Dehydrated human amnion/chorion membrane regulates stem cell activity in vitro

    PubMed Central

    Massee, Michelle; Chinn, Kathryn; Lei, Jennifer; Lim, Jeremy J.; Young, Conan S.

    2015-01-01

    Abstract Human‐derived placental tissues have been shown in randomized clinical trials to be effective for healing chronic wounds, and have also demonstrated the ability to recruit stem cells to the wound site in vitro and in vivo. In this study, PURION® Processed dehydrated human amnion/chorion membrane allografts (dHACM, EpiFix®, MiMedx Group, Marietta, GA) were evaluated for their ability to alter stem cell activity in vitro. Human bone marrow mesenchymal stem cells (BM‐MSCs), adipose derived stem cells (ADSCs), and hematopoietic stem cells (HSCs) were treated with soluble extracts of dHACM tissue, and were evaluated for cellular proliferation, migration, and cytokine secretion. Stem cells were analyzed for cell number by DNA assay after 24 h, closure of an acellular zone using microscopy over 3 days, and soluble cytokine production in the medium of treated stem cells was analyzed after 3 days using a multiplex ELISA array. Treatment with soluble extracts of dHACM tissue stimulated BM‐MSCs, ADSCs, and HSCs to proliferate with a significant increase in cell number after 24 h. dHACM treatment accelerated closure of an acellular zone by ADSCs and BM‐MSCs after 3 days, compared to basal medium. BM‐MSCs, ADSCs, and HSCs also modulated endogenous production of a number of various soluble signals, including regulators of inflammation, mitogenesis, and wound healing. dHACM treatment promoted increased proliferation and migration of ADSCs, BM‐MSCs, and HSCs, along with modulation of secreted proteins from those cells. Therefore, dHACM may impact wound healing by amplifying host stem cell populations and modulating their responses in treated wound tissues. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1495–1503, 2016. PMID:26175122

  20. Dehydrated human amnion/chorion membrane regulates stem cell activity in vitro.

    PubMed

    Massee, Michelle; Chinn, Kathryn; Lei, Jennifer; Lim, Jeremy J; Young, Conan S; Koob, Thomas J

    2016-10-01

    Human-derived placental tissues have been shown in randomized clinical trials to be effective for healing chronic wounds, and have also demonstrated the ability to recruit stem cells to the wound site in vitro and in vivo. In this study, PURION(®) Processed dehydrated human amnion/chorion membrane allografts (dHACM, EpiFix(®) , MiMedx Group, Marietta, GA) were evaluated for their ability to alter stem cell activity in vitro. Human bone marrow mesenchymal stem cells (BM-MSCs), adipose derived stem cells (ADSCs), and hematopoietic stem cells (HSCs) were treated with soluble extracts of dHACM tissue, and were evaluated for cellular proliferation, migration, and cytokine secretion. Stem cells were analyzed for cell number by DNA assay after 24 h, closure of an acellular zone using microscopy over 3 days, and soluble cytokine production in the medium of treated stem cells was analyzed after 3 days using a multiplex ELISA array. Treatment with soluble extracts of dHACM tissue stimulated BM-MSCs, ADSCs, and HSCs to proliferate with a significant increase in cell number after 24 h. dHACM treatment accelerated closure of an acellular zone by ADSCs and BM-MSCs after 3 days, compared to basal medium. BM-MSCs, ADSCs, and HSCs also modulated endogenous production of a number of various soluble signals, including regulators of inflammation, mitogenesis, and wound healing. dHACM treatment promoted increased proliferation and migration of ADSCs, BM-MSCs, and HSCs, along with modulation of secreted proteins from those cells. Therefore, dHACM may impact wound healing by amplifying host stem cell populations and modulating their responses in treated wound tissues. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1495-1503, 2016.

  1. Modeling Electrically Active Viscoelastic Membranes

    PubMed Central

    Roy, Sitikantha; Brownell, William E.; Spector, Alexander A.

    2012-01-01

    The membrane protein prestin is native to the cochlear outer hair cell that is crucial to the ear's amplification and frequency selectivity throughout the whole acoustic frequency range. The outer hair cell exhibits interrelated dimensional changes, force generation, and electric charge transfer. Cells transfected with prestin acquire unique active properties similar to those in the native cell that have also been useful in understanding the process. Here we propose a model describing the major electromechanical features of such active membranes. The model derived from thermodynamic principles is in the form of integral relationships between the history of voltage and membrane resultants as independent variables and the charge density and strains as dependent variables. The proposed model is applied to the analysis of an active force produced by the outer hair cell in response to a harmonic electric field. Our analysis reveals the mechanism of the outer hair cell active (isometric) force having an almost constant amplitude and phase up to 80 kHz. We found that the frequency-invariance of the force is a result of interplay between the electrical filtering associated with prestin and power law viscoelasticity of the surrounding membrane. Paradoxically, the membrane viscoelasticity boosts the force balancing the electrical filtering effect. We also consider various modes of electromechanical coupling in membrane with prestin associated with mechanical perturbations in the cell. We consider pressure or strains applied step-wise or at a constant rate and compute the time course of the resulting electric charge. The results obtained here are important for the analysis of electromechanical properties of membranes, cells, and biological materials as well as for a better understanding of the mechanism of hearing and the role of the protein prestin in this mechanism. PMID:22701528

  2. Membrane Transfer from Mononuclear Cells to Polymorphonuclear Neutrophils Transduces Cell Survival and Activation Signals in the Recipient Cells via Anti-Extrinsic Apoptotic and MAP Kinase Signaling Pathways

    PubMed Central

    Li, Ko-Jen; Wu, Cheng-Han; Shen, Chieh-Yu; Kuo, Yu-Min; Yu, Chia-Li; Hsieh, Song-Chou

    2016-01-01

    The biological significance of membrane transfer (trogocytosis) between polymorphonuclear neutrophils (PMNs) and mononuclear cells (MNCs) remains unclear. We investigated the biological/immunological effects and molecular basis of trogocytosis among various immune cells in healthy individuals and patients with active systemic lupus erythematosus (SLE). By flow cytometry, we determined that molecules in the immunological synapse, including HLA class-I and-II, CD11b and LFA-1, along with CXCR1, are exchanged among autologous PMNs, CD4+ T cells, and U937 cells (monocytes) after cell-cell contact. Small interfering RNA knockdown of the integrin adhesion molecule CD11a in U937 unexpectedly enhanced the level of total membrane transfer from U937 to PMN cells. Functionally, phagocytosis and IL-8 production by PMNs were enhanced after co-culture with T cells. Total membrane transfer from CD4+ T to PMNs delayed PMN apoptosis by suppressing the extrinsic apoptotic molecules, BAX, MYC and caspase 8. This enhancement of activities of PMNs by T cells was found to be mediated via p38- and P44/42-Akt-MAP kinase pathways and inhibited by the actin-polymerization inhibitor, latrunculin B, the clathrin inhibitor, Pitstop-2, and human immunoglobulin G, but not by the caveolin inhibitor, methyl-β-cyclodextrin. In addition, membrane transfer from PMNs enhanced IL-2 production by recipient anti-CD3/anti-CD28 activated MNCs, and this was suppressed by inhibitors of mitogen-activated protein kinase (PD98059) and protein kinase C (Rottlerin). Of clinical significance, decreased total membrane transfer from PMNs to MNCs in patients with active SLE suppressed mononuclear IL-2 production. In conclusion, membrane transfer from MNCs to PMNs, mainly at the immunological synapse, transduces survival and activation signals to enhance PMN functions and is dependent on actin polymerization, clathrin activation, and Fcγ receptors, while membrane transfer from PMNs to MNCs depends on MAP kinase and

  3. A 17-mer Membrane-Active MSI-78 Derivative with Improved Selectivity toward Bacterial Cells.

    PubMed

    Monteiro, Claudia; Pinheiro, Marina; Fernandes, Mariana; Maia, Sílvia; Seabra, Catarina L; Ferreira-da-Silva, Frederico; Reis, Salette; Gomes, Paula; Martins, M Cristina L

    2015-08-01

    Antimicrobial peptides are widely recognized as an excellent alternative to conventional antibiotics. MSI-78, a highly effective and broad spectrum AMP, is one of the most promising AMPs for clinical application. In this study, we have designed shorter derivatives of MSI-78 with the aim of improving selectivity while maintaining antimicrobial activity. Shorter 17-mer derivatives were created by truncating MSI-78 at the N- and/or C-termini, while spanning MSI-78 sequence. Despite the truncations made, we found a 17-mer peptide, MSI-78(4-20) (KFLKKAKKFGKAFVKIL), which was demonstrated to be as effective as MSI-78 against the Gram-positive Staphylococcus strains tested and the Gram-negative Pseudomonas aeruginosa. This shorter derivative is more selective toward bacterial cells as it was less toxic to erythrocytes than MSI-78, representing an improved version of the lead peptide. Biophysical studies support a mechanism of action for MSI-78(4-20) based on the disruption of the bacterial membrane permeability barrier, which in turn leads to loss of membrane integrity and ultimately to cell death. These features point to a mechanism of action similar to the one described for the lead peptide MSI-78. PMID:26066462

  4. A 17-mer Membrane-Active MSI-78 Derivative with Improved Selectivity toward Bacterial Cells.

    PubMed

    Monteiro, Claudia; Pinheiro, Marina; Fernandes, Mariana; Maia, Sílvia; Seabra, Catarina L; Ferreira-da-Silva, Frederico; Reis, Salette; Gomes, Paula; Martins, M Cristina L

    2015-08-01

    Antimicrobial peptides are widely recognized as an excellent alternative to conventional antibiotics. MSI-78, a highly effective and broad spectrum AMP, is one of the most promising AMPs for clinical application. In this study, we have designed shorter derivatives of MSI-78 with the aim of improving selectivity while maintaining antimicrobial activity. Shorter 17-mer derivatives were created by truncating MSI-78 at the N- and/or C-termini, while spanning MSI-78 sequence. Despite the truncations made, we found a 17-mer peptide, MSI-78(4-20) (KFLKKAKKFGKAFVKIL), which was demonstrated to be as effective as MSI-78 against the Gram-positive Staphylococcus strains tested and the Gram-negative Pseudomonas aeruginosa. This shorter derivative is more selective toward bacterial cells as it was less toxic to erythrocytes than MSI-78, representing an improved version of the lead peptide. Biophysical studies support a mechanism of action for MSI-78(4-20) based on the disruption of the bacterial membrane permeability barrier, which in turn leads to loss of membrane integrity and ultimately to cell death. These features point to a mechanism of action similar to the one described for the lead peptide MSI-78.

  5. Membrane in cancer cells

    SciTech Connect

    Galeotti, T.; Cittadini, A.; Neri, G.; Scarpa, A.

    1988-01-01

    This book contains papers presented at a conference on membranes in cancer cells. Topics covered include Oncogenies, hormones, and free-radical processes in malignant transformation in vitro and Superoxide onion may trigger DNA strand breaks in human granulorytes by acting as a membrane target.

  6. Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations

    PubMed Central

    Jansen, J.; De Napoli, I. E; Fedecostante, M.; Schophuizen, C. M. S.; Chevtchik, N. V.; Wilmer, M. J.; van Asbeck, A. H.; Croes, H. J.; Pertijs, J. C.; Wetzels, J. F. M.; Hilbrands, L. B.; van den Heuvel, L. P.; Hoenderop, J. G.; Stamatialis, D.; Masereeuw, R.

    2015-01-01

    The bioartificial kidney (BAK) aims at improving dialysis by developing ‘living membranes’ for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as a fluorescent substrate. Initial ASP+ uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a ‘living membrane’ of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering. PMID:26567716

  7. Microbial products activate monocytic cells through detergent-resistant membrane microdomains.

    PubMed

    Epelman, Slava; Berenger, Byron; Stack, Danuta; Neely, Graham G; Ma, Ling Ling; Mody, Christopher H

    2008-12-01

    Patients with cystic fibrosis suffer recurrent pulmonary infections that are characterized by an overactive yet ineffective and destructive inflammatory response that is associated with respiratory infections by Pseudomonas aeruginosa, a pathogen that produces a number of phlogistic molecules. To better understand this process, we used exoenzyme S (ExoS), one of the key P. aeruginosa-secreted exoproducts, which is known to stimulate cells via the Toll-like receptor (TLR) pathway. We found that ExoS induced proinflammatory cytokine production via the NF-kappaB, Erk1/2, and Src kinase pathways. Because Src kinases are concentrated within cholesterol-containing, detergent-resistant membrane microdomains (DRM) (also called lipid rafts) and DRM act as signaling platforms and amplifiers on the surface of cells, we addressed the role of DRM in ExoS signaling. ExoS bound directly to a subset of DRM and induced the phosphorylation of multiple proteins within DRM, including Src kinases. Disruption of DRM by cholesterol extraction prevented NF-kappaB and Erk 1/2 activation and TNF-alpha production in response to ExoS. Activation of monocytic cells by other TLR and Nod-like receptor agonists, such as lipoteichoic acid, lipopolysaccharide, and peptidoglycan, were also dependent on DRM, and disruption prevented TNF-alpha production. Disruption of DRM did not prevent ExoS binding but did release the Src kinase, Lyn, from the DRM fraction into the detergent-soluble fraction, a site in which Src kinases are not active. These studies show that ExoS, a TLR agonist, requires direct binding to DRM for optimal signaling, which suggests that DRM are possible therapeutic targets in cystic fibrosis.

  8. Antimicrobial activity of syringic acid against Cronobacter sakazakii and its effect on cell membrane.

    PubMed

    Shi, Chao; Sun, Yi; Zheng, Zhiwei; Zhang, Xiaorong; Song, Kaikuo; Jia, Zhenyu; Chen, Yifei; Yang, Miaochun; Liu, Xin; Dong, Rui; Xia, Xiaodong

    2016-04-15

    Syringic acid (SA) has been reported to exhibit antibacterial ability against various microorganisms, but little work has been done on its effect on Cronobacter sakazakii. In this study, minimum inhibitory concentrations (MICs) of SA against various C. sakazakii strains were determined. Moreover, changes in intracellular ATP concentration, intracellular pH (pHin), membrane potential and membrane integrity were measured to evaluate the influence of SA on cell membrane. Finally, field emission scanning electron microscope (FESEM) was used to assess the morphological changes of bacterial cells caused by SA. It was shown that the MICs of SA against all tested C. sakazakii strains were 5mg/mL. SA retarded bacterial growth, and caused cell membrane dysfunction, which was evidenced by intracellular ATP concentration decrease, pHin reduction, cell membrane hyperpolarization and changes in cellular morphology. These findings indicated that SA has potential to be developed as a natural preservative to control C. sakazakii in foods associated with this pathogen and prevent related infections.

  9. Membrane Cells for Brine Electrolysis.

    ERIC Educational Resources Information Center

    Tingle, M.

    1982-01-01

    Membrane cells were developed as alternatives to mercury and diaphragm cells for the electrolysis of brine. Compares the three types of cells, focusing on the advantages and disadvantages of membrane cells. (JN)

  10. Incorporation of Photosynthetic Reaction Centers in the Membrane of Human Cells: Toward a New Tool for Optical Control of Cell Activity

    SciTech Connect

    Pennisi, Cristian P.; Jensen, Poul Erik; Zachar, Vladimir; Greenbaum, Elias; Yoshida, Ken

    2009-01-01

    The Photosystem I (PSI) reaction center is a photosynthetic membrane complex in which light-induced charge separation is accompanied by the generation of an electric potential. It has been recently proposed as a means to confer light sensitivity to cells possessing voltage-activated ion channels, but the feasibility of heterologous incorporation has not been demonstrated. In this work, methods of delivery and detection of PSI in the membrane of human cells are presented. Purified fractions of PSI were reconstituted in proteoliposomes that were used as vehicles for the membrane incorporation. A fluorescent impermeable dye was entrapped in the vesicles to qualitatively analyze the nature of the vesicle cell interaction. After incorporation, the localization and orientation of the complexes in the membrane was studied using immuno-fluorescence microscopy. The results showed complexes oriented as in native membranes, which were randomly distributed in clusters over the entire surface of the cell. Additionally, analysis of cell viability showed that the incorporation process does not damage the cell membrane. Taken together, the results of this work suggest that the mammalian cellular membrane is a reasonable environment for the incorporation of PSI complexes, which opens the possibility of using these molecular photovoltaic structures for optical control of cell activity.

  11. Electrostatics of cell membrane recognition: structure and activity of neutral and cationic rigid push-pull rods in isoelectric, anionic, and polarized lipid bilayer membranes.

    PubMed

    Sakai, N; Gerard, D; Matile, S

    2001-03-21

    Design, synthesis, and structural and functional studies of rigid-rod ionophores of different axial electrostatic asymmetry are reported. The employed design strategy emphasized presence of (a) a rigid scaffold to minimize the conformational complexity, (b) a unimolecular ion-conducting pathway to minimize the suprastructural complexity and monitor the function, (c) an extended fluorophore to monitor structure, (d) variable axial rod dipole, and (e) variable terminal charges to create axial asymmetry. Studies in isoelectric, anionic, and polarized bilayer membranes confirmed a general increase in activity of uncharged rigid push-pull rods in polarized bilayers. The similarly increased activity of cationic rigid push-pull rods with an electrostatic asymmetry comparable to that of alpha-helical bee toxin melittin (positive charge near negative axial dipole terminus) is shown by fluorescence-depth quenching experiments to originate from the stabilization of transmembrane rod orientation by the membrane potential. The reduced activity of rigid push-pull rods having an electrostatic asymmetry comparable to that in alpha-helical natural antibiotics (a positive charge near the positive axial dipole terminus) is shown by structural studies to originate from rod "ejection" by membrane potentials comparable to that found in mammalian plasma membranes. This structural evidence for cell membrane recognition by asymmetric rods is unprecedented and of possible practical importance with regard to antibiotic resistance.

  12. Volumetric deformation of live cells induced by pressure-activated cross-membrane ion transport.

    PubMed

    Hui, T H; Zhou, Z L; Qian, J; Lin, Y; Ngan, A H W; Gao, H

    2014-09-12

    In this work, we developed a method that allows precise control over changes in the size of a cell via hydrostatic pressure changes in the medium. Specifically, we show that a sudden increase, or reduction, in the surrounding pressure, in the physiologically relevant range, triggers cross-membrane fluxes of sodium and potassium ions in leukemia cell lines K562 and HL60, resulting in reversible volumetric deformation with a characteristic time of around 30 min. Interestingly, healthy leukocytes do not respond to pressure shocks, suggesting that the cancer cells may have evolved the ability to adapt to pressure changes in their microenvironment. A model is also proposed to explain the observed cell deformation, which highlights how the apparent viscoelastic response of cells is governed by the microscopic cross-membrane transport.

  13. Volumetric Deformation of Live Cells Induced by Pressure-Activated Cross-Membrane Ion Transport

    NASA Astrophysics Data System (ADS)

    Hui, T. H.; Zhou, Z. L.; Qian, J.; Lin, Y.; Ngan, A. H. W.; Gao, H.

    2014-09-01

    In this work, we developed a method that allows precise control over changes in the size of a cell via hydrostatic pressure changes in the medium. Specifically, we show that a sudden increase, or reduction, in the surrounding pressure, in the physiologically relevant range, triggers cross-membrane fluxes of sodium and potassium ions in leukemia cell lines K562 and HL60, resulting in reversible volumetric deformation with a characteristic time of around 30 min. Interestingly, healthy leukocytes do not respond to pressure shocks, suggesting that the cancer cells may have evolved the ability to adapt to pressure changes in their microenvironment. A model is also proposed to explain the observed cell deformation, which highlights how the apparent viscoelastic response of cells is governed by the microscopic cross-membrane transport.

  14. Fast stack activation procedure and effective long-term storage for high-performance polymer electrolyte membrane fuel cell

    NASA Astrophysics Data System (ADS)

    Yang, Seung Yong; Seo, Dong-Jun; Kim, Myeong-Ri; Seo, Min Ho; Hwang, Sun-Mi; Jung, Yong-Min; Kim, Beom-Jun; Yoon, Young-Gi; Han, Byungchan; Kim, Tae-Young

    2016-10-01

    Time-saving stack activation and effective long-term storage are one of most important issues that must be resolved for the commercialization of polymer electrolyte membrane fuel cell (PEMFC). Herein, we developed the cost-effective stack activation method to finish the whole activation within 30 min and the long-term storage method by using humidified N2 without any significant decrease in cell's performance for 30 days. Specifically, the pre-activation step with the direct injection of DI water into the stack and storage at 65 or 80 °C for 2 h increases the distinctive phase separation between the hydrophobic and hydrophilic regions in Nafion membrane, which significantly reduces the total activation time within 30 min. Additionally, the long-term storage with humidified N2 has no effect on the Pt oxidation and drying of Nafion membrane for 30 days due to its exergonic reaction in the cell. As a result, the high water content in Nafion membrane and the decrease of Pt oxidation are the critical factors that have a strong influence on the activation and long-term storage for high-performance PEMFC.

  15. Fuel cell membrane humidification

    DOEpatents

    Wilson, Mahlon S.

    1999-01-01

    A polymer electrolyte membrane fuel cell assembly has an anode side and a cathode side separated by the membrane and generating electrical current by electrochemical reactions between a fuel gas and an oxidant. The anode side comprises a hydrophobic gas diffusion backing contacting one side of the membrane and having hydrophilic areas therein for providing liquid water directly to the one side of the membrane through the hydrophilic areas of the gas diffusion backing. In a preferred embodiment, the hydrophilic areas of the gas diffusion backing are formed by sewing a hydrophilic thread through the backing. Liquid water is distributed over the gas diffusion backing in distribution channels that are separate from the fuel distribution channels.

  16. Corrugated Membrane Fuel Cell Structures

    SciTech Connect

    Grot, Stephen

    2013-09-30

    One of the most challenging aspects of traditional PEM fuel cell stacks is the difficulty achieving the platinum catalyst utilization target of 0.2 gPt/kWe set forth by the DOE. Good catalyst utilization can be achieved with state-of-the-art catalyst coated membranes (CCM) when low catalyst loadings (<0.3 mg/cm2) are used at a low current. However, when low platinum loadings are used, the peak power density is lower than conventional loadings, requiring a larger total active area and a larger bipolar plate. This results in a lower overall stack power density not meeting the DOE target. By corrugating the fuel cell membrane electrode structure, Ion Power?s goal is to realize both the Pt utilization targets as well as the power density targets of the DOE. This will be achieved by demonstrating a fuel cell single cell (50 cm2) with a twofold increase in the membrane active area over the geometric area of the cell by corrugating the MEA structure. The corrugating structure must be able to demonstrate the target properties of < 10 mOhm-cm2 electrical resistance at > 20 psi compressive strength over the active area, in combination with offering at least 80% of power density that can be achieved by using the same MEA in a flat plate structure. Corrugated membrane fuel cell structures also have the potential to meet DOE power density targets by essentially packaging more membrane area into the same fuel cell volume as compared to conventional stack constructions.

  17. HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death

    PubMed Central

    Jacobsen, A V; Lowes, K N; Tanzer, M C; Lucet, I S; Hildebrand, J M; Petrie, E J; van Delft, M F; Liu, Z; Conos, S A; Zhang, J-G; Huang, D C S; Silke, J; Lessene, G; Murphy, J M

    2016-01-01

    Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3. PMID:26775703

  18. Minimum structural requirements for cell membrane leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s.

    PubMed

    Fujii, Kana; Morita, Daichi; Onoda, Kenji; Kuroda, Teruo; Miyachi, Hiroyuki

    2016-05-01

    Macrocyclic bis(bibenzyl)-type phenolic natural products, found exclusively in bryophytes, exhibit potent antibacterial activity towards methicillin-resistant Staphylococcus aureus (anti-MRSA activity). Here, in order to identify the minimum essential structure for cell membrane leakage-mediated anti-MRSA activity of these compounds, we synthesized acyclic fragment structures and evaluated their anti-MRSA activity. The activities of all of the acyclic fragments tested exhibited similar characteristics to those of the macrocycles, i.e., anti-MRSA bactericidal activity, an enhancing effect on influx and efflux of ethidium bromide (EtBr: fluorescent DNA-binder) in Staphylococcus aureus cells, and bactericidal activity towards a Staphylococcus aureus strain resistant to 2-phenoxyphenol (4). The latter result suggests that they have a different mechanism of action from 4, which is a FabI inhibitor previously proposed to be the minimum active fragment of riccardin-type macrocycles. Thus, cyclic structure is not a necessary condition for cell membrane leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s. PMID:26995530

  19. Clostridium sordellii Lethal-Toxin Autoprocessing and Membrane Localization Activities Drive GTPase Glucosylation Profiles in Endothelial Cells

    PubMed Central

    Craven, Ryan

    2015-01-01

    ABSTRACT Clostridium sordellii infections cause gangrene and edema in humans and gastrointestinal infections in livestock. One of the principle virulence factors is TcsL, a large protein toxin which glucosylates host GTPases to cause cytopathic and cytotoxic effects. TcsL has two enzymatic domains, an N-terminal glucosyltransferase domain (GTD) and an autoprocessing domain responsible for release of the GTD within the cell. The GTD can then use its N-terminal membrane localization domain (MLD) for orientation on membranes and modification of GTPases. This study describes the use of conditionally immortalized murine pulmonary microvascular endothelial cells as a model for the study of TcsL functional activities. Point mutations that disrupt the glucosyltransferase, autoprocessing, or membrane localization activities were introduced into a recombinant version of TcsL, and the activities of these mutants were compared to those of wild-type toxin. We observed that all mutants are defective or impaired in cytotoxicity but differ in their modification of Rac1 and Ras. The data suggest a model where differences in GTPase localization dictate cellular responses to intoxication and highlight the importance of autoprocessing in the function of TcsL. IMPORTANCE Clostridium sordellii is a bacterium that can infect humans and cause serious disease and death. The principle virulence factor associated with clinical symptoms is a large protein toxin known as lethal toxin. The mechanism of lethal-toxin intoxication is assumed to be similar to that of the homologous toxins from C. difficile, but very few studies have been done in the context of endothelial cells, a relevant target in C. sordellii infections. This study was designed to test the role of the lethal-toxin enzymatic activities and membrane localization in endothelial cell toxicity and host substrate modification. PMID:27303685

  20. How Membrane-Active Peptides Get into Lipid Membranes.

    PubMed

    Sani, Marc-Antoine; Separovic, Frances

    2016-06-21

    The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular

  1. How Membrane-Active Peptides Get into Lipid Membranes.

    PubMed

    Sani, Marc-Antoine; Separovic, Frances

    2016-06-21

    The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular

  2. Activation of tumour cell ECM degradation by thrombin-activated platelet membranes: potentially a P-selectin and GPIIb/IIIa-dependent process.

    PubMed

    Pang, J H; Coupland, L A; Freeman, C; Chong, B H; Parish, Christopher R

    2015-06-01

    The promotion of tumour metastasis by platelets may occur through several mechanisms including the induction of a more metastatic phenotype in tumour cells and assisted extravasation of circulating tumour cells. Whilst the mechanisms underlying platelet-assisted extravasation have been extensively studied, much less attention has been paid to the mechanisms underlying platelet promotion of an aggressive phenotype within a tumour cell population. Herein, we demonstrate in vitro that MDA-MB-231 breast carcinoma cells incubated with washed thrombin-activated platelet membranes adopt a Matrigel-degrading phenotype in a dose- and contact time-dependent manner. The same phenotypic change was observed with three other human tumour cell lines of diverse anatomical origin. Moreover, tumour cell lines that had been cultured with washed thrombin-activated platelet membranes had a greater metastatic capacity when injected into mice. This in vivo effect was reliant upon a co-incubation period of >2 h implying a mechanism involving more than platelet membrane binding that occurred within 5 min. Upon further investigation it was found that simultaneous blocking of the platelet-membrane proteins P-selectin and GPIIb/IIIa prevented interactions between platelet membranes and MDA-MB-231 cells but also significantly reduced the ability of tumour cells to degrade Matrigel. These results confirm that platelets induce a more aggressive phenotype in tumour cells but also identify the platelet proteins involved in this effect. P-selectin and GPIIb/IIIa also play a role in assisting tumour cell extravasation and, thus, are ideal targets for the therapeutic intervention of both stages of platelet-assisted metastasis.

  3. The First Cell Membranes

    NASA Technical Reports Server (NTRS)

    Deamer, David; Dworkin, Jason P.; Sandford, Scott A.; Bernstein, Max P.; Allamandola, Louis J.

    2004-01-01

    Organic compounds are synthesized in the interstellar medium and can be delivered to planetary surfaces such as the early Earth, where they mix with endogenous organic mixtures. Some of these compounds are amphiphilic, having polar and non-polar groups on the same molecule. Amphiphilic compounds spontaneously self-assembly into more complex structures such as bimolecular layers, which in turn form closed membranous vesicles. The first forms of cellular life required self-assembled membranes that were likely to be available on the prebiotic Earth. Laboratory simulations show that such vesicles readily encapsulate functional macromolecules, including nucleic acids and polymerases. A goal of future investigations is to fabricate artificial cells as models of the origin of life.

  4. Membrane-active peptides from marine organisms--antimicrobials, cell-penetrating peptides and peptide toxins: applications and prospects.

    PubMed

    Ponnappan, Nisha; Budagavi, Deepthi Poornima; Yadav, Bhoopesh Kumar; Chugh, Archana

    2015-03-01

    Marine organisms are known to be a rich and unique source of bioactive compounds as they are exposed to extreme conditions in the oceans. The present study is an attempt to briefly describe some of the important membrane-active peptides (MAPs) such as antimicrobial peptides (AMPs), cell-penetrating peptides (CPPs) and peptide toxins from marine organisms. Since both AMPs and CPPs play a role in membrane perturbation and exhibit interchangeable role, they can speculatively fall under the broad umbrella of MAPs. The study focuses on the structural and functional characteristics of different classes of marine MAPs. Further, AMPs are considered as a potential remedy to antibiotic resistance acquired by several pathogens. Peptides from marine organisms show novel post-translational modifications such as cysteine knots, halogenation and histidino-alanine bridge that enable these peptides to withstand harsh marine environmental conditions. These unusual modifications of AMPs from marine organisms are expected to increase their half-life in living systems, contributing to their increased bioavailability and stability when administered as drug in in vivo systems. Apart from AMPs, marine toxins with membrane-perturbing properties could be essentially investigated for their cytotoxic effect on various pathogens and their cell-penetrating activity across various mammalian cells. The current review will help in identifying the MAPs from marine organisms with crucial post-translational modifications that can be used as template for designing novel therapeutic agents and drug-delivery vehicles for treatment of human diseases.

  5. Effects of DO levels on surface force, cell membrane properties and microbial community dynamics of activated sludge.

    PubMed

    Ma, Si-Jia; Ding, Li-Li; Huang, Hui; Geng, Jin-Ju; Xu, Ke; Zhang, Yan; Ren, Hong-Qiang

    2016-08-01

    In this paper, we employ atomic force microscopy (AFM), fluorescence recovery after photobleaching (FRAP) technique, phospholipid fatty acids (PLFA) and MiSeq analysis to study the effects of traditional dissolved oxygen (DO) levels (0.71-1.32mg/L, 2.13-3.02mg/L and 4.31-5.16mg/L) on surface force, cell membrane properties and microbial community dynamics of activated sludge. Results showed that low DO level enhanced the surface force and roughness of activated sludge; the medium DO level decreased cell membrane fluidity by reducing the synthesis of branched fatty acids in the cell membrane; high DO level resulted in the highest protein content in the effluent by EEM scanning. Abundance of Micropruina, Zoogloea and Nakamurella increased and Paracoccus and Rudaea decreased with the increase of DO levels. RDA analysis suggested that saturated fatty acids (SFA), anteiso-fatty acids (AFA) and iso-fatty acids (IFA) were closely related to effluent quality as well as some genera. PMID:27187569

  6. Cell Membrane Softening in Cancer Cells

    NASA Astrophysics Data System (ADS)

    Schmidt, Sebastian; Händel, Chris; Käs, Josef

    Biomechanical properties are useful characteristics and regulators of the cell's state. Current research connects mechanical properties of the cytoskeleton to many cellular processes but does not investigate the biomechanics of the plasma membrane. We evaluated thermal fluctuations of giant plasma membrane vesicles, directly derived from the plasma membranes of primary breast and cervical cells and observed a lowered rigidity in the plasma membrane of malignant cells compared to non-malignant cells. To investigate the specific role of membrane rigidity changes, we treated two cell lines with the Acetyl-CoA carboxylase inhibitor Soraphen A. It changed the lipidome of cells and drastically increased membrane stiffness by up regulating short chained membrane lipids. These altered cells had a decreased motility in Boyden chamber assays. Our results indicate that the thermal fluctuations of the membrane, which are much smaller than the fluctuations driven by the cytoskeleton, can be modulated by the cell and have an impact on adhesion and motility.

  7. Diabetes-induced activation of system y+ and nitric oxide synthase in human endothelial cells: association with membrane hyperpolarization.

    PubMed Central

    Sobrevia, L; Cesare, P; Yudilevich, D L; Mann, G E

    1995-01-01

    1. The activity of the human endothelial cell L-arginine transporter (system y+) has been correlated with cGMP production (index of nitric oxide) and prostacyclin (PGI2) release in umbilical vein endothelial cells cultured from normal or gestational diabetic pregnancies. 2. In non-diabetic and diabetic cells, transport of L-arginine was Na+ and pH independent, inhibited by other cationic L-arginine analogues and unaffected by neutral amino acids. 3. Diabetes was associated with an increased Vmax for saturable L-arginine transport (4.6 +/- 0.13 vs. 9.9 +/- 0.5 pmol (microgram protein)-1 min-1, P < 0.01), but had no effect on initial rates of transport for L-serine, L-citrulline, L-leucine or 2-deoxyglucose. 4. In non-diabetic and diabetic cells, elevated K+ resulted in a concentration-dependent inhibition in the initial rates of transport for L-arginine and the membrane potential-sensitive probe tetra[3H]phenylphosphonium (TPP+). 5. When resting membrane potential was measured using the whole-cell patch voltage clamp technique, diabetic cells were hyperpolarized (-78 +/- 0.3 mV) compared with non-diabetic cells (-70 +/- 0.04 mV, P < 0.04). Accumulation of [3H]TPP+ was also increased in diabetic compared with non-diabetic cells. 6. Basal intracellular cGMP levels were elevated 2.5-fold in diabetic cells, and L-NAME (100 microM), an inhibitor of nitric oxide synthase, abolished basal cGMP accumulation in non-diabetic and diabetic cells. 7. Histamine (10 microM) had no effect on L-arginine transport but evoked significant increases in cGMP in non-diabetic and diabetic cells, which were completely inhibited by L-NAME but unaffected by superoxide dismutase. 8. Basal and histamine-stimulated PGI2 release was decreased markedly in diabetic cells. 9. Our findings demonstrate that gestational diabetes is associated with phenotypic changes in fetal endothelial cells, which result in a membrane hyperpolarization, activation of the human endothelial cell L-arginine transporter

  8. A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles.

    PubMed

    Patrick, S; McKenna, J P; O'Hagan, S; Dermott, E

    1996-04-01

    The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacterium Bacteroides fragilis were examined. Outer membrane vesicles are released from the surface of B. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide of B. fragilis confirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations of B. fragilis strains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL); non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence of B. fragilis. PMID:8737489

  9. Computationally efficient simulation of electrical activity at cell membranes interacting with self-generated and externally imposed electric fields

    NASA Astrophysics Data System (ADS)

    Agudelo-Toro, Andres; Neef, Andreas

    2013-04-01

    Objective. We present a computational method that implements a reduced set of Maxwell's equations to allow simulation of cells under realistic conditions: sub-micron cell morphology, a conductive non-homogeneous space and various ion channel properties and distributions. Approach. While a reduced set of Maxwell's equations can be used to couple membrane currents to extra- and intracellular potentials, this approach is rarely taken, most likely because adequate computational tools are missing. By using these equations, and introducing an implicit solver, numerical stability is attained even with large time steps. The time steps are limited only by the time development of the membrane potentials. Main results. This method allows simulation times of tens of minutes instead of weeks, even for complex problems. The extracellular fields are accurately represented, including secondary fields, which originate at inhomogeneities of the extracellular space and can reach several millivolts. We present a set of instructive examples that show how this method can be used to obtain reference solutions for problems, which might not be accurately captured by the traditional approaches. This includes the simulation of realistic magnitudes of extracellular action potential signals in restricted extracellular space. Significance. The electric activity of neurons creates extracellular potentials. Recent findings show that these endogenous fields act back onto the neurons, contributing to the synchronization of population activity. The influence of endogenous fields is also relevant for understanding therapeutic approaches such as transcranial direct current, transcranial magnetic and deep brain stimulation. The mutual interaction between fields and membrane currents is not captured by today's concepts of cellular electrophysiology, including the commonly used activation function, as those concepts are based on isolated membranes in an infinite, isopotential extracellular space. The

  10. Binding and biologic activity of glucagon in liver cell membranes of chronically hyperglucagonemic rats.

    PubMed

    Srikant, C B; Freeman, D; McCorkle, K; Unger, R H

    1977-11-10

    Glucagon binding by liver cell membranes was examined in rats with chronically elevated plasma levels of immunoreactive glucagon (IRG) resulting from insulin deficiency, starvation, or twice daily glucagon injections. The concentration of specific glucagon binding sites was significantly reduced in the three chronically hyperglucagonemic (IRG greater than 125 pg/ml) groups as compared with nondiabetic controls and insulin-treated diabetic control rats with only mild hyperglucagonemia. A reduction in glucagon binding sites did not occur with hyperglucagonemia of 12 h or less. Despite the reduced binding of glucagon in the three chronically hyperglucagonemic groups, the ability of glucagon to stimulate cAMP production was not reduced. It is concluded that while decreased glucagon binding occures in the forms of chronic hyperglucagonemia studied, it is not associated with a reduction in the ability of glucagon to stimulate cAMP production.

  11. NK Cells Respond to Haptens by the Activation of Calcium Permeable Plasma Membrane Channels

    PubMed Central

    Grandclément, Camille; Pick, Horst; Vogel, Horst; Held, Werner

    2016-01-01

    Natural Killer (NK) cells mediate innate immunity to infected and transformed cells. Yet, NK cells can also mount hapten-specific recall responses thereby contributing to contact hypersensitivity (CHS). However, since NK cells lack antigen receptors that are used by the adaptive immune system to recognize haptens, it is not clear if NK cells respond directly to haptens and, if so, what mediates these responses. Here we show that among four haptens the two that are known to induce NK cell-dependent CHS trigger the rapid influx of extracellular Ca2+ into NK cells and lymphocyte cell lines. Thus lymphocytes can respond to haptens independent of antigen presentation and antigen receptors. We identify the Ca2+-permeable cation channel TRPC3 as a component of the lymphocyte response to one of these haptens. These data suggest that the response to the second hapten is based on a distinct mechanism, consistent with the capacity of NK cells to discriminate haptens. These findings raise the possibility that antigen-receptor independent activation of immune cells contributes to CHS. PMID:26963818

  12. Viola tricolor Induces Apoptosis in Cancer Cells and Exhibits Antiangiogenic Activity on Chicken Chorioallantoic Membrane

    PubMed Central

    Sadeghnia, Hamid Reza; Ghorbani Hesari, Taghi; Mortazavian, Seyed Mohsen; Mousavi, Seyed Hadi; Tayarani-Najaran, Zahra

    2014-01-01

    In the present study, the cytotoxic and apoptogenic properties of hydroalcoholic extract and ethyl acetate (EtOAc), n-butanol, and water fractions (0–800 μg/mL) of Viola tricolor were investigated in Neuro2a mouse neuroblastoma and MCF-7 human breast cancer cells. In addition, antiangiogenic effect of EtOAc fraction was evaluated on chicken chorioallantoic membrane (CAM). The quality of EtOAc fraction was also characterized using high performance liquid chromatography (HPLC) fingerprint. Cytotoxicity assay revealed that EtOAc fraction was the most potent among all fractions with maximal effect on MCF-7 and minimal toxicity against normal murine fibroblast L929 cells. Apoptosis induction by EtOAc fraction was confirmed by increased sub-G1 peak of propidium iodide (PI) stained cells. This fraction triggered the apoptotic pathway by increased Bax/Bcl-2 ratio and cleaved caspase-3 level. Moreover, treatment with EtOAc fraction significantly decreased the diameter of vessels on CAM, while the number of newly formed blood vessels was not suppressed significantly. Analysis of quality of EtOAc fraction using HPLC fingerprint showed six major peaks with different retention times. The results of the present study suggest that V. tricolor has potential anticancer property by inducing apoptosis and inhibiting angiogenesis. PMID:25243166

  13. Viola tricolor induces apoptosis in cancer cells and exhibits antiangiogenic activity on chicken chorioallantoic membrane.

    PubMed

    Sadeghnia, Hamid Reza; Ghorbani Hesari, Taghi; Mortazavian, Seyed Mohsen; Mousavi, Seyed Hadi; Tayarani-Najaran, Zahra; Ghorbani, Ahmad

    2014-01-01

    In the present study, the cytotoxic and apoptogenic properties of hydroalcoholic extract and ethyl acetate (EtOAc), n-butanol, and water fractions (0-800 μg/mL) of Viola tricolor were investigated in Neuro2a mouse neuroblastoma and MCF-7 human breast cancer cells. In addition, antiangiogenic effect of EtOAc fraction was evaluated on chicken chorioallantoic membrane (CAM). The quality of EtOAc fraction was also characterized using high performance liquid chromatography (HPLC) fingerprint. Cytotoxicity assay revealed that EtOAc fraction was the most potent among all fractions with maximal effect on MCF-7 and minimal toxicity against normal murine fibroblast L929 cells. Apoptosis induction by EtOAc fraction was confirmed by increased sub-G1 peak of propidium iodide (PI) stained cells. This fraction triggered the apoptotic pathway by increased Bax/Bcl-2 ratio and cleaved caspase-3 level. Moreover, treatment with EtOAc fraction significantly decreased the diameter of vessels on CAM, while the number of newly formed blood vessels was not suppressed significantly. Analysis of quality of EtOAc fraction using HPLC fingerprint showed six major peaks with different retention times. The results of the present study suggest that V. tricolor has potential anticancer property by inducing apoptosis and inhibiting angiogenesis.

  14. Activation of integrin α5 mediated by flow requires its translocation to membrane lipid rafts in vascular endothelial cells.

    PubMed

    Sun, Xiaoli; Fu, Yi; Gu, Mingxia; Zhang, Lu; Li, Dan; Li, Hongliang; Chien, Shu; Shyy, John Y-J; Zhu, Yi

    2016-01-19

    Local flow patterns determine the uneven distribution of atherosclerotic lesions. Membrane lipid rafts and integrins are crucial for shear stress-regulated endothelial function. In this study, we investigate the role of lipid rafts and integrin α5 in regulating the inflammatory response in endothelial cells (ECs) under atheroprone versus atheroprotective flow. Lipid raft proteins were isolated from ECs exposed to oscillatory shear stress (OS) or pulsatile shear stress, and then analyzed by quantitative proteomics. Among 396 proteins redistributed in lipid rafts, integrin α5 was the most significantly elevated in lipid rafts under OS. In addition, OS increased the level of activated integrin α5 in lipid rafts through the regulation of membrane cholesterol and fluidity. Disruption of F-actin-based cytoskeleton and knockdown of caveolin-1 prevented the OS-induced integrin α5 translocation and activation. In vivo, integrin α5 activation and EC dysfunction were observed in the atheroprone areas of low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice, and knockdown of integrin α5 markedly attenuated EC dysfunction in partially ligated carotid arteries. Consistent with these findings, mice with haploinsufficency of integrin α5 exhibited a reduction of atherosclerotic lesions in the regions under atheroprone flow. The present study has revealed an integrin- and membrane lipid raft-dependent mechanotransduction mechanism by which atheroprone flow causes endothelial dysfunction.

  15. Interaction of Defensins with Model Cell Membranes

    NASA Astrophysics Data System (ADS)

    Sanders, Lori K.; Schmidt, Nathan W.; Yang, Lihua; Mishra, Abhijit; Gordon, Vernita D.; Selsted, Michael E.; Wong, Gerard C. L.

    2009-03-01

    Antimicrobial peptides (AMPs) comprise a key component of innate immunity for a wide range of multicellular organisms. For many AMPs, activity comes from their ability to selectively disrupt and lyse bacterial cell membranes. There are a number of proposed models for this action, but the detailed molecular mechanism of selective membrane permeation remains unclear. Theta defensins are circularized peptides with a high degree of selectivity. We investigate the interaction of model bacterial and eukaryotic cell membranes with theta defensins RTD-1, BTD-7, and compare them to protegrin PG-1, a prototypical AMP, using synchrotron small angle x-ray scattering (SAXS). The relationship between membrane composition and peptide induced changes in membrane curvature and topology is examined. By comparing the membrane phase behavior induced by these different peptides we will discuss the importance of amino acid composition and placement on membrane rearrangement.

  16. Active membrane phased array radar

    NASA Technical Reports Server (NTRS)

    Moussessian, Alina; Del Castillo, Linda; Huang, John; Sadowy, Greg; Hoffman, James; Smith, Phil; Hatake, Toshiro; Derksen, Chuck; Lopez, Bernardo; Caro, Ed

    2005-01-01

    We have developed the first membrane-based active phased array in L-band (1.26GHz). The array uses membrane compatible Transmit/Receive (T/R) modules (membrane T/R) for each antenna element. We use phase shifters within each T/R module for electronic beam steering. We will discuss the T/R module design and integration with the membrane, We will also present transmit and receive beam-steering results for the array.

  17. NSOM/QD-based fluorescence-topographic image fusion directly reveals nano-spatial peak-valley polarities of CD69 and CD71 activation molecules on cell-membrane fluctuations during T-cell activation.

    PubMed

    Zhong, Liyun; Zhang, Zhun; Lu, Xiaoxu; Huang, Dan; Chen, Crystal Y; Wang, Richard; Chen, Zheng W

    2011-10-30

    Nano-spatial distribution of cell surface molecules on cell membrane fluctuations during T-cell activation has not been reported. In this study, we innovated application of near-field scanning optical microscopy (NSOM)/quantum dots (QDs)-based nanotechnology through three-dimensional image fusion algorithm to merge the simultaneously obtained dual-color fluorescence information and three-dimensional topography. This novel imaging system made it possible to visualize nano-spatial distribution and organization of early-activation molecules CD69 and late-activation molecules CD71 on cell-membrane fluctuations during T-cell activation. Interestingly, most CD69 molecules were clustered to form 250-500nm nano-domains polarizing predominantly in the peak of the cell-membrane fluctuations. In contrast, although CD71 molecules were also clustered as 250-500nm nano-domains, they polarized dominantly in the valley of the cell-membrane fluctuations. The peak-valley polarities of CD69 nano-domains and CD71 nano-domains implied their different functions. CD69 nano-domains polarizing on membrane-peak fluctuations might serve as transient platforms driving TCR/CD3-induced signaling and activation, whereas CD71 nano-domains distributing in the membrane-valley fluctuations appeared to facilitate iron uptake for increased metabolisms in T-cell activation. Importantly, this NSOM/QD-based fluorescence-topographic image fusion provides a powerful tool to visualize nano-spatial distribution of cell-surface molecules on cell-membrane fluctuations and enable better understanding of distribution-function relationship.

  18. Anaerobic respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment.

    PubMed

    Takahashi, Eiji; Sato, Michihiko

    2014-02-15

    To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we studied the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (ΔO2) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (ΔΦm) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this two-dimensional tissue model, ΔΦm was abolished in cells >500 μm from the oxygen source [the anoxic front (AF)], indicating limitations in diffusional oxygen delivery. This result perfectly matched GFP-determined ΔO2. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain-containing protein (PHD) inhibitor, the AF was expanded to 1,500-2,000 μm from the source. In these cells, tissue ΔO2 was substantially decreased, indicating that PHD pathway activation suppressed mitochondrial respiration. The expansion of the AF and the reduction of ΔO2 were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain ΔΦm, despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding the AF, although tissue ΔO2 remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining ΔΦm in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain ΔΦm in an otherwise anoxic microenvironment by decreasing tissue ΔO2 while activating oxygen-independent electron transport in mitochondria.

  19. Structural and functional effects of benzimidazole/thioether-copper complexes with antitumor activity on cell membranes and molecular models.

    PubMed

    Castillo, Ivan; Suwalsky, Mario; Gallardo, María José; Troncoso, Valentina; Sánchez-Eguía, Brenda N; Santiago-Osorio, Edelmiro; Aguiñiga, Itzen; González-Ugarte, Ana K

    2016-03-01

    Two cytotoxic copper(II) complexes with N-H and N-methylated benzimidazole-derived ligands (Cu-L(2) and Cu-L(2Me)) were synthesized and made to interact with human erythrocytes and molecular models of their plasmatic membranes. The latter consisted in lipid bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), lipids of the types present in the outer and inner monolayers of the human erythrocyte membrane, respectively. Initial assessment of the interaction of the complexes with DMPC and DMPE consisted of X-ray diffraction studies, which showed preferential interactions with the former. Scanning electron microscopy (SEM) of erythrocytes incubated with solutions of the Cu(II) complexes evidenced deformation of the cells to stomatocytes and knizocytes by Cu-L(2) and Cu-L(2Me) due to interactions with the inner and outer leaflets of the cell membranes, respectively. This was further confirmed by real-time observation of the dose-dependent effects of the complexes on live erythrocytes by defocusing microscopy (DM). The combined observations, including the increased antiproliferative activity of the N-methylated complex Cu-L(2Me) over that of Cu-L(2) is rationalized based on the higher lipophilicity of the former. This property would facilitate passive diffusion of Cu-L(2Me) through the cell membrane, particularly in the initial stages when the DMPC-rich outer leaflet is involved. In contrast, the benzimidazole N-H groups of Cu-L(2) may participate in hydrogen bonding with DMPE polar groups; this result is consistent with the formation of stomatocyte induced by the latter complex. PMID:26775279

  20. Rhesus lymphocryptovirus latent membrane protein 2A activates {beta}-catenin signaling and inhibits differentiation in epithelial cells

    SciTech Connect

    Siler, Catherine A.; Raab-Traub, Nancy

    2008-08-01

    Rhesus lymphocryptovirus (LCV) is a {gamma}-herpesvirus closely related to Epstein-Barr virus (EBV). The rhesus latent membrane protein 2A (LMP2A) is highly homologous to EBV LMP2A. EBV LMP2A activates the phosphatidylinositol 3-kinase (PI3K) and {beta}-catenin signaling pathways in epithelial cells and affects differentiation. In the present study, the biochemical and biological properties of rhesus LMP2A in epithelial cells were investigated. The expression of rhesus LMP2A in epithelial cells induced Akt activation, GSK3{beta} inactivation and accumulation of {beta}-catenin in the cytoplasm and nucleus. The nuclear translocation, but not accumulation of {beta}-catenin was dependent on Akt activation. Rhesus LMP2A also impaired epithelial cell differentiation; however, this process was not dependent upon Akt activation. A mutant rhesus LMP2A lacking six transmembrane domains functioned similarly to wild-type rhesus LMP2A indicating that the full number of transmembrane domains is not required for effects on {beta}-catenin or cell differentiation. These results underscore the similarity of LCV to EBV and the suitability of the macaque as an animal model for studying EBV pathogenesis.

  1. Fuel cell with ionization membrane

    NASA Technical Reports Server (NTRS)

    Hartley, Frank T. (Inventor)

    2007-01-01

    A fuel cell is disclosed comprising an ionization membrane having at least one area through which gas is passed, and which ionizes the gas passing therethrough, and a cathode for receiving the ions generated by the ionization membrane. The ionization membrane may include one or more openings in the membrane with electrodes that are located closer than a mean free path of molecules within the gas to be ionized. Methods of manufacture are also provided.

  2. Activation of Na+ channels in cell membrane by capacitive stimulation with silicon chip

    NASA Astrophysics Data System (ADS)

    Schoen, Ingmar; Fromherz, Peter

    2005-11-01

    Sodium channels are the crucial electrical elements of neuronal excitation. As a step toward hybrid neuron-semiconductor devices, we studied the activation of recombinant NaV1.4 sodium channels in human embryonic kidney (HEK293) cells by stimulation from an electrolyte/oxide/silicon (EOS) capacitor. HfO2 was used as an insulator to attain a high capacitance. An effective activation was achieved by decaying voltage ramps at constant intracellular voltage at a depleted NaCl concentration in the bath to enhance the resistance of the cell-chip contact. We were also able to open sodium channels at a NaCl concentration close to physiological conditions. This experiment provides a basis for noninvasive capacitive stimulation of nerve cells with semiconductor chips.

  3. Phosphorylation and activation of the plasma membrane Na+/H+ exchanger (NHE1) during osmotic cell shrinkage.

    PubMed

    Rigor, Robert R; Damoc, Catalina; Phinney, Brett S; Cala, Peter M

    2011-01-01

    The Na(+)/H(+)Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na(+)/H(+) exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na(+)/H(+) exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na(+) transport capacity without affecting transport affinity (K(m)=44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ(32)P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na(+) transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events. PMID:22216214

  4. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.

    PubMed

    Baskin, David S; Ngo, Hop; Didenko, Vladimir V

    2003-08-01

    Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

  5. Specific interaction of tissue-type plasminogen activator (t-PA) with annexin II on the membrane of pancreatic cancer cells activates plasminogen and promotes invasion in vitro

    PubMed Central

    Díaz, V M; Hurtado, M; Thomson, T M; Reventós, J; Paciucci, R

    2004-01-01

    Background: Overexpression of tissue plasminogen activator (t-PA) in pancreatic cancer cells promotes invasion and proliferation in vitro and tumour growth and angiogenesis in vivo. Aims: To understand the mechanisms by which t-PA favours cancer progression, we analysed the surface membrane proteins responsible for binding specifically t-PA and studied the contribution of this interaction to the t-PA promoted invasion of pancreatic cancer cells. Methods: The ability of t-PA to activate plasmin and a fluorogenic plasmin substrate was used to analyse the nature of the binding of active t-PA to cell surfaces. Specific binding was determined in two pancreatic cancer cell lines (SK-PC-1 and PANC-1), and complex formation analysed by co-immunoprecipitation experiments and co-immunolocalisation in tumours. The functional role of the interaction was studied in Matrigel invasion assays. Results: t-PA bound to PANC-1 and SK-PC-1 cells in a specific and saturable manner while maintaining its activity. This binding was competitively inhibited by specific peptides interfering with the interaction of t-PA with annexin II. The t-PA/annexin II interaction on pancreatic cancer cells was also supported by co-immunoprecipitation assays using anti-t-PA antibodies and, reciprocally, with antiannexin II antibodies. In addition, confocal microscopy showed t-PA and annexin II colocalisation in tumour tissues. Finally, disruption of the t-PA/annexin II interaction by a specific hexapeptide significantly decreased the invasive capacity of SK-PC-1 cells in vitro. Conclusion: t-PA specifically binds to annexin II on the extracellular membrane of pancreatic cancer cells where it activates local plasmin production and tumour cell invasion. These findings may be clinically relevant for future therapeutic strategies based on specific drugs that counteract the activity of t-PA or its receptor annexin II, or their interaction at the surface level. PMID:15194650

  6. Proton exchange membrane fuel cell model for aging predictions: Simulated equivalent active surface area loss and comparisons with durability tests

    NASA Astrophysics Data System (ADS)

    Robin, C.; Gérard, M.; Quinaud, M.; d'Arbigny, J.; Bultel, Y.

    2016-09-01

    The prediction of Proton Exchange Membrane Fuel Cell (PEMFC) lifetime is one of the major challenges to optimize both material properties and dynamic control of the fuel cell system. In this study, by a multiscale modeling approach, a mechanistic catalyst dissolution model is coupled to a dynamic PEMFC cell model to predict the performance loss of the PEMFC. Results are compared to two 2000-h experimental aging tests. More precisely, an original approach is introduced to estimate the loss of an equivalent active surface area during an aging test. Indeed, when the computed Electrochemical Catalyst Surface Area profile is fitted on the experimental measures from Cyclic Voltammetry, the computed performance loss of the PEMFC is underestimated. To be able to predict the performance loss measured by polarization curves during the aging test, an equivalent active surface area is obtained by a model inversion. This methodology enables to successfully find back the experimental cell voltage decay during time. The model parameters are fitted from the polarization curves so that they include the global degradation. Moreover, the model captures the aging heterogeneities along the surface of the cell observed experimentally. Finally, a second 2000-h durability test in dynamic operating conditions validates the approach.

  7. Proteomes of Host Cell Membranes Modified by Intracellular Activities of Salmonella enterica*

    PubMed Central

    Vorwerk, Stephanie; Krieger, Viktoria; Deiwick, Jörg; Hensel, Michael; Hansmeier, Nicole

    2015-01-01

    Intracellular pathogens need to establish a growth-stimulating host niche for survival and replication. A unique feature of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium is the creation of extensive membrane networks within its host. An understanding of the origin and function of these membranes is crucial for the development of new treatment strategies. However, the characterization of this compartment is very challenging, and only fragmentary knowledge of its composition and biogenesis exists. Here, we describe a new proteome-based approach to enrich and characterize Salmonella-modified membranes. Using a Salmonella mutant strain that does not form this unique membrane network as a reference, we identified a high-confidence set of host proteins associated with Salmonella-modified membranes. This comprehensive analysis allowed us to reconstruct the interactions of Salmonella with host membranes. For example, we noted that Salmonella redirects endoplasmic reticulum (ER) membrane trafficking to its intracellular niche, a finding that has not been described for Salmonella previously. Our system-wide approach therefore has the potential to rapidly close gaps in our knowledge of the infection process of intracellular pathogens and demonstrates a hitherto unrecognized complexity in the formation of Salmonella host niches. PMID:25348832

  8. Proteomes of host cell membranes modified by intracellular activities of Salmonella enterica.

    PubMed

    Vorwerk, Stephanie; Krieger, Viktoria; Deiwick, Jörg; Hensel, Michael; Hansmeier, Nicole

    2015-01-01

    Intracellular pathogens need to establish a growth-stimulating host niche for survival and replication. A unique feature of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium is the creation of extensive membrane networks within its host. An understanding of the origin and function of these membranes is crucial for the development of new treatment strategies. However, the characterization of this compartment is very challenging, and only fragmentary knowledge of its composition and biogenesis exists. Here, we describe a new proteome-based approach to enrich and characterize Salmonella-modified membranes. Using a Salmonella mutant strain that does not form this unique membrane network as a reference, we identified a high-confidence set of host proteins associated with Salmonella-modified membranes. This comprehensive analysis allowed us to reconstruct the interactions of Salmonella with host membranes. For example, we noted that Salmonella redirects endoplasmic reticulum (ER) membrane trafficking to its intracellular niche, a finding that has not been described for Salmonella previously. Our system-wide approach therefore has the potential to rapidly close gaps in our knowledge of the infection process of intracellular pathogens and demonstrates a hitherto unrecognized complexity in the formation of Salmonella host niches.

  9. Interaction of detergent sclerosants with cell membranes.

    PubMed

    Parsi, Kurosh

    2015-06-01

    Commonly used detergent sclerosants including sodium tetradecyl sulphate (STS) and polidocanol (POL) are clinically used to induce endovascular fibrosis and vessel occlusion. They achieve this by lysing the endothelial lining of target vessels. These agents are surface active (surfactant) molecules that interfere with cell membranes. Surfactants have a striking similarity to the phospholipid molecules of the membrane lipid bilayer. By adsorbing at the cell membrane, surfactants disrupt the normal architecture of the lipid bilayer and reduce the surface tension. The outcome of this interaction is concentration dependent. At high enough concentrations, surfactants solubilise cell membranes resulting in cell lysis. At lower concentrations, these agents can induce a procoagulant negatively charged surface on the external aspect of the cell membrane. The interaction is also influenced by the ionic charge, molecular structure, pH and the chemical nature of the diluent (e.g. saline vs. water). The ionic charge of the surfactant molecule can influence the effect on plasma proteins and the protein contents of cell membranes. STS, an anionic detergent, denatures the tertiary complex of most proteins and in particular the clinically relevant clotting factors. By contrast, POL has no effect on proteins due to its non-ionic structure. These agents therefore exhibit remarkable differences in their interaction with lipid membranes, target cells and circulating proteins with potential implications in a range of clinical applications.

  10. Membrane Elastic Properties and Cell Function

    PubMed Central

    Pontes, Bruno; Ayala, Yareni; Fonseca, Anna Carolina C.; Romão, Luciana F.; Amaral, Racκele F.; Salgado, Leonardo T.; Lima, Flavia R.; Farina, Marcos; Viana, Nathan B.; Moura-Neto, Vivaldo; Nussenzveig, H. Moysés

    2013-01-01

    Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function. PMID:23844071

  11. A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

    PubMed

    Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

    2015-01-01

    The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA.

  12. Membrane permeability of redox active metal chelators: an important element in reducing hydroxyl radical induced NAD+ depletion in neuronal cells.

    PubMed

    Jayasena, T; Grant, R S; Keerthisinghe, N; Solaja, I; Smythe, G A

    2007-03-01

    There is substantial evidence implicating increased production of the hydroxyl radical and oxidative stress in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). Significant amounts of hydroxyl radicals will be produced in the presence of hydrogen peroxide and redox active iron via Fenton chemistry. Increased iron levels within the cytoplasm of vulnerable neurons suggest that this may also be an important site of oxidative activity. We investigated the likelihood that intracellular, rather than extracellular chelation of ferrous or ferric iron may be more effective in reducing hydroxyl radical induced cell damage and preserving NAD(+) levels and cell viability. Using intracellular NAD(H) measurements as an indicator of cell viability we found that membrane permeable ferrous chelators were most efficient in preserving cellular NAD(+) levels. Hydrophilic, ferrous or ferric chelators and lipophilic ferric chelators were essentially ineffective in preventing cellular NAD(+) depletion when added at physiological concentrations. We propose that lipophilic ferrous chelators, due to their actions inside the cell, are effective agents for moderating neuronal damage in conditions such as AD where intracellular oxidative stress plays a significant role in disease pathology. PMID:17210195

  13. Red cell membrane: past, present, and future

    PubMed Central

    Gallagher, Patrick G.

    2008-01-01

    As a result of natural selection driven by severe forms of malaria, 1 in 6 humans in the world, more than 1 billion people, are affected by red cell abnormalities, making them the most common of the inherited disorders. The non-nucleated red cell is unique among human cell type in that the plasma membrane, its only structural component, accounts for all of its diverse antigenic, transport, and mechanical characteristics. Our current concept of the red cell membrane envisions it as a composite structure in which a membrane envelope composed of cholesterol and phospholipids is secured to an elastic network of skeletal proteins via transmembrane proteins. Structural and functional characterization of the many constituents of the red cell membrane, in conjunction with biophysical and physiologic studies, has led to detailed description of the way in which the remarkable mechanical properties and other important characteristics of the red cells arise, and of the manner in which they fail in disease states. Current studies in this very active and exciting field are continuing to produce new and unexpected revelations on the function of the red cell membrane and thus of the cell in health and disease, and shed new light on membrane function in other diverse cell types. PMID:18988878

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

    PubMed

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

    2015-03-01

    Plant cells rely on their cell walls for directed growth and environmental adaptation. Synthesis and remodelling of the cell walls are membrane-related processes. During cell growth and exposure to external stimuli, there is a constant exchange of lipids, proteins, and other cell wall components between the cytosol and the plasma membrane/apoplast. This exchange of material and the localization of cell wall proteins at certain spots in the plasma membrane seem to rely on a particular membrane composition. In addition, sensors at the plasma membrane detect changes in the cell wall architecture, and activate cytoplasmic signalling schemes and ultimately cell wall remodelling. The apoplastic polysaccharide matrix is, on the other hand, crucial for preventing proteins diffusing uncontrollably in the membrane. Therefore, the cell wall-plasma membrane link is essential for plant development and responses to external stimuli. This review focuses on the relationship between the cell wall and plasma membrane, and its importance for plant tissue organization.

  15. Activation of the Ca2+-sensing receptor induces deposition of tight junction components to the epithelial cell plasma membrane

    PubMed Central

    Jouret, François; Wu, Jingshing; Hull, Michael; Rajendran, Vanathy; Mayr, Bernhard; Schöfl, Christof; Geibel, John; Caplan, Michael J.

    2013-01-01

    Summary The Ca2+-sensing receptor (CaSR) belongs to the G-protein-coupled receptor superfamily and plays essential roles in divalent ion homeostasis and cell differentiation. Because extracellular Ca2+ is essential for the development of stable epithelial tight junctions (TJs), we hypothesized that the CaSR participates in regulating TJ assembly. We first assessed the expression of the CaSR in Madin-Darby canine kidney (MDCK) cells at steady state and following manipulations that modulate TJ assembly. Next, we examined the effects of CaSR agonists and antagonists on TJ assembly. Immunofluorescence studies indicate that endogenous CaSR is located at the basolateral pole of MDCK cells. Stable transfection of human CaSR in MDCK cells further reveals that this protein co-distributes with β-catenin on the basolateral membrane. Switching MDCK cells from low-Ca2+ medium to medium containing a normal Ca2+ concentration significantly increases CaSR expression at both the mRNA and protein levels. Exposure of MDCK cells maintained in low-Ca2+ conditions to the CaSR agonists neomycin, Gd3+ or R-568 causes the transient relocation of the tight junction components ZO-1 and occludin to sites of cell–cell contact, while inducing no significant changes in the expression of mRNAs encoding junction-associated proteins. Stimulation of CaSR also increases the interaction between ZO-1 and the F-actin-binding protein I-afadin. This effect does not involve activation of the AMP-activated protein kinase. By contrast, CaSR inhibition by NPS-2143 significantly decreases interaction of ZO-1 with I-afadin and reduces deposition of ZO-1 at the cell surface following a Ca2+ switch from 5 µM to 200 µM [Ca2+]e. Pre-exposure of MDCK cells to the cell-permeant Ca2+ chelator BAPTA-AM, similarly prevents TJ assembly caused by CaSR activation. Finally, stable transfection of MDCK cells with a cDNA encoding a human disease-associated gain-of-function mutant form of the CaSR increases the

  16. Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment

    PubMed Central

    Maza, Paloma K.; Suzuki, Erika

    2016-01-01

    Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation. PMID:27148251

  17. Chicken embryo fibroblasts exposed to weak, time-varying magnetic fields share cell proliferation, adenosine deaminase activity, and membrane characteristics of transformed cells

    SciTech Connect

    Parola, A.H.; Porat, N.; Kiesow, L.A. )

    1993-01-01

    Chicken embryo fibroblasts (CEF) exposed to a sinusoidally varying magnetic field (SVMF) (100 Hz, 700 microT, for 24 h) showed a remarkable rise of segmental rotational relaxation rate of adenosine deaminase (ADA, EC 3.5.4.4) as determined by multifrequency phase fluorometry. Pyrene-labeled, small subunit ADA was applied to cultured (normal) CEF, which have available and abundant ADA complexing protein (ADCP) on their plasma membranes. Sine-wave-modulated fluorometry of the pyrene yielded a profile of phase angle vs. modulation frequency. In SVMF-treated cells and in Rous-sarcoma-virus (RSV) transformed cells the differential phase values at low modulation frequencies of the excitation are remarkably reduced. This effect is magnetic rather than thermal, because the temperature was carefully controlled and monitored; nevertheless to further check this matter we studied CEF, infected by the RSV-Ts68 temperature-sensitive mutant (36 degrees C transformed, 41 degrees C revertant). When grown at 36 degrees C in the SVMF, cells did not show the slightest trend towards reversion, as would be expected had there been local heating. Concomitant with the increased segmental rotational relaxation rate of ADA, there was a decrease in fluorescence lifetime and a slight, yet significant, increase in membrane lipid microfluidity. These biophysical observations prompted us to examine the effect of SVMF on cell proliferation and ADA activity (a malignancy marker): higher rates of cell proliferation and reduced specific activity of ADA were observed.

  18. Innervation of Gill Lateral Cells in the Bivalve Mollusc Crassostrea virginica Affects Cellular Membrane Potential and Cilia Activity

    PubMed Central

    Catapane, Edward J; Nelson, Michael; Adams, Trevon; Carroll, Margaret A

    2016-01-01

    Gill lateral cells of Crassostrea virginica are innervated by the branchial nerve, which contains serotonergic and dopaminergic fibers that regulate cilia beating rate. Terminal release of serotonin or dopamine results in an increase or decrease, respectively, of cilia beating rate in lateral gill cells. In this study we used the voltage sensitive fluorescent probe DiBAC4(3) to quantify changes in gill lateral cell membrane potential in response to electrical stimulation of the branchial nerve or to applications of serotonin and dopamine, and correlate these changes to cilia beating rates. Application of serotonin to gill lateral cells caused prolonged membrane depolarization, similar to plateau potentials, while increasing cilia beating rate. Application of dopamine hyperpolarized the resting membrane while decreasing cilia beating rate. Low frequency (5 Hz) electrical stimulations of the branchial nerve, which cause terminal release of endogenous serotonin, or high frequency (20 Hz) stimulations, which cause terminal release of endogenous dopamine, had the same effects on gill lateral cell membrane potentials and cilia beating rate as the respective applications of serotonin or dopamine. The study shows that innervation of gill lateral cells by the branchial nerve affects membrane potential as well as cilia beating rate, and demonstrates a strong correlation between changes in membrane potential and regulation of cilia beating rate. The study furthers the understanding of serotonin and dopamine signaling in the innervation and regulation of gill cilia in bivalves. The study also shows that voltage sensitive fluorescent probes like DiBAC 4(3) can be successfully used as an alternative to microelectrodes to measure changes in membrane potential of ciliated gill cells and other small cells with fast moving cilia. PMID:27489887

  19. Nonlinear electromagnetic responses of active membrane protein complexes in live cells and organelles

    NASA Astrophysics Data System (ADS)

    Nawarathna, Dharmakirthi

    The response of biological cells to an applied oscillating electric field contains both linear and nonlinear components (eg. induced harmonics). Such noninvasive measurements can be used to study active processes taking place inside the cells. The measurement of induced harmonics is the tool used for the study described here. A highly sensitive superconducting quantum interference device (SQUID) is used to detect the response at low frequencies, which greatly reduces electrode polarization effects. At high frequencies, a four- probe method is used. At low frequencies, harmonic generation by budding yeast cells in response to a sinusoidal electric field is reported, which is seen to be minimal when the field amplitude is less than a threshold value. Surprisingly, sodium metavanadate, an inhibitor of P-type ATPases and glucose, a substrate of P-type ATPase responsible for nonlinear response in yeast, reduces the threshold field amplitude, increasing harmonic generation at low amplitudes while reducing it at large amplitudes. We have thus proposed a model that explicitly introduces a threshold field, similar to those observed in density waves, where fields above threshold drive charge transport through an energy landscape with multiple wells, and in Coulomb blockade tunnel junctions, recently exploited to define the current standard. At high frequencies, the induced harmonics exhibit pronounced features that depend on the specific organism. Budding yeast (S. cerevisiae ) cells produce numerous harmonics. When the second or third harmonic amplitude is plotted vs. applied frequency, we observe two peaks, around 3 kHz and 12 kHz, which are suppressed by the respiratory inhibitor potassium cyanide. We then measured the response to oscillatory electric fields of intact bovine heart mitochondria, a reproducible second harmonic (at ˜3-4 kHz applied frequency) was detected. Further, with coupled mouse mitochondria, an ADP sensitive peak (˜ 12-15 kHz applied frequency) was

  20. The Molecules of the Cell Membrane.

    ERIC Educational Resources Information Center

    Bretscher, Mark S.

    1985-01-01

    Cell membrane molecules form a simple, two-dimensional liquid controlling what enters and leaves the cell. Discusses cell membrane molecular architecture, plasma membranes, epithelial cells, cycles of endocytosis and exocytosis, and other topics. Indicates that some cells internalize, then recycle, membrane area equivalent to their entire surface…

  1. Membrane-to-nucleus signaling links insulin-like growth factor-1- and stem cell factor-activated pathways.

    PubMed

    Hayashi, Yujiro; Asuzu, David T; Gibbons, Simon J; Aarsvold, Kirsten H; Bardsley, Michael R; Lomberk, Gwen A; Mathison, Angela J; Kendrick, Michael L; Shen, K Robert; Taguchi, Takahiro; Gupta, Anu; Rubin, Brian P; Fletcher, Jonathan A; Farrugia, Gianrico; Urrutia, Raul A; Ordog, Tamas

    2013-01-01

    Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes. PMID:24116170

  2. Fabrication of a live cell-containing multilayered polymer hydrogel membrane with micrometer-scale thickness to evaluate pharmaceutical activity.

    PubMed

    Gao, Botao; Konno, Tomohiro; Ishihara, Kazuhiko

    2015-01-01

    We propose a spinning-assisted layer-by-layer method for simple fabrication of a multilayered polymer hydrogel membrane that contains living cells. Hydrogel formation occurred based on the spontaneous cross-linking reaction between two polymers in aqueous solution. A water-soluble 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenylboronic acid groups (PMBV) and poly(vinyl alcohol) (PVA) were used as polymers for hydrogel membrane formation. Changing the number of hydrogel membrane layers, polymer concentration, spinning rate, and processing time for diffusion-dependent gelation of PMBV and PVA facilitated the regulation of the multilayered polymer hydrogel membrane thickness and morphology. We concluded that a multilayered polymer hydrogel membrane prepared using 5.0 wt% PMBV and 5.0 wt% PVA at a spinning rate of 2000 rpm was suitable for precise spatial control of cells in single layers. This multilayered polymer hydrogel membrane was used to prepare a single cell-laden layer to minimize barriers to the diffusion of bioactive compounds while preserving the three-dimensional (3-D) context. The pharmaceutical effects of one of the anticancer agents, paclitaxel, on a human cervical cancer line, HeLa cells, were evaluated in vitro, and the usability of this culture model was demonstrated. PMID:26374190

  3. Activated hepatic stellate cells are dependent on self-collagen, cleaved by membrane type 1 matrix metalloproteinase for their growth.

    PubMed

    Birukawa, Naoko Kubo; Murase, Kazuyuki; Sato, Yasushi; Kosaka, Akemi; Yoneda, Akihiro; Nishita, Hiroki; Fujita, Ryosuke; Nishimura, Miyuki; Ninomiya, Takafumi; Kajiwara, Keiko; Miyazaki, Miyono; Nakashima, Yusuke; Ota, Sigenori; Murakami, Yuya; Tanaka, Yasunobu; Minomi, Kenjiro; Tamura, Yasuaki; Niitsu, Yoshiro

    2014-07-18

    Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVβ1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVβ1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies.

  4. Interaction of peptides with cell membranes: insights from molecular modeling

    NASA Astrophysics Data System (ADS)

    Li, Zhen-lu; Ding, Hong-ming; Ma, Yu-qiang

    2016-03-01

    The investigation of the interaction of peptides with cell membranes is the focus of active research. It can enhance the understanding of basic membrane functions such as membrane transport, fusion, and signaling processes, and it may shed light on potential applications of peptides in biomedicine. In this review, we will present current advances in computational studies on the interaction of different types of peptides with the cell membrane. Depending on the properties of the peptide, membrane, and external environment, the peptide-membrane interaction shows a variety of different forms. Here, on the basis of recent computational progress, we will discuss how different peptides could initiate membrane pores, translocate across the membrane, induce membrane endocytosis, produce membrane curvature, form fibrils on the membrane surface, as well as interact with functional membrane proteins. Finally, we will present a conclusion summarizing recent progress and providing some specific insights into future developments in this field.

  5. A novel bioactive membrane by cell electrospinning.

    PubMed

    Chen, Haiping; Liu, Yuanyuan; Hu, Qingxi

    2015-11-01

    Electrospinning permits fabrication of biodegradable matrices that can resemble the both scale and mechanical behavior of the native extracellular matrix. However, achieving high-cellular density and infiltration of cells within matrices with traditional technique remain challenging and time consuming. The cell electrospinning technique presented in this paper can mitigate the problems associated with these limitations. Cells encapsulated by the material in the cell electrospinning technique survived well and distributed homogenously within the nanofibrous membrane, and their vitality was improved to 133% after being cultured for 28 days. The electrospun nanofibrous membrane has a certain degradation property and favorable cell-membrane interaction that supports the active biocompatibility of the membrane. Its properties are helpful for supporting cell attachment and growth, maintaining phenotypic shape, and secreting an ample amount of extracellular matrix (ECM). This novel membrane may be a potential application within the field of tissue engineering. The ability of cell electrospinning to microintegrate cells into a biodegradable fibrous matrix embodies a novel tissue engineering approach that could be applied to fabricate a high cell density elastic tissue mimetic.

  6. The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across the target cell plasma membrane and displays a contact-dependent membrane disrupting activity.

    PubMed Central

    Håkansson, S; Schesser, K; Persson, C; Galyov, E E; Rosqvist, R; Homblé, F; Wolf-Watz, H

    1996-01-01

    During infection of cultured epithelial cells, surface-located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non-polar yopB mutant strain displays a wild-type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage-like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia-induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB-dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell-to-cell transfer of Yop effector proteins. Images PMID:8918459

  7. High electrical field effects on cell membranes.

    PubMed

    Pliquett, U; Joshi, R P; Sridhara, V; Schoenbach, K H

    2007-05-01

    Electrical charging of lipid membranes causes electroporation with sharp membrane conductance increases. Several recent observations, especially at very high field strength, are not compatible with the simple electroporation picture. Here we present several relevant experiments on cell electrical responses to very high external voltages. We hypothesize that, not only are aqueous pores created within the lipid membranes, but that nanoscale membrane fragmentation occurs, possibly with micelle formation. This effect would produce conductivity increases beyond simple electroporation and display a relatively fast turn-off with external voltage. In addition, material loss can be expected at the anode side of cells, in agreement with published experimental reports at high fields. Our hypothesis is qualitatively supported by molecular dynamics simulations. Finally, such cellular responses might temporarily inactivate voltage-gated and ion-pump activity, while not necessarily causing cell death. This hypothesis also supports observations on electrofusion.

  8. Requirement of translocated lysosomal V1 H+-ATPase for activation of membrane acid sphingomyelinase and raft clustering in coronary endothelial cells

    PubMed Central

    Xu, Ming; Xia, Min; Li, Xiao-Xue; Han, Wei-Qing; Boini, Krishna M.; Zhang, Fan; Zhang, Yang; Ritter, Joseph K; Li, Pin-Lan

    2012-01-01

    Acid sphingomyelinase (ASM) mediates the formation of membrane raft (MR) redox signalosomes in a process that depends on a local acid microenvironment in coronary arterial endothelial cells (CAECs). However, it is not known how this local acid microenvironment is formed and maintained. The present study hypothesized that lysosomal V1 H+-ATPase provides a hospitable acid microenvironment for activation of ASM when lysosomes traffic and fuse into the cell membrane. Confocal microscopy showed that local pH change significantly affected MRs, with more fluorescent patches under low pH. Correspondingly, the ASM product, ceramide, increased locally in the cell membrane. Electron spin resonance assay showed that local pH increase significantly inhibited NADPH oxidase–mediated production of O2−. in CAECs. Direct confocal microscopy demonstrated that Fas ligand resulted in localized areas of decreased pH around CAEC membranes. The inhibitors of both lysosomal fusion and H+-ATPase apparently attenuated FasL-caused pH decrease. V1 H+-ATPase accumulation and activity on cell membranes were substantially suppressed by the inhibitors of lysosomal fusion or H+-ATPase. These results provide the first direct evidence that translocated lysosomal V1 H+-ATPase critically contributes to the formation of local acid microenvironment to facilitate activation of ASM and consequent MR aggregation, forming MR redox signalosomes and mediating redox signaling in CAECs. PMID:22357614

  9. Cell secretion and membrane fusion.

    PubMed

    Jena, Bhanu P

    2005-07-01

    Secretion occurs in all cells of multicellular organisms and involves the delivery of secretory products packaged in membrane-bound vesicles to the cell exterior. Specialized cells for neurotransmission, enzyme secretion or hormone release utilize a highly regulated secretory process. Secretory vesicles are transported to specific sites at the plasma membrane, where they dock and fuse to release their contents. Similar to other cellular processes, cell secretion is found to be highly regulated and a precisely orchestrated event. It has been demonstrated that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized supramolecular structures at the cell plasma membrane. Swelling of secretory vesicles results in a build-up of pressure, allowing expulsion of intravesicular contents. The extent of secretory vesicle swelling dictates the amount of intravesicular contents expelled during secretion. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution into artificial lipid membrane, have been determined. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, has also been resolved. These findings reveal the molecular mechanism of cell secretion.

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

    PubMed

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

    2015-11-01

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

  11. Influence of platelet-activating factor, lyso-platelet-activating factor and edelfosine on Langmuir monolayers imitating plasma membranes of cell lines differing in susceptibility to anti-cancer treatment: the effect of plasmalogen level

    PubMed Central

    Flasiński, Michał; Hąc-Wydro, Katarzyna; Wydro, Paweł; Dynarowicz-Łątka, Patrycja

    2014-01-01

    Three structurally related but differing in biological activities single-chained ether phospholipids (PAF (platelet-activating factor) and lyso-PAF) and an anti-cancer drug (edelfosine (ED)) were investigated in Langmuir monolayers imitating natural membranes. The aim of the undertaken experiments was to study the influence of these lipids on monolayers mimicking plasma membranes of cell lines differing in susceptibility to the anti-cancer activity of ED, i.e. promyelocytic leukaemia cells (HL-60) and promyeloblastic leukaemia cells (K-562). As these cells differ essentially in the cholesterol/phospholipid ratio and plasmalogen concentration in the membrane, we have carried out systematic investigations in artificial systems of various compositions. The results for model leukaemia cell membrane were compared with data acquired for systems imitating normal leucocytes. Our results show that the level of plasmalogens significantly modulates the influence of the single-chained phospholipids on the investigated systems. The experiments confirmed also that the interactions of ether lipids with a model membrane of HL-60 cells (in biological tests sensitive to ED) have opposite character when compared with K-562, being resistant to ED. Moreover, the values of the parameters characterizing monolayers serving as membrane models (strength of interactions, monolayers fluidity and morphology) proved both sensitivity of these cells to ED and lack of their susceptibility towards PAF. Interestingly, it has been found that lyso-PAF, which is usually described as an inactive precursor of PAF, displays a stronger effect on HL-60 model membranes than ED. PMID:24694892

  12. Influence of platelet-activating factor, lyso-platelet-activating factor and edelfosine on Langmuir monolayers imitating plasma membranes of cell lines differing in susceptibility to anti-cancer treatment: the effect of plasmalogen level.

    PubMed

    Flasiński, Michał; Hąc-Wydro, Katarzyna; Wydro, Paweł; Dynarowicz-Łątka, Patrycja

    2014-06-01

    Three structurally related but differing in biological activities single-chained ether phospholipids (PAF (platelet-activating factor) and lyso-PAF) and an anti-cancer drug (edelfosine (ED)) were investigated in Langmuir monolayers imitating natural membranes. The aim of the undertaken experiments was to study the influence of these lipids on monolayers mimicking plasma membranes of cell lines differing in susceptibility to the anti-cancer activity of ED, i.e. promyelocytic leukaemia cells (HL-60) and promyeloblastic leukaemia cells (K-562). As these cells differ essentially in the cholesterol/phospholipid ratio and plasmalogen concentration in the membrane, we have carried out systematic investigations in artificial systems of various compositions. The results for model leukaemia cell membrane were compared with data acquired for systems imitating normal leucocytes. Our results show that the level of plasmalogens significantly modulates the influence of the single-chained phospholipids on the investigated systems. The experiments confirmed also that the interactions of ether lipids with a model membrane of HL-60 cells (in biological tests sensitive to ED) have opposite character when compared with K-562, being resistant to ED. Moreover, the values of the parameters characterizing monolayers serving as membrane models (strength of interactions, monolayers fluidity and morphology) proved both sensitivity of these cells to ED and lack of their susceptibility towards PAF. Interestingly, it has been found that lyso-PAF, which is usually described as an inactive precursor of PAF, displays a stronger effect on HL-60 model membranes than ED.

  13. Enhanced desalination performance of membrane capacitive deionization cells by packing the flow chamber with granular activated carbon.

    PubMed

    Bian, Yanhong; Yang, Xufei; Liang, Peng; Jiang, Yong; Zhang, Changyong; Huang, Xia

    2015-11-15

    A new design of membrane capacitive deionization (MCDI) cell was constructed by packing the cell's flow chamber with granular activated carbon (GAC). The GAC packed-MCDI (GAC-MCDI) delivered higher (1.2-2.5 times) desalination rates than the regular MCDI at all test NaCl concentrations (∼ 100-1000 mg/L). The greatest performance enhancement by packed GAC was observed when treating saline water with an initial NaCl concentration of 100 mg/L. Several different GAC materials were tested and they all exhibited similar enhancement effects. Comparatively, packing the MCDI's flow chamber with glass beads (GB; non-conductive) and graphite granules (GG; conductive but with lower specific surface area than GAC) resulted in inferior desalination performance. Electrochemical impedance spectroscopy (EIS) analysis showed that the GAC-MCDI had considerably smaller internal resistance than the regular MCDI (∼ 19.2 ± 1.2 Ω versus ∼ 1222 ± 15 Ω at 100 mg/L NaCl). The packed GAC also decreased the ionic resistance across the flow chamber (∼ 1.49 ± 0.05 Ω versus ∼ 1130 ± 12 Ω at 100 mg/L NaCl). The electric double layer (EDL) formed on the GAC surface was considered to store salt ions during electrosorption, and facilitate the ion transport in the flow chamber because of the higher ion conductivity in the EDLs than in the bulk solution, thereby enhancing the MCDI's desalination rate.

  14. Artesunate reduces chicken chorioallantoic membrane neovascularisation and exhibits antiangiogenic and apoptotic activity on human microvascular dermal endothelial cell.

    PubMed

    Huan-huan, Chen; Li-Li, You; Shang-Bin, Li

    2004-08-10

    Artesunate (ART), a semi-synthetic derivative of artemisinin extracted from the Chinese herb Artemisia annua, is a safe and effective antimalarial drug. ART has now been analyzed for its anti-angiogenic activity in vivo and in vitro. The anti-angiogenic effect in vivo was evaluated on chicken chorioallantoic membrane (CAM) neovascularisation model. ART started to significantly inhibit CAM angiogenesis at a low concentration of 10 nm/100 microl/egg, and completely inhibited the angiogenesis at 80 nm/100 microl/egg. The inhibitory effect of in vitro angiogenesis was tested on the models of proliferation and differentiation of human microvascular dermal endothelial cell line, an important representive of endothelial cells, as well as immunocytochemistry assay for two major VEGF receptors (Flt-1 and KDR/flk-1) expressions. The results showed that ART could remarkably inhibit proliferation and differentiation of endothelial cells in a dose-dependent form in a range of 12.5-100 microM. ART also could reduce Flt-1 and KDR/flk-1 expressions in a range of 0.1-0.5 microM. Furthermore, we examined the apoptosis of human microvascular dermal endothelial cell line induced by ART. The apoptosis was detected by morphological assay of ethidium bromide (EB)/acridine orange (AO) dual staining as well as DNA fragmentation assay of TUNEL labeling and quantified by flowcytometric PI assay. Our results suggest that the antiangiogenic effect induced by ART might occur by the induction of cellular apoptosis. These findings and the known low toxicity indicated ART might be a promising candidate for angiogenesis inhibitors. PMID:15219940

  15. Dielectric breakdown of cell membranes.

    PubMed

    Zimmermann, U; Pilwat, G; Riemann, F

    1974-11-01

    With human and bovine red blood cells and Escherichia coli B, dielectric breakdown of cell membranes could be demonstrated using a Coulter Counter (AEG-Telefunken, Ulm, West Germany) with a hydrodynamic focusing orifice. In making measurements of the size distributions of red blood cells and bacteria versus increasing electric field strength and plotting the pulse heights versus the electric field strength, a sharp bend in the otherwise linear curve is observed due to the dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated yields a value of about 1.6 V for the membrane potential at which dielectric breakdown occurs with modal volumes of red blood cells and bacteria. The same value is also calculated for red blood cells by applying the capacitor spring model of Crowley (1973. Biophys. J. 13:711). The corresponding electric field strength generated in the membrane at breakdown is of the order of 4 . 10(6) V/cm and, therefore, comparable with the breakdown voltages for bilayers of most oils. The critical detector voltage for breakdown depends on the volume of the cells. The volume-dependence predicted by Laplace theory with the assumption that the potential generated across the membrane is independent of volume, could be verified experimentally. Due to dielectric breakdown the red blood cells lose hemoglobin completely. This phenomenon was used to study dielectric breakdown of red blood cells in a homogeneous electric field between two flat platinum electrodes. The electric field was applied by discharging a high voltage storage capacitor via a spark gap. The calculated value of the membrane potential generated to produce dielectric breakdown in the homogeneous field is of the same order as found by means of the Coulter Counter. This indicates that mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter could also be excluded as a hemolysing mechanism. The detector

  16. Staphylococcus aureus α-toxin-mediated cation entry depolarizes membrane potential and activates p38 MAP kinase in airway epithelial cells.

    PubMed

    Eiffler, Ina; Behnke, Jane; Ziesemer, Sabine; Müller, Christian; Hildebrandt, Jan-Peter

    2016-09-01

    Membrane potential (Vm)-, Na(+)-, or Ca(2+)-sensitive fluorescent dyes were used to analyze changes in Vm or intracellular ion concentrations in airway epithelial cells treated with Staphylococcus aureus α-toxin (Hla), a major virulence factor of pathogenic strains of these bacteria. Gramicidin, a channel-forming peptide causing membrane permeability to monovalent cations, a mutated form of Hla, rHla-H35L, which forms oligomers in the plasma membranes of eukaryotic cells but fails to form functional transmembrane pores, or the cyclodextrin-derivative IB201, a blocker of the Hla pore, were used to investigate the permeability of the pore. Na(+) as well as Ca(2+) ions were able to pass the Hla pore and accumulated in the cytosol. The pore-mediated influx of calcium ions was blocked by IB201. Treatment of cells with recombinant Hla resulted in plasma membrane depolarization as well as in increases in the phosphorylation levels of paxillin (signaling pathway mediating disruption of the actin cytoskeleton) and p38 MAP kinase (signaling pathway resulting in defensive actions). p38 MAP kinase phosphorylation, but not paxillin phosphorylation, was elicited by treatment of cells with gramicidin. Although treatment of cells with rHla-H35L resulted in the formation of membrane-associated heptamers, none of these cellular effects were observed in our experiments. This indicates that formation of functional Hla-transmembrane pores is required to induce the cell physiological changes mediated by α-toxin. Specifically, the changes in ion equilibria and plasma membrane potential are important activators of p38 MAP kinase, a signal transduction module involved in host cell defense. PMID:27496896

  17. Staphylococcus aureus α-toxin-mediated cation entry depolarizes membrane potential and activates p38 MAP kinase in airway epithelial cells.

    PubMed

    Eiffler, Ina; Behnke, Jane; Ziesemer, Sabine; Müller, Christian; Hildebrandt, Jan-Peter

    2016-09-01

    Membrane potential (Vm)-, Na(+)-, or Ca(2+)-sensitive fluorescent dyes were used to analyze changes in Vm or intracellular ion concentrations in airway epithelial cells treated with Staphylococcus aureus α-toxin (Hla), a major virulence factor of pathogenic strains of these bacteria. Gramicidin, a channel-forming peptide causing membrane permeability to monovalent cations, a mutated form of Hla, rHla-H35L, which forms oligomers in the plasma membranes of eukaryotic cells but fails to form functional transmembrane pores, or the cyclodextrin-derivative IB201, a blocker of the Hla pore, were used to investigate the permeability of the pore. Na(+) as well as Ca(2+) ions were able to pass the Hla pore and accumulated in the cytosol. The pore-mediated influx of calcium ions was blocked by IB201. Treatment of cells with recombinant Hla resulted in plasma membrane depolarization as well as in increases in the phosphorylation levels of paxillin (signaling pathway mediating disruption of the actin cytoskeleton) and p38 MAP kinase (signaling pathway resulting in defensive actions). p38 MAP kinase phosphorylation, but not paxillin phosphorylation, was elicited by treatment of cells with gramicidin. Although treatment of cells with rHla-H35L resulted in the formation of membrane-associated heptamers, none of these cellular effects were observed in our experiments. This indicates that formation of functional Hla-transmembrane pores is required to induce the cell physiological changes mediated by α-toxin. Specifically, the changes in ion equilibria and plasma membrane potential are important activators of p38 MAP kinase, a signal transduction module involved in host cell defense.

  18. Sex and parental hypertension as predictors of worsened red blood cell membrane enzyme activities in type 1 insulin-dependent diabetic subjects.

    PubMed

    Finotti, P; Piccoli, A

    1993-01-01

    The possibility that distinct genetic factors may concur, in association with diabetes, to increase susceptibility to vascular morbidity, including hypertension, has been evaluated in ninety-four normotensive insulin-dependent diabetic patients by testing both the frequency and prevalence of hypertension in parents and by measuring membrane red blood cell enzyme activities. Parental hypertension was present in a significantly higher proportion of diabetic compared to control subjects. A significant decrease in basal membrane red blood cell (Na(+)-K+), (Mg2+) and (Ca2+) ATPase activities was also related to the disease and was apparently uninfluenced by short--or long term metabolic control. In contrast with what was observed in the control group, sex caused in diabetic subjects significant variations in red blood cell enzyme activities, with women showing the lowest mean basal values of all enzyme activities. Parental hypertension turned out to be an independent risk factor in significantly reducing red blood cell enzyme activities both in diabetic and control subjects. However, whereas in diabetic subjects sex interacted strongly with parental hypertension in causing reduction of enzyme activities, in controls the effect of parental hypertension was sex-independent and significantly reduced basal enzyme activities, thus rendering subjects similar to diabetics. It is concluded that both sex and parental hypertension in association with diabetes, are predictors of further damage to red blood cell enzyme activities, which may thus be linked to increased risk of susceptibility towards vascular complications. PMID:8389303

  19. Detailed search for protein kinase(s) involved in plasma membrane H+-ATPase activity regulation of yeast cells.

    PubMed

    Pereira, Renata R; Castanheira, Diogo; Teixeira, Janaina A; Bouillet, Leoneide E M; Ribeiro, Erica M C; Trópia, Maria M J; Alvarez, Florencia; Correa, Lygia F M; Mota, Bruno E F; Conceição, Luis Eduardo F R; Castro, Ieso M; Brandão, Rogelio L

    2015-03-01

    This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.

  20. Coriolus versicolor mushroom polysaccharides exert immunoregulatory effects on mouse B cells via membrane Ig and TLR-4 to activate the MAPK and NF-κB signaling pathways.

    PubMed

    Yang, Shu-fa; Zhuang, Tai-feng; Si, Yan-mei; Qi, Ke-yan; Zhao, Juan

    2015-03-01

    This study aimed to characterize the immunopotentiating effects and immune receptors for Coriolus versicolor mushroom polysaccharides (CVP), a Chinese medicinal fungus that exerts anti-tumor activities by enhancing host immunity. Proliferation assays were used to determine whether CVP could activate splenocytes. Flow cytometry analysis and IgM and IgG detection were used to characterize CVP-binding cells. Immune receptors were analyzed in immunoprecipitation and western blot assays. The downstream signaling pathways were identified by western blotting or immunostaining. CVP significantly stimulated the proliferation of mouse splenocytes. Fluorescence-labeled CVP (fl-CVP) selectively stained mouse B cells, but not T cells. CVP induced the production of IgM and IgG1 with or without exogenous IL-4. Membrane Ig (B cell antigen-receptor, BCR) was identified as a CVP-binding protein in immunoprecipitation and western blot experiments. CVP-induced B cell proliferation could be significantly inhibited by anti-mouse immunoglobulin (Ig) blocking antibody (Fab) or in cells from TLR4-mutant mice (C3H/HeJ). Phosphorylation of ERK-1/2 and p38 MAPK were clearly increased in a time-dependent manner, as was the nuclear translocation of the cytosolic NF-κB p65 subunit after CVP stimulation. Together, we demonstrate that CVP can bind and induce B cell activation using membrane Ig and TLR-4 as potential immune receptors. CVP activates mouse B cells through the MAPK and NF-κB signaling pathways.

  1. Investigation of Ion Channel Activities of Gramicidin A in the Presence of Ionic Liquids Using Model Cell Membranes

    PubMed Central

    Ryu, Hyunil; Lee, Hwankyu; Iwata, Seigo; Choi, Sangbaek; Ki Kim, Moon; Kim, Young-Rok; Maruta, Shinsaku; Min Kim, Sun; Jeon, Tae-Joon

    2015-01-01

    Ionic liquids (ILs) are considered to be green solvents because of their non-volatility. Although ILs are relatively safe in the atmospheric environment, they may be toxic in other environments. Our previous research showed that the cytotoxicity of ILs to biological organisms is attributable to interference with cell membranes by IL insertion. However, the effects of ILs on ion channels, which play important roles in cell homeostasis, have not been comprehensively studied to date. In this work, we studied the interactions between ILs and lipid bilayer membranes with gramicidin A ion channels. We used two methods, namely electrical and fluorescence measurements of ions that permeate the membrane. The lifetimes of channels were increased by all the ILs tested in this work via stabilizing the compressed structure of the lipid bilayer and the rate of ion flux through gA channels was decreased by changing the membrane surface charge. The former effect, which increased the rate of ion flux, was dominant at high salt concentrations, whereas the latter, which decreased the rate of ion flux, was dominant at low salt concentrations. The effects of ILs increased with increasing concentration and alkyl chain length. The experimental results were further studied using molecular dynamics simulations. PMID:26189604

  2. Investigation of Ion Channel Activities of Gramicidin A in the Presence of Ionic Liquids Using Model Cell Membranes.

    PubMed

    Ryu, Hyunil; Lee, Hwankyu; Iwata, Seigo; Choi, Sangbaek; Kim, Moon Ki; Kim, Young-Rok; Maruta, Shinsaku; Kim, Sun Min; Jeon, Tae-Joon

    2015-07-20

    Ionic liquids (ILs) are considered to be green solvents because of their non-volatility. Although ILs are relatively safe in the atmospheric environment, they may be toxic in other environments. Our previous research showed that the cytotoxicity of ILs to biological organisms is attributable to interference with cell membranes by IL insertion. However, the effects of ILs on ion channels, which play important roles in cell homeostasis, have not been comprehensively studied to date. In this work, we studied the interactions between ILs and lipid bilayer membranes with gramicidin A ion channels. We used two methods, namely electrical and fluorescence measurements of ions that permeate the membrane. The lifetimes of channels were increased by all the ILs tested in this work via stabilizing the compressed structure of the lipid bilayer and the rate of ion flux through gA channels was decreased by changing the membrane surface charge. The former effect, which increased the rate of ion flux, was dominant at high salt concentrations, whereas the latter, which decreased the rate of ion flux, was dominant at low salt concentrations. The effects of ILs increased with increasing concentration and alkyl chain length. The experimental results were further studied using molecular dynamics simulations.

  3. High-expression β(1) adrenergic receptor/cell membrane chromatography method based on a target receptor to screen active ingredients from traditional Chinese medicines.

    PubMed

    Yue, Yuan; Xue, Hui; Wang, Xin; Yang, Qian; Song, Yanhong; Li, Xiaoni

    2014-02-01

    β-Adrenergic receptors are important targets for drug discovery. We have developed a new β1 -adrenergic receptor cell membrane chromatography (β1 AR-CMC) with offline ultra-performance LC (UPLC) and MS method for screening active ingredients from traditional Chinese medicines. In this study, Chinese hamster ovary-S cells with high β1 AR expression levels were established and used to prepare a cell membrane stationary phase in a β1 AR-CMC model. The retention fractions were separated and identified by the UPLC-MS system. The screening results found that isoimperatorin from Rhizoma et Radix Notopterygii was the targeted component that could act on β1 AR in similar manner of metoprolol as a control drug. In addition, the biological effects of active component were also investigated in order to search for a new type of β1 AR antagonist. It will be a useful method for drug discovery as a leading compound resource.

  4. Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity.

    PubMed Central

    Szabò, I; Brutsche, S; Tombola, F; Moschioni, M; Satin, B; Telford, J L; Rappuoli, R; Montecucco, C; Papini, E; Zoratti, M

    1999-01-01

    The vacuolating toxin VacA, a major determinant of Helicobacter pylori-associated gastric diseases, forms anion-selective channels in artificial planar lipid bilayers. Here we show that VacA increases the anion permeability of the HeLa cell plasma membrane and determines membrane depolarization. Electrophysiological and pharmacological approaches indicated that this effect is due to the formation of low-conductance VacA pores in the cell plasma membrane and not to the opening of Ca(2+)- or volume-activated chloride channels. VacA-dependent increase of current conduction both in artificial planar lipid bilayers and in the cellular system was effectively inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), while2-[(2-cyclopentenyl-6,7dichloro-2, 3-dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy]acetic acid (IAA-94) was less effective. NPPB inhibited and partially reversed the vacuolation of HeLa cells and the increase of ion conductivity of polarized Madine Darby canine kidney cell monolayers induced by VacA, while IAA-94 had a weaker effect. We conclude that pore formation by VacA accounts for plasma membrane permeabilization and is required for both cell vacuolation and increase of trans-epithelial conductivity. PMID:10523296

  5. Membrane Cholesterol Affects Stimulus-Activity Coupling in Type 1, but not Type 2, CCK Receptors: Use of Cell Lines with Elevated Cholesterol

    PubMed Central

    Harikumar, Kaleeckal G.; Potter, Ross M.; Patil, Achyut; Echeveste, Valerie

    2013-01-01

    The lipid microenvironment of membrane proteins can affect their structure, function, and regulation. We recently described differential effects of acute modification of membrane cholesterol on the function of type 1 and 2 cholecystokinin (CCK) receptors. We now explore the regulatory impact of chronic cholesterol modification on these receptors using novel receptor-bearing cell lines with elevated membrane cholesterol. Stable CCK1R and CCK2R expression was established in clonal lines of 25RA cells having gain-of-function in SCAP [sterol regulatory element binding protein (SREBP) cleavage-activating protein] and SRD15 cells having deficiencies in Insig-1 and Insig-2 enzymes affecting HMG CoA reductase and SREBP. Increased cholesterol in the plasma membrane of these cells was directly demonstrated, and receptor binding and signaling characteristics were shown to reflect predicted effects on receptor function. In both environments, both types of CCK receptors were internalized and recycled normally in response to agonist occupation. No differences in receptor distribution within the membrane were appreciated at the light microscopic level in these CHO-derived cell lines. Fluorescence anisotropy was studied for these receptors occupied by fluorescent agonist and antagonist, as well as when tagged with YFP. These studies demonstrated increased anisotropy of the agonist ligand occupying the active state of the CCK1R in a cholesterol-enriched environment, mimicking fluorescence of the uncoupled, inactive state of this receptor, while there was no effect of increasing cholesterol on fluorescence at the CCK2R. These cell lines should be quite useful for examining the functional characteristics of potential drugs that might be used in an abnormal lipid environment. PMID:23306829

  6. The peculiar N- and (-termini of trichogin GA IV are needed for membrane interaction and human cell death induction at doses lacking antibiotic activity.

    PubMed

    Tavano, Regina; Malachin, Giulia; De Zotti, Marta; Peggion, Cristina; Biondi, Barbara; Forrnaggio, Fernando; Papini, Emanuele

    2015-01-01

    Peptaibiotics, non-ribosomally synthetized peptides from various ascomycetes, are uniquely characterized by dialkylated a-amino acids, a rigid heli cal conformation, and membrane permeation properties. Although generally considered as antimicrobial peptides, peptaibiotics may display other toxicological properties, and their function is in many cases unknown. With the goal to define the biological activity and selectivity of the peptaibiotictrichogin GA IV from the human opportunist Trichodenna longibrachiatum we analyzed its membrane interaction,cytotoxic activity and antibacterial effect. Trichogin GA IV effectively killed several types of healthy and neoplastic human cells at doses (EC 50%= 4-6 ~) lacking antibiotic effects on both Gram- and Gram+ bacteria(MIC > 64 ~ ). The peptaibiotic distinctive (-terminal primary alcohol was found to cooperate with theN-terminal n-octanoyl group to permeate the membrane phospholipid bilayer and to mediate effective binding and active endocytosis of trichogin GA IV in eukaryotic cells, two steps essential for cell death induction.Replacement of one Gly with Lys plus the simultaneous esterification of the (-terminus, strongly increased trichogin GA IV anti-Gram+ activity (MIC 1-4 ~ ). but further mitigated its cytotoxicity on human cells. PMID:25306964

  7. Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Sánchez-Mir, Laura; Franco, Alejandro; Martín-García, Rebeca; Madrid, Marisa; Vicente-Soler, Jero; Soto, Teresa; Gacto, Mariano; Pérez, Pilar

    2014-01-01

    The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade. PMID:24820419

  8. Tunicamycin depresses P-glycoprotein glycosylation without an effect on its membrane localization and drug efflux activity in L1210 cells.

    PubMed

    Sereš, Mário; Cholujová, Dana; Bubenčíkova, Tatiana; Breier, Albert; Sulová, Zdenka

    2011-01-01

    P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump.

  9. Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

    PubMed Central

    Šereš, Mário; Cholujová, Dana; Bubenčíkova, Tatiana; Breier, Albert; Sulová, Zdenka

    2011-01-01

    P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump. PMID:22174631

  10. CeO2 nanocubes-graphene oxide as durable and highly active catalyst support for proton exchange membrane fuel cell.

    PubMed

    Lei, M; Wang, Z B; Li, J S; Tang, H L; Liu, W J; Wang, Y G

    2014-01-01

    Rapid degradation of cell performance still remains a significant challenge for proton exchange membrane fuel cell (PEMFC). In this work, we develop novel CeO2 nanocubes-graphene oxide nanocomposites as durable and highly active catalyst support for proton exchange membrane fuel cell. We show that the use of CeO2 as the radical scavenger in the catalysts remarkably improves the durability of the catalyst. The catalytic activity retention of Pt-graphene oxide-8 wt.% CeO2 nanocomposites reaches as high as 69% after 5000 CV-cycles at a high voltage range of 0.8-1.23 V, in contrast to 19% for that of the Pt-graphene oxide composites. The excellent durability of the Pt-CeO2 nanocubes-graphene oxide catalyst is attributed to the free radical scavenging activity of CeO2, which significantly slows down the chemical degradation of Nafion binder in catalytic layers, and then alleviates the decay of Pt catalysts, resulting in the excellent cycle life of Pt-CeO2-graphene oxide nanocomposite catalysts. Additionally, the performance of single cell assembled with Nafion 211 membrane and Pt-CeO2-graphene oxide catalysts with different CeO2 contents in the cathode as well as the Pt-C catalysts in the anode are also recorded and discussed in this study.

  11. CeO2 nanocubes-graphene oxide as durable and highly active catalyst support for proton exchange membrane fuel cell

    PubMed Central

    Lei, M.; Wang, Z. B.; Li, J. S.; Tang, H. L.; Liu, W. J.; Wang, Y. G.

    2014-01-01

    Rapid degradation of cell performance still remains a significant challenge for proton exchange membrane fuel cell (PEMFC). In this work, we develop novel CeO2 nanocubes-graphene oxide nanocomposites as durable and highly active catalyst support for proton exchange membrane fuel cell. We show that the use of CeO2 as the radical scavenger in the catalysts remarkably improves the durability of the catalyst. The catalytic activity retention of Pt-graphene oxide-8 wt.% CeO2 nanocomposites reaches as high as 69% after 5000 CV-cycles at a high voltage range of 0.8–1.23 V, in contrast to 19% for that of the Pt-graphene oxide composites. The excellent durability of the Pt-CeO2 nanocubes-graphene oxide catalyst is attributed to the free radical scavenging activity of CeO2, which significantly slows down the chemical degradation of Nafion binder in catalytic layers, and then alleviates the decay of Pt catalysts, resulting in the excellent cycle life of Pt-CeO2-graphene oxide nanocomposite catalysts. Additionally, the performance of single cell assembled with Nafion 211 membrane and Pt-CeO2-graphene oxide catalysts with different CeO2 contents in the cathode as well as the Pt-C catalysts in the anode are also recorded and discussed in this study. PMID:25491655

  12. Enzymatically active Rho and Rac small-GTPases are involved in the establishment of the vacuolar membrane after Toxoplasma gondii invasion of host cells

    PubMed Central

    2013-01-01

    Background GTPases are the family of hydrolases that bind and hydrolyze guanosine triphosphate. The large Immunity-related GTPases and the small GTPase ADP-ribosylation factor-6 in host cells are known to accumulate on the parasitophorous vacuole membrane (PVM) of Toxoplasma gondii and play critical roles in this parasite infection, but these GTPases cannot explain the full extent of infection. Results In this research, RhoA and Rac1 GTPases from the host cell were found to accumulate on the PVM regardless of the virulence of the T. gondii strains after T. gondii invasion, and this accumulation was dependent on their GTPase activity. The real-time micrography of T. gondii tachyzoites invading COS-7 cells overexpressing CFP-RhoA showed that this GTPase was recruited to the PVM at the very beginning of the invasion through the host cell membrane or from the cytosol. Host cell RhoA and Rac1 were also activated after T. gondii tachyzoites invasion, which was needed for host cell cytoskeleton reorganization to facilitate intracellular pathogens invasion. The decisive domains for the RhoA accumulation on the PVM included the GTP/Mg2+ binding site, the mDia effector interaction site, the G1 box, the G2 box and the G5 box, respectively, which were related to the binding of GTP for enzymatic activity and mDia for the regulation of microtubules. The recruited CFP-RhoA on the PVM could not be activated by epithelial growth factor (EGF) and no translocation was observed, unlike the unassociated RhoA in the host cell cytosol that migrated to the cell membrane towards the EGF activation spot. This result supported the hypothesis that the recruited RhoA or Rac1 on the PVM were in the GTP-bound active form. Wild-type RhoA or Rac1 overexpressed cells had almost the same infection rates by T. gondii as the mock-treated cells, while RhoA-N19 or Rac1-N17 transfected cells and RhoA, Rac1 or RhoA + Rac1 siRNA-treated cells showed significantly diminished infection rates compared to mock

  13. The kinase, SH3, and SH2 domains of Lck play critical roles in T-cell activation after ZAP-70 membrane localization.

    PubMed Central

    Yamasaki, S; Takamatsu, M; Iwashima, M

    1996-01-01

    Antigenic stimulation of the T-cell antigen receptor initiates signal transduction through the immunoreceptor tyrosine-based activation motifs (ITAMs). When its two tyrosines are phosphorylated, ITAM forms a binding site for ZAP-70, one of the cytoplasmic protein tyrosine kinases essential for T-cell activation. The signaling process that follows ZAP-70 binding to ITAM has been analyzed by the construction of fusion proteins that localize ZAP-70 to the plasma membrane. We found that membrane-localized forms of ZAP-70 induce late signaling events such as activation of nuclear factor of activated T cells without any stimulation. This activity was observed only when Lck was expressed and functional. In addition, each mutation that affects the function of Lck in the kinase, Src homology 2 (SH2), and SH3 domains greatly impaired the signaling ability of the chimeric protein. Therefore, Lck functions in multiple manners in T-cell activation for the steps following ZAP-70 binding to ITAM. PMID:8943371

  14. GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

    PubMed

    Walker, M W; Bobak, D A; Tsai, S C; Moss, J; Vaughan, M

    1992-02-15

    ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.

  15. Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12.

    PubMed

    Milner, J L; Grothe, S; Wood, J M

    1988-10-15

    Proline porter II is rapidly activated when nongrowing bacteria are subjected to a hyperosmotic shift (Grothe, S., Krogsrud, R. L., McClellan, D. J., Milner, J. L., and Wood, J. M. (1986) J. Bacteriol. 166, 253-259). Proline porter II was active in membrane vesicles prepared from bacteria grown under optimal conditions, nutritional stress, or osmotic stress. That activity was: (i) dependent on the presence of the energy sources phenazine methosulphate plus ascorbate or D-lactate; (ii) observed only when a hyperosmotic shift accompanied the transport measurement; (iii) inhibited by glycine betaine in a manner analogous to that observed in whole cells; and (iv) eliminated by lesions in proP. Membrane vesicles were able to transport serine but not glutamine and serine transport was reduced by the hyperosmotic shift. In whole cells, proline porter II activity was supported by glucose and by D-lactate in a strain defective for proline porters I and III and the F1F0-ATPase. Glucose energized proline uptake was eliminated by carbonyl cyanide m-chlorophenylhydrazone and KCN as was serine uptake. These results suggested that proline porter II was respiration-dependent and probably ion-linked. Activation of proline porter II in whole cells by sucrose or NaCl was sustained over 30 min, whereas activation by glycerol was transient. Proline porter II was activated by NaCl and sucrose with a half-time of approximately 1 min in both whole cells and membrane vesicles. Thus, activation of proline porter II was reversible. It occurred at a rate comparable to that of K+ influx and much more rapid than the genetic regulatory responses that follow a hyperosmotic shift. PMID:3049595

  16. Strategies for cell membrane functionalization

    PubMed Central

    Armstrong, James PK

    2016-01-01

    The ability to rationally manipulate and augment the cytoplasmic membrane can be used to overcome many of the challenges faced by conventional cellular therapies and provide innovative opportunities when combined with new biotechnologies. The focus of this review is on emerging strategies used in cell functionalization, highlighting both pioneering approaches and recent developments. These will be discussed within the context of future directions in this rapidly evolving field. PMID:27229904

  17. Progestin-mediated activation of MAPK and AKT in nuclear progesterone receptor negative breast epithelial cells: The role of membrane progesterone receptors.

    PubMed

    Salazar, Monica; Lerma-Ortiz, Alejandra; Hooks, Grace M; Ashley, Amanda K; Ashley, Ryan L

    2016-10-10

    Progesterone (P4), a steroid produced during estrous cycles and gestation for maintenance of pregnancy, also plays key roles in breast development to allow lactation post-parturition. Progestins (P4 and related steroids) are also implicated in breast cancer etiology. Hormone replacement therapy containing both estrogen and progestins increases breast cancer incidence while estrogen hormone therapy lowers breast cancer risk. P4 signaling via nuclear P4 receptors (PRs) has been extensively studied in breast cancer, however, progestin signaling via non-classical membrane bound progestin receptors (MPRs and PGRMC1) remains unclear. Moreover, P4 metabolites and synthetic progestins may bind membrane progestin receptors. We hypothesized that PR-negative breast epithelial cells express non-classical progestin receptors, which activate intracellular signaling pathways differently depending on nature of progestin. Therefore, our objectives for the current study were to determine expression of MPRs and PGRMC1 in two PR-negative non-tumorigenic breast epithelial cell lines, assess progestin-mediated signaling and biological functions. We determined five MPR isoforms and PGRMC1 were present in MCF10A cells and all progestin receptors but MPRβ in MCF12A cells. MCF10A and MCF12A cells were treated with P4, select P4 metabolites (5αP and 3αHP), medroxyprogesterone acetate (MPA), or a specific MPR-Agonist (MPR-Ag) and phosphorylation of ERK, p38, JNK, and AKT was characterized following treatment. To our knowledge this is the first report of ERK and JNK activation in MCF10A and MCF12A cells with P4, P4 metabolites, MPA, and MPR-Ag. Activation of ERK and JNK in cells treated with MPR-Ag implicates MPRs may serve as the receptors responsible for their activation. In contrast, p38 activation varied with cell type and with progestin treatment. P4 and MPA promoted AKT phosphorylation in the MCF12A cell line only whereas no activation was observed in MCF10A cells. Interestingly

  18. Cu2+ ions interact with cell membranes.

    PubMed

    Suwalsky, M; Ungerer, B; Quevedo, L; Aguilar, F; Sotomayor, C P

    1998-07-01

    The influence of Cu2+ ions on the physical properties of resealed human erythrocyte membranes was studied by fluorescence spectroscopy. A net ordering effect was observed at the hydrophobic-hydrophilic interface both in the bulk as well as in the lipid-protein boundary. The explanation for this result was found by X-ray diffraction performed in multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Cu2+ did not significantly affect the structure of DMPE; however, DMPC polar head and hydrocarbon chain arrangements were perturbed at low but reordered at high Cu2+ concentrations. These effects were respectively explained in terms of a limited and extended interaction between Cu2+ ions and DMPC PO4 groups. Thus, the ordering effect in the erythrocyte membrane could be based on the interaction of this cation with phosphatidylcholine phosphate groups located in its outer leaflet. This binding, besides producing a decrease of membrane fluidity, might also induce a change in its electric field. These two effects should affect the activity of membrane proteins, particularly of ion channels. In fact, it was found that increasing concentrations of Cu2+ ions applied to either the mucosal or serosal surface of the isolated toad skin elicited a dose-dependent decrease of the short-circuit current (SCC) and of the potential difference (PD). These results lead to the conclusion that Cu2+ ions inhibited Na+ transport across the epithelial cell membranes. PMID:9720309

  19. Rack1 is required for Vangl2 membrane localization and planar cell polarity signaling while attenuating canonical Wnt activity

    PubMed Central

    Li, Shuangding; Esterberg, Robert; Lachance, Veronik; Ren, Dongdong; Radde-Gallwitz, Kristen; Chi, Fanglu; Parent, Jean-Luc; Fritz, Andreas; Chen, Ping

    2011-01-01

    The vertebrate planar cell polarity (PCP) pathway shares molecular components with the β-catenin–mediated canonical Wnt pathway but acts through membrane complexes containing Vang or Frizzled to orient neighboring cells coordinately. The molecular interactions underlying the action of Vang in PCP signaling and specification, however, are yet to be delineated. Here, we report the identification of Rack1 as an interacting protein of a vertebrate Vang protein, Vangl2. We demonstrate that Rack1 is required in zebrafish for PCP-regulated processes, including oriented cell division, cellular polarization, and convergent extension during gastrulation. We further show that the knockdown of Rack1 affects membrane localization of Vangl2 and that the Vangl2-interacting domain of Rack1 has a dominant-negative effect on Vangl2 localization and gastrulation. Moreover, Rack1 antagonizes canonical Wnt signaling. Together, our data suggest that Rack1 regulates the localization of an essential PCP protein and acts as a molecular switch to promote PCP signaling. PMID:21262816

  20. Physical principles of membrane remodelling during cell mechanoadaptation

    PubMed Central

    Kosmalska, Anita Joanna; Casares, Laura; Elosegui-Artola, Alberto; Thottacherry, Joseph Jose; Moreno-Vicente, Roberto; González-Tarragó, Víctor; del Pozo, Miguel Ángel; Mayor, Satyajit; Arroyo, Marino; Navajas, Daniel; Trepat, Xavier; Gauthier, Nils C.; Roca-Cusachs, Pere

    2015-01-01

    Biological processes in any physiological environment involve changes in cell shape, which must be accommodated by their physical envelope—the bilayer membrane. However, the fundamental biophysical principles by which the cell membrane allows for and responds to shape changes remain unclear. Here we show that the 3D remodelling of the membrane in response to a broad diversity of physiological perturbations can be explained by a purely mechanical process. This process is passive, local, almost instantaneous, before any active remodelling and generates different types of membrane invaginations that can repeatedly store and release large fractions of the cell membrane. We further demonstrate that the shape of those invaginations is determined by the minimum elastic and adhesive energy required to store both membrane area and liquid volume at the cell–substrate interface. Once formed, cells reabsorb the invaginations through an active process with duration of the order of minutes. PMID:26073653

  1. Active membrane having uniform physico-chemically functionalized ion channels

    DOEpatents

    Gerald, II, Rex E; Ruscic, Katarina J; Sears, Devin N; Smith, Luis J; Klingler, Robert J; Rathke, Jerome W

    2012-09-24

    The present invention relates to a physicochemically-active porous membrane for electrochemical cells that purports dual functions: an electronic insulator (separator) and a unidirectional ion-transporter (electrolyte). The electrochemical cell membrane is activated for the transport of ions by contiguous ion coordination sites on the interior two-dimensional surfaces of the trans-membrane unidirectional pores. One dimension of the pore surface has a macroscopic length (1 nm-1000 .mu.m) and is directed parallel to the direction of an electric field, which is produced between the cathode and the anode electrodes of an electrochemical cell. The membrane material is designed to have physicochemical interaction with ions. Control of the extent of the interactions between the ions and the interior pore walls of the membrane and other materials, chemicals, or structures contained within the pores provides adjustability of the ionic conductivity of the membrane.

  2. Correlation of cell membrane dynamics and cell motility

    PubMed Central

    2011-01-01

    Background Essential events of cell development and homeostasis are revealed by the associated changes of cell morphology and therefore have been widely used as a key indicator of physiological states and molecular pathways affecting various cellular functions via cytoskeleton. Cell motility is a complex phenomenon primarily driven by the actin network, which plays an important role in shaping the morphology of the cells. Most of the morphology based features are approximated from cell periphery but its dynamics have received none to scant attention. We aim to bridge the gap between membrane dynamics and cell states from the perspective of whole cell movement by identifying cell edge patterns and its correlation with cell dynamics. Results We present a systematic study to extract, classify, and compare cell dynamics in terms of cell motility and edge activity. Cell motility features extracted by fitting a persistent random walk were used to identify the initial set of cell subpopulations. We propose algorithms to extract edge features along the entire cell periphery such as protrusion and retraction velocity. These constitute a unique set of multivariate time-lapse edge features that are then used to profile subclasses of cell dynamics by unsupervised clustering. Conclusions By comparing membrane dynamic patterns exhibited by each subclass of cells, correlated trends of edge and cell movements were identified. Our findings are consistent with published literature and we also identified that motility patterns are influenced by edge features from initial time points compared to later sampling intervals. PMID:22372978

  3. Different activities of the reovirus FAST proteins and influenza hemagglutinin in cell-cell fusion assays and in response to membrane curvature agents

    SciTech Connect

    Clancy, Eileen K.; Barry, Chris; Ciechonska, Marta; Duncan, Roy

    2010-02-05

    The reovirus fusion-associated small transmembrane (FAST) proteins evolved to induce cell-cell, rather than virus-cell, membrane fusion. It is unclear whether the FAST protein fusion reaction proceeds in the same manner as the enveloped virus fusion proteins. We now show that fluorescence-based cell-cell and cell-RBC hemifusion assays are unsuited for detecting lipid mixing in the absence of content mixing during FAST protein-mediated membrane fusion. Furthermore, membrane curvature agents that inhibit hemifusion or promote pore formation mediated by influenza hemagglutinin had no effect on p14-induced cell-cell fusion, even under conditions of limiting p14 concentrations. Standard assays used to detect fusion intermediates induced by enveloped virus fusion proteins are therefore not applicable to the FAST proteins. These results suggest the possibility that the nature of the fusion intermediates or the mechanisms used to transit through the various stages of the fusion reaction may differ between these distinct classes of viral fusogens.

  4. Conjugation of succinic acid to non-ionogenic amphiphilic polymers modulates their interaction with cell plasma membrane and reduces cytotoxic activity.

    PubMed

    Bondar, O V; Sagitova, A V; Badeev, Y V; Shtyrlin, Y G; Abdullin, T I

    2013-09-01

    Pluronic block copolymers L61 and L121 were reacted with succinic anhydride to produce, respectively, their mono- and bisderivatives with succinic acid. The critical micelle concentration of Pluronics decreased after modification. The modification of Pluronic L61 promoted its association with the plasma membrane of human cells and increased membrane damage, while the membranotropic activity of modified Pluronic L121 reduced compared to the initial copolymer. Modified Pluronics interfered with the viability, apoptosis induction and metabolism of A549 cells and skin fibroblasts to a much lesser extent presumably due to the introduction of succinic acid residue inhibited intracellular penetration of copolymers. Modified Pluronic L121 promoted the cellular uptake of doxorubicin and rhodamine 123 in A549 cells attributed to the inhibition of membrane P-glycoprotein. Our study provides an approach to assessing the mechanism of interaction of amphiphilic polymers with living cells and demonstrates that Pluronic-succinic acid conjugates can be used as safe and efficient modulators of intracellular drug delivery.

  5. Fuel-Cell Structure Prevents Membrane Drying

    NASA Technical Reports Server (NTRS)

    Mcelroy, J.

    1986-01-01

    Embossed plates direct flows of reactants and coolant. Membrane-type fuel-cell battery has improved reactant flow and heat removal. Compact, lightweight battery produces high current and power without drying of membranes.

  6. Fluid transport by active elastic membranes

    NASA Astrophysics Data System (ADS)

    Evans, Arthur A.; Lauga, Eric

    2011-09-01

    A flexible membrane deforming its shape in time can self-propel in a viscous fluid. Alternatively, if the membrane is anchored, its deformation will lead to fluid transport. Past work in this area focused on situations where the deformation kinematics of the membrane were prescribed. Here we consider models where the deformation of the membrane is not prescribed, but instead the membrane is internally forced. Both the time-varying membrane shape and the resulting fluid motion result then from a balance between prescribed internal active stresses, internal passive resistance, and external viscous stresses. We introduce two specific models for such active internal forcing: one where a distribution of active bending moments is prescribed, and one where active inclusions exert normal stresses on the membrane by pumping fluid through it. In each case, we asymptotically calculate the membrane shape and the fluid transport velocities for small forcing amplitudes, and recover our results using scaling analysis.

  7. Vesicle associated membrane protein (VAMP)-7 and VAMP-8, but not VAMP-2 or VAMP-3, are required for activation-induced degranulation of mature human mast cells.

    PubMed

    Sander, Leif E; Frank, Simon P C; Bolat, Seza; Blank, Ulrich; Galli, Thierry; Bigalke, Hans; Bischoff, Stephan C; Lorentz, Axel

    2008-03-01

    Mediator release from mast cells (MC) is a crucial step in allergic and non-allergic inflammatory disorders. However, the final events in response to activation leading to membrane fusion and thereby facilitating degranulation have hitherto not been analyzed in human MC. Soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNARE) represent a highly conserved family of proteins that have been shown to mediate intracellular membrane fusion events. Here, we show that mature MC isolated from human intestinal tissue express soluble N-ethylmaleide sensitive factor attachment protein (SNAP)-23, Syntaxin (STX)-1B, STX-2, STX-3, STX-4, and STX-6 but not SNAP-25. Furthermore, we found that primary human MC express substantial amounts of vesicle associated membrane protein (VAMP)-3, VAMP-7 and VAMP-8 and, in contrast to previous reports about rodent MC, only low levels of VAMP-2. Furthermore, VAMP-7 and VAMP-8 were found to translocate to the plasma membrane and interact with SNAP-23 and STX-4 upon activation. Inhibition of SNAP-23, STX-4, VAMP-7 or VAMP-8, but not VAMP-2 or VAMP-3, resulted in a markedly reduced high-affinity IgE receptor-mediated histamine release. In summary, our data show that mature human MC express a specific pattern of SNARE and that VAMP-7 and VAMP-8, but not VAMP-2, are required for rapid degranulation.

  8. Actinide transport across cell membranes.

    PubMed

    Bulman, R A; Griffin, R J

    1980-01-01

    Protactinium uptake into the normal liver does not exceed 3%, but when the phospholipid levels in the liver are elevated by administration of thioacetamide this uptake increases to 31%. Phosphatidic acid, which is absent from the normal liver, has been shown to extract protactinium into organic solvents. However, phosphatidylserine, a component of normal liver cell membranes, does not extract protactinium. It might be conjectured that this is why so little protactinium is taken up by the normal liver. The hypothesis is advanced that phosphatidylserine, which is known to complex plutonium, americium and curium, may regulate the uptake of these elements by liver.

  9. Actinide transport across cell membranes.

    PubMed

    Bulman, R A; Griffin, R J

    1980-01-01

    Protactinium uptake into the normal liver does not exceed 3%, but when the phospholipid levels in the liver are elevated by administration of thioacetamide this uptake increases to 31%. Phosphatidic acid, which is absent from the normal liver, has been shown to extract protactinium into organic solvents. However, phosphatidylserine, a component of normal liver cell membranes, does not extract protactinium. It might be conjectured that this is why so little protactinium is taken up by the normal liver. The hypothesis is advanced that phosphatidylserine, which is known to complex plutonium, americium and curium, may regulate the uptake of these elements by liver. PMID:7373293

  10. The application of Dow Chemical's perfluorinated membranes in proton-exchange membrane fuel cells

    NASA Technical Reports Server (NTRS)

    Eisman, G. A.

    1989-01-01

    Dow Chemical's research activities in fuel cells revolve around the development of perfluorosulfonic acid membranes useful as the proton transport medium and separator. Some of the performance characteristics which are typical for such membranes are outlined. The results of tests utilizing a new experimental membrane useful in proton-exchange membrane fuel cells are presented. The high voltage at low current densities can lead to higher system efficiencies while, at the same time, not sacrificing other critical properties pertinent to membrane fuel cell operation. A series of tests to determine response times indicated that on-off cycles are on the order of 80 milliseconds to reach 90 percent of full power. The IR free voltage at 100 amps/sq ft was determined and the results indicating a membrane/electrode package resistance to be .15 ohm-sq cm at 100 amps/sq ft.

  11. Chemical degradation mechanisms of membranes for alkaline membrane fuel cells

    SciTech Connect

    Choe, Yoong-Kee; Henson, Neil J.; Kim, Yu Seung

    2015-12-31

    Chemical degradation mechanisms of membranes for alkaline membrane fuel cells have been investigated using density functional theory (DFT). We have elucidated that the aryl-ether moiety of membranes is one of the weakest site against attack of hydroxide ions. The results of DFT calculations for hydroxide initiated aryl-ether cleavage indicated that the aryl-ether cleavage occurred prior to degradation of cationic functional group. Such a weak nature of the aryl-ether group arises from the electron deficiency of the aryl group as well as the low bond dissociation energy. The DFT results suggests that removal of the aryl-ether group in the membrane should enhance the stability of membranes under alkaline conditions. In fact, an ether fee poly(phenylene) membrane exhibits excellent stability against the attack from hydroxide ions.

  12. In-membrane micro fuel cell

    DOEpatents

    Omosebi, Ayokunle; Besser, Ronald

    2016-09-06

    An in-membrane micro fuel cell comprises an electrically-insulating membrane that is permissive to the flow of cations, such as protons, and a pair of electrodes deposited on channels formed in the membrane. The channels are arranged as conduits for fluids, and define a membrane ridge between the channels. The electrodes are porous and include catalysts for promoting the liberation of a proton and an electron from a chemical species and/or or the recombination of a proton and an electron with a chemical specie. The fuel cell may be provided a biosensor, an electrochemical sensor, a microfluidic device, or other microscale devices fabricated in the fuel cell membrane.

  13. Calcium efflux activity of plasma membrane Ca2+ ATPase-4 (PMCA4) mediates cell cycle progression in vascular smooth muscle cells.

    PubMed

    Afroze, Talat; Yang, Ge; Khoshbin, Amir; Tanwir, Mansoor; Tabish, Taha; Momen, Abdul; Husain, Mansoor

    2014-03-01

    We explored the role played by plasma membrane calcium ATPase-4 (PMCA4) and its alternative splice variants in the cell cycle of vascular smooth muscle cells (VSMC). A novel variant (PMCA4e) was discovered. Quantitative real-time-PCR-quantified PMCA4 splice variant proportions differed in specific organs. The PMCA4a:4b ratio in uninjured carotid arteries (∼1:1) was significantly reduced by wire denudation injury (to ∼1:3) by modulation of alternative splicing, as confirmed by novel antibodies against PMCA4a/e and PMCA4b. Laser capture microdissection localized this shift to the media and adventitia. Primary carotid VSMC from PMCA4 knock-out (P4KO) mice showed impaired [(3)H]thymidine incorporation and G1 phase arrest as compared with wild type (P4WT). Electroporation of expression constructs encoding PMCA4a, PMCA4b, and a PMCA4b mutant lacking PDZ binding rescued this phenotype of P4KO cells, whereas a mutant with only 10% of normal Ca(2+) efflux activity could not. Microarray of early G1-synchronized VSMC showed 39-fold higher Rgs16 (NFAT (nuclear factor of activated T-cells) target; MAPK inhibitor) and 69-fold higher Decorin (G1 arrest marker) expression in P4KO versus P4WT. Validation by Western blot also revealed decreased levels of Cyclin D1 and NFATc3 in P4KO. Microarrays of P4KO VSMC rescued by PMCA4a or PMCA4b expression showed reversal of perturbed Rgs16, Decorin, and NFATc3 expression levels. However, PMCA4a rescue caused a 44-fold reduction in AP-2β, a known anti-proliferative transcription factor, whereas PMCA4b rescue resulted in a 50-fold reduction in p15 (Cyclin D1/Cdk4 inhibitor). We conclude that Ca(2+) efflux activity of PMCA4 underlies G1 progression in VSMC and that PMCA4a and PMCA4b differentially regulate specific downstream mediators. PMID:24448801

  14. Current density and catalyst-coated membrane resistance distribution of hydro-formed metallic bipolar plate fuel cell short stack with 250 cm2 active area

    NASA Astrophysics Data System (ADS)

    Haase, S.; Moser, M.; Hirschfeld, J. A.; Jozwiak, K.

    2016-01-01

    An automotive fuel cell with an active area of 250 cm2 is investigated in a 4-cell short stack with a current and temperature distribution device next to the bipolar plate with 560 current and 140 temperature segments. The electrical conductivities of the bipolar plate and gas diffusion layer assembly are determined ex-situ with this current scan shunt module. The applied fuel cell consists of bipolar plates constructed of 75-μm-thick, welded stainless-steel foils and a graphitic coating. The electrical conductivities of the bipolar plate and gas diffusion layer assembly are determined ex-situ with this module with a 6% deviation in in-plane conductivity. The current density distribution is evaluated up to 2.4 A cm-2. The entire cell's investigated volumetric power density is 4.7 kW l-1, and its gravimetric power density is 4.3 kW kg-1 at an average cell voltage of 0.5 V. The current density distribution is determined without influencing the operating cell. In addition, the current density distribution in the catalyst-coated membrane and its effective resistivity distribution with a finite volume discretisation of Ohm's law are evaluated. The deviation between the current density distributions in the catalyst-coated membrane and the bipolar plate is determined.

  15. In-situ electrochemically active surface area evaluation of an open-cathode polymer electrolyte membrane fuel cell stack

    NASA Astrophysics Data System (ADS)

    Torija, Sergio; Prieto-Sanchez, Laura; Ashton, Sean J.

    2016-09-01

    The ability to evaluate the electrochemically active surface area (ECSA) of fuel cell electrodes is crucial toward characterising designs and component suites in-situ, particularly when evaluating component durability in endurance testing, since it is a measure of the electrode area available to take part in the fuel cell reactions. Conventional methods to obtain the ECSA using cyclic voltammetry, however, rely on potentiostats that cannot be easily scaled to simultaneously evaluate all cells in a fuel cell stack of practical size, which is desirable in fuel cell development. In-situ diagnostics of an open-cathode fuel cell stack are furthermore challenging because the cells do not each possess an enclosed cathode compartment; instead, the cathodes are rather open to the environment. Here we report on a diagnostic setup that allows the electrochemically active surface area of each cell anode or cathode in an open-cathode fuel cell stack to be evaluated in-situ and simultaneously, with high resolution and reproducibility, using an easily scalable chronopotentiometry methodology and a gas-tight stack enclosure.

  16. Nonhumidified High-Temperature Membranes Developed for Proton Exchange Membrane Fuel Cells

    NASA Technical Reports Server (NTRS)

    Kinder, James D.

    2005-01-01

    Fuel cells are being considered for a wide variety of aerospace applications. One of the most versatile types of fuel cells is the proton-exchange-membrane (PEM) fuel cell. PEM fuel cells can be easily scaled to meet the power and space requirements of a specific application. For example, small 100-W PEM fuel cells are being considered for personal power for extravehicular activity suit applications, whereas larger PEM fuel cells are being designed for primary power in airplanes and in uninhabited air vehicles. Typically, PEM fuel cells operate at temperatures up to 80 C. To increase the efficiency and power density of the fuel cell system, researchers are pursuing methods to extend the operating temperature of the PEM fuel cell to 180 C. The most widely used membranes in PEM fuel cells are Nafion 112 and Nafion 117--sulfonated perfluorinated polyethers that were developed by DuPont. In addition to their relatively high cost, the properties of these membranes limit their use in a PEM fuel cell to around 80 C. The proton conductivity of Nafion membranes significantly decreases above 80 C because the membrane dehydrates. The useful operating range of Nafion-based PEM fuel cells can be extended to over 100 C if ancillary equipment, such as compressors and humidifiers, is added to maintain moisture levels within the membrane. However, the addition of these components reduces the power density and increases the complexity of the fuel cell system.

  17. Evaluation of potential implication of membrane estrogen binding sites on ERE-dependent transcriptional activity and intracellular estrogen receptor-alpha regulation in MCF-7 breast cancer cells.

    PubMed

    Seo, Hye Sook; Leclercq, Guy

    2002-01-01

    The potential involvement of membrane estrogen binding sites in the induction of ERE-dependent transcriptional activity as well as in the regulation of intracellular estrogen receptor alpha (ER-alpha) level under estradiol (E2) stimulation was investigated. Our approach relied upon the use of two DCC-treated E2-BSA (bovine serum albumin) solutions (E2-6-BSA and E2-17-BSA). The absence of detectable free E2 in these solutions was established. Both E2-BSA conjugates led to a transient dose-dependent stimulation of the expression of ERE-luciferase (LUC) reporter gene in MVLN cells (MCF-7 cells stably transfected with a pVit-tk-LUC reporter plasmid), a property not recorded with free E2, which maintained enhanced transcriptional activity during the whole experiment. A very low concentration of E2 (10 pM) synergistically acted with E2-BSA conjugates. Hence, ERE-dependent transcriptional activity induced by these conjugates appeared to result from their known interactions with membrane estrogen binding sites. Anti-estrogens (AEs: 4-OH-TAM and RU 58,668), which antagonize genomic ER responses, abrogated the luciferase activity induced by E2-BSA conjugates, confirming a potential relationship between membrane-related signals and intracellular ER. Moreover, induction of luciferase was recorded when the cells were exposed to IBMX (3-isobutyl-1-methylxanthine) and cyclic nucleotides (cAMP/cGMP), suggesting the implication of the latter in the signal transduction pathway leading to the expression of the reporter gene. Growth factors (IGF-I, EGF and TGF-alpha) also slightly stimulated luciferase and synergistically acted with 10 pM E2, or 1 microM E2-BSA conjugates, in agreement with the concept of a cross-talk between steroids and peptides acting on the cell membrane. Remarkably, E2-BSA conjugates, IBMX and all investigated growth factors failed to down-regulate intracellular ER in MCF-7 cells, indicating the need for a direct intracellular interaction of the ligand with the

  18. Self-enhanced targeted delivery of a cell wall- and membrane-active antibiotics, daptomycin, against staphylococcal pneumonia.

    PubMed

    Jiang, Hong; Xiong, Meimei; Bi, Qiuyan; Wang, Ying; Li, Chong

    2016-07-01

    Considering that some antibacterial agents can identify the outer structure of pathogens like cell wall and/or cell membrane, we explored a self-enhanced targeted delivery strategy by which a small amount of the antibiotic molecules were modified on the surface of carriers as targeting ligands of certain bacteria while more antibiotic molecules were loaded inside the carriers, and thus has the potential to improve the drug concentration at the infection site, enhance efficacy and reduce potential toxicity. In this study, a novel targeted delivery system against methicillin-resistant Staphylococcus aureus (MRSA) pneumonia was constructed with daptomycin, a lipopeptide antibiotic, which can bind to the cell wall of S. aureus via its hydrophobic tail. Daptomycin was conjugated with N-hydroxysuccinimidyl-polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphoethanolamine to synthesize a targeting compound (Dapt-PEG-DSPE) which could be anchored on the surface of liposomes, while additional daptomycin molecules were encapsulated inside the liposomes. These daptomycin-modified, daptomycin-loaded liposomes (DPD-L[D]) showed specific binding to MRSA as detected by flow cytometry and good targeting capabilities in vivo to MRSA-infected lungs in a pneumonia model. DPD-L[D] exhibited more favorable antibacterial efficacy against MRSA than conventional PEGylated liposomal daptomycin both in vitro and in vivo. Our study demonstrates that daptomycin-modified liposomes can enhance MRSA-targeted delivery of encapsulated antibiotic, suggesting a novel drug delivery approach for existing antimicrobial agents. PMID:27471672

  19. Live-cell imaging of receptors around postsynaptic membranes.

    PubMed

    Tanaka, Hiromitsu; Fujii, Shumpei; Hirano, Tomoo

    2014-01-01

    This protocol describes how to image the trafficking of glutamate receptors around excitatory postsynaptic membrane formed on an adhesion protein-coated glass surface. The protocol was developed to clarify how receptors move during the induction of synaptic plasticity. Dissociated neurons are cultured on a coverslip coated with neurexin, which induces the formation of postsynaptic membrane-like structures on the glass surface. A glutamate receptor tagged with a fluorescent protein is then transfected into neurons, and it is observed with total internal reflection fluorescence microscopy. The whole process takes about 3 weeks. Changes in the amount of cell-surface receptors caused by neuronal activities can be quantified, and individual exocytosis events of receptors can be clearly observed around the pseudo-postsynaptic membrane. This protocol has potential applications for studies of movements of membrane proteins around other specialized regions of the cell membrane, such as the inhibitory postsynaptic membrane, the presynaptic membrane or the immunological synapses.

  20. Nanoparticles meet cell membranes: probing nonspecific interactions using model membranes.

    PubMed

    Chen, Kai Loon; Bothun, Geoffrey D

    2014-01-21

    Nanotoxicity studies have shown that both carbon-based and inorganic engineered nanoparticles can be toxic to microorganisms. Although the pathways for cytotoxicity are diverse and dependent upon the nature of the engineered nanoparticle and the chemical environment, numerous studies have provided evidence that direct contact between nanoparticles and bacterial cell membranes is necessary for cell inactivation or damage, and may in fact be a primary mechanism for cytotoxicity. The propensities for nanoparticles to attach to and disrupt cell membranes are still not well understood due to the heterogeneous and dynamic nature of biological membranes. Model biological membranes can be employed for systematic investigations of nanoparticle-membrane interactions. In this article, current and emerging experimental approaches to identify the key parameters that control the attachment of ENPs on model membranes and the disruption of membranes by ENPs will be discussed. This critical information will help enable the "safe-by-design" production of engineered nanoparticles that are nontoxic or biocompatible, and also allow for the design of antimicrobial nanoparticles for environmental and biomedical applications.

  1. Polymer electrolyte membrane assembly for fuel cells

    NASA Technical Reports Server (NTRS)

    Yen, Shiao-Ping S. (Inventor); Kindler, Andrew (Inventor); Yavrouian, Andre (Inventor); Halpert, Gerald (Inventor)

    2002-01-01

    An electrolyte membrane for use in a fuel cell can contain sulfonated polyphenylether sulfones. The membrane can contain a first sulfonated polyphenylether sulfone and a second sulfonated polyphenylether sulfone, wherein the first sulfonated polyphenylether and the second sulfonated polyphenylether sulfone have equivalent weights greater than about 560, and the first sulfonated polyphenylether and the second sulfonated polyphenylether sulfone also have different equivalent weights. Also, a membrane for use in a fuel cell can contain a sulfonated polyphenylether sulfone and an unsulfonated polyphenylether sulfone. Methods for manufacturing a membrane electrode assemblies for use in fuel cells can include roughening a membrane surface. Electrodes and methods for fabricating such electrodes for use in a chemical fuel cell can include sintering an electrode. Such membranes and electrodes can be assembled into chemical fuel cells.

  2. Polymer electrolyte membrane assembly for fuel cells

    NASA Technical Reports Server (NTRS)

    Yen, Shiao-Ping S. (Inventor); Kindler, Andrew (Inventor); Yavrouian, Andre (Inventor); Halpert, Gerald (Inventor)

    2000-01-01

    An electrolyte membrane for use in a fuel cell can contain sulfonated polyphenylether sulfones. The membrane can contain a first sulfonated polyphenylether sulfone and a second sulfonated polyphenylether sulfone, wherein the first sulfonated polyphenylether and the second sulfonated polyphenylether sulfone have equivalent weights greater than about 560, and the first sulfonated polyphenylether and the second sulfonated polyphenylether sulfone also have different equivalent weights. Also, a membrane for use in a fuel cell can contain a sulfonated polyphenylether sulfone and an unsulfonated polyphenylether sulfone. Methods for manufacturing a membrane electrode assemblies for use in fuel cells can include roughening a membrane surface. Electrodes and methods for fabricating such electrodes for use in a chemical fuel cell can include sintering an electrode. Such membranes and electrodes can be assembled into chemical fuel cells.

  3. Serum factors, cell membrane CD14, and beta2 integrins are not required for activation of bovine macrophages by lipopolysaccharide.

    PubMed Central

    Jungi, T W; Sager, H; Adler, H; Brcic, M; Pfister, H

    1997-01-01

    The role of serum factors such as lipopolysaccharide (LPS)-binding protein (LBP) and of macrophage-expressed CD14 and beta2 integrins in the activation of bovine macrophages by LPS was investigated. Macrophage activation was determined by measuring tumor necrosis factor production, NO generation, and upregulation of procoagulant activity by LPS (Escherichia coli O55:B5) at concentrations of 100 pg/ml to 100 ng/ml. The 50% effective dose for LPS was 1 order of magnitude higher than that for activating human macrophages. Macrophages were activated by LPS in the presence of serum or in the presence of albumin demonstrated to be free of LBP. The capacity to react to LPS in the absence of LBP was not due to the acquisition of LBP during a previous culture in serum. It was then established which CD14-specific antibodies block LPS binding to monocytes. Among the CD14-specific antibodies recognizing bovine mononuclear phagocytes (60bca, 3C10, My4, CAM36, VPM65, CMRF31, and TUK4), the first four blocked the binding of LPS-fluorescein isothiocyanate to bovine monocytes at low concentrations. Anti-CD14 antibodies did not block LPS-mediated activation of bovine bone marrow-derived macrophages, monocyte-derived macrophages, and alveolar macrophages. This was observed in experiments in which anti-CD14 concentrations exceeded the 50% inhibitory dose by >30-fold (3C10 and My4) or >300-fold (60bca), as defined in the binding assay described above. Monocyte-derived macrophages from an animal deficient in beta2 integrins and control macrophages were activated by similar concentrations of LPS, suggesting that beta2 integrins are not important bovine LPS receptors. Thus, in bovine macrophages, LPS recognition pathways which are independent of exogenous LBP, of membrane-expressed CD14, and of beta2 integrins may exist. PMID:9284122

  4. Fuel cell ion-exchange membrane investigation

    NASA Technical Reports Server (NTRS)

    Toy, M. S.

    1972-01-01

    The present deficiencies in the fluorocarbon sulfonic acid membrane used as the solid polymer electrolyte in the H2/O2 fuel cell are studied. Considered are: Adhesives selection, elastomeric formulations, scavenger exploration, and membrane characterization. The significant data are interpreted and recommendations are given for both short and long range further investigations in two of the four major areas: membrane adhesives and membrane stabilization.

  5. Restoration of caveolin-1 expression suppresses growth, membrane-type-4 metalloproteinase expression and metastasis-associated activities in colon cancer cells.

    PubMed

    Nimri, Lili; Barak, Hossei; Graeve, Lutz; Schwartz, Betty

    2013-11-01

    Caveolin-1 (cav-1) and flotillin-1 are two major structural proteins associated with lipid rafts in mammalian cells. The membrane-type matrix metalloproteinases (MT-MMPs) are expressed at the cell surface, hydrolyze extracellular matrix, and play an important role in cancer cell migration and metastasis. Expression of cav-1, flotillin-1, and MT4-MMP in lysates and lipid rafts of LS174T and HM-7 colon cancer cells was determined. The impact of restoration of cav-1 expression on proliferation, adhesion, motility in vitro, and growth of implanted tumors in vivo was characterized. Cav-1 is not expressed in lipid rafts of the highly metastatic colon cancer cell line (HM-7), but expressed in cytosolic fractions of the parental lower metastatic cell line (LS174T). In contrast, MT4-MMP was expressed in lipid rafts of HM-7 cells but not in LS174T cells. Overexpression of cav-1 in HM-7 cells down-regulate proliferation, viability, wound closure, adhesion to laminin, invasion, and development of filopodial and lamellipodial structures in a dose-dependent manner. Cav-1 positive HM-7 clones ceased to express MT4-MMP in their lipid rafts. Comparative proteomic analyses of lipid rafts from cav-1 positive and cav-1 negative cells demonstrated de novo expression of flotillin-1 only on the cells expressing cav-1. Xenografting control cells devoid of cav-1 in nude mice induced development of bigger tumors expressing higher levels of proliferating cell nuclear antigen as compared to mice injected with cells expressing the highest cav-1 levels. We conclude that cav-1 orchestrates and reorganize several proteins in lipid rafts, activities directly associated with reduced tumorigenic and metastatic ability of colon cancer cells.

  6. Membrane stretching triggers mechanosensitive Ca2+ channel activation in Chara.

    PubMed

    Kaneko, Toshiyuki; Takahashi, Naoya; Kikuyama, Munehiro

    2009-03-01

    In order to confirm that mechanosensitive Ca(2+) channels are activated by membrane stretching, we stretched or compressed the plasma membrane of Chara by applying osmotic shrinkage or swelling of the cell by varying the osmotic potential of the bathing medium. Aequorin studies revealed that treatments causing membrane stretching induced a transient but large increase in cytoplasmic concentration of Ca(2+) (Delta[Ca(2+)](c)). However, the observed Delta[Ca(2+)](c) decreased during the treatments, resulting in membrane compression. A second experiment was carried out to study the relationship between changes in membrane potential (DeltaE(m)) and stretching or compression of the plasma membrane. Significant DeltaE(m) values, often accompanied by an action potential, were observed during the initial exchange of the bathing medium from a hypotonic medium to a hypertonic one (plasmolysis). DeltaE(m) appears to be triggered by a partial stretching of the membrane as it was peeled from the cell wall. After plasmolysis, other exchanges from hypertonic to hypotonic media, with their accompanying membrane stretching, always induced large DeltaE(m) values and were often accompanied by an action potential. By contrast, action potentials were scarcely observed during other exchanges from hypotonic to hypertonic solutions (=membrane compression). Thus, we concluded that activation of the mechanosensitive channels is triggered by membrane stretching in Chara.

  7. Membrane stretching triggers mechanosensitive Ca2+ channel activation in Chara.

    PubMed

    Kaneko, Toshiyuki; Takahashi, Naoya; Kikuyama, Munehiro

    2009-03-01

    In order to confirm that mechanosensitive Ca(2+) channels are activated by membrane stretching, we stretched or compressed the plasma membrane of Chara by applying osmotic shrinkage or swelling of the cell by varying the osmotic potential of the bathing medium. Aequorin studies revealed that treatments causing membrane stretching induced a transient but large increase in cytoplasmic concentration of Ca(2+) (Delta[Ca(2+)](c)). However, the observed Delta[Ca(2+)](c) decreased during the treatments, resulting in membrane compression. A second experiment was carried out to study the relationship between changes in membrane potential (DeltaE(m)) and stretching or compression of the plasma membrane. Significant DeltaE(m) values, often accompanied by an action potential, were observed during the initial exchange of the bathing medium from a hypotonic medium to a hypertonic one (plasmolysis). DeltaE(m) appears to be triggered by a partial stretching of the membrane as it was peeled from the cell wall. After plasmolysis, other exchanges from hypertonic to hypotonic media, with their accompanying membrane stretching, always induced large DeltaE(m) values and were often accompanied by an action potential. By contrast, action potentials were scarcely observed during other exchanges from hypotonic to hypertonic solutions (=membrane compression). Thus, we concluded that activation of the mechanosensitive channels is triggered by membrane stretching in Chara. PMID:19234734

  8. Fuel cell and membrane therefore

    DOEpatents

    Aindow, Tai-Tsui

    2016-08-09

    A fuel cell includes first and second flow field plates, and an anode electrode and a cathode electrode between the flow field plates. A polymer electrolyte membrane (PEM) is arranged between the electrodes. At least one of the flow field plates influences, at least in part, an in-plane anisotropic physical condition of the PEM that varies in magnitude between a high value direction and a low value direction. The PEM has an in-plane physical property that varies in magnitude between a high value direction and a low value direction. The PEM is oriented with its high value direction substantially aligned with the high value direction of the flow field plate.

  9. Influence of the nuclear membrane, active transport, and cell shape on the Hes1 and p53-Mdm2 pathways: insights from spatio-temporal modelling.

    PubMed

    Sturrock, Marc; Terry, Alan J; Xirodimas, Dimitris P; Thompson, Alastair M; Chaplain, Mark A J

    2012-07-01

    There are many intracellular signalling pathways where the spatial distribution of the molecular species cannot be neglected. These pathways often contain negative feedback loops and can exhibit oscillatory dynamics in space and time. Two such pathways are those involving Hes1 and p53-Mdm2, both of which are implicated in cancer. In this paper we further develop the partial differential equation (PDE) models of Sturrock et al. (J. Theor. Biol., 273:15-31, 2011) which were used to study these dynamics. We extend these PDE models by including a nuclear membrane and active transport, assuming that proteins are convected in the cytoplasm towards the nucleus in order to model transport along microtubules. We also account for Mdm2 inhibition of p53 transcriptional activity. Through numerical simulations we find ranges of values for the model parameters such that sustained oscillatory dynamics occur, consistent with available experimental measurements. We also find that our model extensions act to broaden the parameter ranges that yield oscillations. Hence oscillatory behaviour is made more robust by the inclusion of both the nuclear membrane and active transport. In order to bridge the gap between in vivo and in silico experiments, we investigate more realistic cell geometries by using an imported image of a real cell as our computational domain. For the extended p53-Mdm2 model, we consider the effect of microtubule-disrupting drugs and proteasome inhibitor drugs, obtaining results that are in agreement with experimental studies.

  10. Hyperpolization-activated Ca(2+) channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba.

    PubMed

    Wang, Fang; Jia, Juanjuan; Wang, Yufang; Wang, Weixia; Chen, Yuling; Liu, Ting; Shang, Zhonglin

    2014-09-01

    Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5'-O-(3-thio) triphosphate (ATPγS), 3'-O-(4-benzoyl) benzoyl adenosine 5'-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5'-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca(2+) channel blockers GdCl3 and LaCl3. A hyperpolarization-activated Ca(2+) channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca(2+) channel significantly. Extracellular ATP-promoted Ca(2+) channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca(2+) channels.

  11. Proton Exchange Membranes for Fuel Cells

    SciTech Connect

    Devanathan, Ramaswami

    2010-11-01

    Proton exchange membrane, also known as polymer electrolyte membrane, fuel cells (PEMFCs) offer the promise of efficient conversion of chemical energy of fuel, such as hydrogen or methanol, into electricity with minimal pollution. Their widespread use to power zero-emission automobiles as part of a hydrogen economy can contribute to enhanced energy security and reduction in greenhouse gas emissions. However, the commercial viability of PEMFC technology is hindered by high cost associated with the membrane electrode assembly (MEA) and poor membrane durability under prolonged operation at elevated temperature. Membranes for automotive fuel cell applications need to perform well over a period comparable to the life of an automotive engine and under heavy load cycling including start-stop cycling under sub-freezing conditions. The combination of elevated temperature, changes in humidity levels, physical stresses and harsh chemical environment contribute to membrane degradation. Perfluorinated sulfonic acid (PFSA)-based membranes, such as Nafion®, have been the mainstay of PEMFC technology. Their limitations, in terms of cost and poor conductivity at low hydration, have led to continuing research into membranes that have good proton conductivity at elevated temperatures above 120 °C and under low humidity conditions. Such membranes have the potential to avoid catalyst poisoning, simplify fuel cell design and reduce the cost of fuel cells. Hydrocarbon-based membranes are being developed as alternatives to PFSA membranes, but concerns about chemical and mechanical stability and durability remain. Novel anhydrous membranes based on polymer gels infused with protic ionic liquids have also been recently proposed, but considerable fundamental research is needed to understand proton transport in novel membranes and evaluate durability under fuel cell operating conditions. In order to advance this promising technology, it is essential to rationally design the next generation

  12. Detecting Nanodomains in Living Cell Membrane by Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    He, Hai-Tao; Marguet, Didier

    2011-05-01

    Cell membranes actively participate in numerous cellular functions. Inasmuch as bioactivities of cell membranes are known to depend crucially on their lateral organization, much effort has been focused on deciphering this organization on different length scales. Within this context, the concept of lipid rafts has been intensively discussed over recent years. In line with its ability to measure diffusion parameters with great precision, fluorescence correlation spectroscopy (FCS) measurements have been made in association with innovative experimental strategies to monitor modes of molecular lateral diffusion within the plasma membrane of living cells. These investigations have allowed significant progress in the characterization of the cell membrane lateral organization at the suboptical level and have provided compelling evidence for the in vivo existence of raft nanodomains. We review these FCS-based studies and the characteristic structural features of raft nanodomains. We also discuss the findings in regards to the current view of lipid rafts as a general membrane-organizing principle.

  13. THE IN VIVO PROTEIN SYNTHETIC ACTIVITIES OF FREE VERSUS MEMBRANE-BOUND RIBONUCLEOPROTEIN IN A PLASMA-CELL TUMOR OF THE MOUSE

    PubMed Central

    Kuff, E. L.; Hymer, W. C.; Shelton, E.; Roberts, N. E.

    1966-01-01

    Cytoplasmic extracts of the transplantable RPC-20 plasma-cell tumor were fractionated by sucrose density gradient centrifugation. Four major fractions were distinguished: (a) microsomes and mitochondria; (b) membrane-free polyribosomes; (c) free monomeric ribosomes; and (d) soluble fraction. The fractions were analyzed for RNA and lipid phosphorus, and their particulate components were characterized by electron microscopy. Particular attention was paid to the problem of membrane contamination of the free polyribosome fraction. It was shown that this contamination was small in relation with the total content of ribosomes in the fraction, and that it consisted primarily of smooth-surfaced membranes which were not physically associated with the polyribosomes themselves. In vivo incorporation studies were carried out by injecting tumor-bearing animals intravenously with leucine-C14, removing the tumors at various times thereafter, and determining the distribution of protein radioactivity among the gradient-separated cytoplasmic fractions. The free polyribosome and the microsome-mitochondria fractions constituted active centers for protein synthesis. It was shown that nascent protein of the free polyribosome fractions was not associated significantly with the contaminating membranes. The kinetics of labeling during incorporation times up to 11 min suggested that protein synthesized on the free polyribosomes was rapidly transferred in vivo to the soluble fraction of the cell, while protein synthesized by the microsomes and mitochondria remained localized within these elements. It was estimated that the free polyribosome fraction and the microsome-mitochondria fraction accounted for approximately equal proportions of the total cytoplasmic protein synthesis in vivo. PMID:5920197

  14. Membrane, electrochemical cell, and electrolysis process

    SciTech Connect

    Bissot, Th.C.; Grot, W.G.; Resnick, P.R.

    1984-03-20

    An ion exchange membrane which comprises a layer of fluorinated polymer which has carboxylic functional groups, a second layer of fluorinated polymer which has sulfonic or carboxylic functional groups at a surface layer, and a web of support material therein, and which has channels in the membrane which extend from window areas of the membrane to blind areas of the membrane occluded by members of the support material, is described. Precursor membrane which contains both reinforcement members and sacrificial members, and from which the ion exchange membrane is made, is also described. The ion exchange membrane can be used to separate the compartments of a chloralkali cell, and such a cell operates at low voltage, high current efficiency, and low power consumption.

  15. Src kinase and Syk activation initiate PI3K signaling by a chimeric latent membrane protein 1 in Epstein-Barr virus (EBV)+ B cell lymphomas.

    PubMed

    Hatton, Olivia; Lambert, Stacie L; Krams, Sheri M; Martinez, Olivia M

    2012-01-01

    The B lymphotrophic γ-herpesvirus EBV is associated with a variety of lymphoid- and epithelial-derived malignancies, including B cell lymphomas in immunocompromised and immunosuppressed individuals. The primary oncogene of EBV, latent membrane protein 1 (LMP1), activates the PI3K/Akt pathway to induce the autocrine growth factor, IL-10, in EBV-infected B cells, but the mechanisms underlying PI3K activation remain incompletely understood. Using small molecule inhibition and siRNA strategies in human B cell lines expressing a chimeric, signaling-inducible LMP1 protein, nerve growth factor receptor (NGFR)-LMP1, we show that NGFR-LMP1 utilizes Syk to activate PI3K/Akt signaling and induce IL-10 production. NGFR-LMP1 signaling induces phosphorylation of BLNK, a marker of Syk activation. Whereas Src kinases are often required for Syk activation, we show here that PI3K/Akt activation and autocrine IL-10 production by NGFR-LMP1 involves the Src family kinase Fyn. Finally, we demonstrate that NGFR-LMP1 induces phosphorylation of c-Cbl in a Syk- and Fyn-dependent fashion. Our results indicate that the EBV protein LMP1, which lacks the canonical ITAM required for Syk activation, can nevertheless activate Syk, and the Src kinase Fyn, resulting in downstream c-Cbl and PI3K/Akt activation. Fyn, Syk, and PI3K/Akt antagonists thus may present potential new therapeutic strategies that target the oncogene LMP1 for treatment of EBV+ B cell lymphomas.

  16. Does ATP cross the cell plasma membrane.

    PubMed Central

    Chaudry, I. H.

    1982-01-01

    Although there is an abundance of evidence which indicates that ATP is released as well as taken up by cells, the concept that ATP cannot cross the cell membrane has tended to prevail. This article reviews the evidence for the release as well as uptake of ATP by cells. The evidence presented by various investigators clearly indicates that ATP can cross the cell membrane and suggests that the release and uptake of ATP are physiological processes. PMID:7051582

  17. Favorable effect of in-situ generated platinum in the membrane on fuel cell membrane durability

    NASA Astrophysics Data System (ADS)

    Macauley, Natalia; Wong, Ka Hung; Watson, Mark; Kjeang, Erik

    2015-12-01

    The overall lifetime of polymer electrolyte fuel cells is often determined by the membrane durability. Platinum, which may dissolve from the catalyst layers during fuel cell operation and deposit in the membrane, has been shown to have both positive and negative effects on membrane stability. In the present work, we analyze what specific conditions are required in order to reach a favorable, membrane stabilizing effect with the controlled use of platinum in the membrane. Using accelerated membrane durability testing, field operated membrane samples, and electron microscopy, we demonstrate that a high platinum concentration with specific particle shapes and sizes is essential for enhanced membrane stability. Specifically, star shaped and dendritic particles with high particle density and high surface area are shown to be preferable. These particles contain high levels of Pt(111) and are expected to have high catalytic activity toward peroxide quenching and crossover gas consumption, thereby mitigating chemical membrane degradation. On the other hand, small, dispersed cubic particles are found to have no effect or the opposite, negative effect on membrane stability.

  18. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    NASA Astrophysics Data System (ADS)

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  19. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    DOE PAGES

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-21

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolutionmore » inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. We find our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. Lastly, these observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.« less

  20. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    PubMed Central

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture. PMID:26790980

  1. A membrane vesicle/ribosome preparation from Serratia marcescens elicits peritoneal exudate cells expressing both tumoricidal and bactericidal activity.

    PubMed

    McCall, C; Weimer, L; Baldwin, S; Riches, D W; Canono, B; Campbell, P A

    1992-08-01

    A biological response modifier called ImuVert, derived from the bacterium Serratia marcescens, produced long-lasting elevation of peritoneal exudate cell (PEC) numbers after intraperitoneal injection into mice. These cells had enhanced ability to phagocytose both latex beads and opsonized Listeria monocytogenes. PEC harvested 2-14 days after a single injection of ImuVert killed L. monocytogenes, and ImuVert protected mice from infection by L. monocytogenes, measured both by LD50 and bacterial growth in vivo. Cells harvested 7 and 14 days after ImuVert injection also were tumoricidal, measured as killing of P815 mastocytoma cells, and ImuVert induced macrophages to express tumoricidal properties in vitro. These data suggest that ImuVert has a unique ability to induce a chronic inflammatory response, as other agents do not induce such a long-lasting influx of bactericidal inflammatory cells that also show tumoricidal activity. The consequences of this response appear to include protection from infection by certain bacteria.

  2. Dengue Virus Activates Membrane TRAIL Relocalization and IFN-α Production by Human Plasmacytoid Dendritic Cells In Vitro and In Vivo

    PubMed Central

    Gandini, Mariana; Gras, Christophe; Azeredo, Elzinandes Leal; Pinto, Luzia Maria de Oliveira; Smith, Nikaïa; Despres, Philippe; da Cunha, Rivaldo Venâncio; de Souza, Luiz José

    2013-01-01

    Background Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro. Methods & Findings Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production Conclusions This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease. PMID:23755314

  3. Interaction investigations of crustacean β-GBP recognition toward pathogenic microbial cell membrane and stimulate upon prophenoloxidase activation.

    PubMed

    Sivakamavalli, Jeyachandran; Selvaraj, Chandrabose; Singh, Sanjeev Kumar; Vaseeharan, Baskaralingam

    2014-04-01

    In invertebrates, crustaceans' immune system consists of pattern recognition receptors (PRRs) instead of immunoglobulin's, which involves in the microbial recognition and initiates the protein-ligand interaction between hosts and pathogens. In the present study, PRRs namely β-1,3 glucan binding protein (β-GBP) from mangrove crab Episesarma tetragonum and its interactions with the pathogens such as bacterial and fungal outer membrane proteins (OMP) were investigated through microbial aggregation and computational interaction studies. Molecular recognition and microbial aggregation results of Episesarma tetragonum β-GBP showed the specific binding affinity toward the fungal β-1,3 glucan molecule when compared to other bacterial ligands. Because of this microbial recognition, prophenoloxidase activity was enhanced and triggers the innate immunity inside the host animal. Our findings disclose the role of β-GBP in molecular recognition, host-pathogen interaction through microbial aggregation, and docking analysis. In vitro results were concurred with the in silico docking, and molecular dynamics simulation analysis. This study would be helpful to understand the molecular mechanism of β-GBP and update the current knowledge on the PRRs of crustaceans.

  4. The application of Dow Chemical's perfluorinated membranes in proton-exchange membrane fuel cells

    NASA Technical Reports Server (NTRS)

    Eisman, G. A.

    1989-01-01

    Dow Chemical's research activities in fuel cell devices revolves around the development and subsequent investigation of the perfluorinated inomeric membrane separator useful in proton-exchange membrane systems. Work is currently focusing on studying the effects of equivalent weight, thickness, water of hydration, pretreatment procedures, as well as the degree of water management required for a given membrane separator in the cell. The presentation will include details of certain aspects of the above as well as some of the requirements for high and low power generation.

  5. Polymer synthesis toward fuel cell membrane materials

    NASA Astrophysics Data System (ADS)

    Rebeck, Nathaniel T.

    Fuel cells are a promising technology that will be part of the future energy landscape. New membranes for alkaline and proton exchange membrane fuel cells are needed to improve the performance, simplify the system, and reduce cost. Polymer chemistry can be applied to develop new polymers and to assemble polymers into improved membranes that need less water, have increased performance and are less expensive, thereby removing the deficiencies of current membranes. Nucleophilic aromatic substitution polymerization typically produces thermally stable engineering polymers that can be easily functionalized. New functional monomers were developed to explore new routes to novel functional polymers. Sulfonamides were discovered as new activating groups for polymerization of high molecular weight thermooxidatively stable materials with sulfonic acid latent functionality. While the sulfonamide functional polymers could be produced, the sulfonamide group proved to be too stable to convert into a sulfonic acid after reaction. The reactivity of 2-aminophenol was investigated to search for a new class of ion conducting polymer materials. Both the amine and the phenol groups are found to be reactive in a nucleophilic aromatic substitution, however not to the extent to allow the formation of high molecular weight polymer materials. Layer-by-layer films were assembled from aqueous solutions of poly(styrene sulfonate) and trimethylammonium functionalized poly(phenylene oxide). The deposition conditions were adjusted to increase the free charge carrier content, and chloride conductivites reached almost 30 mS/cm for the best films. Block and random poly(phenylene oxide) copolymers were produced from 2,6-dimethylphenol and 2,6-diphenylphenol and the methyl substituted repeat units were functionalized with trimethylammonium bromide. The block copolymers displayed bromide conductivities up to 26 mS/cm and outperformed the random copolymers, indicating that morphology has an effect on ion

  6. The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

    PubMed

    Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

    2015-04-21

    A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

  7. Energy efficiency enhancement of ethanol electrooxidation on Pd-CeO(2)/C in passive and active polymer electrolyte-membrane fuel cells.

    PubMed

    Bambagioni, Valentina; Bianchini, Claudio; Chen, Yanxin; Filippi, Jonathan; Fornasiero, Paolo; Innocenti, Massimo; Lavacchi, Alessandro; Marchionni, Andrea; Oberhauser, Werner; Vizza, Francesco

    2012-07-01

    Pd nanoparticles have been generated by performing an electroless procedure on a mixed ceria (CeO(2))/carbon black (Vulcan XC-72) support. The resulting material, Pd-CeO(2)/C, has been characterized by means of transmission electron microscopy (TEM), inductively coupled plasma atomic emission spectroscopy (ICP-AES), and X-ray diffraction (XRD) techniques. Electrodes coated with Pd-CeO(2)/C have been scrutinized for the oxidation of ethanol in alkaline media in half cells as well as in passive and active direct ethanol fuel cells (DEFCs). Membrane electrode assemblies have been fabricated using Pd-CeO(2)/C anodes, proprietary Fe-Co cathodes, and Tokuyama anion-exchange membranes. The monoplanar passive and active DEFCs have been fed with aqueous solutions of 10 wt% ethanol and 2 M KOH, supplying power densities as high as 66 mW cm(-2) at 25 °C and 140 mW cm(-2) at 80 °C. A comparison with a standard anode electrocatalyst containing Pd nanoparticles (Pd/C) has shown that, at even metal loading and experimental conditions, the energy released by the cells with the Pd-CeO(2)/C electrocatalyst is twice as much as that supplied by the cells with the Pd/C electrocatalyst. A cyclic voltammetry study has shown that the co-support ceria contributes to the remarkable decrease of the onset oxidation potential of ethanol. It is proposed that ceria promotes the formation at low potentials of species adsorbed on Pd, Pd(I)-OH(ads), that are responsible for ethanol oxidation.

  8. Moderate activation of autophagy regulates the intracellular calcium ion concentration and mitochondrial membrane potential in beta-amyloid-treated PC12 cells.

    PubMed

    Xue, Zhongfeng; Guo, Yalei; Fang, Yongqi

    2016-04-01

    Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease. Aggregated beta-amyloid (Aβ) disturbs Ca(2+) homeostasis and causes mitochondrial dysfunction and finally underlies AD. Recent evidence suggests that autophagy initiation by Beclin-1 protein might be involved in the pathogenesis of AD. However, the effects of Beclin-1 dependent autophagy on intracellular calcium ion concentration ([Ca(2+)]i) and mitochondrial membrane potential (MMP) is unclear. The effects of Beclin-1 dependent autophagy that were activated by a gradient concentration of autophagy activator rapamycin or inhibited by autophagy inhibitor 3-methyladenine (3-MA) on cell viability and cell morphology were examined. Pretreatment with rapamycin significantly up-regulated the expression of Beclin-1 in response to Aβ1-42 application, but after pretreatment with 3-MA it was significantly down-regulated. Moderate activation of Beclin-1 dependent autophagy had an up regulation effect on cell viability and could maintain the original morphology of cells. Furthermore, rapamycin or 3-MA on [Ca(2+)]i and MMP in Aβ1-42 treatment of PC12 cells were evaluated. We also report that PC12 cells treated with Aβ1-42 showed an increase in [Ca(2+)]i but a decrease in MMP when compared to the normal control. However the application of rapamycin prior to this prevented the increase in [Ca(2+)]i and the decrease in MMP in response to Aβ1-42. When 3-MA was applied this exacerbated the effect of Aβ1-42 on the [Ca(2+)]i and the MMP. This shows that moderate activation of Beclin-1 dependent autophagy by rapamycin can modulate Ca(2+) homeostasis and maintain MMP in response to Aβ1-42 induced cytotoxicity and so may have a preventive function in AD. PMID:26923671

  9. [Membrane type 1 matrix metalloproteinase (MT1-MMP) and the regulators of its activity as invasive factors in squamous cell cervical carcinomas].

    PubMed

    Timoshenko, O S; Gureeva, T A; Kugaevskaia, E V; Solov'eva, N I

    2014-01-01

    Membrane type 1 matrix metalloproteinase (MT1MMP) is one of matrix metalloproteinases (MMP), which play а key role in tumor invasion and metastasis. The aim of this study was to elucidate the peculiarities of expression of MT1MMP and endogenous regulators of its activity: the activator - furin and the inhibitor - TIMP-2, as invasive factors of squamous cell cervical carcinomas (SCC). The study was carried out using 11 specimens of SCC and 11 specimens of morphologically normal tissue adjacent to the tumor. It was shown that the increase of MT1-MMP and furin expression and low of TIMP-2 expression makes the main contribution to the destructive (invasive) potential of SCC. Moreover, substantial expression of MT1-MMP was registered in the specimens of morphologically normal adjoining to tumor tissue. This expression was found to make an additional contribution to the destructive potential of the cervical tumor.

  10. Reassessing ecdysteroidogenic cells from the cell membrane receptors' perspective.

    PubMed

    Alexandratos, Alexandros; Moulos, Panagiotis; Nellas, Ioannis; Mavridis, Konstantinos; Dedos, Skarlatos G

    2016-01-01

    Ecdysteroids secreted by the prothoracic gland (PG) cells of insects control the developmental timing of their immature life stages. These cells have been historically considered as carrying out a single function in insects, namely the biochemical conversion of cholesterol to ecdysteroids and their secretion. A growing body of evidence shows that PG cells receive multiple cues during insect development so we tested the hypothesis that they carry out more than just one function in insects. We characterised the molecular nature and developmental profiles of cell membrane receptors in PG cells of Bombyx mori during the final larval stage and determined what receptors decode nutritional, developmental and physiological signals. Through iterative approaches we identified a complex repertoire of cell membrane receptors that are expressed in intricate patterns and activate previously unidentified signal transduction cascades in PG cells. The expression patterns of some of these receptors explain precisely the mechanisms that are known to control ecdysteroidogenesis. However, the presence of receptors for the notch, hedgehog and wingless signalling pathways and the expression of innate immunity-related receptors such as phagocytosis receptors, receptors for microbial ligands and Toll-like receptors call for a re-evaluation of the role these cells play in insects. PMID:26847502

  11. Reassessing ecdysteroidogenic cells from the cell membrane receptors’ perspective

    PubMed Central

    Alexandratos, Alexandros; Moulos, Panagiotis; Nellas, Ioannis; Mavridis, Konstantinos; Dedos, Skarlatos G.

    2016-01-01

    Ecdysteroids secreted by the prothoracic gland (PG) cells of insects control the developmental timing of their immature life stages. These cells have been historically considered as carrying out a single function in insects, namely the biochemical conversion of cholesterol to ecdysteroids and their secretion. A growing body of evidence shows that PG cells receive multiple cues during insect development so we tested the hypothesis that they carry out more than just one function in insects. We characterised the molecular nature and developmental profiles of cell membrane receptors in PG cells of Bombyx mori during the final larval stage and determined what receptors decode nutritional, developmental and physiological signals. Through iterative approaches we identified a complex repertoire of cell membrane receptors that are expressed in intricate patterns and activate previously unidentified signal transduction cascades in PG cells. The expression patterns of some of these receptors explain precisely the mechanisms that are known to control ecdysteroidogenesis. However, the presence of receptors for the notch, hedgehog and wingless signalling pathways and the expression of innate immunity-related receptors such as phagocytosis receptors, receptors for microbial ligands and Toll-like receptors call for a re-evaluation of the role these cells play in insects. PMID:26847502

  12. Modeling the cathode in a proton exchange membrane fuel cell using density functional theory How the carbon support can affect durability and activity of a platinum catalyst

    NASA Astrophysics Data System (ADS)

    Groves, Michael Nelson

    The current global energy and environmental challenges need to be addressed by developing a new portfolio of clean power producing devices. The proton exchange membrane fuel cell has the potential to be included and can fit into a variety of niches ranging from portable electronics to stationary residential applications. One of the many barriers to commercial viability is the cost of the cathode layer which requires too much platinum metal to achieve a comparable power output as well as would need to be replaced more frequently when compared to conventional sources for most applications. Using density functional theory, an ab initio modeling technique, these durability and activity issues are examined for platinum catalysts on graphene and carbon nanotube supports. The carbon supports were also doped by replacing individual carbon atoms with other second row elements (beryllium, boron, nitrogen, and oxygen) and the effect on the platinum-surface interaction along with the interaction between the platinum and the oxygen reduction reaction intermediates are discussed. Keywords: proton exchange membrane fuel cell, density functional theory, platinum catalyst, oxygen reduction reaction, doped carbon surfaces

  13. Visualizing active membrane protein complexes by electron cryotomography

    PubMed Central

    Gold, Vicki A.M.; Ieva, Raffaele; Walter, Andreas; Pfanner, Nikolaus; van der Laan, Martin; Kühlbrandt, Werner

    2014-01-01

    Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future. PMID:24942077

  14. Nuclear myosin I regulates cell membrane tension.

    PubMed

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-08-02

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension.

  15. Nuclear myosin I regulates cell membrane tension

    PubMed Central

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  16. Nuclear myosin I regulates cell membrane tension.

    PubMed

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  17. Proton conducting membrane for fuel cells

    DOEpatents

    Colombo, Daniel G.; Krumpelt, Michael; Myers, Deborah J.; Kopasz, John P.

    2007-03-27

    An ion conducting membrane comprising dendrimeric polymers covalently linked into a network structure. The dendrimeric polymers have acid functional terminal groups and may be covalently linked via linking compounds, cross-coupling reactions, or copolymerization reactions. The ion conducting membranes may be produced by various methods and used in fuel cells.

  18. Proton conducting membrane for fuel cells

    DOEpatents

    Colombo, Daniel G.; Krumpelt, Michael; Myers, Deborah J.; Kopasz, John P.

    2005-12-20

    An ion conducting membrane comprising dendrimeric polymers covalently linked into a network structure. The dendrimeric polymers have acid functional terminal groups and may be covalently linked via linking compounds, cross-coupling reactions, or copolymerization reactions. The ion conducting membranes may be produced by various methods and used in fuel cells.

  19. The role of cell membranes in the regulation of lignification in pine cells

    NASA Technical Reports Server (NTRS)

    Hendrix, D. L.

    1978-01-01

    The identity of pine cell membranes bearing PAL enzyme activity, the isolation of a plasma membrane preparation from pine cells for testing as a regulatory barrier in lignification, and the measurement of the geopotential effect in pine stems are presented. A model to describe and predict the interaction of gravity and lignification of higher plants was developed.

  20. Structural model of active Bax at the membrane.

    PubMed

    Bleicken, Stephanie; Jeschke, Gunnar; Stegmueller, Carolin; Salvador-Gallego, Raquel; García-Sáez, Ana J; Bordignon, Enrica

    2014-11-20

    Bax plays a central role in the mitochondrial pathway of apoptosis. Upon activation, cytosolic Bax monomers oligomerize on the surface of mitochondria and change conformation concertedly to punch holes into the outer membrane. The subsequent release of cytochrome c initiates cell death. However, the structure of membrane-inserted Bax and its mechanism of action remain largely unknown. Here, we propose a 3D model of active Bax at the membrane based on double electron-electron resonance (DEER) spectroscopy in liposomes and isolated mitochondria. We show that active Bax is organized at the membrane as assemblies of dimers. In addition to a stable dimerization domain, each monomer contains a more flexible piercing domain involved in interdimer interactions and pore formation. The most important structural change during Bax activation is the opening of the hairpin formed by helices 5 and 6, which adopts a clamp-like conformation central to the mechanism of mitochondrial permeabilization. PMID:25458844

  1. Structural Model of Active Bax at the Membrane

    PubMed Central

    Bleicken, Stephanie; Jeschke, Gunnar; Stegmueller, Carolin; Salvador-Gallego, Raquel; García-Sáez, Ana J.; Bordignon, Enrica

    2016-01-01

    Bax plays a central role in the mitochondrial pathway of apoptosis. Upon activation, cytosolic Bax monomers oligomerize on the surface of mitochondria and change conformation concertedly to punch holes into the outer membrane. The subsequent release of cytochrome c initiates cell death. However, the structure of membrane-inserted Bax and its mechanism of action remain largely unknown. Here, we propose a 3D model of active Bax at the membrane based on double electron-electron resonance (DEER) spectroscopy in liposomes and isolated mitochondria. We show that active Bax is organized at the membrane as assemblies of dimers. In addition to a stable dimerization domain, each monomer contains a more flexible piercing domain involved in interdimer interactions and pore formation. The most important structural change during Bax activation is the opening of the hairpin formed by helices 5 and 6, which adopts a clamp-like conformation central to the mechanism of mitochondrial permeabilization. PMID:25458844

  2. Membrane damage as first and DNA as the secondary target for anti-candidal activity of antimicrobial peptide P7 derived from cell-penetrating peptide ppTG20 against Candida albicans.

    PubMed

    Li, Lirong; Song, Fengxia; Sun, Jin; Tian, Xu; Xia, Shufang; Le, Guowei

    2016-06-01

    P7, a peptide analogue derived from cell-penetrating peptide ppTG20, possesses antibacterial and antitumor activities without significant hemolytic activity. In this study, we investigated the antifungal effect of P7 and its anti-Candida acting mode in Candida albicans. P7 displayed antifungal activity against the reference C. albicans (MIC = 4 μM), Aspergilla niger (MIC = 32 μM), Aspergillus flavus (MIC = 8 μM), and Trichopyton rubrum (MIC = 16 μM). The effect of P7 on the C. albicans cell membrane was examined by investigating the calcein leakage from fungal membrane models made of egg yolk l-phosphatidylcholine/ergosterol (10 : 1, w/w) liposomes. P7 showed potent leakage effects against fungal liposomes similar to Melittin-treated cells. C. albicans protoplast regeneration assay demonstrated that P7 interacted with the C. albicans plasma membrane. Flow cytometry of the plasma membrane potential and integrity of C. albicans showed that P7 caused 60.9 ± 1.8% depolarization of the membrane potential of intact C. albicans cells and caused 58.1 ± 3.2% C. albicans cell membrane damage. Confocal laser scanning microscopy demonstrated that part of FITC-P7 accumulated in the cytoplasm. DNA retardation analysis was also performed, which showed that P7 interacted with C. albicans genomic DNA after penetrating the cell membrane, completely inhibiting the migration of genomic DNA above the weight ratio (peptide : DNA) of 6. Our results indicated that the plasma membrane was the primary target, and DNA was the secondary intracellular target of the mode of action of P7 against C. albicans. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27197902

  3. NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells.

    PubMed

    Valdés, Juan Antonio; Gaggero, Eduardo; Hidalgo, Jorge; Leal, Nancy; Jaimovich, Enrique; Carrasco, M Angélica

    2008-03-01

    Depolarization of skeletal muscle cells triggers intracellular Ca2+ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K+-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c-fos, c-jun, and egr-1 through IP3-dependent Ca2+ release, whereas NF-kappa B activation is elicited by both ryanodine and IP3 receptor-mediated Ca2+ signals. We have further shown that field stimulation with electrical pulses results in an NF-kappa B activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K+ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca2+ source for NFAT activation is through release by ryanodine receptors and extracellular Ca2+ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-kappa B and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K+-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca2+ release and triggered by the same voltage sensor but activating different intracellular release channels. PMID:18184878

  4. Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells

    PubMed Central

    Rodríguez-García, Ruddi; López-Montero, Iván; Mell, Michael; Egea, Gustavo; Gov, Nir S.; Monroy, Francisco

    2015-01-01

    Erythrocytes are flexible cells specialized in the systemic transport of oxygen in vertebrates. This physiological function is connected to their outstanding ability to deform in passing through narrow capillaries. In recent years, there has been an influx of experimental evidence of enhanced cell-shape fluctuations related to metabolically driven activity of the erythroid membrane skeleton. However, no direct observation of the active cytoskeleton forces has yet been reported to our knowledge. Here, we show experimental evidence of the presence of temporally correlated forces superposed over the thermal fluctuations of the erythrocyte membrane. These forces are ATP-dependent and drive enhanced flickering motions in human erythrocytes. Theoretical analyses provide support for a direct force exerted on the membrane by the cytoskeleton nodes as pulses of well-defined average duration. In addition, such metabolically regulated active forces cause global membrane softening, a mechanical attribute related to the functional erythroid deformability. PMID:26083919

  5. A Facile Approach to Functionalize Cell Membrane-Coated Nanoparticles

    PubMed Central

    Zhou, Hao; Fan, Zhiyuan; Lemons, Pelin K.; Cheng, Hao

    2016-01-01

    Convenient strategies to provide cell membrane-coated nanoparticles (CM-NPs) with multi-functionalities beyond the natural function of cell membranes would dramatically expand the application of this emerging class of nanomaterials. We have developed a facile approach to functionalize CM-NPs by chemically modifying live cell membranes prior to CM-NP fabrication using a bifunctional linker, succinimidyl-[(N-maleimidopropionamido)-polyethyleneglycol] ester (NHS-PEG-Maleimide). This method is particularly suitable to conjugate large bioactive molecules such as proteins on cell membranes as it establishes a strong anchorage and enable the control of linker length, a critical parameter for maximizing the function of anchored proteins. As a proof of concept, we show the conjugation of human recombinant hyaluronidase, PH20 (rHuPH20) on red blood cell (RBC) membranes and demonstrate that long linker (MW: 3400) is superior to short linker (MW: 425) for maintaining enzyme activity, while minimizing the changes to cell membranes. When the modified membranes were fabricated into RBC membrane-coated nanoparticles (RBCM-NPs), the conjugated rHuPH20 can assist NP diffusion more efficiently than free rHuPH20 in matrix-mimicking gels and the pericellular hyaluronic acid matrix of PC3 prostate cancer cells. After quenching the unreacted chemical groups with polyethylene glycol, we demonstrated that the rHuPH20 modification does not reduce the ultra-long blood circulation time of RBCM-NPs. Therefore, this surface engineering approach provides a platform to functionlize CM-NPs without sacrificing the natural function of cell membranes. PMID:27217834

  6. Nonlinear electro-mechanobiological behavior of cell membrane during electroporation

    NASA Astrophysics Data System (ADS)

    Deng, Peigang; Lee, Yi-Kuen; Lin, Ran; Zhang, Tong-Yi

    2012-07-01

    A nonlinear electroporation (EP) model is proposed to study the electro-mechanobiological behavior of cell membrane during EP, by taking the nonlinear large deformation of the membrane into account. The proposed model predicts the critical transmembrane potential and the activation energy for EP, the equilibrium pore size, and the resealing process of the pore. Single-cell EP experiments using a micro EP chip were conducted on chicken red blood cells at different temperatures to determine the activation energy and the critical transmembrane potential for EP. The experimental results are in good agreement with the theoretical predictions.

  7. Epstein-Barr Virus-Encoded Latent Membrane Protein 1 Impairs G2 Checkpoint in Human Nasopharyngeal Epithelial Cells through Defective Chk1 Activation

    PubMed Central

    Deng, Wen; Pang, Pei Shin; Tsang, Chi Man; Hau, Pok Man; Yip, Yim Ling; Cheung, Annie L. M.; Tsao, Sai Wah

    2012-01-01

    Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development. PMID:22761726

  8. Catalytic membranes for fuel cells

    DOEpatents

    Liu, Di-Jia; Yang, Junbing; Wang, Xiaoping

    2011-04-19

    A fuel cell of the present invention comprises a cathode and an anode, one or both of the anode and the cathode including a catalyst comprising a bundle of longitudinally aligned graphitic carbon nanotubes including a catalytically active transition metal incorporated longitudinally and atomically distributed throughout the graphitic carbon walls of said nanotubes. The nanotubes also include nitrogen atoms and/or ions chemically bonded to the graphitic carbon and to the transition metal. Preferably, the transition metal comprises at least one metal selected from the group consisting of Fe, Co, Ni, Mn, and Cr.

  9. Ambidextrous binding of cell and membrane bilayers by soluble matrix metalloproteinase-12.

    PubMed

    Koppisetti, Rama K; Fulcher, Yan G; Jurkevich, Alexander; Prior, Stephen H; Xu, Jia; Lenoir, Marc; Overduin, Michael; Van Doren, Steven R

    2014-11-21

    Matrix metalloproteinases (MMPs) regulate tissue remodelling, inflammation and disease progression. Some soluble MMPs are inexplicably active near cell surfaces. Here we demonstrate the binding of MMP-12 directly to bilayers and cellular membranes using paramagnetic NMR and fluorescence. Opposing sides of the catalytic domain engage spin-labelled membrane mimics. Loops project from the β-sheet interface to contact the phospholipid bilayer with basic and hydrophobic residues. The distal membrane interface comprises loops on the other side of the catalytic cleft. Both interfaces mediate MMP-12 association with vesicles and cell membranes. MMP-12 binds plasma membranes and is internalized to hydrophobic perinuclear features, the nuclear membrane and inside the nucleus within minutes. While binding of TIMP-2 to MMP-12 hinders membrane interactions beside the active site, TIMP-2-inhibited MMP-12 binds vesicles and cells, suggesting compensatory rotation of its membrane approaches. MMP-12 association with diverse cell membranes may target its activities to modulate innate immune responses and inflammation.

  10. Advanced membrane electrode assemblies for fuel cells

    SciTech Connect

    Kim, Yu Seung; Pivovar, Bryan S

    2014-02-25

    A method of preparing advanced membrane electrode assemblies (MEA) for use in fuel cells. A base polymer is selected for a base membrane. An electrode composition is selected to optimize properties exhibited by the membrane electrode assembly based on the selection of the base polymer. A property-tuning coating layer composition is selected based on compatibility with the base polymer and the electrode composition. A solvent is selected based on the interaction of the solvent with the base polymer and the property-tuning coating layer composition. The MEA is assembled by preparing the base membrane and then applying the property-tuning coating layer to form a composite membrane. Finally, a catalyst is applied to the composite membrane.

  11. Advanced membrane electrode assemblies for fuel cells

    DOEpatents

    Kim, Yu Seung; Pivovar, Bryan S.

    2012-07-24

    A method of preparing advanced membrane electrode assemblies (MEA) for use in fuel cells. A base polymer is selected for a base membrane. An electrode composition is selected to optimize properties exhibited by the membrane electrode assembly based on the selection of the base polymer. A property-tuning coating layer composition is selected based on compatibility with the base polymer and the electrode composition. A solvent is selected based on the interaction of the solvent with the base polymer and the property-tuning coating layer composition. The MEA is assembled by preparing the base membrane and then applying the property-tuning coating layer to form a composite membrane. Finally, a catalyst is applied to the composite membrane.

  12. Spatial coordination of chloroplast and plasma membrane activities in Chara cells and its disruption through inactivation of 14-3-3 proteins.

    PubMed

    Bulychev, A A; van den Wijngaard, P W J; de Boer, A H

    2005-01-01

    In Chara corallina cells exposed to continuous light, external pH (pH(o)) and photosystem II (PSII) photochemical yield show correlated banding patterns. Photosynthetic activity is low in cell regions producing alkaline zones and high in the acid regions. We addressed the question whether (and how) photosynthetic activity and plasma membrane (PM) H+-pumping and H+-conductance are coupled in the different bands. First, PM H+-pump activity was stimulated with fusicoccin. This resulted in a more acidic pH in the acid bands without disturbing the correlation of photosynthetic electron transport and H+ fluxes across the PM. Next, H+-pump activity was reduced through microinjection of a phosphorylated peptide matching the canonical 14-3-3 binding motif RSTpSTP in the acid cell region. Microinjection induced a rapid (~5 min) rise in pH(o) by ca. 1.0 unit near the injection site, whereas the injection of the non-phosphorylated peptide had no effect. This pH rise confirms the supposed inhibition of the H+-pump upon the detachment of 14-3-3 proteins from the H+-ATPase. However, the PSII yield in the cell regions corresponding to the new alkaline peak remained high, which violated the normal inverse relations between the pH(o) and PSII photochemical yield. We conclude that the injection of the competitive inhibitor of the H+-ATPase disrupts the balanced operation of PM H+-transport and photosynthetic electron flow and promotes electron flow through alternative pathways.

  13. The Prostate Specific Membrane Antigen Regulates the Expression of IL-6 and CCL5 in Prostate Tumour Cells by Activating the MAPK Pathways1

    PubMed Central

    Colombatti, Marco; Fracasso, Giulio; Scupoli, Maria Teresa; Cingarlini, Sara; Poffe, Ornella; Naim, Hassan Y.; Heine, Martin; Tridente, Giuseppe; Mainiero, Fabrizio; Ramarli, Dunia

    2009-01-01

    The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-κB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis. PMID:19242540

  14. Bilayer Membrane Modulation of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Structure and Proteolytic Activity.

    PubMed

    Cerofolini, Linda; Amar, Sabrina; Lauer, Janelle L; Martelli, Tommaso; Fragai, Marco; Luchinat, Claudio; Fields, Gregg B

    2016-01-01

    Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis. PMID:27405411

  15. Bilayer Membrane Modulation of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Structure and Proteolytic Activity

    PubMed Central

    Cerofolini, Linda; Amar, Sabrina; Lauer, Janelle L.; Martelli, Tommaso; Fragai, Marco; Luchinat, Claudio; Fields, Gregg B.

    2016-01-01

    Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis. PMID:27405411

  16. Helicobacter pylori Disrupts Host Cell Membranes, Initiating a Repair Response and Cell Proliferation

    PubMed Central

    Lin, Li-Ling; Huang, Hsuan-Cheng; Ogihara, Satoshi; Wang, Jin-Town; Wu, Meng-Chuan; McNeil, Paul L.; Chen, Chiung-Nien; Juan, Hsueh-Fen

    2012-01-01

    Helicobacter pylori (H. pylori), the human stomach pathogen, lives on the inner surface of the stomach and causes chronic gastritis, peptic ulcer, and gastric cancer. Plasma membrane repair response is a matter of life and death for human cells against physical and biological damage. We here test the hypothesis that H. pylori also causes plasma membrane disruption injury, and that not only a membrane repair response but also a cell proliferation response are thereby activated. Vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA) have been considered to be major H. pylori virulence factors. Gastric cancer cells were infected with H. pylori wild type (vacA+/cagA+), single mutant (ΔvacA or ΔcagA) or double mutant (ΔvacA/ΔcagA) strains and plasma membrane disruption events and consequent activation of membrane repair components monitored. H. pylori disrupts the host cell plasma membrane, allowing localized dye and extracellular Ca2+ influx. Ca2+-triggered members of the annexin family, A1 and A4, translocate, in response to injury, to the plasma membrane, and cell surface expression of an exocytotic maker of repair, LAMP-2, increases. Additional forms of plasma membrane disruption, unrelated to H. pylori exposure, also promote host cell proliferation. We propose that H. pylori activation of a plasma membrane repair is pro-proliferative. This study might therefore provide new insight into potential mechanisms of H. pylori-induced gastric carcinogenesis. PMID:22949854

  17. Kinetics and mechanism of cell membrane electrofusion.

    PubMed Central

    Abidor, I G; Sowers, A E

    1992-01-01

    A new quantitative approach to study cell membrane electrofusion has been developed. Erythrocyte ghosts were brought into close contact using dielectrophoresis and then treated with one square or even exponentially decaying fusogenic pulse. Individual fusion events were followed by lateral diffusion of the fluorescent lipid analogue 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) from originally labeled to unlabeled adjacent ghosts. It was found that ghost fusion can be described as a first-order rate process with corresponding rate constants; a true fusion rate constant, k(f), for the square waveform pulse and an effective fusion rate constant, k(ef), for the exponential pulse. Compared with the fusion yield, the fusion rate constants are more fundamental characteristics of the fusion process and have implications for its mechanisms. Values of k(f) for rabbit and human erythrocyte ghosts were obtained at different electric field strength and temperatures. Arrhenius k(f) plots revealed that the activation energy of ghost electrofusion is in the range of 6-10 kT. Measurements were also made with the rabbit erythrocyte ghosts exposed to 42 degrees C for 10 min (to disrupt the spectrin network) or 0.1-1.0 mM uranyl acetate (to stabilize the bilayer lipid matrix of membranes). A correlation between the dependence of the fusion and previously published pore-formation rate constants for all experimental conditions suggests that the cell membrane electrofusion process involve pores formed during reversible electrical breakdown. A statistical analysis of fusion products (a) further supports the idea that electrofusion is a stochastic process and (b) shows that the probability of ghost electrofusion is independent of the presence of Dil as a label as well as the number of fused ghosts. PMID:1617138

  18. Prism-patterned Nafion membrane for enhanced water transport in polymer electrolyte membrane fuel cell

    NASA Astrophysics Data System (ADS)

    Kim, Sang Moon; Kang, Yun Sik; Ahn, Chiyeong; Jang, Segeun; Kim, Minhyoung; Sung, Yung-Eun; Yoo, Sung Jong; Choi, Mansoo

    2016-06-01

    Here, we report a simple and effective strategy to enhance the performance of the polymer electrolyte membrane fuel cell by imprinting prism-patterned arrays onto the Nafion membrane, which provides three combined effects directly related to the device performance. First, a locally thinned membrane via imprinted micro prism-structures lead to reduced membrane resistance, which is confirmed by electrochemical impedance spectroscopy. Second, increments of the geometrical surface area of the prism-patterned Nafion membrane compared to a flat membrane result in the increase in the electrochemical active surface area. Third, the vertically asymmetric geometry of prism structures in the cathode catalyst layer lead to enhanced water transport, which is confirmed by oxygen gain calculation. To explain the enhanced water transport, we propose a simple theoretical model on removal of water droplets existing in the asymmetric catalyst layer. These three combined effects achieved via incorporating prism patterned arrays into the Nafion membrane effectively enhance the performance of the polymer electrolyte membrane fuel cell.

  19. Photothermal nanoblade for patterned cell membrane cutting

    PubMed Central

    Wu, Ting-Hsiang; Teslaa, Tara; Teitell, Michael A.; Chiou, Pei-Yu

    2010-01-01

    We report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized and specifically shaped explosive vapor bubble. Rapid bubble expansion and collapse punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The membrane cutting pattern is controlled by the metallic nanostructure configuration, laser pulse polarization, and energy. Highly controllable, sub-micron sized circular hole pairs to half moon-like, or cat-door shaped, membrane cuts were realized in glutaraldehyde treated HeLa cells. PMID:21164656

  20. Photothermal nanoblade for patterned cell membrane cutting.

    PubMed

    Wu, Ting-Hsiang; Teslaa, Tara; Teitell, Michael A; Chiou, Pei-Yu

    2010-10-25

    We report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized and specifically shaped explosive vapor bubble. Rapid bubble expansion and collapse punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The membrane cutting pattern is controlled by the metallic nanostructure configuration, laser pulse polarization, and energy. Highly controllable, sub-micron sized circular hole pairs to half moon-like, or cat-door shaped, membrane cuts were realized in glutaraldehyde treated HeLa cells. PMID:21164656

  1. Photothermal nanoblade for patterned cell membrane cutting.

    PubMed

    Wu, Ting-Hsiang; Teslaa, Tara; Teitell, Michael A; Chiou, Pei-Yu

    2010-10-25

    We report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized and specifically shaped explosive vapor bubble. Rapid bubble expansion and collapse punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The membrane cutting pattern is controlled by the metallic nanostructure configuration, laser pulse polarization, and energy. Highly controllable, sub-micron sized circular hole pairs to half moon-like, or cat-door shaped, membrane cuts were realized in glutaraldehyde treated HeLa cells.

  2. Active Nuclear Import of Membrane Proteins Revisited.

    PubMed

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast. PMID:26473931

  3. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

    Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

  4. Cd and Hg ions stimulate cell membrane potassium conductance

    SciTech Connect

    Jungwirth, A.; Paulmichl, M.; Lang, F. )

    1989-02-09

    Intracellular microelectrodes have been applied to study the effect of cadmium (Cd) and mercury (Hg) ions on cultured renal epitheloid Madin Darby Canine Kidney (MDCK) cells. Within 10 seconds Cd and within 50 seconds Hg hyperpolarize the cell membrane from - 53 {plus minus} 1 mV to - 68 {plus minus} 1 mV and - 67 {plus minus} 1 mV, resp., increase the potassium selectivity of the cell membrane (tk) from 0.33 {plus minus} 0.02 to 0.64 {plus minus} 0.03 and 0.77 {plus minus} 0.02, resp., and reduce the apparent cell membrane resistance from 40 {plus minus} 2 MOhm to 27 {plus minus} 2 MOhm and 22 {plus minus} 2 MOhm, resp.. Thus, both, Cd and Hg hyperpolarize the cell membrane by enhancement of the potassium conductance. The concentration required to elicit half maximal hyperpolarization is some 400 nmol/1 for either, Cd or Hg. Barium (1 mmol/1) depolarizes the cell membrane to - 34 {plus minus} 1 mV and virtually abolishes tk in the absence of Cd and Hg. In the presence of barium Cd leads to a transient, Hg to a sustained reappearance of tk and hyperpolarization. Thus, the Cd induced potassium conductance is blocked by barium with delay, the Hg induced potassium conductance is insensitive to barium. Quinidine (1 mmol/1) depolarizes the cell membrane to - 3 {plus minus}1 mV and abolishes the effect of both, Cd and Hg. In the nominal absence of extracellular calcium Cd leads to transient, Hg to sustained increase of tk and hyperpolarization of the cell membrane. In conclusion, both, CD and Hg at the low concentrations encountered during Cd and Hg intoxication enhance potassium conductance of MDCK cell membranes. However, the channels activated apparently differ.

  5. Lysophosphatidylcholine cell depolarization: increased membrane permeability for use in the determination of cell membrane potentials

    SciTech Connect

    Gallo, R.L.; Wersto, R.P.; Notter, R.H.; Finkelstein, J.N.

    1984-12-01

    Current techniques for the determination of cellular membrane potentials based on the uptake of a radiolabeled lipophilic cation, (3H)triphenylmethylphosphonium, and the cyanine dye, DiOC5(3), were analyzed in terms of the proportions of these probes which are accumulated due to potential-dependent and potential-independent forces. Measurements were made of probe uptake in two model systems: rabbit type II pneumocytes and human promyelocytic HL60 cells. For both cell types, the membrane potential-independent component of triphenylmethylphosphonium uptake was found to be a function of several variables, including the length of exposure of the cells to the transport facilitator tetraphenylboron, the concentration of tetraphenylboron, and the integrity of the cell membrane. To accurately determine the magnitude of the potential-independent component of probe uptake by type II and HL60 cells, the cell-permeabilizing agent lysophosphatidylcholine was used. The ability of lysophosphatidylcholine to depolarize cell membranes and accurately predict membrane potential-independent accumulation was found to be equal to or superior to several other techniques commonly used to achieve membrane depolarization (e.g. gramicidin, valinomycin plus high external potassium). Lysophosphatidylcholine cell treatment was found to be a simple, rapid, and accurate technique to increase cell membrane permeability and allow equilibration of intra- and extracellular ions. The method is shown to be useful for determining membrane potential-independent accumulation of both radiolabeled and fluorescent probes of membrane potential.

  6. Membrane Composition Tunes the Outer Hair Cell Motor

    NASA Astrophysics Data System (ADS)

    Rajagopalan, L.; Sfondouris, J.; Oghalai, J. S.; Pereira, F. A.; Brownell, W. E.

    2009-02-01

    Cholesterol and docosahexaenoic acid (DHA), an ω-3 fatty acid, affect membrane mechanical properties in different ways and modulate the function of membrane proteins. We have probed the functional consequence of altering cholesterol and DHA levels in the membranes of OHCs and prestin expressing HEK cells. Large, dynamic and reversible changes in prestin-associated charge movement and OHC motor activity result from altering the concentration of membrane cholesterol. Increasing membrane cholesterol shifts the q/V function ~ 50 mV in the hyperpolarizing direction, possibly a response related to increases in membrane stiffness. The voltage shift is linearly related to total membrane cholesterol. Increasing cholesterol also decreases the total charge moved in a linear fashion. Decreasing membrane cholesterol shifts the q/V function ~ 50 mV in the depolarizing direction with little or no effect on the amount of charge moved. In vivo increases in membrane cholesterol transiently increase but ultimately lead to decreases in DPOAE. Docosahexaenoic acid shifts the q/V function in the hyperpolarizing direction < 15 mV and increases total charge moved. Tuning of cochlear function by membrane cholesterol contributes to the exquisite temporal and frequency processing of mammalian hearing by optimizing the cochlear amplifier.

  7. Direct liquid-feed fuel cell with membrane electrolyte and manufacturing thereof

    NASA Technical Reports Server (NTRS)

    Narayanan, Sekharipuram (Inventor); Surampudi, Subbarao (Inventor); Halpert, Gerald (Inventor)

    1999-01-01

    An improved direct liquid-feed fuel cell having a solid membrane electrolyte for electrochemical reactions of an organic fuel. Improvements in interfacing of the catalyst layer and the membrane and activating catalyst materials are disclosed.

  8. Vesicle trafficking and cell surface membrane patchiness.

    PubMed

    Tang, Q; Edidin, M

    2001-07-01

    Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited. PMID:11423406

  9. Acute and chronic effects of opioids on delta and mu receptor activation of G proteins in NG108-15 and SK-N-SH cell membranes.

    PubMed

    Breivogel, C S; Selley, D E; Childers, S R

    1997-04-01

    To compare activation of G proteins by opioid receptors, opioid agonist-stimulated guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP gamma S) binding in the presence of excess GDP was assayed in membranes from NG108-15 (delta) and SK-N-SH (primarily mu) cells. Basal [35S]GTP gamma S binding consisted of a single class of low-affinity sites (KD 400-500 nM). Addition of agonists produced a high-affinity site 100-300-fold higher in affinity than the basal site. The receptor/transducer amplification factor (ratio of activated G protein Bmax to opioid receptor Bmax) was 10-fold higher for SK-N-SH mu receptors than for NG108-15 delta receptors. Chronic delta agonist ([D-Ser2]-Leu-enkephalin-Thr; DSLET) treatment of NG108-15 cells resulted in an 80% loss of DSLET-stimulated [35S]-GTP gamma S binding within 1 h. Morphine treatment of SK-N-SH cells decreased mu agonist ([D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin; DAMGO)-stimulated [35S]GTP gamma S binding by 45% after 16 h, with no effect after 1 h. Loss of agonist response was due to a decrease in the Bmax of activated G proteins with no change in the KD. These results provide a quantitative description of G protein activation occurring on acute and chronic exposure to opioid agonists.

  10. Redistribution of Cholesterol in Model Lipid Membranes in Response to the Membrane-Active Peptide Alamethicin

    NASA Astrophysics Data System (ADS)

    Heller, William; Qian, Shuo

    2013-03-01

    The cellular membrane is a heterogeneous, dynamic mixture of molecules and macromolecules that self-assemble into a tightly-regulated functional unit that provides a semipermeable barrier between the cell and its environment. Among the many compositional differences between mammalian and bacterial cell membranes that impact its physical properties, one key difference is cholesterol content, which is more prevalent in mammals. Cholesterol is an amphiphile that associates with membranes and serves to maintain its fluidity and permeability. Membrane-active peptides, such as the alpha-helical peptide alamethicin, interact with membranes in a concentration- and composition-dependent manner to form transmembrane pores that are responsible for the lytic action of the peptide. Through the use of small-angle neutron scattering and deuterium labeling, it was possible to observe a redistribution of the lipid and cholesterol in unilamellar vesicles in response to the presence of alamethicin at a peptide-to-lipid ratio of 1/200. The results demonstrate that the membrane remodeling powers of alamethicin reach beyond the membrane thinning effect to altering the localization of specific components in the bilayer, complementing the accepted two-state mechanism of pore formation. Research was supported by U. S. DOE-OBER (CSMB; FWP ERKP291) and the U. S. DOE-BES Scientific User Facilities Division (ORNL's SNS and HFIR).

  11. Penetration of Cell Membranes and Synthetic Lipid Bilayers by Nanoprobes

    PubMed Central

    Angle, Matthew R.; Wang, Andrew; Thomas, Aman; Schaefer, Andreas T.; Melosh, Nicholas A.

    2014-01-01

    Nanoscale devices have been proposed as tools for measuring and controlling intracellular activity by providing electrical and/or chemical access to the cytosol. Unfortunately, nanostructures with diameters of 50–500 nm do not readily penetrate the cell membrane, and rationally optimizing nanoprobes for cell penetration requires real-time characterization methods that are capable of following the process of membrane penetration with nanometer resolution. Although extensive work has examined the rupture of supported synthetic lipid bilayers, little is known about the applicability of these model systems to living cell membranes with complex lipid compositions, cytoskeletal attachment, and membrane proteins. Here, we describe atomic force microscopy (AFM) membrane penetration experiments in two parallel systems: live HEK293 cells and stacks of synthetic lipid bilayers. By using the same probes in both systems, we were able to clearly identify membrane penetration in synthetic bilayers and compare these events with putative membrane penetration events in cells. We examined membrane penetration forces for three tip geometries and 18 chemical modifications of the probe surface, and in all cases the median forces required to penetrate cellular and synthetic lipid bilayers with nanoprobes were greater than 1 nN. The penetration force was sensitive to the probe's sharpness, but not its surface chemistry, and the force did not depend on cell surface or cytoskeletal properties, with cells and lipid stacks yielding similar forces. This systematic assessment of penetration under various mechanical and chemical conditions provides insights into nanoprobe-cell interactions and informs the design of future intracellular nanoprobes. PMID:25418094

  12. Fibronectin coating of oxygenator membranes enhances endothelial cell attachment

    PubMed Central

    2013-01-01

    Background Extracorporeal membrane oxygenation (ECMO) can replace the lungs’ gas exchange capacity in refractory lung failure. However, its limited hemocompatibility, the activation of the coagulation and complement system as well as plasma leakage and protein deposition hamper mid- to long-term use and have constrained the development of an implantable lung assist device. In a tissue engineering approach, lining the blood contact surfaces of the ECMO device with endothelial cells might overcome these limitations. As a first step towards this aim, we hypothesized that coating the oxygenator’s gas exchange membrane with proteins might positively influence the attachment and proliferation of arterial endothelial cells. Methods Sheets of polypropylene (PP), polyoxymethylpentene (TPX) and polydimethylsiloxane (PDMS), typical material used for oxygenator gas exchange membranes, were coated with collagen, fibrinogen, gelatin or fibronectin. Tissue culture treated well plates served as controls. Endothelial cell attachment and proliferation were analyzed for a period of 4 days by microscopic examination and computer assisted cell counting. Results Endothelial cell seeding efficiency is within range of tissue culture treated controls for fibronectin treated surfaces only. Uncoated membranes as well as all other coatings lead to lower cell attachment. A confluent endothelial cell layer develops on fibronectin coated PDMS and the control surface only. Conclusions Fibronectin increases endothelial cells’ seeding efficiency on different oxygenator membrane material. PDMS coated with fibronectin shows sustained cell attachment for a period of four days in static culture conditions. PMID:23356939

  13. Investigation of the effects of 2.1 GHz microwave radiation on mitochondrial membrane potential (ΔΨm), apoptotic activity and cell viability in human breast fibroblast cells.

    PubMed

    Esmekaya, Meric Arda; Seyhan, Nesrin; Kayhan, Handan; Tuysuz, Mehmet Zahid; Kurşun, Ayşe Canseven; Yağcı, Münci

    2013-01-01

    In the present study we aimed to investigate the effects of 2.1 GHz Wideband Code Division Multiple Access (W-CDMA) modulated Microwave (MW) Radiation on cell survival and apoptotic activity of human breast fibroblast cells. The cell cultures were exposed to W-CDMA modulated MW at 2.1 GHz at a SAR level of 0.607 W/kg for 4 and 24 h. The cell viability was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The percentage of apoptotic cells was analyzed by Annexin V-FITC and PI staining. 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to measure Mitochondrial Membrane Potential (ΔΨm). sFasL and Fas/APO-1 protein levels were determined by ELISA method. 2.1 GHz MW radiation was shown to be able to inhibit cell proliferation and induce apoptosis in human breast fibroblast cells. The cell viability of MW-exposed cells was decreased significantly. The percentages of Annexin V-FITC positive cells were higher in MW groups. ΔΨm was decreased significantly due to MW radiation exposure. However, neither sFas nor FasL level was significantly changed in MW-exposed fibroblast cells. The results of this study showed that 2.1 GHz W-CDMA modulated MW radiation-induced apoptotic cell death via the mitochondrial pathway.

  14. Fuel cell subassemblies incorporating subgasketed thrifted membranes

    DOEpatents

    Iverson, Eric J.; Pierpont, Daniel M.; Yandrasits, Michael A.; Hamrock, Steven J.; Obradovich, Stephan J.; Peterson, Donald G.

    2013-03-01

    A fuel cell roll good subassembly is described that includes a plurality of individual electrolyte membranes. One or more first subgaskets are attached to the individual electrolyte membranes. Each of the first subgaskets has at least one aperture and the first subgaskets are arranged so the center regions of the individual electrolyte membranes are exposed through the apertures of the first subgaskets. A second subgasket comprises a web having a plurality of apertures. The second subgasket web is attached to the one or more first subgaskets so the center regions of the individual electrolyte membranes are exposed through the apertures of the second subgasket web. The second subgasket web may have little or no adhesive on the subgasket surface facing the electrolyte membrane.

  15. Fuel cell subassemblies incorporating subgasketed thrifted membranes

    DOEpatents

    Iverson, Eric J; Pierpont, Daniel M; Yandrasits, Michael A; Hamrock, Steven J; Obradovich, Stephan J; Peterson, Donald G

    2014-01-28

    A fuel cell roll good subassembly is described that includes a plurality of individual electrolyte membranes. One or more first subgaskets are attached to the individual electrolyte membranes. Each of the first subgaskets has at least one aperture and the first subgaskets are arranged so the center regions of the individual electrolyte membranes are exposed through the apertures of the first subgaskets. A second subgasket comprises a web having a plurality of apertures. The second subgasket web is attached to the one or more first subgaskets so the center regions of the individual electrolyte membranes are exposed through the apertures of the second subgasket web. The second subgasket web may have little or no adhesive on the subgasket surface facing the electrolyte membrane.

  16. Activation of ATP-sensitive potassium channels in rat pancreatic beta-cells by linoleic acid through both intracellular metabolites and membrane receptor signalling pathway.

    PubMed

    Zhao, Yu-Feng; Pei, Jianming; Chen, Chen

    2008-09-01

    ATP-sensitive potassium channels (K(ATP) channels) determine the excitability of pancreatic beta-cells and importantly regulate glucose-stimulated insulin secretion (GSIS). Long-chain free fatty acids (FFAs) decrease GSIS after long-term exposure to beta-cells, but the effects of exogenous FFAs on K(ATP) channels are not yet well clarified. In this study, the effects of linoleic acid (LA) on membrane potential (MP) and K(ATP) channels were observed in primary cultured rat pancreatic beta-cells. LA (20 microM) induced hyperpolarization of MP and opening of K(ATP) channels, which was totally reversed and inhibited by tolbutamide, a K(ATP) channel blocker. Inhibition of LA metabolism by acyl-CoA synthetase inhibitor, triacsin C (10 microM), partially inhibited LA-induced opening of K(ATP) channels by 64%. The non-FFA G protein-coupled receptor (GPR) 40 agonist, GW9508 (40 microM), induced an opening of K(ATP) channels, which was similar to that induced by LA under triacsin C treatment. Blockade of protein kinases A and C did not influence the opening of K(ATP) channels induced by LA and GW9508, indicating that these two protein kinase pathways are not involved in the action of LA on K(ATP) channels. The present study demonstrates that LA induces hyperpolarization of MP by activating K(ATP) channels via both intracellular metabolites and activation of GPR40. It indicates that not only intracellular metabolites of FFAs but also GPR40-mediated pathways take part in the inhibition of GSIS and beta-cell dysfunction induced by FFAs.

  17. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    PubMed

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

    2016-03-29

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

  18. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    PubMed

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

    2016-03-29

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

  19. Effect of heat treatment on the activity and stability of carbon supported PtMo alloy electrocatalysts for hydrogen oxidation in proton exchange membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Hassan, Ayaz; Carreras, Alejo; Trincavelli, Jorge; Ticianelli, Edson Antonio

    2014-02-01

    The effect of heat treatment on the activity, stability and CO tolerance of PtMo/C catalysts was studied, due to their applicability in the anode of proton exchange membrane fuel cells (PEMFCs). To this purpose, a carbon supported PtMo (60:40) alloy electrocatalyst was synthesized by the formic acid reduction method, and samples of this catalyst were heat-treated at various temperatures ranging between 400 and 700 °C. The samples were characterized by temperature programmed reduction (TPR), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), Transmission electron microscopy (TEM), X-ray absorption spectroscopy (XAS), cyclic voltammetry (CV), scanning electron microscopy (SEM) and wavelength dispersive X-ray spectroscopy (WDS). Cyclic voltammetry was used to study the stability, and polarization curves were used to investigate the performance of all materials as CO tolerant anode on a PEM single cell text fixture. The catalyst treated at 600 °C, for which the average crystallite size was 16.7 nm, showed the highest hydrogen oxidation activity in the presence of CO, giving an overpotential induced by CO contamination of 100 mV at 1 Acm-2. This catalyst also showed a better stability up to 5000 potential cycles of cyclic voltammetry, as compared to the untreated catalyst. CV, SEM and WDS results indicated that a partial dissolution of Mo and its migration/diffusion from the anode to the cathode occurs during the single cell cycling. Polarization results showed that the catalytic activity and the stability can be improved by a heat treatment, in spite of a growth of the catalyst particles.

  20. Cell membrane softening in human breast and cervical cancer cells

    NASA Astrophysics Data System (ADS)

    Händel, Chris; Schmidt, B. U. Sebastian; Schiller, Jürgen; Dietrich, Undine; Möhn, Till; Kießling, Tobias R.; Pawlizak, Steve; Fritsch, Anatol W.; Horn, Lars-Christian; Briest, Susanne; Höckel, Michael; Zink, Mareike; Käs, Josef A.

    2015-08-01

    Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

  1. Organelle morphogenesis by active membrane remodeling

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, N.; Ipsen, John H.; Rao, Madan; Kumar, P. B. Sunil

    Intracellular organelles are subject to a steady flux of lipids and proteins through active, energy consuming transport processes. Active fission and fusion are promoted by GTPases, e.g., Arf-Coatamer and the Rab-Snare complexes, which both sense and generate local membrane curvature. Here we investigate through Dynamical Triangulation Monte Carlo simulations, the role that these active processes play in determining the morphology and compositional segregation in closed membranes. Our results suggest that the ramified morphologies of organelles observed in-vivo are a consequence of driven nonequilibrium processes rather than equilibrium forces.

  2. [Kinetics of inhibitory effect of calix[4]arene C-90 on activity of transporting plasma membrane Ca2+, Mg2+-ATPase of smooth muscle cells].

    PubMed

    Veklich, T O; Shkrabak, O A; Mazur, Iu Iu; Rodik, R V; Kal'chenko, V I; Kosterin, S O

    2014-01-01

    In experiments on the suspension of myometrium cell plasma membrane, processed by 0.1% digitonin, the inhibitory action of calix[4]arene C-90 (5,11,17,23-tetra(threeftor)methyl(phenilsulphonilimino)-methylamino-25,26,27,28-tetrapropoxy-calix[4]arene) on the activity of Ca2+,Mg2+-ATPase was investigated. The authors also examined the influence of calix[4]arene in different concentration on affinity of enzyme (Ca2,Mg2+-ATPase) for the ATP and ions of Mg and Ca, and its influence on cooperative effect and maximum velocity of ATP hydrolysis. It is shown that calix[4]arene does not influence the affinity of Ca2+,Mg2+-ATPase for the ATP, which means that these two compounds have different binding centers. Also calix[4]arene has no influence on affinity and cooperative effect of Ca ions, if it is used in concentration lower than 50 μM. Calix[4]arene slightly increases coefficient of Ca2+,Mg2+-ATPase activation by magnesium chloride. In all three cases, where ATP, Mg and Ca ions are used to test the impact of calix[4]arene, maximum velocity of ATP hydrolysis significantly decreases. All these results clarify that calix[4]arene implements its inhibitory action through mechanism of uncompetitive inhibition of Ca2+,Mg2+-ATPase activity.

  3. Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

    PubMed

    Zhao, Yingxin; Valbuena, Gustavo; Walker, David H; Gazi, Michal; Hidalgo, Marylin; DeSousa, Rita; Oteo, Jose Antonio; Goez, Yenny; Brasier, Allan R

    2016-01-01

    Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from

  4. A Journey of Cytolethal Distending Toxins through Cell Membranes

    PubMed Central

    Boesze-Battaglia, Kathleen; Alexander, Desiree; Dlakić, Mensur; Shenker, Bruce J.

    2016-01-01

    The multifunctional role of lipids as structural components of membranes, signaling molecules, and metabolic substrates makes them an ideal partner for pathogens to hijack host cell processes for their own survival. The properties and composition of unique membrane micro-domains such as membrane rafts make these regions a natural target for pathogens as it affords them an opportunity to hijack cell signaling and intracellular trafficking pathways. Cytolethal distending toxins (Cdts), members of the AB2 family of toxins are comprised of three subunits, the active, CdtB unit, and the binding, CdtA-CdtC unit. Cdts are cyclomodulins leading to cell cycle arrest and apoptosis in a wide variety of cell types. Cdts from several species share a requirement for membrane rafts, and often cholesterol specifically for cell binding and CdtB mediated cytotoxicity. In this review we focus on how host–cell membrane bilayer organization contributes to the cell surface association, internalization, and action of bacteria derived cytolethal distending toxins (Cdts), with an emphasis on Aggregatibacter actinomycetemcomitans Cdt. PMID:27559534

  5. Modulating activity of vancomycin and daptomycin on the expression of autolysis cell-wall turnover and membrane charge genes in hVISA and VISA strains.

    PubMed

    Cafiso, Viviana; Bertuccio, Taschia; Spina, Daniela; Purrello, Simona; Campanile, Floriana; Di Pietro, Cinzia; Purrello, Michele; Stefani, Stefania

    2012-01-01

    Glycopeptides are still the gold standard to treat MRSA (Methicillin Resistant Staphylococcus aureus) infections, but their widespread use has led to vancomycin-reduced susceptibility [heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-Staphylococcus aureus (VISA)], in which different genetic loci (regulatory, autolytic, cell-wall turnover and cell-envelope positive charge genes) are involved. In addition, reduced susceptibility to vancomycin can influence the development of resistance to daptomycin. Although the phenotypic and molecular changes of hVISA/VISA have been the focus of different papers, the molecular mechanisms responsible for these different phenotypes and for the vancomycin and daptomycin cross-resistance are not clearly understood. The aim of our study was to investigate, by real time RT-PCR, the relative quantitative expression of genes involved in autolysis (atl-lytM), cell-wall turnover (sceD), membrane charges (mprF-dltA) and regulatory mechanisms (agr-locus-graRS-walKR), in hVISA and VISA cultured with or without vancomycin and daptomycin, in order to better understand the molecular basis of vancomycin-reduced susceptibility and the modulating activity of vancomycin and daptomycin on the expression of genes implicated in their reduced susceptibility mechanisms. Our results show that hVISA and VISA present common features that distinguish them from Vancomycin-Susceptible Staphylococcus aureus (VSSA), responsible for the intermediate glycopeptide resistance i.e. an increased cell-wall turnover, an increased positive cell-wall charge responsible for a repulsion mechanism towards vancomycin and daptomycin, and reduced agr-functionality. Indeed, VISA emerges from hVISA when VISA acquires a reduced autolysis caused by a down-regulation of autolysin genes, atl/lytM, and a reduction of the net negative cell-envelope charge via dltA over-expression. Vancomycin and daptomycin, acting in a similar manner in h

  6. The Anti-angiogenic Peptide Anginex Disrupts the Cell Membrane

    PubMed Central

    Pilch, Jan; Franzin, Carla M.; Knowles, Lynn M.; Ferrer, Fernando J.; Marassi, Francesca M.; Ruoslahti, Erkki

    2010-01-01

    Anginex is a synthetic beta-sheet peptide with anti-angiogenic and anti-tumor activity. When added to cultured endothelial cells at concentrations ranging from 2.5 μM to 25 μM, anginex induced cell death, which was reflected by a strong increase of subdiploid cells and fragments, loss of cellular ATP, and LDH release. Cytotoxicity remained the same whether cells were treated with anginex at 4 °C or at 37 °C. At low temperatures, fluorescein-conjugated anginex accumulated on the endothelial surface, but did not reach into the cytoplasm, indicating that the cell membrane is the primary target for the peptide. Within minutes of treatment, anginex caused endothelial cells to take up propidium iodide and undergo depolarization, both parameters characteristic for permeabilization of the cell membrane. This process was amplified when cells were activated with hydrogen peroxide. Red blood cell membranes were essentially unaffected by anginex. Anginex bound lipid bilayers with high affinity and with a clear preference for anionic over zwitterionic phospholipids. Structural studies by circular dichroism and solid-state nuclear magnetic resonance showed that anginex forms a beta-sheet and adopts a unique and highly ordered conformation upon binding to lipid membranes. This is consistent with lipid micellization or the formation of pore-forming beta-barrels. The data suggest that the cytotoxicity of anginex stems from its ability to target and disrupt the endothelial cell membrane, providing a possible explanation for the angiostatic activity of the peptide. PMID:16403516

  7. Thermoelasticity of red blood cell membrane.

    PubMed Central

    Waugh, R; Evans, E A

    1979-01-01

    The elastic properties of the human red blood cell membrane have been measured as functions of temperature. The area compressibility modulus and the elastic shear modulus, which together characterize the surface elastic behavior of the membrane, have been measured over the temperature range of 2-50 degrees C with micropipette aspiration of flaccid and osmotically swollen red cells. In addition, the fractional increase in membrane surface area from 2-50 degrees C has been measured to give a value for the thermal area expansivity. The value of the elastic shear modulus at 25 degrees C was measured to be 6.6 X 10(-3) dyne/cm. The change in the elastic shear modulus with temperature was -6 X 10(-5) dyne/cm degrees C. Fractional forces were shown to be only on the order of 10-15%. The area compressibility modulus at 25 degrees C was measured to be 450 dyne/cm. The change in the area compressibility modulus with temperature was -6 dyne/cm degrees C. The thermal area expansivity for red cell membrane was measured to be 1.2 X 10(-3)/degrees C. With this data and thermoelastic relations the heat of expansion is determined to be 110-200 ergs/cm2; the heat of extension is 2 X 10(-2) ergs/cm2 for unit extension of the red cell membrane. The heat of expansion is of the order anticipated for a lipid bilayer idealized as twice the behavior of a monolayer at an oil-water interface. The observation that the heat of extension is positive demonstrates that the entropy of the material increases with extension, and that the dominant mechanism of elastic energy storage is energetic. Assuming that the red cell membrane shear rigidity is associated with "spectrin," unit extension of the membrane increases the configurational entropy of spectrin by 500 cal/mol. Images FIGURE 3 PMID:262408

  8. Active membrane cholesterol as a physiological effector.

    PubMed

    Lange, Yvonne; Steck, Theodore L

    2016-09-01

    Sterols associate preferentially with plasma membrane sphingolipids and saturated phospholipids to form stoichiometric complexes. Cholesterol in molar excess of the capacity of these polar bilayer lipids has a high accessibility and fugacity; we call this fraction active cholesterol. This review first considers how active cholesterol serves as an upstream regulator of cellular sterol homeostasis. The mechanism appears to utilize the redistribution of active cholesterol down its diffusional gradient to the endoplasmic reticulum and mitochondria, where it binds multiple effectors and directs their feedback activity. We have also reviewed a broad literature in search of a role for active cholesterol (as opposed to bulk cholesterol or lipid domains such as rafts) in the activity of diverse membrane proteins. Several systems provide such evidence, implicating, in particular, caveolin-1, various kinds of ABC-type cholesterol transporters, solute transporters, receptors and ion channels. We suggest that this larger role for active cholesterol warrants close attention and can be tested easily.

  9. Cell migration does not produce membrane flow

    PubMed Central

    1990-01-01

    We have previously reported that rearward migration of surface particles on slowly moving cells is not driven by membrane flow (Sheetz, M. P., S. Turney, H. Qian, and E. L. Elson. 1989. Nature (Lond.). 340:284-288) and recent photobleaching measurements have ruled out any rapid rearward lipid flow (Lee, J., M. Gustafsson, D. E. Magnussen, and K. Jacobson. 1990. Science (Wash. DC.) 247:1229-1233). It was not possible, however, to conclude from those studies that a slower or tank-tread membrane lipid flow does not occur. Therefore, we have used the technology of single particle tracking to examine the movements of diffusing particles on rapidly locomoting fish keratocytes where the membrane current is likely to be greatest. The keratocytes had a smooth lamellipodial surface on which bound Con A-coated gold particles were observed either to track toward the nuclear region (velocity of 0.35 +/- 0.15 micron/s) or to diffuse randomly (apparent diffusion coefficient of [3.5 +/- 2.0] x 10(-10) cm2/s). We detected no systematic drift relative to the cell edge of particles undergoing random diffusion even after the cell had moved many micrometers. The average net particle displacement was 0.01 +/- 2.7% of the cell displacement. These results strongly suggest that neither the motions of membrane proteins driven by the cytoskeleton nor other possible factors produce a bulk flow of membrane lipid. PMID:2211827

  10. Alternate Fuel Cell Membranes for Energy Independence

    SciTech Connect

    Storey, Robson, F.; Mauritz, Kenneth, A.; Patton, Derek, L.; Savin, Daniel, A.

    2012-12-18

    The overall objective of this project was the development and evaluation of novel hydrocarbon fuel cell (FC) membranes that possess high temperature performance and long term chemical/mechanical durability in proton exchange membrane (PEM) fuel cells (FC). The major research theme was synthesis of aromatic hydrocarbon polymers of the poly(arylene ether sulfone) (PAES) type containing sulfonic acid groups tethered to the backbone via perfluorinated alkylene linkages and in some cases also directly attached to the phenylene groups along the backbone. Other research themes were the use of nitrogen-based heterocyclics instead of acid groups for proton conduction, which provides high temperature, low relative humidity membranes with high mechanical/thermal/chemical stability and pendant moieties that exhibit high proton conductivities in the absence of water, and synthesis of block copolymers consisting of a proton conducting block coupled to poly(perfluorinated propylene oxide) (PFPO) blocks. Accomplishments of the project were as follows: 1) establishment of a vertically integrated program of synthesis, characterization, and evaluation of FC membranes, 2) establishment of benchmark membrane performance data based on Nafion for comparison to experimental membrane performance, 3) development of a new perfluoroalkyl sulfonate monomer, N,N-diisopropylethylammonium 2,2-bis(p-hydroxyphenyl) pentafluoropropanesulfonate (HPPS), 4) synthesis of random and block copolymer membranes from HPPS, 5) synthesis of block copolymer membranes containing high-acid-concentration hydrophilic blocks consisting of HPPS and 3,3'-disulfonate-4,4'-dichlorodiphenylsulfone (sDCDPS), 6) development of synthetic routes to aromatic polymer backbones containing pendent 1H-1,2,3-triazole moieties, 7) development of coupling strategies to create phase-separated block copolymers between hydrophilic sulfonated prepolymers and commodity polymers such as PFPO, 8) establishment of basic performance

  11. Red cell membrane lipids in hemoglobinopathies.

    PubMed

    Kuypers, Frans A

    2008-11-01

    The complex mixture of lipids and proteins of the red blood cell membrane is well maintained during the life of the cell. Lipid analysis of the red cell reveals hundreds of phospholipid molecular species and cholesterol that differ with respect to their (polar) head group, and (apolar) side chains. These molecules move rapidly in the plane, as well as across the lipid bilayer. This dynamic movement is highly organized. In the plane of the bilayer, areas enriched in certain lipids accommodate protein structure and modulate function. While lipids move across the bilayer, the organization is highly asymmetric. Amino phospholipids are mainly found on the inside and choline containing phospholipids on the outside. Both the composition and organization of the red cell membrane is maintained throughout the life of the red cell by an intricate mechanism that involves enzymes, transporters and cytosolic factors. Key proteins that maintain red blood cell lipid organization have recently been identified. Alterations in these mechanisms, as the result of the globin mutations in sickle cell disease or thalassemia will lead to loss of membrane viability, apoptosis during erythropoiesis, early demise of the cell in the circulation, and when these cells are not removed appropriately their presence has pathologic consequences.

  12. Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species.

    PubMed Central

    Lafleur, M A; Hollenberg, M D; Atkinson, S J; Knäuper, V; Murphy, G; Edwards, D R

    2001-01-01

    Thrombin, a critical enzyme in the coagulation cascade, has also been associated with angiogenesis and activation of the zymogen form of matrix metalloproteinase-2 (MMP-2 or gelatinase-A). We show that thrombin activated pro-MMP-2 in a dose- and time-dependent manner in cultured human umbilical-vein endothelial cells (HUVECs) to generate a catalytically active 63 kDa protein that accumulated as the predominant form in the conditioned medium. This 63 kDa thrombin-activated MMP-2 is distinct from the 62 kDa species found following concanavalin A or PMA stimulated pro-MMP-2 activation. Hirudin and leupeptin blocked thrombin-induced pro-MMP-2 activation, demonstrating that the proteolytic activity of thrombin is essential. However, activation was also dependent upon membrane-type-MMP (MT-MMP) action, since it was blocked by EDTA, o-phenanthroline, hydroxamate metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-4, but not TIMP-1. Thrombin inefficiently cleaved recombinant 72 kDa pro-MMP-2, but efficiently cleaved the 64 kDa MT-MMP-processed intermediate form in the presence of cells. Thrombin also rapidly (within 1 h) increased cellular MT-MMP activity, and at longer time points (>6 h) it increased expression of MT1-MMP mRNA and protein. Thus signalling via proteinase-activated receptors (PARs) may play a role in thrombin-induced MMP-2 activation, though this does not appear to involve PAR1, PAR2, or PAR4 in HUVECs. These results indicate that in HUVECs the activation of pro-MMP-2 by thrombin involves increased MT-MMP activity and preferential cleavage of the MT-MMP-processed 64 kDa MMP-2 form in the presence of cells. The integration of these proteinase systems in the vascular endothelium may be important during thrombogenesis and tissue remodelling associated with neovascularization. PMID:11415441

  13. Modification and evaluation of fuel cell membranes

    NASA Astrophysics Data System (ADS)

    Nalawade, Amol Prataprao

    The primary goals of this study were modification of existing NafionRTM membranes and characterization of newly developed hydrocarbon-based membranes for high temperature fuel cell applications. Various NafionRTM/silicate nanocomposites were formulated via in situ sol-gel reactions for tetraethylorthosilicate. Different silicate composition profiles generated across membrane cross-sections were investigated by EDAX/ESEM. Composite water uptake, proton conductivity and fuel cell performance were comparable to that of unmodified Nafion RTM. Tafel analysis showed better electrode kinetics for composites having more silicate in the middle and less or no silicate at electrolyte-electrode interfaces. All composites showed reduced fuel cross-over and superior mechanical as well as chemical durability than unmodified NafionRTM. Poly(cyclohexadiene) (PCHD) materials were characterized in the interest of developing alternative low-cost proton exchange membranes. All cross-linked sulfonated (xsPCHD) membranes showed significantly higher water uptake at 80 °C and higher proton conductivity at 120 °C at all relative humidities (RH), compared to the current benchmark membrane, NafionRTM. A xsPCHD-poly(ethylene glycol) (PEG) copolymer and a xsPCHD-PEG blend surpassed the DOE target by exhibiting proton conductivities of 141.44 and 322.40 mS/cm, respectively, at 50 % RH. Although the PCHD-based PEMs exhibited thermal stability up to 150 °C, they showed poor mechanical properties which would cause poor membrane durability during fuel cell operation. Atomic force microscopy studies demonstrated nanophase separated morphology of xsPCHD having a higher degree of connectedness of hydrophilic domains in the copolymer and blends relative to the xsPCHD homopolymer. Broadband dielectric spectroscopy (BDS) was used to study sub-Tg relaxations in annealed poly(2,5-benzimidazole) (ABPBI) fuel cell precursor materials. A trend in degree of connectivity of charge migration pathways and

  14. Effects of chronic kidney disease on blood cells membrane properties.

    PubMed

    Kaderjakova, Z; Lajdova, I; Horvathova, M; Morvova, M; Sikurova, L

    2012-10-01

    Chronic kidney disease (CKD) is progressive loss of renal function associated among others with increased intracellular calcium concentration. The purpose of this study was to identify the effects of CKD on cell membrane properties such as human red blood cell Ca(2+) ATPase activity, lymphocyte plasma membrane P2X(7) receptor expression and function. This could help us in elucidating the origin of increased calcium concentration in blood cells. We found out Ca(2+) ATPase activity is decreased in early stage CKD patients resulting in altered calcium removal from cytoplasm. By means of flow cytometry we assessed that P2X(7) receptor expression on lymphocyte membrane is 1.5 fold increased for CKD patients. Moreover, we detected an increased uptake of ethidium bromide through this receptor in CKD at basal conditions. It means CKD lymphocyte membranes contain more receptors which are more permeable thus allowing increased calcium influx from extracellular milieu. Finally, we can state alterations in blood cell membranes are closely linked to CKD and may be responsible for intracellular calcium accumulation.

  15. Membrane proteins of dense lysosomes from Chinese hamster ovary cells

    SciTech Connect

    Chance, S.C.

    1987-01-01

    In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

  16. Cell or Cell Membrane-Based Drug Delivery Systems

    PubMed Central

    Tan, Songwei; Wu, Tingting; Zhang, Dan; Zhang, Zhiping

    2015-01-01

    Natural cells have been explored as drug carriers for a long period. They have received growing interest as a promising drug delivery system (DDS) until recently along with the development of biology and medical science. The synthetic materials, either organic or inorganic, are found to be with more or less immunogenicity and/or toxicity. The cells and extracellular vesicles (EVs), are endogenous and thought to be much safer and friendlier. Furthermore, in view of their host attributes, they may achieve different biological effects and/or targeting specificity, which can meet the needs of personalized medicine as the next generation of DDS. In this review, we summarized the recent progress in cell or cell membrane-based DDS and their fabrication processes, unique properties and applications, including the whole cells, EVs and cell membrane coated nanoparticles. We expect the continuing development of this cell or cell membrane-based DDS will promote their clinic applications. PMID:26000058

  17. [Studies on potassium transport through glial cell membranes (author's transl)].

    PubMed

    Coles, J A; Gardner-Medwin, A R; Tsacopoulos, M

    1980-04-01

    The retina of the honeybee drone is used as a model for the study of ion movements across the membranes of the glial cells caused by changes in the extracellular potassium concentration. The values found for changes in extracellular potential suggest that at least some of the potassium that enters glial cells in an active region of tissue is associated with an efflux of potassium from parts of the glial syncytium not affected by an increase in extracellular potassium concentration. In addition, it appears that ions other than K+ cross the glial membrane.

  18. Accumulation of Multipotent Progenitor Cells on Polymethylpentene Membranes During Extracorporeal Membrane Oxygenation.

    PubMed

    Lehle, Karla; Friedl, Lucas; Wilm, Julius; Philipp, Alois; Müller, Thomas; Lubnow, Matthias; Schmid, Christof

    2016-06-01

    Multipotent progenitor cells were mobilized during pediatric extracorporeal membrane oxygenation (ECMO). We hypothesize that these cells also adhered onto polymethylpentene (PMP) fibers within the membrane oxygenator (MO) during adult ECMO support. Mononuclear cells were removed from the surface of explanted PMP-MOs (n = 16). Endothelial-like outgrowth and mesenchymal-like cells were characterized by flow cytometric analysis using different surface markers. Spindle-shaped attaching cells were identified early, but without proliferative activity. After long-term cultivation palisading type or cobblestone-type outgrowth cells with high proliferative activity appeared and were characterized as (i) leukocytoid CD45+/CD31+ (CD133+/VEGFR-II+/CD90+/CD14+/CD146dim/CD105dim); (ii) endothelial-like CD45-/CD31+ (VEGF-RII+/CD146+/CD105+/CD133-/CD14-/CD90-); and (iii) mesenchymal-like cells CD45-/CD31- (CD105+/CD90+/CD133dim/VEGFR-II-/CD146-/CD14-). The distribution of the cell populations depended on the MO and cultivation time. Endothelial-like cells formed capillary-like structures and did uptake Dil-acetylated low-density lipoprotein. Endothelial- and mesenchymal-like cells adhered on the surface of PMP-MOs. Further research is needed to identify the clinical relevance of these cells.

  19. Hypercompliant Apical Membranes of Bladder Umbrella Cells

    PubMed Central

    Mathai, John C.; Zhou, Enhua H.; Yu, Weiqun; Kim, Jae Hun; Zhou, Ge; Liao, Yi; Sun, Tung-Tien; Fredberg, Jeffrey J.; Zeidel, Mark L.

    2014-01-01

    Urinary bladder undergoes dramatic volume changes during filling and voiding cycles. In the bladder the luminal surface of terminally differentiated urothelial umbrella cells is almost completely covered by plaques. These plaques (500 to 1000 nm) are made of a family of proteins called uroplakins that are known to form a tight barrier to prevent leakage of water and solutes. Electron micrographs from previous studies show these plaques to be interconnected by hinge regions to form structures that appear rigid, but these same structures must accommodate large changes in cell shape during voiding and filling cycles. To resolve this paradox, we measured the stiffness of the intact, living urothelial apical membrane and found it to be highly deformable, even more so than the red blood cell membrane. The intermediate cells underlying the umbrella cells do not have uroplakins but their membranes are an order of magnitude stiffer. Using uroplakin knockout mouse models we show that cell compliance is conferred by uroplakins. This hypercompliance may be essential for the maintenance of barrier function under dramatic cell deformation during filling and voiding of the bladder. PMID:25229135

  20. Membrane electrode assembly for a fuel cell

    NASA Technical Reports Server (NTRS)

    Prakash, Surya (Inventor); Narayanan, Sekharipuram R. (Inventor); Atti, Anthony (Inventor); Olah, George (Inventor); Smart, Marshall C. (Inventor)

    2006-01-01

    A catalyst ink for a fuel cell including a catalytic material and poly(vinylidene fluoride). The ink may be applied to a substrate to form an electrode, or bonded with other electrode layers to form a membrane electrode assembly (MEA).

  1. Age related alterations of adrenoreceptor activity in erythrocyte membrane.

    PubMed

    Lomsadze, G; Khetsuriani, R; Arabuli, M; Intskirveli, N; Sanikidze, T

    2011-06-01

    The aim of the study was the investigation of age-related functional alterations of adrenoreceptors and the effect of agonist and antagonist drugs on age related adrenoreceptor activity in erythrocyte membrane. The impact of isopropanol and propanol on functional activity β- adrenergic receptors in red blood cell membrane were studied in 50 practically healthy men--volunteers. (I group--75-89 years old, II group--22-30 years old). The EPR signals S1 and S2 were registered in red blood cell membrane samples after incubation with isopropanol and propanol respectively. It was found that decreasing sensitivity (functional activity) of red blood cells membrane adrenoreceptors comes with aging (S1old

  2. Nanoporous membrane-sealed microfluidic devices for improved cell viability.

    PubMed

    Masand, Shirley N; Mignone, Lindsay; Zahn, Jeffrey D; Shreiber, David I

    2011-12-01

    Cell-laden microfluidic devices have broad potential in various biomedical applications, including tissue engineering and drug discovery. However, multiple difficulties encountered while culturing cells within devices affecting cell viability, proliferation, and behavior has complicated their use. While active perfusion systems have been used to overcome the diffusive limitations associated with nutrient delivery into microchannels to support longer culture times, these systems can result in non-uniform oxygen and nutrient delivery and subject cells to shear stresses, which can affect cell behavior. Additionally, histological analysis of cell cultures within devices is generally laborious and yields inconsistent results due to difficulties in delivering labeling agents in microchannels. Herein, we describe a simple, cost-effective approach to preserve cell viability and simplify labeling within microfluidic networks without the need for active perfusion. Instead of bonding a microfluidic network to glass, PDMS, or other solid substrate, the network is bonded to a semi-permeable nanoporous membrane. The membrane-sealed devices allow free exchange of proteins, nutrients, buffers, and labeling reagents between the microfluidic channels and culture media in static culture plates under sterile conditions. The use of the semi-permeable membrane dramatically simplifies microniche cell culturing while avoiding many of the complications which arise from perfusion systems.

  3. Oxytocin binding by myoepithelial cell membranes from involuted mammary tissue.

    PubMed

    Ruberti, A; Olins, G M; Eakle, K A; Bremel, R D

    1983-04-29

    Oxytocin binding activity of myoepithelial cell membranes from mammary tissue was measured under a variety of different experimental conditions. Mammary tissue from non-lactating rats bound oxytocin with a Kd of 9.2 +/- 1.6 nM (+/- S.E.) and indicates that receptors are retained by the myoepithelial cells in a non-lactating state. Ovariectomy of non-lactating rats did not depress the binding activity of the membranes. Administration of the estrogenic compounds estradiol-17 beta and diethylstibestrol at doses which affect uterine weight and are known to increase uterine oxytocin binding did not influence the binding activity of the myoepithelial cells. This indicates that the oxytocin receptors of the mammary gland are not under the same endocrine control as the uterine receptors. PMID:6303330

  4. Selectivity of Direct Methanol Fuel Cell Membranes.

    PubMed

    Aricò, Antonino S; Sebastian, David; Schuster, Michael; Bauer, Bernd; D'Urso, Claudia; Lufrano, Francesco; Baglio, Vincenzo

    2015-11-24

    Sulfonic acid-functionalized polymer electrolyte membranes alternative to Nafion(®) were developed. These were hydrocarbon systems, such as blend sulfonated polyetheretherketone (s-PEEK), new generation perfluorosulfonic acid (PFSA) systems, and composite zirconium phosphate-PFSA polymers. The membranes varied in terms of composition, equivalent weight, thickness, and filler and were investigated with regard to their methanol permeation characteristics and proton conductivity for application in direct methanol fuel cells. The behavior of the membrane electrode assemblies (MEA) was investigated in fuel cell with the aim to individuate a correlation between membrane characteristics and their performance in a direct methanol fuel cell (DMFC). The power density of the DMFC at 60 °C increased according to a square root-like function of the membrane selectivity. This was defined as the reciprocal of the product between area specific resistance and crossover. The power density achieved at 60 °C for the most promising s-PEEK-based membrane-electrode assembly (MEA) was higher than the benchmark Nafion(®) 115-based MEA (77 mW·cm(-2) vs. 64 mW·cm(-2)). This result was due to a lower methanol crossover (47 mA·cm(-2) equivalent current density for s-PEEK vs. 120 mA·cm(-2) for Nafion(®) 115 at 60 °C as recorded at OCV with 2 M methanol) and a suitable area specific resistance (0.15 Ohm cm² for s-PEEK vs. 0.22 Ohm cm² for Nafion(®) 115).

  5. Selectivity of Direct Methanol Fuel Cell Membranes

    PubMed Central

    Aricò, Antonino S.; Sebastian, David; Schuster, Michael; Bauer, Bernd; D’Urso, Claudia; Lufrano, Francesco; Baglio, Vincenzo

    2015-01-01

    Sulfonic acid-functionalized polymer electrolyte membranes alternative to Nafion® were developed. These were hydrocarbon systems, such as blend sulfonated polyetheretherketone (s-PEEK), new generation perfluorosulfonic acid (PFSA) systems, and composite zirconium phosphate–PFSA polymers. The membranes varied in terms of composition, equivalent weight, thickness, and filler and were investigated with regard to their methanol permeation characteristics and proton conductivity for application in direct methanol fuel cells. The behavior of the membrane electrode assemblies (MEA) was investigated in fuel cell with the aim to individuate a correlation between membrane characteristics and their performance in a direct methanol fuel cell (DMFC). The power density of the DMFC at 60 °C increased according to a square root-like function of the membrane selectivity. This was defined as the reciprocal of the product between area specific resistance and crossover. The power density achieved at 60 °C for the most promising s-PEEK-based membrane-electrode assembly (MEA) was higher than the benchmark Nafion® 115-based MEA (77 mW·cm−2 vs. 64 mW·cm−2). This result was due to a lower methanol crossover (47 mA·cm−2 equivalent current density for s-PEEK vs. 120 mA·cm−2 for Nafion® 115 at 60 °C as recorded at OCV with 2 M methanol) and a suitable area specific resistance (0.15 Ohm cm2 for s-PEEK vs. 0.22 Ohm cm2 for Nafion® 115). PMID:26610582

  6. Elisidepsin Interacts Directly with Glycosylceramides in the Plasma Membrane of Tumor Cells to Induce Necrotic Cell Death

    PubMed Central

    Molina-Guijarro, José Manuel; García, Carolina; Macías, Álvaro; García-Fernández, Luis Francisco; Moreno, Cristina; Reyes, Fernando; Martínez-Leal, Juan Fernando; Fernández, Rogelio; Martínez, Valentín; Valenzuela, Carmen; Lillo, M. Pilar; Galmarini, Carlos M.

    2015-01-01

    Plasma membrane integrity is essential for cell life. Any major break on it immediately induces the death of the affected cell. Different molecules were described as disrupting this cell structure and thus showing antitumor activity. We have previously defined that elisidepsin (Irvalec®, PM02734) inserts and self-organizes in the plasma membrane of tumor cells, inducing a rapid loss of membrane integrity, cell permeabilization and necrotic death. Here we show that, in sensitive HCT-116 colorectal cells, all these effects are consequence of the interaction of elisidepsin with glycosylceramides in the cell membrane. Of note, an elisidepsin-resistant subline (HCT-116-Irv) presented reduced levels of glycosylceramides and no accumulation of elisidepsin in the plasma membrane. Consequently, drug treatment did not induce the characteristic necrotic cell death. Furthermore, GM95, a mutant derivative from B16 mouse melanoma cells lacking ceramide glucosyltransferase (UGCG) activity and thus the synthesis of glycosylceramides, was also resistant to elisidepsin. Over-expression of UGCG gene in these deficient cells restored glycosylceramides synthesis, rendering them sensitive to elisidepsin, at a similar level than parental B16 cells. These results indicate that glycosylceramides act as membrane targets of elisidepsin, facilitating its insertion in the plasma membrane and the subsequent membrane permeabilization that leads to drug-induced cell death. They also indicate that cell membrane lipids are a plausible target for antineoplastic therapy. PMID:26474061

  7. Aluminum and temperature alteration of cell membrane permeability of Quercus rubra

    SciTech Connect

    Junping Chen; Sucoff, E.I.; Stadelmann, E.J. )

    1991-06-01

    Al toxicity is the major factor limiting plant growth in acid soils. This report extends research on Al-induced changes in membrane behavior of intact root cortex cells of Northern red oak (Quercus rubra). Membrane permeability was determined by the plasmometric method for individual intact cells at temperatures from 2 or 4 to 35 C. Al (0.37 millimolar) significantly increased membrane permeability to urea and monoethyl urea and decreased permeability to water. Al significantly altered the activation energy required to transport water (+ 32%), urea (+ 9%), and monoethyl urea ({minus}7%) across cell membranes. Above 9 C, Al increased the lipid partiality of the cell membranes; below 7 C, Al decreased it. Al narrowed by 6 C the temperature range over which plasmolysis occurred without membrane damage. These changes in membrane behavior are explainable if Al reduced membrane lipid fluidity and kink frequency and increases packing density and the occurrence of straight lipid chains.

  8. Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells.

    PubMed

    Nakano, Miyako; Saldanha, Rohit; Göbel, Anja; Kavallaris, Maria; Packer, Nicolle H

    2011-11-01

    Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

  9. Collective charge excitations along cell membranes

    NASA Astrophysics Data System (ADS)

    Manousakis, E.

    2005-07-01

    A significant part of the thin layers of counter-ions adjacent to the exterior and interior surfaces of a cell membrane form quasi-two-dimensional (2D) layers of mobile charge. Collective charge density oscillations, known as plasmon modes, in these 2D charged systems of counter-ions are predicted in the present paper. This is based on a calculation of the self-consistent response of this system to a fast electric field fluctuation. The possibility that the membrane channels might be using these excitations to carry out fast communication is suggested and experiments are proposed to reveal the existence of such excitations.

  10. Understanding the transport processes in polymer electrolyte membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Cheah, May Jean

    Polymer electrolyte membrane (PEM) fuel cells are energy conversion devices suitable for automotive, stationary and portable applications. An engineering challenge that is hindering the widespread use of PEM fuel cells is the water management issue, where either a lack of water (resulting in membrane dehydration) or an excess accumulation of liquid water (resulting in fuel cell flooding) critically reduces the PEM fuel cell performance. The water management issue is addressed by this dissertation through the study of three transport processes occurring in PEM fuel cells. Water transport within the membrane is a combination of water diffusion down the water activity gradient and the dragging of water molecules by protons when there is a proton current, in a phenomenon termed electro-osmotic drag, EOD. The impact of water diffusion and EOD on the water flux across the membrane is reduced due to water transport resistance at the vapor/membrane interface. The redistribution of water inside the membrane by EOD causes an overall increase in the membrane resistance that regulates the current and thus EOD, thereby preventing membrane dehydration. Liquid water transport in the PEM fuel cell flow channel was examined at different gas flow regimes. At low gas Reynolds numbers, drops transitioned into slugs that are subsequently pushed out of the flow channel by the gas flow. The slug volume is dependent on the geometric shape, the surface wettability and the orientation (with respect to gravity) of the flow channel. The differential pressure required for slug motion primarily depends on the interfacial forces acting along the contact lines at the front and the back of the slug. At high gas Reynolds number, water is removed as a film or as drops depending on the flow channel surface wettability. The shape of growing drops at low and high Reynolds number can be described by a simple interfacial energy minimization model. Under flooding conditions, the fuel cell local current

  11. Membrane potential genesis in Nitella cells, mitochondria, and thylakoids.

    PubMed

    Kitasato, Hiroshi

    2003-10-01

    The resting membrane potential of Nitella cells shifts in parallel with the change in H+ equilibrium potential, but is not equal to the H+ equilibrium potential. The deviation of the membrane potential from the H+ equilibrium potential depends on the extrusion rate of H+ by the electrogenic H+-pump. The activity of the electrogenic H+-pump was formulated in terms of the change in the free energy of ATP hydrolysis. The deviation of membrane potential from the H+ equilibrium potential induces a passive H+ flow. The passive inward H+ current may be coupled with Cl- uptake. The coupling rate of H+,Cl- co-transport was discussed. The membrane potential of mitochondria was electrochemically formulated in terms of oxidation-reduction H2/H+ half-cells spontaneously formed at the inner and outer boundaries of each trans-membrane electron-conducting pathway. The membrane potential formed by a pair of H2/H+ redox cells is pH-sensitive in its nature, but deviates from the H+ equilibrium potential to an extent that depends on the logarithm of the ratio of H2 concentrations at the inner and outer boundaries. The membrane potential of thylakoids is considered to be primarily due to the electromotive force of photocells embedded in the thylakoid membrane, as far as the anode and cathode of each photocell are in contact with the inner and outer solutions, respectively. The light-induced electronic current yields oxygen at the inner boundary and causes an increase in the H2 pool at the outer boundary of the electron-conducting pathway, which has no shunting plastoquinone chain between these two boundaries.

  12. Aging of cell membranes: facts and theories.

    PubMed

    Zs-Nagy, Imre

    2014-01-01

    This chapter is intended to outline the main results of a research trend realized by the author during the last 45 years, focused on the main role played by the cell membrane in the aging process. It is a very wide field; therefore, the reader cannot expect in this limited space a detailed description, but will be given a wide, interdisciplinary insight into the main facts and theories regarding cellular aging. The central idea described here is the concept called the membrane hypothesis of aging (MHA). The history, the chemical roots, physicochemical facts, biophysical processes, as well as the obligatory biochemical consequences are all touched in by indicating the most important sources of detailed knowledge for those who are more interested in the basic biology of the aging process. This chapter covers also the available anti-aging interventions on the cell membrane by means of the centrophenoxine treatment based on the MHA. It also briefly interprets the possibilities of a just developing anti-aging method by using the recombinant human growth hormone, essential basis of which is the species specificity, and the general presence of receptors of this hormone in the plasma membrane of all types of cells.

  13. Fe(III) and Fe(II) ions different effects on Enterococcus hirae cell growth and membrane-associated ATPase activity

    SciTech Connect

    Vardanyan, Zaruhi; Trchounian, Armen

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Fe{sup 3+} stimulates but Fe{sup 2+} suppresses Enterococcus hirae wild-type and atpD mutant growth. Black-Right-Pointing-Pointer Fe ions change oxidation-reduction potential drop during cell growth. Black-Right-Pointing-Pointer Fe{sup 3+} and Fe{sup 2+} have opposite effects on a membrane-associated ATPase activity. Black-Right-Pointing-Pointer These effects are either in the presence of F{sub 0}F{sub 1} inhibitor or non-functional F{sub 0}F{sub 1}. Black-Right-Pointing-Pointer Fe ions decrease protons and coupled potassium ions fluxes across the membrane. -- Abstract: Enterococcus hirae is able to grow under anaerobic conditions during glucose fermentation (pH 8.0) which is accompanied by acidification of the medium and drop in its oxidation-reduction potential (E{sub h}) from positive values to negative ones (down to {approx}-200 mV). In this study, iron (III) ions (Fe{sup 3+}) have been shown to affect bacterial growth in a concentration-dependent manner (within the range of 0.05-2 mM) by decreasing lag phase duration and increasing specific growth rate. While iron(II) ions (Fe{sup 2+}) had opposite effects which were reflected by suppressing bacterial growth. These ions also affected the changes in E{sub h} values during bacterial growth. It was revealed that ATPase activity with and without N,N Prime -dicyclohexylcarbodiimide (DCCD), an inhibitor of the F{sub 0}F{sub 1}-ATPase, increased in the presence of even low Fe{sup 3+} concentration (0.05 mM) but decreased in the presence of Fe{sup 2+}. It was established that Fe{sup 3+} and Fe{sup 2+} both significantly inhibited the proton-potassium exchange of bacteria, but stronger effects were in the case of Fe{sup 2+} with DCCD. Such results were observed with both wild-type ATCC9790 and atpD mutant (with defective F{sub 0}F{sub 1}) MS116 strains but they were different with Fe{sup 3+} and Fe{sup 2+}. It is suggested that the effects of Fe{sup 3+} might be due to

  14. Photomodification of the electrical properties of the plasma membrane: a comparison between 6 different membrane-active photosensitizers.

    PubMed

    Killig, F; Kunz, L; Stark, G

    2001-05-01

    The present study deals with photomodification of the electrical properties of the plasma membrane of an epithelial cell line (opossum kidney (OK) cells). The effect of photofrin II (previously investigated) is compared with that of 5 other membrane-active sensitizers: sulfonated Zn-phthalocyanine, merocyanine 540, rose bengal, methylene blue and protoporphyrin IX (an endogenous sensitizer induced by addition of its biosynthetic precursor 5-aminolaevulinic acid). The study was performed in order to investigate whether photomodification of the ion transport properties of the plasma membrane by membrane-active sensitizers is a general and early event in cellular photosensitization. The changes in the electrical properties were monitored by application of the whole-cell and the inside-out configuration of the patch-clamp technique. Illumination in the presence of the compounds (apart from merocyanine 540) gave rise to similar changes of the electrical properties of the membrane: depolarization of the membrane potential, inactivation of a large-conductance, Ca2+-dependent K+-channel (maxi-KCa), and a strong increase of the leak conductance of the membrane. This similarity indicates the general character of the functional photomodifications by membrane-active sensitizers previously reported for photofrin II.

  15. TIR fluorescence screening of cell membranes

    NASA Astrophysics Data System (ADS)

    Bruns, Thomas; Strauss, Wolfgang S.; Sailer, Reinhard; Wagner, Michael; Schneckenburger, Herbert

    2005-03-01

    A novel setup for fluorescence measurements of surfaces of biological samples, in particular cell membranes, is described. The method is based on multiple total internal reflection (TIR) of a laser beam on the surface of a multi-well plate, such that 96 individual samples are excited simultaneously. Main prerequisites are an appropriate thickness and high transmission of the glass bottom, a non-cytotoxic adhesive, and appropriate glass rods for TIR illumination. Fluorescence from cell surface is detected simultaneously using an integrating CCD camera and appropriate optical filters. For validation of the system, transfected cells expressing a fluorescent membrane protein are used. In addition, intracellular translocation of green fluorescent protein kinase c upon stimulation is examined. The method appears well suitable for high throughput screening (HTS), since neither washing of the samples nor any re-adjustment of the equipment after changing of individual plates are necessary.

  16. Microfluidic microbial fuel cells: from membrane to membrane free

    NASA Astrophysics Data System (ADS)

    Yang, Yang; Ye, Dingding; Li, Jun; Zhu, Xun; Liao, Qiang; Zhang, Biao

    2016-08-01

    Microfluidic microbial fuel cells (MMFCs) are small carbon-neutral devices that use self-organized bacteria to degrade organic substrates and harness energy from the waste water. Conventional MMFCs have made great strides in the past decade and have overcome some limitations, such as high capital costs and low energy output. A co-laminar flow MFC has been first proposed in 2011 with the potential to be an attractively power source to niche applications. Co-laminar MFCs typically operate without any physical membranes separating the reactants, and bacterial ecosystems can be easily manipulated by regulating the inlet conditions. This paper highlights recent accomplishments in the development of co-laminar MFCs, emphasizing basic principles, mass transport and fluid dynamics including boundary layer theory, entrance conditions and mixing zone issues. Furthermore, the development of current techniques, major challenges and the potential research directions are discussed.

  17. Depolarization of cell membrane is associated with an increase in ciliary beat frequency (CBF).

    PubMed

    Mao, H; Wong, L B

    1995-10-24

    We hypothesize that activation of muscarinic cholinergic receptors depolarizes the cell membrane of the mammalian ciliated cells which in turn causes an increase of CBF. To test this hypothesis, a di-8-ANEPPS fluorescence photon counting and nonstationary heterodyne laser light scattering system was developed to measure cell membrane potential (psi) and CBF in cultured ovine tracheal ciliated cells simultaneously. Carbachol dose dependently depolarized the cell membrane with a corresponding stimulation of CBF. The carbachol induced depolarization of cell membrane and increases of CBF were inhibited by prior application of either atropine or verapamil or amiloride. These novel data suggest that depolarization of the cell membrane and the corresponding stimulation of CBF caused by the activation of muscarinic receptors of the mammalian ciliated cells are dependent on the influx of either extracellular Ca2+ or Na+. PMID:7488025

  18. Upregulation of bcl-2 by the Epstein-Barr virus latent membrane protein LMP1: a B-cell-specific response that is delayed relative to NF-kappa B activation and to induction of cell surface markers.

    PubMed Central

    Rowe, M; Peng-Pilon, M; Huen, D S; Hardy, R; Croom-Carter, D; Lundgren, E; Rickinson, A B

    1994-01-01

    An ability of the Epstein-Barr virus latent membrane protein LMP1 to enhance the survival of infected B cells through upregulation of the bcl-2 oncogene was first suggested by experiments involving gene transfection and the selection of stable LMP1+ clones (S. Henderson, M. Rowe, C. Gregory, F. Wang, E. Kieff, and A. Rickinson, Cell 65:1107-1115, 1991). However, it was not possible to ascertain whether Bcl-2 upregulation was a specific consequence of LMP1 expression or an artifact of the selection procedure whereby rare Bcl-2+ cells already present in the starting population might best be able to tolerate the potentially toxic effects of LMP1. We therefore reexamined this issue by using two different experimental approaches that allowed LMP1-induced effects to be monitored immediately following expression of the viral protein and in the absence of selective pressures; activation of the NF-kappa B transcription factor and upregulation of the cell adhesion molecule ICAM-1 were used as early indices of LMP1 function. In the first approach, stable clones of two B-cell lines carrying an LMP1 gene under the control of an inducible metallothionein promoter were induced to express LMP1 in all cells. Activation of NK-kappa B and upregulation of ICAM-1 occurred within 24 h and were followed at 48 to 72 h by upregulation of Bcl-2. In the second approach, we tested the generality of this phenomenon by transiently expressing LMP1 from a strong constitutively active promoter in a range of different cell types. All six B-cell lines tested showed NF-kappa B activation in response to LMP1 expression, and this was followed in five of six lines by expression of ICAM-1 and Bcl-2. In the same experiments, all three non-B-cell lines showed NF-kappa B activation and ICAM-1 upregulation but never any effect upon Bcl-2. We therefore conclude that Bcl-2 upregulation is part of the panoply of cellular changes induced by LMP1 but that the effect is cell type specific. Our data also suggest

  19. Interactions of Model Cell Membranes with Nanoparticles

    NASA Astrophysics Data System (ADS)

    D'Angelo, S. M.; Camesano, T. A.; Nagarajan, R.

    2011-12-01

    The same properties that give nanoparticles their enhanced function, such as high surface area, small size, and better conductivity, can also alter the cytotoxicity of nanomaterials. Ultimately, many of these nanomaterials will be released into the environment, and can cause cytotoxic effects to environmental bacteria, aquatic organisms, and humans. Previous results from our laboratory suggest that nanoparticles can have a detrimental effect on cells, depending on nanoparticle size. It is our goal to characterize the properties of nanomaterials that can result in membrane destabilization. We tested the effects of nanoparticle size and chemical functionalization on nanoparticle-membrane interactions. Gold nanoparticles at 2, 5,10, and 80 nm were investigated, with a concentration of 1.1x1010 particles/mL. Model cell membranes were constructed of of L-α-phosphatidylcholine (egg PC), which has negatively charged lipid headgroups. A quartz crystal microbalance with dissipation (QCM-D) was used to measure frequency changes at different overtones, which were related to mass changes corresponding to nanoparticle interaction with the model membrane. In QCM-D, a lipid bilayer is constructed on a silicon dioxide crystal. The crystals, oscillate at different harmonic frequencies depending upon changes in mass or energy dissipation. When mass is added to the crystal surface, such as through addition of a lipid vesicle solution, the frequency change decreases. By monitoring the frequency and dissipation, we could verify that a supported lipid bilayer (SLB) formed on the silica surface. After formation of the SLB, the nanoparticles can be added to the system, and the changes in frequency and dissipation are monitored in order to build a mechanistic understanding of nanoparticle-cell membrane interactions. For all of the smaller nanoparticles (2, 5, and 10 nm), nanoparticle addition caused a loss of mass from the lipid bilayer, which appears to be due to the formation of holes

  20. Sputter-deposited fuel cell membranes and electrodes

    NASA Technical Reports Server (NTRS)

    Narayanan, Sekharipuram R. (Inventor); Jeffries-Nakamura, Barbara (Inventor); Chun, William (Inventor); Ruiz, Ron P. (Inventor); Valdez, Thomas I. (Inventor)

    2001-01-01

    A method for preparing a membrane for use in a fuel cell membrane electrode assembly includes the steps of providing an electrolyte membrane, and sputter-depositing a catalyst onto the electrolyte membrane. The sputter-deposited catalyst may be applied to multiple sides of the electrolyte membrane. A method for forming an electrode for use in a fuel cell membrane electrode assembly includes the steps of obtaining a catalyst, obtaining a backing, and sputter-depositing the catalyst onto the backing. The membranes and electrodes are useful for assembling fuel cells that include an anode electrode, a cathode electrode, a fuel supply, and an electrolyte membrane, wherein the electrolyte membrane includes a sputter-deposited catalyst, and the sputter-deposited catalyst is effective for sustaining a voltage across a membrane electrode assembly in the fuel cell.

  1. Chemical Modulation of the Biological Activity of Reutericyclin: a Membrane-Active Antibiotic from Lactobacillus reuteri

    PubMed Central

    Cherian, Philip T.; Wu, Xiaoqian; Maddox, Marcus M.; Singh, Aman P.; Lee, Richard E.; Hurdle, Julian G.

    2014-01-01

    Whilst the development of membrane-active antibiotics is now an attractive therapeutic concept, progress in this area is disadvantaged by poor knowledge of the structure-activity relationship (SAR) required for optimizing molecules to selectively target bacteria. This prompted us to explore the SAR of the Lactobacillus reuteri membrane-active antibiotic reutericyclin, modifying three key positions about its tetramic acid core. The SAR revealed that lipophilic analogs were generally more active against Gram-positive pathogens, but introduction of polar and charged substituents diminished their activity. This was confirmed by cytometric assays showing that inactive compounds failed to dissipate the membrane potential. Radiolabeled substrate assays indicated that dissipation of the membrane potential by active reutericyclins correlated with inhibition of macromolecular synthesis in cells. However, compounds with good antibacterial activities also showed cytotoxicity against Vero cells and hemolytic activity. Although this study highlights the challenge of optimizing membrane-active antibiotics, it shows that by increasing antibacterial potency the selectivity index could be widened, allowing use of lower non-cytotoxic doses. PMID:24739957

  2. MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration.

    PubMed

    Aranda, Juan F; Reglero-Real, Natalia; Kremer, Leonor; Marcos-Ramiro, Beatriz; Ruiz-Sáenz, Ana; Calvo, María; Enrich, Carlos; Correas, Isabel; Millán, Jaime; Alonso, Miguel A

    2011-04-15

    Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes. PMID:21325632

  3. Fuel cell membranes and crossover prevention

    DOEpatents

    Masel, Richard I.; York, Cynthia A.; Waszczuk, Piotr; Wieckowski, Andrzej

    2009-08-04

    A membrane electrode assembly for use with a direct organic fuel cell containing a formic acid fuel includes a solid polymer electrolyte having first and second surfaces, an anode on the first surface and a cathode on the second surface and electrically linked to the anode. The solid polymer electrolyte has a thickness t:.gtoreq..times..times..times..times. ##EQU00001## where C.sub.f is the formic acid fuel concentration over the anode, D.sub.f is the effective diffusivity of the fuel in the solid polymer electrolyte, K.sub.f is the equilibrium constant for partition coefficient for the fuel into the solid polymer electrolyte membrane, I is Faraday's constant n.sub.f is the number of electrons released when 1 molecule of the fuel is oxidized, and j.sub.f.sup.c is an empirically determined crossover rate of fuel above which the fuel cell does not operate.

  4. Feedback regulation between plasma membrane tension and membrane-bending proteins organizes cell polarity during leading edge formation.

    PubMed

    Tsujita, Kazuya; Takenawa, Tadaomi; Itoh, Toshiki

    2015-06-01

    Tension applied to the plasma membrane (PM) is a global mechanical parameter involved in cell migration. However, how membrane tension regulates actin assembly is unknown. Here, we demonstrate that FBP17, a membrane-bending protein and an activator of WASP/N-WASP-dependent actin nucleation, is a PM tension sensor involved in leading edge formation. In migrating cells, FBP17 localizes to short membrane invaginations at the leading edge, while diminishing from the cell rear in response to PM tension increase. Conversely, following reduced PM tension, FBP17 dots randomly distribute throughout the cell, correlating with loss of polarized actin assembly on PM tension reduction. Actin protrusive force is required for the polarized accumulation, indicating a role for FBP17-mediated activation of WASP/N-WASP in PM tension generation. In vitro experiments show that FBP17 membrane-bending activity depends on liposomal membrane tension. Thus, FBP17 is the local activator of actin polymerization that is inhibited by PM tension in the feedback loop that regulates cell migration.

  5. Restoration of "normal" membrane function in CLL cells by patients' serum.

    PubMed

    Lewis, C M; Pegrum, G D

    1977-02-01

    Direct labelling of cell surface protein with 125I has enabled measurements to be made of lymphocyte membrane turnover. In CLL cells this is evidently much retarded in comparison with normal lymphocytes and it was thought that slow replacement of cell surface proteins might be responsible both for lack of membrane mobility and poor immunological responses. Serum from CLL patients has been found to enhance viability of the leukaemic cells in culture and the interaction of these serum factors with the CLL cell membrane gives rise to rapid replacement of surface proteins. Thus the rate of membrane turnover in vivo would be comparable with that of normal lymphocytes. Lack of cell-surface mobility and poor membrane biosynthesis can not be held responsible for poor immunological activity in vivo and it is suggested that autoimmune antibodies binding to the surface of CLL cells may spatially inhibit activation by ligands.

  6. Membrane catalyst layer for fuel cells

    DOEpatents

    Wilson, Mahlon S.

    1993-01-01

    A gas reaction fuel cell incorporates a thin catalyst layer between a solid polymer electrolyte (SPE) membrane and a porous electrode backing. The catalyst layer is preferably less than about 10 .mu.m in thickness with a carbon supported platinum catalyst loading less than about 0.35 mgPt/cm.sup.2. The film is formed as an ink that is spread and cured on a film release blank. The cured film is then transferred to the SPE membrane and hot pressed into the surface to form a catalyst layer having a controlled thickness and catalyst distribution. Alternatively, the catalyst layer is formed by applying a Na.sup.+ form of a perfluorosulfonate ionomer directly to the membrane, drying the film at a high temperature, and then converting the film back to the protonated form of the ionomer. The layer has adequate gas permeability so that cell performance is not affected and has a density and particle distribution effective to optimize proton access to the catalyst and electronic continuity for electron flow from the half-cell reaction occurring at the catalyst.

  7. Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells.

    PubMed

    Chauveau, Anne; Aucher, Anne; Eissmann, Philipp; Vivier, Eric; Davis, Daniel M

    2010-03-23

    Membrane nanotubes are membranous tethers that physically link cell bodies over long distances. Here, we present evidence that nanotubes allow human natural killer (NK) cells to interact functionally with target cells over long distances. Nanotubes were formed when NK cells contacted target cells and moved apart. The frequency of nanotube formation was dependent on the number of receptor/ligand interactions and increased on NK cell activation. Most importantly, NK cell nanotubes contained a submicron scale junction where proteins accumulated, including DAP10, the signaling adaptor that associates with the activating receptor NKG2D, and MHC class I chain-related protein A (MICA), a cognate ligand for NKG2D, as occurs at close intercellular synapses between NK cells and target cells. Quantitative live-cell fluorescence imaging suggested that MICA accumulated at small nanotube synapses in sufficient numbers to trigger cell activation. In addition, tyrosine-phosphorylated proteins and Vav-1 accumulated at such junctions. Functionally, nanotubes could aid the lysis of distant target cells either directly or by moving target cells along the nanotube path into close contact for lysis via a conventional immune synapse. Target cells moving along the nanotube path were commonly polarized such that their uropods faced the direction of movement. This is the opposite polarization than for normal cell migration, implying that nanotubes can specifically drive target cell movement. Finally, target cells that remained connected to an NK cell by a nanotube were frequently lysed, whereas removing the nanotube using a micromanipulator reduced lysis of these target cells.

  8. A review of radiation-grafted polymer electrolyte membranes for alkaline polymer electrolyte membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Zhou, Tianchi; Shao, Rong; Chen, Song; He, Xuemei; Qiao, Jinli; Zhang, Jiujun

    2015-10-01

    The past two decades have witnessed many efforts to develop radiation-grafted alkaline membranes for alkaline PEM fuel cell applications, as such membranes have certain advantages over other kinds of alkaline membranes, including well-controlled composition, functionality, and other promising properties. To facilitate research and development in this area, the present paper reviews radiation-grafted alkaline membranes. We examine their synthesis/fabrication/characterization, membrane material selection, and theoretical approaches for fundamental understanding. We also present detailed examinations of their application in fuel cell in terms of the working principles of the radiation grafting process, the fabrication of MEAs using radiation-grafted membranes, the membranes' corresponding performance in alkaline PEM fuel cells, as well as performance optimization. The paper also summarizes the challenges and mitigation strategies for radiation-grafted alkaline membranes and their application in PEM fuel cells, presenting an overall picture of the technology as it presently stands.

  9. Human T Cell Crosstalk Is Induced by Tumor Membrane Transfer

    PubMed Central

    Uzana, Ronny; Eisenberg, Galit; Merims, Sharon; Frankenburg, Shoshana; Pato, Aviad; Yefenof, Eitan; Engelstein, Roni; Peretz, Tamar

    2015-01-01

    Trogocytosis is a contact-dependent unidirectional transfer of membrane fragments between immune effector cells and their targets, initially detected in T cells following interaction with professional antigen presenting cells (APC). Previously, we have demonstrated that trogocytosis also takes place between melanoma-specific cytotoxic T lymphocytes (CTLs) and their cognate tumors. In the present study, we took this finding a step further, focusing on the ability of melanoma membrane-imprinted CD8+ T cells to act as APCs (CD8+T-APCs). We demonstrate that, following trogocytosis, CD8+T-APCs directly present a variety of melanoma derived peptides to fraternal T cells with the same TCR specificity or to T cells with different TCRs. The resulting T cell-T cell immune synapse leads to (1) Activation of effector CTLs, as determined by proliferation, cytokine secretion and degranulation; (2) Fratricide (killing) of CD8+T-APCs by the activated CTLs. Thus, trogocytosis enables cross-reactivity among CD8+ T cells with interchanging roles of effectors and APCs. This dual function of tumor-reactive CTLs may hint at their ability to amplify or restrict reactivity against the tumor and participate in modulation of the anti-cancer immune response. PMID:25671577

  10. Pseudomonas aeruginosa cells adapted to benzalkonium chloride show resistance to other membrane-active agents but not to clinically relevant antibiotics.

    PubMed

    Loughlin, M F; Jones, M V; Lambert, P A

    2002-04-01

    Our objective was to determine whether strains of Pseudomonas aeruginosa can adapt to growth in increasing concentrations of the disinfectant benzalkonium chloride (BKC), and whether co-resistance to clinically relevant antimicrobial agents occurs. Attempts were made to determine what phenotypic alterations accompanied resistance and whether these explained the mechanism of resistance. Strains were serially passaged in increasing concentrations of BKC in static nutrient broth cultures. Serotyping and genotyping were used to determine purity of the cultures. Two strains were examined for cross-resistance to other disinfectants and antibiotics by broth dilution MIC determination. Alterations in outer membrane proteins and lipopolysaccharide (LPS) expressed were examined by SDS-PAGE. Cell surface hydrophobicity and charge, uptake of disinfectant and proportion of specific fatty acid content of outer and cytoplasmic membranes were determined. Two P. aeruginosa strains showed a stable increase in resistance to BKC. Co-resistance to other quaternary ammonium compounds was observed in both strains; chloramphenicol and polymyxin B resistance were observed in one and a reduction in resistance to tobramycin observed in the other. However, no increased resistance to other biocides (chlorhexidine, triclosan, thymol) or antibiotics (ceftazidime, imipenem, ciprofloxacin, tobramycin) was detected. Characteristics accompanying resistance included alterations in outer membrane proteins, uptake of BKC, cell surface charge and hydrophobicity, and fatty acid content of the cytoplasmic membrane, although no evidence was found for alterations in LPS. Each of the two strains had different alterations in phenotype, indicating that such adaptation is unique to each strain of P. aeruginosa and does not result from a single mechanism shared by the whole species. PMID:11909837

  11. Durability aspects of polymer electrolyte membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Sethuraman, Vijay Anand

    In order for the successful adoption of proton exchange membrane (PEM) fuel cell technology, it is imperative that durability is understood, quantified and improved. A number of mechanisms are known to contribute to PEMFC membrane electrode assembly (MEA) performance degradation. In this dissertation, we show, via experiments, some of the various processes that degrade the proton exchange membrane in a PEM fuel cell; and catalyst poisoning due to hydrogen sulfide (H2S) and siloxane. The effect of humidity on the chemical stability of two types of membranes, [i.e., perfluorosulfonic acid type (PFSA, NafionRTM 112) and biphenyl sulfone hydrocarbon type, (BPSH-35)] was studied by subjecting the MEAs to open-circuit voltage (OCV) decay and potential cycling tests at elevated temperatures and low inlet gas relative humidities. The BPSH-35 membranes showed poor chemical stability in ex situ Fenton tests compared to that of NafionRTM membranes. However, under fuel cell conditions, BPSH-35 MEAs outperformed NafionRTM 112 MEAs in both the OCV decay and potential cycling tests. For both membranes, (i) at a given temperature, membrane degradation was more pronounced at lower humidities and (ii) at a given relative humidity operation, increasing the cell temperature accelerated membrane degradation. Mechanical stability of these two types of membranes was also studied using relative humidity (RH) cycling. Hydrogen peroxide (H2O2) formation rates in a proton exchange membrane (PEM) fuel cell were estimated by studying the oxygen reduction reaction (ORR) on a rotating ring disc electrode (RRDE). Fuel cell conditions were replicated by depositing a film of Pt/Vulcan XC-72 catalyst onto the disk and by varying the temperature, dissolved O2 concentration and the acidity levels in HClO4. The HClO4 acidity was correlated to ionomer water activity and hence fuel cell humidity. H 2O2 formation rates showed a linear dependence on oxygen concentration and square dependence on water

  12. Regulation of Cell Contraction and Membrane Ruffling by Distinct Signals in Migratory Cells

    PubMed Central

    Cheresh, David A.; Leng, Jie; Klemke, Richard L.

    1999-01-01

    Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively. PMID:10477763

  13. Fabrication and evaluation of novel zeolite membranes to control the neoplastic activity and anti-tumoral drug treatments in human breast cancer cells. Part 1: Synthesis and characterization of Pure Zeolite Membranes and Mixed Matrix Membranes for adhesion and growth of cancer cells.

    PubMed

    Tavolaro, Palmira; Martino, Guglielmo; Andò, Sebastiano; Tavolaro, Adalgisa

    2016-12-01

    Novel pure and hybrid zeolite membranes were prepared with appropriate different physicochemical characteristics such as frameworks, hydrophilicity, crystal size, chemical composition, acid-base properties (Point of Zero Charge, PZC) and surface morphology and used in inorganic cell/scaffold constructs. Because the control of cell interactions, as the adhesion, proliferation, remodelling and mobility, is important for differentiation and progression of tumors, this work focused on response of cancer cells adhered and grown on synthesized zeolite surfaces in order to study the influence of these scaffolds in controlled conditions. We have selected the MCF-7 and MDA-MB-231 human breast cancer cell line as model tumor cell lines. This study showed that all the zeolite membranes synthesized are excellent scaffolds because they are very selective materials to support the adhesion and growth of neoplastic cells. All zeolite scaffolds were characterized by FESEM, FTIR ATR, XRD, AFM, PZC and contact angle analyses. Cell adhesion, viability and morphology were measured by count, MTT assay and FESEM microphotography analysis, at various incubation times. PMID:27612784

  14. Fabrication and evaluation of novel zeolite membranes to control the neoplastic activity and anti-tumoral drug treatments in human breast cancer cells. Part 1: Synthesis and characterization of Pure Zeolite Membranes and Mixed Matrix Membranes for adhesion and growth of cancer cells.

    PubMed

    Tavolaro, Palmira; Martino, Guglielmo; Andò, Sebastiano; Tavolaro, Adalgisa

    2016-12-01

    Novel pure and hybrid zeolite membranes were prepared with appropriate different physicochemical characteristics such as frameworks, hydrophilicity, crystal size, chemical composition, acid-base properties (Point of Zero Charge, PZC) and surface morphology and used in inorganic cell/scaffold constructs. Because the control of cell interactions, as the adhesion, proliferation, remodelling and mobility, is important for differentiation and progression of tumors, this work focused on response of cancer cells adhered and grown on synthesized zeolite surfaces in order to study the influence of these scaffolds in controlled conditions. We have selected the MCF-7 and MDA-MB-231 human breast cancer cell line as model tumor cell lines. This study showed that all the zeolite membranes synthesized are excellent scaffolds because they are very selective materials to support the adhesion and growth of neoplastic cells. All zeolite scaffolds were characterized by FESEM, FTIR ATR, XRD, AFM, PZC and contact angle analyses. Cell adhesion, viability and morphology were measured by count, MTT assay and FESEM microphotography analysis, at various incubation times.

  15. Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides.

    PubMed

    Grage, Stephan L; Afonin, Sergii; Kara, Sezgin; Buth, Gernot; Ulrich, Anne S

    2016-01-01

    Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state (2)H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of

  16. Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides

    PubMed Central

    Grage, Stephan L.; Afonin, Sergii; Kara, Sezgin; Buth, Gernot; Ulrich, Anne S.

    2016-01-01

    Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state 2H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of a

  17. Large-Aperture Membrane Active Phased-Array Antennas

    NASA Technical Reports Server (NTRS)

    Karasik, Boris; McGrath, William; Leduc, Henry

    2009-01-01

    Large-aperture phased-array microwave antennas supported by membranes are being developed for use in spaceborne interferometric synthetic aperture radar systems. There may also be terrestrial uses for such antennas supported on stationary membranes, large balloons, and blimps. These antennas are expected to have areal mass densities of about 2 kg/sq m, satisfying a need for lightweight alternatives to conventional rigid phased-array antennas, which have typical areal mass densities between 8 and 15 kg/sq m. The differences in areal mass densities translate to substantial differences in total mass in contemplated applications involving aperture areas as large as 400 sq m. A membrane phased-array antenna includes patch antenna elements in a repeating pattern. All previously reported membrane antennas were passive antennas; this is the first active membrane antenna that includes transmitting/receiving (T/R) electronic circuits as integral parts. Other integral parts of the antenna include a network of radio-frequency (RF) feed lines (more specifically, a corporate feed network) and of bias and control lines, all in the form of flexible copper strip conductors on flexible polymeric membranes. Each unit cell of a prototype antenna (see Figure 1) contains a patch antenna element and a compact T/R module that is compatible with flexible membrane circuitry. There are two membrane layers separated by a 12.7-mm air gap. Each membrane layer is made from a commercially available flexible circuit material that, as supplied, comprises a 127-micron-thick polyimide dielectric layer clad on both sides with 17.5-micron-thick copper layers. The copper layers are patterned into RF, bias, and control conductors. The T/R module is located on the back side of the ground plane and is RF-coupled to the patch element via a slot. The T/R module is a hybrid multilayer module assembled and packaged independently and attached to the membrane array. At the time of reporting the information for

  18. Hemoglobin s polymerization and red cell membrane changes.

    PubMed

    Kuypers, Frans A

    2014-04-01

    Different pathways lead from the simple point mutation in hemoglobin to the membrane changes that characterize the altered interaction of the sickle red blood cell with its environment, including endothelial cells, white blood cells, and platelets. Polymerization and oxidation-induced damage to both lipid and protein components of the red cell membrane, as well as the generation of bioreactive membrane material (microparticles), has a profound effect on all tissues and organs, and defines the vasculopathy of the patient with sickle cell disease.

  19. Membrane-Bound TRAIL Supplements Natural Killer Cell Cytotoxicity Against Neuroblastoma Cells

    PubMed Central

    Sheard, Michael A.; Asgharzadeh, Shahab; Liu, Yin; Lin, Tsen-Yin; Wu, Hong-Wei; Ji, Lingyun; Groshen, Susan; Lee, Dean A.; Seeger, Robert C.

    2013-01-01

    Neuroblastoma cells have been reported to be resistant to death induced by soluble, recombinant forms of TRAIL (CD253/TNFSF10) due to low or absent expression of caspase-8 and/or TRAIL-receptor 2 (TRAIL-R2/DR5/CD262/TNFRSF10b). However, their sensitivity to membrane-bound TRAIL on natural killer (NK) cells is not known. Comparing microarray gene expression and response to NK cell-mediated cytotoxicity, we observed a correlation between TRAIL-R2 expression and the sensitivity of fourteen neuroblastoma cell lines to the cytotoxicity of NK cells activated with IL-2 plus IL-15. Even though most NK cytotoxicity was dependent upon perforin, the cytotoxicity was supplemented by TRAIL in fourteen of seventeen (82%) neuroblastoma cell lines as demonstrated using an anti-TRAIL neutralizing antibody. Similarly, a recently developed NK cell expansion system employing IL-2 plus lethally irradiated K562 feeder cells constitutively expressing membrane-bound IL-21 (K562 clone 9.mbIL21) resulted in activated NK cells derived from normal healthy donors and neuroblastoma patients that also utilized TRAIL to supplement cytotoxicity. Exogenous IFNγ up-regulated expression of caspase-8 in three of four neuroblastoma cell lines and increased the contribution of TRAIL to NK cytotoxicity against two of the three lines; however, relatively little inhibition of cytotoxicity was observed when activated NK cells were treated with an anti-IFNγ neutralizing antibody. Constraining the binding of anti-TRAIL neutralizing antibody to membrane-bound TRAIL but not soluble TRAIL indicated that membrane-bound TRAIL alone was responsible for essentially all of the supplemental cytotoxicity. Together, these findings support a role for membrane-bound TRAIL in the cytotoxicity of NK cells against neuroblastoma cells. PMID:23719242

  20. Fuel cell membrane hydration and fluid metering

    DOEpatents

    Jones, Daniel O.; Walsh, Michael M.

    2003-01-01

    A hydration system includes fuel cell fluid flow plate(s) and injection port(s). Each plate has flow channel(s) with respective inlet(s) for receiving respective portion(s) of a given stream of reactant fluid for a fuel cell. Each injection port injects a portion of liquid water directly into its respective flow channel. This serves to hydrate at least corresponding part(s) of a given membrane of the corresponding fuel cell(s). The hydration system may be augmented by a metering system including flow regulator(s). Each flow regulator meters an injecting at inlet(s) of each plate of respective portions of liquid into respective portion(s) of a given stream of fluid by corresponding injection port(s).

  1. Fuel cell membrane hydration and fluid metering

    DOEpatents

    Jones, Daniel O.; Walsh, Michael M.

    1999-01-01

    A hydration system includes fuel cell fluid flow plate(s) and injection port(s). Each plate has flow channel(s) with respective inlet(s) for receiving respective portion(s) of a given stream of reactant fluid for a fuel cell. Each injection port injects a portion of liquid water directly into its respective flow channel in order to mix its respective portion of liquid water with the corresponding portion of the stream. This serves to hydrate at least corresponding part(s) of a given membrane of the corresponding fuel cell(s). The hydration system may be augmented by a metering system including flow regulator(s). Each flow regulator meters an injecting at inlet(s) of each plate of respective portions of liquid into respective portion(s) of a given stream of fluid by corresponding injection port(s).

  2. Cell membrane-camouflaged nanoparticles for drug delivery.

    PubMed

    Luk, Brian T; Zhang, Liangfang

    2015-12-28

    Nanoparticles can preferentially accumulate at sites of action and hold great promise to improve the therapeutic index of many drugs. While conventional methods of nanocarrier-mediated drug delivery have focused on primarily synthetic approaches, engineering strategies that combine synthetic nanoparticles with natural biomaterials have recently gained much attention. In particular, cell membrane-camouflaged nanoparticles are a new class of biomimetic nanoparticles that combine the unique functionalities of cellular membranes and engineering versatility of synthetic nanomaterials for effective delivery of therapeutic agents. Herein, we report on the recent progress on cell membrane-coated nanoparticles for drug delivery. In particular, we highlight three areas: (i) prolonging systemic circulation via cell membrane coating, (ii) cell-specific targeting via cell membrane coating, and (iii) applications of cell membrane coating for drug delivery. The cell membrane-camouflaged nanoparticle platform has emerged as a novel delivery strategy with the potential to improve the therapeutic efficacy for the treatment of a variety of diseases.

  3. Membrane Purification Cell for Aluminum Recycling

    SciTech Connect

    David DeYoung; James Wiswall; Cong Wang

    2011-11-29

    Recycling mixed aluminum scrap usually requires adding primary aluminum to the scrap stream as a diluent to reduce the concentration of non-aluminum constituents used in aluminum alloys. Since primary aluminum production requires approximately 10 times more energy than melting scrap, the bulk of the energy and carbon dioxide emissions for recycling are associated with using primary aluminum as a diluent. Eliminating the need for using primary aluminum as a diluent would dramatically reduce energy requirements, decrease carbon dioxide emissions, and increase scrap utilization in recycling. Electrorefining can be used to extract pure aluminum from mixed scrap. Some example applications include producing primary grade aluminum from specific scrap streams such as consumer packaging and mixed alloy saw chips, and recycling multi-alloy products such as brazing sheet. Electrorefining can also be used to extract valuable alloying elements such as Li from Al-Li mixed scrap. This project was aimed at developing an electrorefining process for purifying aluminum to reduce energy consumption and emissions by 75% compared to conventional technology. An electrolytic molten aluminum purification process, utilizing a horizontal membrane cell anode, was designed, constructed, operated and validated. The electrorefining technology could also be used to produce ultra-high purity aluminum for advanced materials applications. The technical objectives for this project were to: - Validate the membrane cell concept with a lab-scale electrorefining cell; - Determine if previously identified voltage increase issue for chloride electrolytes holds for a fluoride-based electrolyte system; - Assess the probability that voltage change issues can be solved; and - Conduct a market and economic analysis to assess commercial feasibility. The process was tested using three different binary alloy compositions (Al-2.0 wt.% Cu, Al-4.7 wt.% Si, Al-0.6 wt.% Fe) and a brazing sheet scrap composition (Al-2

  4. Macrophages engulf endothelial cell membrane particles preceding pupillary membrane capillary regression

    PubMed Central

    Poché, Ross A.; Hsu, Chih-Wei; McElwee, Melissa L.; Burns, Alan R.; Dickinson, Mary E.

    2015-01-01

    Programmed capillary regression and remodeling are essential developmental processes. However, the cellular and molecular mechanisms that regulate vessel regression are only beginning to be understood. Here, using in vivo, dynamic, confocal imaging of mouse transgenic reporters as well as static confocal and electron microscopy, we studied the embryonic development and postnatal regression of the transient mouse pupillary membrane (PM) vasculature. This approach allowed us to directly observe the precise temporal sequence of cellular events preceding and during the elimination of the PM from the mouse eye. Imaging of Tcf/Lef-H2B::GFP Wnt-reporter mice uncovered that, unlike the hyaloid vasculature of the posterior eye, a PM endothelial cell (EC) Wnt/β-catenin response is unlikely to be part of the regression mechanism. Live imaging of EC and macrophage dynamics revealed highly active Csf1r-GFP+ macrophages making direct contact with the Flk1-myr::mCherry+ vessel surface and with membrane protrusions or filopodia extending from the ECs. Flk1-myr::mCherry+ EC membrane particles were observed on and around ECs as well as within macrophages. Electron microscopy studies confirmed that they were in phagosomes within macrophages, indicating that the macrophages engulfed the membrane particles. Interestingly, EC plasma membrane uptake by PM macrophages did not correlate with apoptosis and was found shortly after vessel formation at mid-gestation stages in the embryo; long before vessel regression begins during postnatal development. Additionally, genetic ablation of macrophages showed that EC membrane particles were still shed in the absence of macrophages suggesting that macrophages do not induce the formation or release of EC microparticles. These studies have uncovered a novel event during programmed capillary regression in which resident macrophages scavenge endothelial cell microparticles released from the PM vessels. This finding suggests that there may be an

  5. Membrane electrolytic cell for minimizing hypochlorite and chlorate formation

    SciTech Connect

    Fair, D. L.; Justice, D. D.; Woodard Jr., K. E.

    1985-07-09

    An electrolytic cell for the electrolysis of an alkali metal chloride brine is comprised of an anode compartment and a cathode compartment separated by a cation exchange membrane. The anode is comprised of an unflattened expanded structure of a valve metal selected from the group consisting of titanium, tantalum, niobium, and alloys thereof. At least one side of the anode has as the electrochemically active surface an electrodeposited layer of a valve metal oxide. A plurality of cracks traverse the electrodeposited layer and a coating of a platinum metal group oxide covers the electrodeposited layer and substantially fills the cracks. The cationic exchange membrane is comprised of a laminated structure having a first surface adapted to contact an anolyte in which the ion exchange groups are predominately sulfonic acid groups. The first surface is also in contact with the electrochemically active surface of the anode. A second surface of the cation exchange membrane, adapted to contact a catholyte, has ion exchange groups which are predominately carboxylic acid groups. The cathode positioned in the cathode compartment is spaced apart from the cation exchange membrane. The cell operates with both a low chlorine overvoltage and a low oxygen overvoltage. During electrolysis of alkali metal chloride brines, the formation of hypochlorite and chlorate ions is minimized and the alkali metal hydroxides produced have low chlorate concentrations and are suitable for use without further treatment in chlorate-sensitive applications. Spent brine treatment is simplified and at reduced costs.

  6. Phosphoinositide phosphorylation and hydrolysis in pancreatic islet cell membrane

    SciTech Connect

    Dunlop, M.E.; Malaisse, W.J.

    1986-02-01

    Membranes were isolated from dispersed rat pancreatic islet cells by attachment to Sephadex beads. When these membranes were exposed to (gamma-32P)ATP, formation of 32P-labeled phosphatidate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate was observed. Carbamylcholine, added 10 s prior to lipid extraction, caused a dose-related fall in 32P-labeled phospholipids. The effect of the cholinergic agent was suppressed by atropine, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and verapamil, and simulated, in part, by an increase in Ca2+ concentration. When the membranes were derived from islet cells prelabeled with (U-14C)arachidonate, carbamylcholine stimulation, in addition to decreasing labeled polyphosphoinositides, was accompanied by an increased production of labeled diacylglycerol, without a concomitant increase in labeled phosphatidylinositol. These results indicate that activation of a plasma membrane-associated phospholipase C directed against polyphosphoinositides represents a primary event in the functional response of the pancreatic beta cell to cholinergic agents.

  7. Biological activities of Eikenella corrodens outer membrane and lipopolysaccharide.

    PubMed Central

    Progulske, A; Mishell, R; Trummel, C; Holt, S C

    1984-01-01

    Highly purified preparations of the outer membrane and lipopolysaccharide (LPS) of Eikenella corrodens strain ATCC 23834 and the outer membrane fraction (OMF) of strain 470 were tested in in vitro biological assays. The OMFs of both strains were found to be mitogenic for BDF and C3H/HeJ murine splenocytes. The E. corrodens LPS was mitogenic for BDF spleen cells; however, doses of LPS as high as 50 micrograms/ml failed to stimulate C3H/HeJ cells. When incubated with T-lymphocyte-depleted C3H/HeJ splenocytes, the strain 23834 OMF demonstrated significant mitogenic activity, indicating that the OMF is a B-cell mitogen by a mechanism other than that elicited by conventional LPS. The E. corrodens 23834 OMF and LPS were stimulators of bone resorption when tested in organ cultures of fetal rat long bones. In contrast, the strain 470 OMF was only weakly stimulatory. Both OMFs and LPSs demonstrated "endotoxic" activity, since as little as 0.062 micrograms of E. corrodens LPS and 0.015 micrograms of the OMFs induced gelation in the Limulus amebocyte clotting assay. Thus, despite having a "nonclassical" LPS biochemistry, the E. corrodens LPS elicits classical endotoxic activities. These results also indicate that the surface structures of E. corrodens have significant biological activities as measured in vitro. The expression of such activities in vivo may play an important role in the pathogenesis of periodontitis as well as other E. corrodens infections. PMID:6360893

  8. Detection of focal adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer.

    PubMed

    Seong, Jihye; Ouyang, Mingxing; Kim, Taejin; Sun, Jie; Wen, Po-Chao; Lu, Shaoying; Zhuo, Yue; Llewellyn, Nicholas M; Schlaepfer, David D; Guan, Jun-Lin; Chien, Shu; Wang, Yingxiao

    2011-07-26

    Proper subcellular localization of focal adhesion kinase (FAK) is crucial for many cellular processes. It remains, however, unclear how FAK activity is regulated at subcellular compartments. To visualize the FAK activity at different membrane microdomains, we develop a fluorescence resonance energy transfer (FRET)-based FAK biosensor, and target it into or outside of detergent-resistant membrane (DRM) regions at the plasma membrane. Here we show that, on cell adhesion to extracellular matrix proteins or stimulation by platelet-derived growth factor (PDGF), the FRET responses of DRM-targeting FAK biosensor are stronger than that at non-DRM regions, suggesting that FAK activation can occur at DRM microdomains. Further experiments reveal that the PDGF-induced FAK activation is mediated and maintained by Src activity, whereas FAK activation on cell adhesion is independent of, and in fact essential for the Src activation. Therefore, FAK is activated at membrane microdomains with distinct activation mechanisms in response to different physiological stimuli.

  9. Surrogate or conventional light chains are required for membrane immunoglobulin mu to activate the precursor B cell transition [published erratum appears in J Exp Med 1997 Jan 6;185(1):183

    PubMed Central

    1996-01-01

    To examine the role of light chains in early B cell development we combined RAG-1 and lambda 5 mutations to produce mice that expressed neither conventional nor surrogate light chains (RAG-1-/-, lambda 5-/- ). Unique heavy and light chain genes were then introduced into the double and single mutant backgrounds. Membrane immunoglobulin (Ig)mu (mIg mu) associated with Ig alpha-Ig beta but was unable to activate the pre-B cell transition in RAG-1-/-lambda 5-/- mice. Either lambda 5 or kappa light chains were sufficient to complement this deficiency. Therefore light chains are absolutely required for a functional Ig signaling module in early B cell development. Our data provide direct evidence for the existence of two pathways for induction of early B cell development: one which is activated through surrogate light chains and mIg mu, and an alternative pathway which uses conventional light chains and mIg mu. PMID:8920890

  10. Human amniotic membrane-derived epithelial stem cells display anticancer activity in BALB/c female nude mice bearing disseminated breast cancer xenografts.

    PubMed

    Kang, Nam-Hee; Yi, Bo-Rim; Lim, So Yoon; Hwang, Kyung-A; Baek, Young Seok; Kang, Kyung-Sun; Choi, Kyung-Chul

    2012-06-01

    Breast cancer is one of the most common malignant tumors and the leading cause of mortality among women. In this study, we propose a human stem cell transplantation strategy, an important method for treating various cancers, as a potential breast cancer therapy. To this end, we used human amniotic membrane-derived epithelial stem cells (hAECs) as a cell source for performing human stem cell transplantation. hAECs have multipotent differentiation abilities and possess high proliferative potential. We transplanted hAECs into female BALB/c nude mice bearing tumors originating from MDA-MB-231 breast cancer cells. Co-culturred hAECs and MDA-MB-231 cells at a ratio of 1:4 or 1:8 (tumor cells to stem cells) inhibited breast cancer cell growth by 67.29 and 67.33%, respectively. In the xenograft mouse model, tumor volumes were significantly decreased by 5-flurouracil (5-FU) treatment and two different ratios of hAECs (1:4 and 1:8) by 84.33, 73.88 and 56.89%, respectively. Treatment of nude mice with hAECs (1:4) produced remarkable antitumor effects without any side-effects (e.g., weight loss, death and bruising) compared to the mice that received only 5-FU treatment. Tumor progression was significantly reduced by hAEC treatment compared to the xenograft model. On the other hand, breast tissues (e.g., the epidermis, dermis and reticular layer) appeared to be well-maintained following treatment with hAECs. Taken together, these results provide strong evidence that hAECs can be used as a safe and effective cancer-targeting cytotherapy for treating breast cancer.

  11. Microfabrication of High-Resolution Porous Membranes for Cell Culture

    PubMed Central

    Kim, Monica Y.; Li, David Jiang; Pham, Long K.; Wong, Brandon G.

    2014-01-01

    Microporous membranes are widely utilized in cell biology to study cell-cell signaling and cell migration. However, the thickness and low porosity of commercial track-etched membranes limit the quality of cell imaging and the degree of cell-cell contact that can be achieved on such devices. We employ photolithography-based microfabrication to achieve porous membranes with pore diameter as small as 0.9 μm, up to 40% porosity, and less than 5% variation in pore size. Through the use of a soap release layer, membranes as thin as 1 μm can be achieved. The thin membranes minimally disrupt contrast enhancement optics, thus allowing good quality imaging of unlabeled cells under white light, unlike commercial membranes. In addition, the polymer membrane materials display low autofluorescence even after patterning, facilitating high quality fluorescence microscopy. Finally, confocal imaging suggests that substantial cell-cell contact is possible through the pores of these thin membranes. This membrane technology can enhance existing uses of porous membranes in cell biology as well as enable new types of experiments. PMID:24567663

  12. Membrane tension feedback on shape and motility of eukaryotic cells

    NASA Astrophysics Data System (ADS)

    Winkler, Benjamin; Aranson, Igor S.; Ziebert, Falko

    2016-04-01

    In the framework of a phase field model of a single cell crawling on a substrate, we investigate how the properties of the cell membrane affect the shape and motility of the cell. Since the membrane influences the cell dynamics on multiple levels and provides a nontrivial feedback, we consider the following fundamental interactions: (i) the reduction of the actin polymerization rate by membrane tension; (ii) area conservation of the cell's two-dimensional cross-section vs. conservation of the circumference (i.e. membrane inextensibility); and (iii) the contribution from the membrane's bending energy to the shape and integrity of the cell. As in experiments, we investigate two pertinent observables - the cell's velocity and its aspect ratio. We find that the most important effect is the feedback of membrane tension on the actin polymerization. Bending rigidity has only minor effects, visible mostly in dynamic reshaping events, as exemplified by collisions of the cell with an obstacle.

  13. Influence of estrogenic pesticides on membrane integrity and membrane transfer of monosaccharide into the human red cell

    SciTech Connect

    Ingermann, R.L. )

    1989-09-01

    Some natural and synthetic estrogens inhibit carrier-mediated transport of glucose into human red blood cells and membrane vesicles from the placenta. The inhibitory action of these estrogens on transport appears to be a direct effect at the membrane and does not involve receptor binding and protein synthesis. It is not clear, however, whether such inhibition is a common feature among estrogenic agents. Several chlorinated hydrocarbon pesticides have been shown to possess estrogenic activity. These pesticides could have inhibitory effects on the human sodium-independent glucose transporter. Owing to the apparent importance of this membrane transporter in human tissues, direct interaction of hormones and xenobiotics with the glucose transporter is of fundamental significance. Some pesticides have been shown to alter membrane structure directly and alter the passive permeability of membranes. Whether the estrogenic pesticides influence passive diffusion of sugars across membranes has not been established. Finally, preliminary observations have suggested that some estrogens and pesticides have lytic effects on intact cells. Consequently, this study focuses on the ability of several estrogens and estrogenic pesticides to disrupt the cell membrane, influence the monosaccharide transporter, and alter the rate of monosaccharide permeation through the membrane by simple diffusion.

  14. PYR/PYL/RCAR Abscisic Acid Receptors Regulate K+ and Cl− Channels through Reactive Oxygen Species-Mediated Activation of Ca2+ Channels at the Plasma Membrane of Intact Arabidopsis Guard Cells1[W][OPEN

    PubMed Central

    Wang, Yizhou; Chen, Zhong-Hua; Zhang, Ben; Hills, Adrian; Blatt, Michael R.

    2013-01-01

    The discovery of the START family of abscisic acid (ABA) receptors places these proteins at the front of a protein kinase/phosphatase signal cascade that promotes stomatal closure. The connection of these receptors to Ca2+ signals evoked by ABA has proven more difficult to resolve, although it has been implicated by studies of the pyrbactin-insensitive pyr1/pyl1/pyl2/pyl4 quadruple mutant. One difficulty is that flux through plasma membrane Ca2+ channels and Ca2+ release from endomembrane stores coordinately elevate cytosolic free Ca2+ concentration ([Ca2+]i) in guard cells, and both processes are facilitated by ABA. Here, we describe a method for recording Ca2+ channels at the plasma membrane of intact guard cells of Arabidopsis (Arabidopsis thaliana). We have used this method to resolve the loss of ABA-evoked Ca2+ channel activity at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant and show the consequent suppression of [Ca2+]i increases in vivo. The basal activity of Ca2+ channels was not affected in the mutant; raising the concentration of Ca2+ outside was sufficient to promote Ca2+ entry, to inactivate current carried by inward-rectifying K+ channels and to activate current carried by the anion channels, both of which are sensitive to [Ca2+]i elevations. However, the ABA-dependent increase in reactive oxygen species (ROS) was impaired. Adding the ROS hydrogen peroxide was sufficient to activate the Ca2+ channels and trigger stomatal closure in the mutant. These results offer direct evidence of PYR/PYL/RCAR receptor coupling to the activation by ABA of plasma membrane Ca2+ channels through ROS, thus affecting [Ca2+]i and its regulation of stomatal closure. PMID:23899646

  15. Polybenzimidazole-multiwall carbon nanotubes composite membranes for polymer electrolyte membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Guerrero Moreno, Nayibe; Gervasio, Dominic; Godínez García, Andrés; Pérez Robles, Juan Francisco

    2015-12-01

    Polymer membranes are prepared as a composite of polybenzimidazole and non-functionalized multiwall carbon nanotubes (PBI-CNT) and polybenzimidazole (PBI) only. Each is doped with H3PO4 (PA) and used as a proton exchange membrane (PEM) as the electrolyte in a fuel cell. The proton conductivities at 180 °C for the doped PBI membrane (PBIPA) and the doped PBI-CNT membrane (PBICNTPA) are 6.3 × 10-2 and 7.4 × 10-2 Scm-1 respectively. A single fuel cell having these membranes as electrolyte has a Pt catalyzed hydrogen gas fed anode and a similar oxygen cathode without humidification of feed gases; the cell with the PBICNTPA membrane has higher open circuit voltage (0.96 V) than that with a PBIPA membrane (0.8 V) at 180 °C. The mechanical stability of the membrane improves with CNTs addition. The tensile strength of the composite PBI-CNT membrane with 1 wt.% CNTs loading is 32% higher and the Young's Modulus is 147% higher than the values for a membrane of PBI alone. The improvement in conductivity and mechanical properties in the composite membrane due to the CNT addition indicates that a PBI-CNT membrane is a good alternative as a membrane electrolyte in a PEMFC.

  16. Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy.

    PubMed

    Sun, Shiyu; Zhao, Guangxu; Huang, Yibing; Cai, Mingjun; Shan, Yuping; Wang, Hongda; Chen, Yuxin

    2016-01-01

    In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers. PMID:27363513

  17. Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy.

    PubMed

    Sun, Shiyu; Zhao, Guangxu; Huang, Yibing; Cai, Mingjun; Shan, Yuping; Wang, Hongda; Chen, Yuxin

    2016-07-01

    In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers.

  18. Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy

    PubMed Central

    Sun, Shiyu; Zhao, Guangxu; Huang, Yibing; Cai, Mingjun; Shan, Yuping; Wang, Hongda; Chen, Yuxin

    2016-01-01

    In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers. PMID:27363513

  19. Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

    SciTech Connect

    Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B. Palo Alto Veterans Administration Medical Center, CA ); Rubin, D.H. )

    1988-04-01

    Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 (VP3); 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. To determine whether trypsin treatment affected rotavirus internalization, the authors studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 mediated {sup 51}Cr, ({sup 14}C)choline, and ({sup 3}H)inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.

  20. Cell membrane stretch in osteoclasts triggers a self-reinforcing Ca2+ entry pathway.

    PubMed

    Wiltink, A; Nijweide, P J; Scheenen, W J; Ypey, D L; Van Duijn, B

    1995-03-01

    Many cell types respond to mechanical membrane perturbation with intracellular Ca2+ responses. Stretch-activated (SA) ion channels may be involved in such responses. We studied the occurrence as well as the underlying mechanisms of cell membrane stretch-evoked responses in fetal chicken osteoclasts using separate and simultaneous patch-clamp and Ca2+ imaging measurements. In the present paper, evidence is presented showing that such responses involve a self-reinforcing mechanism including SA channel activity, Ca(2+)-activated K+ (KCa) channel activity, membrane potential changes and local and general intracellular Ca2+ ([Ca2+]i) increases. The model we propose is that during membrane stretch, both SA channels and KCa channels open at membrane potential values near the resting membrane potential. SA channel characterization showed that these SA channels are permeable to Ca2+. During membrane stretch, Ca2+ influx through SA channels and hyperpolarization due to KCa channel activity serve as positive feedback, leading ultimately to a Ca2+ wave and cell membrane hyperpolarization. This self-reinforcing mechanism is turned off upon SA channel closure after cessation of membrane stretch. We suggest that this Ca2+ entry mechanism plays a role in regulation of osteoclast activity.

  1. Driving mechanisms of passive and active transport across cellular membranes as the mechanisms of cell metabolism and development as well as the mechanisms of cellular distance reactions on hormonal expression and the immune response.

    PubMed

    Ponisovskiy, M R

    2011-01-01

    The article presents mechanisms of cell metabolism, cell development, cell activity, and maintenance of cellular stability. The literature is reviewed from the point of view of these concepts. The balance between anabolic and catabolic processes induces chemical potentials in the extracellular and intracellular media. The chemical potentials of these media are defined as the driving forces of both passive and active transport of substances across cellular membranes. The driving forces of substance transport across cellular membranes as in cellular metabolism and in immune responses and hormonal expressions are considered in the biochemical and biophysical models, reflecting the mechanisms for maintenance of stability of the internal medium and internal energy of an organism. The interactions of passive transport and active transport of substances across cellular walls promote cell proliferation, as well as the mechanism of cellular capacitors, promoting remote reactions across distance for hormonal expression and immune responses. The offered concept of cellular capacitors has given the possibility to explain the mechanism of remote responses of cells to new situations, resulting in the appearance of additional agents. The biophysical model develops an explanation of some cellular functions: cellular membrane action have been identified with capacitor action, based on the similarity of the structures and as well as on similarity of biophysical properties of electric data that confirm the action of the compound-specific interactions of cells within an organism, promoting hormonal expressions and immune responses to stabilize the thermodynamic system of an organism. Comparison of a cellular membrane action to a capacitor has given the possibility for the explanations of exocytosis and endocytosis mechanisms, internalization of the receptor-ligand complex, selection as a receptor reaction to a ligand by immune responses or hormonal effects, reflecting cellular

  2. Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer

    PubMed Central

    Husain, Kabir; Iljazi, Elda; Bhat, Abrar; Bieling, Peter; Mullins, R. Dyche; Rao, Madan; Mayor, Satyajit

    2016-01-01

    The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization. PMID:26929326

  3. Influence of water and membrane microstructure on the transport properties of proton exchange membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Siu, Ana Rosa

    Proton transport in proton exchange membranes (PEMs) depends on interaction between water and acid groups covalently bound to the polymer. Although the presence of water is important in maintaining the PEM's functions, a thorough understanding of this topic is still lacking. The objective of this work is to provide a better understanding of how the nature water, confined to ionic domains of the polymer, influences the membrane's ability to transport protons, methanol and water. Understanding this topic will facilitate development of new materials with favorable transport properties for fuel cells use. Five classes of polymer membranes were used in this work: polyacrylonitrile-graft-poly(styrenesulfonic) acid (PAN-g-macPSSA); poly(vinylidene difluoride) irradiation-graft-poly(styrenesulfonic) acid (PVDF-g-PSSA); poly(ethylenetetrafluoroethylene) irradiation-graft-poly(styrenesulfonic) acid (ETFE-gPSSA); PVDF-g-PSSA with hydroxyethylmethacrylate (HEMA); and perfluorosulfonic acid membrane (Nafion). The nature of water within the polymers (freezable versus non-freezable states) was measured by systematically freezing samples, and observing the temperature at which water freezes and the amount of heat released in the process. Freezing water-swollen membranes resulted in a 4-fold decrease in the proton conductivity of the PEM. Activation energies of proton transport before and after freezing were ˜ 0.15 eV and 0.5 eV, consistent with proton transport through liquid water and bound water, respectively. Reducing the content of water in membrane samples decreased the amount of freezable and non-freezable water. Calorimetric measurements of membranes in various degrees of hydration showed that water molecules became non-freezable when lambda, (water molecules per sulfonic acid group) was less than ˜14. Proton conduction through membranes containing only non-freezable water was demonstrated to be feasible. Diffusion experiments showed that the permeability of methanol

  4. T cell glycolipid-enriched membrane domains are constitutively assembled as membrane patches that translocate to immune synapses.

    PubMed

    Jordan, Stephen; Rodgers, William

    2003-07-01

    In T cells, glycolipid-enriched membrane (GEM) domains, or lipid rafts, are assembled into immune synapses in response to Ag presentation. However, the properties of T cell GEM domains in the absence of stimulatory signals, such as their size and distribution in the plasma membrane, are less clear. To address this question, we used confocal microscopy to measure GEM domains in unstimulated T cells expressing a GEM-targeted green fluorescent protein molecule. Our experiments showed that the GEM domains were assembled into membrane patches that were micrometers in size, as evidenced by a specific enrichment of GEM-associated molecules and resistance of the patches to extraction by Triton X-100. However, treatment of cells with latrunculin B disrupted the patching of the GEM domains and their resistance to Triton X-100. Similarly, the patches were coenriched with F-actin, and actin occurred in the detergent-resistant GEM fraction of T cells. Live-cell imaging showed that the patches were mobile and underwent translocation in the plasma membrane to immune synapses in stimulated T cells. Targeting of GEM domains to immune synapses was found to be actin-dependent, and required phosphatidylinositol 3-kinase activity and myosin motor proteins. We conclude from our results that T cell GEM domains are constitutively assembled by the actin cytoskeleton into micrometer-sized membrane patches, and that GEM domains and the GEM-enriched patches can function as a vehicle for targeting molecules to immune synapses.

  5. Functional disturbance of the cell membrane in hypertension.

    PubMed

    Swales, J D

    1990-12-01

    Hypertension is associated with a variety of disturbances of cell membrane function. These associations have been attributed to changes in vascular smooth muscle sodium, calcium or PH, although direct evidence is conflicting in this context. While observations have been confounded by poor matching of the populations and technical shortcomings, it seems likely that genuine associations do exist. It is suggested that their significance can only be established by identifying the tissue abnormality responsible for elevated blood pressure. Evidence is cited that this abnormality is a structural increase in the resistance wall: lumen ratio, secondary to increased autonomic activity. A cell membrane disturbance could therefore give rise to hypertension, either through increased trophic responsiveness of the resistance vasculature or increased sympathetic drive to the resistance vessels. There is more direct evidence for the latter than the former.

  6. Collaboration between primitive cell membranes and soluble catalysts

    PubMed Central

    Adamala, Katarzyna P.; Engelhart, Aaron E.; Szostak, Jack W.

    2016-01-01

    One widely held model of early life suggests primitive cells consisted of simple RNA-based catalysts within lipid compartments. One possible selective advantage conferred by an encapsulated catalyst is stabilization of the compartment, resulting from catalyst-promoted synthesis of key membrane components. Here we show model protocell vesicles containing an encapsulated enzyme that promotes the synthesis of simple fatty acid derivatives become stabilized to Mg2+, which is required for ribozyme activity and RNA synthesis. Thus, protocells capable of such catalytic transformations would have enjoyed a selective advantage over other protocells in high Mg2+ environments. The synthetic transformation requires both the catalyst and vesicles that solubilize the water-insoluble precursor lipid. We suggest that similar modified lipids could have played a key role in early life, and that primitive lipid membranes and encapsulated catalysts, such as ribozymes, may have acted in conjunction with each other, enabling otherwise-impossible chemical transformations within primordial cells. PMID:26996603

  7. Collaboration between primitive cell membranes and soluble catalysts.

    PubMed

    Adamala, Katarzyna P; Engelhart, Aaron E; Szostak, Jack W

    2016-01-01

    One widely held model of early life suggests primitive cells consisted of simple RNA-based catalysts within lipid compartments. One possible selective advantage conferred by an encapsulated catalyst is stabilization of the compartment, resulting from catalyst-promoted synthesis of key membrane components. Here we show model protocell vesicles containing an encapsulated enzyme that promotes the synthesis of simple fatty acid derivatives become stabilized to Mg(2+), which is required for ribozyme activity and RNA synthesis. Thus, protocells capable of such catalytic transformations would have enjoyed a selective advantage over other protocells in high Mg(2+) environments. The synthetic transformation requires both the catalyst and vesicles that solubilize the water-insoluble precursor lipid. We suggest that similar modified lipids could have played a key role in early life, and that primitive lipid membranes and encapsulated catalysts, such as ribozymes, may have acted in conjunction with each other, enabling otherwise-impossible chemical transformations within primordial cells. PMID:26996603

  8. Cellular reactions of osteoblast-like cells to a novel nanocomposite membrane for guided bone regeneration

    NASA Astrophysics Data System (ADS)

    Meng, Yao; Liu, Man; Wang, Shao-An; Mo, An-Chun; Huang, Cui; Zuo, Yi; Li, Ji-Dong

    2008-11-01

    This study investigated the bioactivity and biocompatibility of hydroxyapatite nanoparticles (n-HA)/Polyamide-66 (PA66) nanocomposite membrane and expanded-polytetrafluoroethylene (e-PTFE) membrane (as control) to MG63 osteoblast-like cells. The attachment and proliferation of the cells on the porous surface of nHA/PA66 membrane and the surface of e-PTFE membrane were evaluated by scanning electron microscope (SEM) observation and the MTT assay. The bioactivity of the cells on the surface of the two membranes was evaluated by testing cell viability and alkaline phosphatase (ALP) activities. The results suggested that the bioresponse of MG63 osteoblast-like cells on the porous surface of nHA/PA66 membrane was better than the bioresponse on the opposite surface of e-PTFE membrane. Because of a better cell attachment manner, there is a potential utilization of the guided bone regeneration (GBR) membrane to substitute nHA/PA66 membrane for e-PTFE membrane.

  9. Membrane thickness is an important variable in membrane scaffolds: Influence of chitosan membrane structure on the behavior of cells

    PubMed Central

    Uygun, Basak E.; Bou-Akl, Therese; Albanna, Mohammad

    2009-01-01

    Cell and tissue responses to polymeric materials are orchestrated in part by the conformations of adsorbed plasma proteins. Thus, the chemical properties of a polymer membrane that govern protein adsorption behaviour can play an important role in determining the biological properties of tissue engineered scaffolds derived from that polymer. In this study, we explored the role of membrane thickness as a factor influencing cell adhesion and proliferation on chitosan membranes with and without covalently-attached glycosaminoglycans. Rat mesenchymal stem cells cultured on chitosan membranes of various thicknesses demonstrated significantly improved cell adhesion, spreading and proliferation as membrane thickness was increased. Hepatocytes displayed increased spreading on the substrate with increasing membrane thickness similar to MSCs. Increased thickness reduced the overall crystallinity of the membrane, and the data indicate that the improved cellular responses were likely due to enhanced adsorption of serum vitronectin, presumably due to reduced membrane crystallinity. These results demonstrate that membrane thickness is an important design variable that can be manipulated in chitosan-based scaffolds to achieve enhanced cell spreading, proliferation and function. PMID:19925888

  10. Ezrin is a Major Regulator of Membrane Tension in Epithelial Cells

    PubMed Central

    Rouven Brückner, Bastian; Pietuch, Anna; Nehls, Stefan; Rother, Jan; Janshoff, Andreas

    2015-01-01

    Plasma membrane tension is responsible for a variety of cellular functions such as motility, cell division, and endocytosis. Since membrane tension is dominated by the attachment of the actin cortex to the inner leaflet of the plasma membrane, we investigated the importance of ezrin, a major cross-linker of the membrane-cytoskeleton interface, for cellular mechanics of confluent MDCK II cells. For this purpose, we carried out ezrin depletion experiments and also enhanced the number of active ezrin molecules at the interface. Mechanical properties were assessed by force indentation experiments followed by membrane tether extraction. PIP2 micelles were injected into individual living cells to reinforce the linkage between plasma membrane and actin-cortex, while weakening of this connection was reached by ezrin siRNA and administration of the inhibitors neomycin and NSC 668394, respectively. We observed substantial stiffening of cells and an increase in membrane tension after addition of PIP2 micelles. In contrast, reduction of active ezrin led to a decrease of membrane tension accompanied by loss of excess surface area, increase in cortical tension, remodelling of actin cytoskeleton, and reduction of cell height. The data confirm the importance of the ezrin-mediated connection between plasma membrane and cortex for cellular mechanics and cell morphology. PMID:26435322

  11. High cell-surface density of HER2 deforms cell membranes.

    PubMed

    Chung, Inhee; Reichelt, Mike; Shao, Lily; Akita, Robert W; Koeppen, Hartmut; Rangell, Linda; Schaefer, Gabriele; Mellman, Ira; Sliwkowski, Mark X

    2016-01-01

    Breast cancers (BC) with HER2 overexpression (referred to as HER2 positive) progress more aggressively than those with normal expression. Targeted therapies against HER2 can successfully delay the progression of HER2-positive BC, but details of how this overexpression drives the disease are not fully understood. Using single-molecule biophysical approaches, we discovered a new effect of HER2 overexpression on disease-relevant cell biological changes in these BC. We found HER2 overexpression causes deformation of the cell membranes, and this in turn disrupts epithelial features by perturbing cell-substrate and cell-cell contacts. This membrane deformation does not require receptor signalling activities, but results from the high levels of HER2 on the cell surface. Our finding suggests that early-stage morphological alterations of HER2-positive BC cells during cancer progression can occur in a physical and signalling-independent manner. PMID:27599456

  12. 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

  13. Aldosterone secretion, measurements of membrane potential and intracellular potassium activity in the isolated adrenal zone glomerulosa.

    PubMed

    Wiederholt, M; Hampel, J; Belkien, L; Oelkers, W

    1984-09-01

    Cell membrane potential and intracellular potassium activity (microelectrodes filled with ion-sensitive liquid ion exchanger) were measured in the zona glomerulosa of superfused hemi-adrenals of rats kept on different diets. Simultaneously, samples of the superfusate were collected and analyzed by radioimmunoassay for aldosterone content. Cell membrane potential and intracellular potassium activity were not influenced by high sodium, low sodium or high potassium diet. However, aldosterone secretion significantly changed. These results suggest that membrane potential and intracellular potassium activity per se may not be linked to changes in aldosterone secretion.

  14. Activation of mitogen-activated protein kinase by membrane-targeted Raf chimeras is independent of raft localization.

    PubMed

    Chen, X; Resh, M D

    2001-09-14

    Binding of proteins to the plasma membrane can be achieved with various membrane targeting motifs, including combinations of fatty acids, isoprenoids, and basic domains. In this study, we investigate whether attachment of different membrane targeting motifs influences the signaling capacity of membrane-bound signal transduction proteins by directing the proteins to different membrane microdomains. We used c-Raf-1 as a model for a signaling protein that is activated when membrane-bound. Three different membrane targeting motifs from K-Ras, Fyn, and Src proteins were fused to the N or C terminus of Raf-1. The ability of the modified Rafs to initiate MAPK signaling was then investigated. All three modified Raf-1 constructs activated MAPK to nearly equivalent levels. The extent of localization of the Raf-1 constructs to membrane microdomains known as rafts did not correlate with the level of MAPK activation. Moreover, treatment of cells with the raft disrupting drug methyl-beta-cyclodextrin (MbetaCD) caused activation of MAPK to levels equivalent to those achieved with membrane-targeted Raf constructs. The use of pharmacological agents as well as dominant negative mutants revealed that MAPK activation by MbetaCD proceeds via a phosphoinositide 3-kinase-dependent mechanism that is Ras/Raf-independent. We conclude that cholesterol depletion from the plasma membrane by MbetaCD constitutes an alternative pathway for activating MAPK.

  15. The relevance of membrane models to understand nanoparticles-cell membrane interactions

    NASA Astrophysics Data System (ADS)

    Rascol, Estelle; Devoisselle, Jean-Marie; Chopineau, Joël

    2016-02-01

    Over the past two decades, numerous types of nanoparticles (NPs) have been developed for medical applications; however only a few nanomedicines are actually available on the market. One reason is the lack of understanding and data concerning the NP fate and their behavior upon contact with biological media and cell membranes. Biomimetic membrane models are interesting tools to approach and understand NPs-cell membrane interactions. The use of these models permits one to control physical and chemical parameters and to rapidly compare membrane types and the influence of different media conditions. The interactions between NPs and cell membranes can be qualified and quantified using analytical and modeling methods. In this review, the major studies concerning NPs-cell membrane models and associated methods are described. The advantages and drawbacks for each method are compared for the different models. The key mechanisms of interactions between NPs and cell membranes are revealed using cell membrane models and are interrogated in comparison with the NP behavior in cellulo or in vivo. Investigating the interactions between NPs and cell membrane models is now proposed as an intermediate step between physicochemical characterization of NPs and biological assays.

  16. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen.

    PubMed

    Lopez, Jodie; Bittame, Amina; Massera, Céline; Vasseur, Virginie; Effantin, Grégory; Valat, Anne; Buaillon, Célia; Allart, Sophie; Fox, Barbara A; Rommereim, Leah M; Bzik, David J; Schoehn, Guy; Weissenhorn, Winfried; Dubremetz, Jean-François; Gagnon, Jean; Mercier, Corinne; Cesbron-Delauw, Marie-France; Blanchard, Nicolas

    2015-12-15

    Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation.

  17. Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

    PubMed

    Jäger, Jens; Keese, Susanne; Roessle, Manfred; Steinert, Michael; Schromm, Andra B

    2015-05-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells.

  18. Spatio-temporal patterns of photosystem II activity and plasma-membrane proton flows in Chara corallina cells exposed to overall and local illumination.

    PubMed

    Bulychev, Alexander; Vredenberg, Wim

    2003-11-01

    Pulse-amplitude modulated microfluorometry and an extracellular pH microprobe were used to examine light-induced spatial heterogeneity of photosynthetic and H(+)-transporting activities in cells of Chara corallina Klein ex Willd. Subcellular domains featuring different PSII photochemical activities were found to conform to alternate alkaline and acid zones produced near the cell surface, with peaks of PSII activity correlating with the position of acid zones. Buffers eliminated pH variations near the cell surface but did not destroy the variations in PSII photochemical yield (deltaF/Fm'). When a dark-adapted cell was exposed to actinic light, the PSII effective yield decreased within 5-15 min in the alkaline regions but rose after the initial decline in the acid regions. The light-induced decrease in deltaF/Fm' in the alkaline regions occurred prior to or synchronously with the steep rise in local pH. The kinetics of deltaF/Fm', Fm', and F observed in alkaline regions under overall illumination of Chara cells were replaced by those typical of acid regions, when the illumination area size was restricted to 1.5-2 mm. The data show that photoinduced patterns in photosynthetic activity are not predetermined by the particular structural organization of alkaline and acid cell regions but are subject to dynamic changes.

  19. The three dimensionality of cell membranes: lamellar to cubic membrane transition as investigated by electron microscopy.

    PubMed

    Chong, Ketpin; Deng, Yuru

    2012-01-01

    Biological membranes are generally perceived as phospholipid bilayer structures that delineate in a lamellar form the cell surface and intracellular organelles. However, much more complex and highly convoluted membrane organizations are ubiquitously present in many cell types under certain types of stress, states of disease, or in the course of viral infections. Their occurrence under pathological conditions make such three-dimensionally (3D) folded and highly ordered membranes attractive biomarkers. They have also stimulated great biomedical interest in understanding the molecular basis of their formation. Currently, the analysis of such membrane arrangements, which include tubulo-reticular structures (TRS) or cubic membranes of various subtypes, is restricted to electron microscopic methods, including tomography. Preservation of membrane structures during sample preparation is the key to understand their true 3D nature. This chapter discusses methods for appropriate sample preparations to successfully examine and analyze well-preserved highly ordered membranes by electron microscopy. Processing methods and analysis conditions for green algae (Zygnema sp.) and amoeba (Chaos carolinense), mammalian cells in culture and primary tissue cells are described. We also discuss methods to identify cubic membranes by transmission electron microscopy (TEM) with the aid of a direct template matching method and by computer simulation. A 3D analysis of cubic cell membrane topology by electron tomography is described as well as scanning electron microscopy (SEM) to investigate surface contours of isolated mitochondria with cubic membrane arrangement.

  20. The membrane proteome of the mouse lens fiber cell

    PubMed Central

    Wilmarth, Phillip A.; David, Larry L.

    2009-01-01

    Purpose Fiber cells of the ocular lens are bounded by a highly specialized plasma membrane. Despite the pivotal role that membrane proteins play in the physiology and pathophysiology of the lens, our knowledge of the structure and composition of the fiber cell plasma membrane remains fragmentary. In the current study, we utilized mass spectrometry-based shotgun proteomics to provide a comprehensive survey of the mouse lens fiber cell membrane proteome. Methods Membranes were purified from young mouse lenses and subjected to MudPIT (Multidimensional protein identification technology) analysis. The resulting proteomic data were analyzed further by reference to publically available microarray databases. Results More than 200 membrane proteins were identified by MudPIT, including Type I, Type II, Type III (multi-pass), lipid-anchored, and GPI-anchored membrane proteins, in addition to membrane-associated cytoskeletal elements and extracellular matrix components. The membrane proteins of highest apparent abundance included Mip, Lim2, and the lens-specific connexin proteins Gja3, Gja8, and Gje1. Significantly, many proteins previously unsuspected in the lens were also detected, including proteins with roles in cell adhesion, solute transport, and cell signaling. Conclusions The MudPIT technique constitutes a powerful technique for the analysis of the lens membrane proteome and provides valuable insights into the composition of the lens fiber cell unit membrane. PMID:19956408

  1. Cell Adhesion and Growth on the Anodized Aluminum Oxide Membrane.

    PubMed

    Park, Jeong Su; Moon, Dalnim; Kim, Jin-Seok; Lee, Jin Seok

    2016-03-01

    Nanotopological cues are popular tools for in vivo investigation of the extracellular matrix (ECM) and cellular microenvironments. The ECM is composed of multiple components and generates a complex microenvironment. The development of accurate in vivo methods for the investigation of ECM are important for disease diagnosis and therapy, as well as for studies on cell behavior. Here, we fabricated anodized aluminum oxide (AAO) membranes using sulfuric and oxalic acid under controlled voltage and temperature. The membranes were designed to possess three different pore and interpore sizes, AAO-1, AAO-2, and AAO-3 membranes, respectively. These membranes were used as tools to investigate nanotopology-signal induced cell behavior. Cancerous cells, specifically, the OVCAR-8 cell-line, were cultured on porous AAO membranes and the effects of these membranes on cell shape, proliferation, and viability were studied. AAO-1 membranes bearing small sized pores were found to maintain the spreading shape of the cultured cells. Cells cultured on AAO-2 and AAO-3 membranes, bearing large pore-sized AAO membranes, changed shape from spreading to rounding. Furthermore, cellular area decreased when cells were cultured on all three AAO membranes that confirmed decreased levels of focal adhesion kinase (FAK). Additionally, OVCAR-8 cells exhibited increased proliferation on AAO membranes possessing various pore sizes, indicating the importance of the nanosurface structure in regulating cell behaviors, such as cell proliferation. Our results suggest that porous-AAO membranes induced nanosurface regulated cell behavior as focal adhesion altered the intracellular organization of the cytoskeleton. Our results may find potential applications as tools in in vivo cancer research studies. PMID:27280255

  2. Synthesis of Nanogels via Cell Membrane-Templated Polymerization.

    PubMed

    Zhang, Jianhua; Gao, Weiwei; Fang, Ronnie H; Dong, Anjie; Zhang, Liangfang

    2015-09-01

    The synthesis of biomimetic hydrogel nanoparticles coated with a natural cell membrane is described. Compared to the existing strategy of wrapping cell membranes onto pre-formed nanoparticle substrates, this new approach forms the cell membrane-derived vesicles first, followed by growing nanoparticle cores in situ. It adds significant controllability over the nanoparticle properties and opens unique opportunities for a broad range of biomedical applications. PMID:26044721

  3. Synthesis of Nanogels via Cell Membrane-Templated Polymerization

    PubMed Central

    Zhang, Jianhua; Gao, Weiwei; Fang, Ronnie H.; Dong, Anjie

    2015-01-01

    The synthesis of biomimetic hydrogel nanoparticles coated with natural cell membrane is described. Compared to existing strategy of wrapping cell membrane onto pre-formed nanoparticle substrates, this new approach forms the cell membrane-derived vesicles first, followed by growing nanoparticle cores in situ. It adds significant controllability over the nanoparticle properties and opens unique opportunities for a broad range of biomedical applications. PMID:26044721

  4. Cell Adhesion and Growth on the Anodized Aluminum Oxide Membrane.

    PubMed

    Park, Jeong Su; Moon, Dalnim; Kim, Jin-Seok; Lee, Jin Seok

    2016-03-01

    Nanotopological cues are popular tools for in vivo investigation of the extracellular matrix (ECM) and cellular microenvironments. The ECM is composed of multiple components and generates a complex microenvironment. The development of accurate in vivo methods for the investigation of ECM are important for disease diagnosis and therapy, as well as for studies on cell behavior. Here, we fabricated anodized aluminum oxide (AAO) membranes using sulfuric and oxalic acid under controlled voltage and temperature. The membranes were designed to possess three different pore and interpore sizes, AAO-1, AAO-2, and AAO-3 membranes, respectively. These membranes were used as tools to investigate nanotopology-signal induced cell behavior. Cancerous cells, specifically, the OVCAR-8 cell-line, were cultured on porous AAO membranes and the effects of these membranes on cell shape, proliferation, and viability were studied. AAO-1 membranes bearing small sized pores were found to maintain the spreading shape of the cultured cells. Cells cultured on AAO-2 and AAO-3 membranes, bearing large pore-sized AAO membranes, changed shape from spreading to rounding. Furthermore, cellular area decreased when cells were cultured on all three AAO membranes that confirmed decreased levels of focal adhesion kinase (FAK). Additionally, OVCAR-8 cells exhibited increased proliferation on AAO membranes possessing various pore sizes, indicating the importance of the nanosurface structure in regulating cell behaviors, such as cell proliferation. Our results suggest that porous-AAO membranes induced nanosurface regulated cell behavior as focal adhesion altered the intracellular organization of the cytoskeleton. Our results may find potential applications as tools in in vivo cancer research studies.

  5. Plasma membrane calcium pump activity is affected by the membrane protein concentration. Evidence for the involvement of the actin cytoskeleton

    PubMed Central

    Vanagas, Laura; Rossi, Rolando C.; Caride, Ariel J.; Filoteo, Adelaida G.; Strehler, Emanuel E.; Rossi, Juan Pablo F.C.

    2007-01-01

    Plasma membrane calcium pumps (PMCAs) are integral membrane proteins that actively expel Ca2+ from the cell. Specific Ca2+-ATPase activity of erythrocyte membranes increased steeply up to 1.5–5 times when the membrane protein concentration decreased from 50 μg/ml to 1 μg/ml. The activation by dilution was also observed for ATP-dependent Ca2+ uptake into vesicles from Sf9 over-expressing the PMCA 4b isoform, confirming that it is a property of the PMCA. Dilution of the protein did not modify the activation by ATP, Ca2+ or Ca2+-calmodulin. Treatment with non-ionic detergents did not abolish the dilution effect, suggesting that it was not due to resealing of the membrane vesicles. Pre-incubation of erythrocyte membranes with Cytochalasin D under conditions that promote actin polymerization abolished the dilution effect. Highly-purified, micellar PMCA showed no dilution effect and was not affected by Cytochalasin D. Taken together, these results suggest that the concentration-dependent behavior of the PMCA activity was due to interactions with cytoskeletal proteins. The dilution effect was also observed with different PMCA isoforms, indicating that this is a general phenomenon for all PMCAs. PMID:17481573

  6. Role of membrane biophysics in Alzheimer's–related cell pathways

    PubMed Central

    Zhu, Donghui; Bungart, Brittani L.; Yang, Xiaoguang; Zhumadilov, Zhaxybay; Lee, James C-M.; Askarova, Sholpan

    2015-01-01

    Cellular membrane alterations are commonly observed in many diseases, including Alzheimer's disease (AD). Membrane biophysical properties, such as membrane molecular order, membrane fluidity, organization of lipid rafts, and adhesion between membrane and cytoskeleton, play an important role in various cellular activities and functions. While membrane biophysics impacts a broad range of cellular pathways, this review addresses the role of membrane biophysics in amyloid-β peptide aggregation, Aβ-induced oxidative pathways, amyloid precursor protein processing, and cerebral endothelial functions in AD. Understanding the mechanism(s) underlying the effects of cell membrane properties on cellular processes should shed light on the development of new preventive and therapeutic strategies for this devastating disease. PMID:26074758

  7. Cholera Toxin and Cell Growth: Role of Membrane Gangliosides

    PubMed Central

    Hollenberg, Morley D.; Fishman, Peter H.; Bennett, Vann; Cuatrecasas, Pedro

    1974-01-01

    The binding of cholera toxin to three transformed mouse cell lines derived from the same parent strain, and the effects of the toxin on DNA synthesis and adenylate cyclase activity, vary in parallel with the ganglioside composition of the cells. TAL/N cells of early passage, which contain large quantities of gangliosides GM3, GM2, GM1, and GDla, as well as the glycosyltransferases necessary for the synthesis of these gangliosides, bind the most cholera toxin and are the most sensitive to its action. TAL/N cells of later passage, which lack chemically detectable GM1 and GDla and which have no UDP-Gal:GM2 galactosyltransferase activity, are intermediate in binding and response to the toxin. SVS AL/N cells, which lack GM2 in addition to GM1 and GDla and which have little detectable UDP-GalNAc:GM3N-acetylgalactosaminyltransferase activity, bind the least amount of toxin. The SVS AL/N cells are the least responsive to inhibition of DNA synthesis and stimulation of adenylate cyclase activity by cholera toxin. Gangliosides (especially GM1), which appear to be the natural membrane receptors for cholera toxin, may normally have important roles in the regulation of cell growth and cAMP-mediated responses. PMID:4530298

  8. [The sodium-potassium-chloride cotransport of the cell membrane].

    PubMed

    Urazaev, A Kh

    1998-01-01

    Discovery and active exploration of the furosemid-sensitive derived-active co-transport of sodium-potassium-chlorine ions took place in the end of 1970-es-1980-es. This transportation mechanism was discovered in various types of cells, both of plant and of animal origin. This review describes properties of the transportation process, which was most comprehensive explored in experiments with erythrocytes, epithelium cells and muscles. The review covers the following properties: anion and cation selectivity of the chlorine transportation, its sensitivity to the specific blocking agents (furocemid, bumetanid, etc.), stoichiometry of the transportation process, etc. For energy source, the chlorine transportation is based on transmembrane electrochemical gradient for sodium ions. The article provides the most recent results of investigation of the chemical nature of the molecule of the chlorine membrane transport. Based on various studies, the molecule of this protein weighs from 120 to 200 kD, includes about 1200 amino acid residua, and forms long cytoplasmatic NH2 and COOH-termini. The gene encoding the amino acid sequence has been cloned. The article discusses the issues of regulation of the chlorine transportation. Humoral control of intensity of the chlorine transportation has been mostly studied in experiments with plain muscles, the issues related to nervous regulation--with only skeleton muscle fibers. The article provides specific data on the mechanisms of the above types of the physiological regulation of active chlorine transportation. In general, the humoral factors, which increase the intracellular concentration of cAMF stimulate chlorine transportation. On the contrary, the hormones, which increase concentration of cGMF in cytoplasm reduce its activity in plain muscles. The discussion of the mechanisms of the nervous controls of the chlorine transportation in the skeleton muscles includes the original results of the author. These results indicate that the

  9. Hydrocarbon-based polymer electrolyte cerium composite membranes for improved proton exchange membrane fuel cell durability

    NASA Astrophysics Data System (ADS)

    Lee, Hyejin; Han, Myungseong; Choi, Young-Woo; Bae, Byungchan

    2015-11-01

    Hydrocarbon-based cerium composite membranes were prepared for proton exchange membrane fuel cell applications to increase oxidative stability. Different amounts of cerium ions were impregnated in sulfonated poly(arylene ether sulfone) (SPES) membranes and their physicochemical properties were investigated according to the cerium content. Field-emission scanning electron microscopy and inductively coupled plasma analyses confirmed the presence of cerium ions in the composite membranes and 1H NMR indicated the successful coordination of sulfonic acid groups with the metal ions. Increasing amounts of cerium ions resulted in decreases in the proton conductivity and water uptake, but enhanced oxidative stability. The oxidative stability of the composite membranes was proven via a hydrogen peroxide exposure experiment which mimicked fuel cell operating conditions. In addition, more than 2200 h was achieved with the composite membrane under in situ accelerated open circuit voltage (OCV) durability testing (DOE protocol), whereas the corresponding pristine SPES membrane attained only 670 h.

  10. Impedance study of membrane dehydration and compression in proton exchange membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Le Canut, Jean-Marc; Latham, Ruth; Mérida, Walter; Harrington, David A.

    Electrochemical impedance spectroscopy (EIS) is used to measure drying and rehydration in proton exchange membrane fuel cells running under load. The hysteresis between forward and backward acquisition of polarization curves is shown to be largely due to changes in the membrane resistance. Drying tests are carried out with hydrogen and simulated reformate (hydrogen and carbon dioxide), and quasi-periodic drying and rehydration conditions are studied. The membrane hydration state is clearly linked to the high-frequency arc in the impedance spectrum, which increases in size for dry conditions indicating an increase in membrane resistance. Changes in impedance spectra as external compression is applied to the cell assembly show that EIS can separate membrane and interfacial effects, and that changes in membrane resistance dominate. Reasons for the presence of a capacitance in parallel with the membrane resistance are discussed.

  11. Ambidextrous Binding of Cell and Membrane Bilayers by Soluble Matrix Metalloproteinase-12

    PubMed Central

    Koppisetti, Rama K.; Fulcher, Yan G.; Jurkevich, Alexander; Prior, Stephen H.; Xu, Jia; Lenoir, Marc; Overduin, Michael; Van Doren, Steven R.

    2014-01-01

    Matrix metalloproteinases (MMPs) regulate tissue remodeling, inflammation, and disease progression. Some soluble MMPs are inexplicably active near cell surfaces. Here, we demonstrate binding of MMP-12 directly to bilayers and cellular membranes using paramagnetic NMR and fluorescence. Opposing sides of the catalytic domain engage spin-labeled membrane mimics. Loops project from the β-sheet interface to contact the phospholipid bilayer with basic and hydrophobic residues. The distal membrane interface comprises loops on the other side of the catalytic cleft. Both interfaces mediate MMP-12 association with vesicles and cell membranes. MMP-12 binds plasma membranes and is internalized to hydrophobic perinuclear features, the nuclear membrane, and inside the nucleus within minutes. While binding of TIMP-2 to MMP-12 hinders membrane interactions beside the active site, TIMP-2-inhibited MMP-12 binds vesicles and cells, suggesting compensatory rotation of its membrane approaches. MMP-12 association with diverse cell membranes may target its activities to modulate innate immune responses and inflammation. PMID:25412686

  12. Development of composite membranes of PVA-TEOS doped KOH for alkaline membrane fuel cell

    NASA Astrophysics Data System (ADS)

    Haryadi, Sugianto, D.; Ristopan, E.

    2015-12-01

    Anion exchange membranes (AEMs) play an important role in separating fuel and oxygen (or air) in the Alkaline Membrane Fuel Cells. Preparation of hybrid organic inorganic materials of Polyvinylalcohol (PVA) - Tetraethylorthosilicate (TEOS) composite membrane doped KOH for direct alcohol alkaline fuel cell application has been investigated. The sol-gel method has been used to prepare the composite membrane of PVA-TEOS through crosslinking step and catalyzed by concentrated of hydrochloric acid. The gel solution was cast on the membrane plastic plate to obtain membrane sheets. The dry membranes were then doped by immersing in various concentrations of KOH solutions for about 4 hours. Investigations of the cross-linking process and the presence of hydroxyl group were conducted by FTIR as shown for frequency at about 1600 cm-1 and 3300 cm-1 respectively. The degree of swelling in ethanol decreased as the KOH concentration for membrane soaking process increased. The ion exchange capacity (IEC) of the membrane was 0.25meq/g. This composite membranes display significant ionic conductivity of 3.23 x 10-2 S/cm in deionized water at room temperature. In addition, the morphology observation by scanning electron microscope (SEM) of the membrane indicates that soaking process of membrane in KOH increased thermal resistant.

  13. Development of composite membranes of PVA-TEOS doped KOH for alkaline membrane fuel cell

    SciTech Connect

    Haryadi, Sugianto, D.; Ristopan, E.

    2015-12-29

    Anion exchange membranes (AEMs) play an important role in separating fuel and oxygen (or air) in the Alkaline Membrane Fuel Cells. Preparation of hybrid organic inorganic materials of Polyvinylalcohol (PVA) - Tetraethylorthosilicate (TEOS) composite membrane doped KOH for direct alcohol alkaline fuel cell application has been investigated. The sol-gel method has been used to prepare the composite membrane of PVA-TEOS through crosslinking step and catalyzed by concentrated of hydrochloric acid. The gel solution was cast on the membrane plastic plate to obtain membrane sheets. The dry membranes were then doped by immersing in various concentrations of KOH solutions for about 4 hours. Investigations of the cross-linking process and the presence of hydroxyl group were conducted by FTIR as shown for frequency at about 1600 cm{sup −1} and 3300 cm{sup −1} respectively. The degree of swelling in ethanol decreased as the KOH concentration for membrane soaking process increased. The ion exchange capacity (IEC) of the membrane was 0.25meq/g. This composite membranes display significant ionic conductivity of 3.23 x 10{sup −2} S/cm in deionized water at room temperature. In addition, the morphology observation by scanning electron microscope (SEM) of the membrane indicates that soaking process of membrane in KOH increased thermal resistant.

  14. Membrane tether formation from voltage-clamped outer hair cells using optical tweezers

    NASA Astrophysics Data System (ADS)

    Qian, Feng; Ermilov, Sergey A.; Murdock, David R.; Brownell, William E.; Anvari, Bahman

    2004-06-01

    Outer hair cells contribute an active mechanical feedback to the vibrations of the cochlear structures resulting in the high sensitivity and frequency selectivity of normal hearing. We have designed and implemented a novel experimental setup that combines optical tweezers with patch-clamp apparatus to investigate the electromechanical properties of cellular plasma membranes. A micron-size bead trapped by the optical tweezers is brought in contact with the membrane of a voltage-clamped cell, and subsequently moved away to form a plasma membrane tether. Bead displacement during tether elongation is monitored by a quadrant photodetector to obtain time-resolved measurements of the tethering force. Salient information associated with the mechanical properties of the membrane tether can thus be obtained. Tethers can be pulled from the cell membrane at different holding potentials, and the tether force response can be measured while changing transmembrane potential. Experimental results from outer hair cells and human embryonic kidney cells are presented.

  15. Dissociation of membrane binding and lytic activities of the lymphocyte pore-forming protein (perforin).

    PubMed

    Young, J D; Damiano, A; DiNome, M A; Leong, L G; Cohn, Z A

    1987-05-01

    Granules isolated from CTL and NK cells contain a cytolytic pore-forming protein (PFP/perforin). At low temperatures (on ice), PFP binds to erythrocyte membranes without producing hemolysis. Hemolysis occurs when the PFP-bound erythrocytes are warmed up to 37 degrees C, which defines a temperature-dependent, lytic (pore-formation) step distinct from the membrane-binding event. Ca2+ and neutral pH are required for both membrane binding and pore formation by PFP. Serum, LDL, HDL, and heparin inhibit the hemolytic activity of PFP by blocking its binding to lipid membranes. Lysis by PFP that has bound to erythrocyte membranes is no longer susceptible to the effect of these inhibitors. The hemolytic activities associated with intact granules and solubilized PFP show different requirements for Ca2+ and pH, indicating that cytolysis produced by isolated granules may involve an additional step, possibly fusion of granules with membranes. It is suggested that three distinct Ca2+- and pH-dependent events may be involved during cell killing by CTL and NK cells: fusion of cytoplasmic granules of effector cells with their plasma membrane, releasing PFP from cells; binding of the released PFP to target membranes; and insertion of monomers and the subsequent formation of lytic pores in the target membrane. The serum-mediated inhibition of membrane binding by PFP could prevent the accidental injury of bystander cells by cell-released PFP, but would allow cytolysis to proceed to completion once PFP has bound to the target membrane. PMID:3494808

  16. Active water management at the cathode of a planar air-breathing polymer electrolyte membrane fuel cell using an electroosmotic pump

    NASA Astrophysics Data System (ADS)

    Fabian, T.; O'Hayre, R.; Litster, S.; Prinz, F. B.; Santiago, J. G.

    In a typical air-breathing fuel cell design, ambient air is supplied to the cathode by natural convection and dry hydrogen is supplied to a dead-ended anode. While this design is simple and attractive for portable low-power applications, the difficulty in implementing effective and robust water management presents disadvantages. In particular, excessive flooding of the open-cathode during long-term operation can lead to a dramatic reduction of fuel cell power. To overcome this limitation, we report here on a novel air-breathing fuel cell water management design based on a hydrophilic and electrically conductive wick in conjunction with an electroosmotic (EO) pump that actively pumps water out of the wick. Transient experiments demonstrate the ability of the EO-pump to "resuscitate" the fuel cell from catastrophic flooding events, while longer term galvanostatic measurements suggest that the design can completely eliminate cathode flooding using less than 2% of fuel cell power, and lead to stable operation with higher net power performance than a control design without EO-pump. This demonstrates that active EO-pump water management, which has previously only been demonstrated in forced-convection fuel cell systems, can also be applied effectively to miniaturized (<5 W) air-breathing fuel cell systems.

  17. Selective effect of cell membrane on synaptic neurotransmission

    NASA Astrophysics Data System (ADS)

    Postila, Pekka A.; Vattulainen, Ilpo; Róg, Tomasz

    2016-01-01

    Atomistic molecular dynamics simulations were performed with 13 non-peptidic neurotransmitters (NTs) in three different membrane environments. The results provide compelling evidence that NTs are divided into membrane-binding and membrane-nonbinding molecules. NTs adhere to the postsynaptic membrane surface whenever the ligand-binding sites of their synaptic receptors are buried in the lipid bilayer. In contrast, NTs that have extracellular ligand-binding sites do not have a similar tendency to adhere to the membrane surface. This finding is a seemingly simple yet important addition to the paradigm of neurotransmission, essentially dividing it into membrane-independent and membrane-dependent mechanisms. Moreover, the simulations also indicate that the lipid composition especially in terms of charged lipids can affect the membrane partitioning of NTs. The revised paradigm, highlighting the importance of cell membrane and specific lipids for neurotransmission, should to be of interest to neuroscientists, drug industry and the general public alike.

  18. Condurango (Gonolobus condurango) Extract Activates Fas Receptor and Depolarizes Mitochondrial Membrane Potential to Induce ROS-dependent Apoptosis in Cancer Cells in vitro

    PubMed Central

    Bishayee, Kausik; Mondal, Jesmin; Sikdar, Sourav; Khuda-Bukhsh, Anisur Rahman

    2015-01-01

    Objectives: Condurango (Gonolobus condurango) extract is used by complementary and alternative medicine (CAM) practitioners as a traditional medicine, including homeopathy, mainly for the treatment of syphilis. Condurango bark extract is also known to reduce tumor volume, but the underlying molecular mechanisms still remain unclear. Methods: Using a cervical cancer cell line (HeLa) as our model, the molecular events behind condurango extract’s (CE’s) anticancer effect were investigated by using flow cytometry, immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Other included cell types were prostate cancer cells (PC3), transformed liver cells (WRL-68), and peripheral blood mononuclear cells (PBMCs). Results: Condurango extract (CE) was found to be cytotoxic against target cells, and this was significantly deactivated in the presence of N-acetyl cysteine (NAC), a scavenger of reactive oxygen species (ROS), suggesting that its action could be mediated through ROS generation. CE caused an increase in the HeLa cell population containing deoxyribonucleic acid (DNA) damage at the G zero/Growth 1 (G0/G1) stage. Further, CE increased the tumor necrosis factor alpha (TNF-α) and the fas receptor (FasR) levels both at the ribonucleic acid (RNA) and the protein levels, indicating that CE might have a cytotoxic mechanism of action. CE also triggered a sharp decrease in the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB ) both at the RNA and the protein levels, a possible route to attenuation of B-cell lymphoma 2 (Bcl-2), and caused an opening of the mitochondrial membrane’s permeability transition (MPT) pores, thus enhancing caspase activities. Conclusion: Overall, our results suggest possible pathways for CE mediated cytotoxicity in model cancer cells. PMID:26389000

  19. Efficient activation of human T cells of both CD4 and CD8 subsets by urease-deficient recombinant Mycobacterium bovis BCG that produced a heat shock protein 70-M. tuberculosis-derived major membrane protein II fusion protein.

    PubMed

    Mukai, Tetsu; Tsukamoto, Yumiko; Maeda, Yumi; Tamura, Toshiki; Makino, Masahiko

    2014-01-01

    For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8(+) T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8(+) T cells and perforin-producing effector CD8(+) T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.

  20. Activity inhibition and its mitigation in high temperature proton exchange membrane fuel cells: The role of phosphoric acid, ammonium trifluoromethanesulfonate, and polyvinylidene difluoride

    NASA Astrophysics Data System (ADS)

    Holst-Olesen, Kaspar; Nesselberger, Markus; Perchthaler, Markus; Hacker, Viktor; Arenz, Matthias

    2014-12-01

    In the presented work we systematically study the influence of phosphoric acid, ammonium trifluoromethanesulfonate (ATFMS), and polyvinylidene difluoride (PVDF) on the oxygen reduction reaction (ORR) activity of carbon supported, Pt based catalysts. The influence of phosphoric acid is investigated in a mixed solution of perchloric acid with small amounts of phosphoric acid added. Thin-film rotating disk electrode (TF-RDE) measurements show that such a mixed electrolyte is advantageous as the oxygen reduction reaction (ORR) is inhibited without influencing the oxygen solubility in the electrolyte. In contrast to previous reports it is seen when investigating additives that ATFMS acts as a catalyst poison; whereas the results provide evidence of a better performance in case of the PVDF incorporated catalysts as compared to reference samples without PVDF. The technological relevance of the PVDF improvements and its stability over prolonged time was validated by membrane electrode assembly (MEA) tests.

  1. Interaction of gentamicin polycation with model and cell membranes.

    PubMed

    Kovács, Eugenia; Savopol, Tudor; Iordache, Maria-Minodora; Săplăcan, Lavinia; Sobaru, Iuliana; Istrate, Claudia; Mingeot-Leclercq, Marie-Paule; Moisescu, Mihaela-Georgeta

    2012-10-01

    The interaction of positively-charged antibiotic gentamicin with cell membranes was studied to determine if any changes in membrane organization were induced by the drug. Opossum kidney epithelia (OK) cells were used as models of eukaryotic cells. Two methods were used: laurdan fluorescence spectroscopy and fluorescence anisotropy recordings on 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) labeled cell suspensions. Both methods showed an altered membrane hydration and fluidity of gentamicin treated cells. Liposomes prepared from dimyristoyl-phosphatidylcholine (DMPC) mixed with cardiolipin, which mimics the heterogeneous charge composition of the natural cell membrane, were used to determine the effect of gentamicin on artificial bilayers. The membrane lipid packing as revealed by generalized polarization (GP) and fluorescence anizotropy variation with increasing temperature was studied. It was found that the generalized polarization of liposomal membranes containing a negatively charged lipid (cardiolipin) is higher in the presence of gentamicin; in the membrane of living cell (OK), gentamicin induces, on the contrary, a decrease of general polarization. Considering the role of membrane organization in the function of transmembrane channels and receptors, our findings suggest hypotheses that may explain the permeation of gentamicin through the living cell membrane by using these channels.

  2. Increased phorbol 12,13-dibutyrate (PDBu) receptor function associated with sickle red cell membrane ghosts

    SciTech Connect

    Ramachandran, M.; Nair, C.N.; Abraham, E.C.

    1987-05-01

    The biological receptor for tumor-promoting phorbol esters has been identified as the CaS /phospholipid dependent enzyme, protein kinase C. In the red cell, this enzyme is mainly cytosolic but becomes translocated to the membrane if the cellular CaS is allowed to rise. Since cellular CaS in sickle red cells is high, it was reasoned that this enzyme may become more membrane-bound. In fact, the authors noticed a four-fold increase in the binding of TH-PDBu by membrane ghosts isolated from sickle red cells compared to normal red cells (pmoles PDBu bound/mg protein; normal = 0.3 vs sickle cell = 1.4). Attempts to assay the enzyme directly as phospholipid-activated TSP incorporation into the acid-precipitable membrane proteins also indicated a two-fold increase in the radiolabelling of sickle cell membrane ghosts. Autophosphorylation of membrane proteins and analysis of the phosphorylation profile by SDS-PAGE and autoradiography revealed phosphorylation predominantly of bands 3, 4.1 and 4.9 which are known protein kinase C substrates for the red cell enzyme. The increased membrane-associated protein kinase C in sickle red cells may have a bearing on the altered membrane properties reported in this condition.

  3. Graphene-doped electrospun nanofiber membrane electrodes and proton exchange membrane fuel cell performance

    NASA Astrophysics Data System (ADS)

    Wei, Meng; Jiang, Min; Liu, Xiaobo; Wang, Min; Mu, Shichun

    2016-09-01

    A rational electrode structure can allow proton exchange membrane (PEM) fuel cells own high performance with a low noble metal loading and an optimal transport pathway for reaction species. In this study, we develop a graphene doped polyacrylonitile (PAN)/polyvinylident fluoride (PVDF) (GPP) electrospun nanofiber electrode with improved electrical conductivity and high porosity, which could enhance the triple reaction boundary and promote gas and water transport throughout the porous electrode. Thus the increased electrochemical active surface area (ECSA) of Pt catalysts and fuel cell performance can be expected. As results, the ECSA of hot-pressed electrospun electrodes with 2 wt% graphene oxide (GO) is up to 84.3 m2/g, which is greatly larger than that of the conventional electrode (59.5 m2/g). Significantly, the GPP nanofiber electrospun electrode with Pt loading of 0.2 mg/cm2 exhibits higher fuel cell voltage output and stability than the conventional electrode.

  4. Fluctuations of coupled fluid and solid membranes with application to red blood cells

    NASA Astrophysics Data System (ADS)

    Auth, Thorsten; Safran, S. A.; Gov, Nir S.

    2007-11-01

    The fluctuation spectra and the intermembrane interaction of two membranes at a fixed average distance are investigated. Each membrane can either be a fluid or a solid membrane, and in isolation, its fluctuations are described by a bare or a wave-vector-dependent bending modulus, respectively. The membranes interact via their excluded-volume interaction; the average distance is maintained by an external, homogeneous pressure. For strong coupling, the fluctuations can be described by a single, effective membrane that combines the elastic properties. For weak coupling, the fluctuations of the individual, noninteracting membranes are recovered. The case of a composite membrane consisting of one fluid and one solid membrane can serve as a microscopic model for the plasma membrane and cytoskeleton of the red blood cell. We find that, despite the complex microstructure of bilayers and cytoskeletons in a real cell, the fluctuations with wavelengths λ≳400nm are well described by the fluctuations of a single, polymerized membrane (provided that there are no inhomogeneities of the microstructure). The model is applied to the fluctuation data of discocytes (“normal” red blood cells), a stomatocyte, and an echinocyte. The elastic parameters of the membrane and an effective temperature that quantifies active, metabolically driven fluctuations are extracted from the experiments.

  5. Characterization and fuel cell testing of radiation-grafted PSI membranes

    SciTech Connect

    Brack, H.P.; Koebel, M.M.; Tsukada, A.; Huslage, J.; Buechi, F.; Geiger, F.; Rota, M.; Scherer, G.G.

    2000-07-01

    The authors have demonstrated earlier the useful performance of PSI radiation-grafted membranes in terms of the current-voltage characteristics of 30 cm{sup 2} active area fuel cells containing these membranes and their long-term testing over 6,000 h at 60 C. They report here on testing of PSI radiation-grafted membranes in these fuel cells at 80 C and in short stacks comprised of two or four 100 cm{sup 2} active area cells. The in-situ degradation of membranes has been investigated by characterizing membranes both before testing in fuel cells and post-mortem after testing in fuel cells. Characterization was accomplished by means of ion-exchange capacity and infrared and Raman spectroscopic measurements. In addition, a rapid screening method for ex-situ testing of the oxidative stability of proton-conducting membranes was developed in this work. Comparison of the initial screening test results concerning the oxidative stability of some perfluorinated, partially-fluorinated, and non-fluorinated membranes compare well qualitatively with the relative stability of these same membranes during their long-term testing in fuel cells.

  6. One-way membrane trafficking of SOS in receptor-triggered Ras activation.

    PubMed

    Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

    2016-09-01

    SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis. PMID:27501536

  7. One-way membrane trafficking of SOS in receptor-triggered Ras activation.

    PubMed

    Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

    2016-09-01

    SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

  8. Roles of membrane trafficking in plant cell wall dynamics

    PubMed Central

    Ebine, Kazuo; Ueda, Takashi

    2015-01-01

    The cell wall is one of the characteristic components of plant cells. The cell wall composition differs among cell types and is modified in response to various environmental conditions. To properly generate and modify the cell wall, many proteins are transported to the plasma membrane or extracellular space through membrane trafficking, which is one of the key protein transport mechanisms in eukaryotic cells. Given the diverse composition and functions of the cell wall in plants, the transport of the cell wall components and proteins that are involved in cell wall-related events could be specialized for each cell type, i.e., the machinery for cell wall biogenesis, modification, and maintenance could be transported via different trafficking pathways. In this review, we summarize the recent progress in the current understanding of the roles and mechanisms of membrane trafficking in plant cells and focus on the biogenesis and regulation of the cell wall. PMID:26539200

  9. Conductivity Measurements of Synthesized Heteropoly Acid Membranes for Proton Exchange Membrane Fuel Cells

    SciTech Connect

    Record, K.A.; Haley, B.T.; Turner, J.

    2006-01-01

    Fuel cell technology is receiving attention due to its potential to be a pollution free method of electricity production when using renewably produced hydrogen as fuel. In a Proton Exchange Membrane (PEM) fuel cell H2 and O2 react at separate electrodes, producing electricity, thermal energy, and water. A key component of the PEM fuel cell is the membrane that separates the electrodes. DuPont’s Nafion® is the most commonly used membrane in PEM fuel cells; however, fuel cell dehydration at temperatures near 100°C, resulting in poor conductivity, is a major hindrance to fuel cell performance. Recent studies incorporating heteropoly acids (HPAs) into membranes have shown an increase in conductivity and thus improvement in performance. HPAs are inorganic materials with known high proton conductivities. The primary objective of this work is to measure the conductivity of Nafion, X-Ionomer membranes, and National Renewable Energy Laboratory (NREL) Developed Membranes that are doped with different HPAs at different concentrations. Four-point conductivity measurements using a third generation BekkTech conductivity test cell are used to determine membrane conductivity. The effect of multiple temperature and humidification levels is also examined. While the classic commercial membrane, Nafion, has a conductivity of approximately 0.10 S/cm, measurements for membranes in this study range from 0.0030 – 0.58 S/cm, depending on membrane type, structure of the HPA, and the relative humidity. In general, the X-ionomer with H6P2W21O71 HPA gave the highest conductivity and the Nafion with the 12-phosphotungstic (PW12) HPA gave the lowest. The NREL composite membranes had conductivities on the order of 0.0013 – 0.025 S/cm.

  10. Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

    PubMed Central

    Zhao, Yuting; Liu, Jianglan; Yang, Changsong; Capraro, Benjamin R.; Baumgart, Tobias; Bradley, Ryan P.; Ramakrishnan, N.; Xu, Xiaowei; Radhakrishnan, Ravi; Svitkina, Tatyana; Guo, Wei

    2013-01-01

    Dynamic shape changes of the plasma membrane are fundamental to many processes ranging from morphogenesis and cell migration to phagocytosis and viral propagation. Here we demonstrate that Exo70, a component of the exocyst complex, induces tubular membrane invaginations towards the lumen of synthetic vesicles in vitro and generates protrusions on the surface of cells. Biochemical analyses using Exo70 mutants and independent molecular dynamics simulations based on Exo70 structure demonstrate that Exo70 generates negative membrane curvature through an oligomerization-based mechanism. In cells, the membrane-deformation function of Exo70 is required for protrusion formation and directional cell migration. Exo70 thus represents a membrane-bending protein that may couple actin dynamics and plasma membrane remodeling for morphogenesis. PMID:23948253

  11. Engineered nanoparticles mimicking cell membranes for toxin neutralization.

    PubMed

    Fang, Ronnie H; Luk, Brian T; Hu, Che-Ming J; Zhang, Liangfang

    2015-08-01

    Protein toxins secreted from pathogenic bacteria and venomous animals rely on multiple mechanisms to overcome the cell membrane barrier to inflict their virulence effect. A promising therapeutic concept toward developing a broadly applicable anti-toxin platform is to administer cell membrane mimics as decoys to sequester these virulence factors. As such, lipid membrane-based nanoparticulates are an ideal candidate given their structural similarity to cellular membranes. This article reviews the virulence mechanisms employed by toxins at the cell membrane interface and highlights the application of cell-membrane mimicking nanoparticles as toxin decoys for systemic detoxification. In addition, the implication of particle/toxin nanocomplexes in the development of toxoid vaccines is discussed.

  12. Membrane tension and cytoskeleton organization in cell motility

    NASA Astrophysics Data System (ADS)

    Sens, Pierre; Plastino, Julie

    2015-07-01

    Cell membrane shape changes are important for many aspects of normal biological function, such as tissue development, wound healing and cell division and motility. Various disease states are associated with deregulation of how cells move and change shape, including notably tumor initiation and cancer cell metastasis. Cell motility is powered, in large part, by the controlled assembly and disassembly of the actin cytoskeleton. Much of this dynamic happens in close proximity to the plasma membrane due to the fact that actin assembly factors are membrane-bound, and thus actin filaments are generally oriented such that their growth occurs against or near the membrane. For a long time, the membrane was viewed as a relatively passive scaffold for signaling. However, results from the last five years show that this is not the whole picture, and that the dynamics of the actin cytoskeleton are intimately linked to the mechanics of the cell membrane. In this review, we summarize recent findings concerning the role of plasma membrane mechanics in cell cytoskeleton dynamics and architecture, showing that the cell membrane is not just an envelope or a barrier for actin assembly, but is a master regulator controlling cytoskeleton dynamics and cell polarity.

  13. Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells.

    PubMed

    Gioia, Magda; Vindigni, Giulia; Testa, Barbara; Raniolo, Sofia; Fasciglione, Giovanni Francesco; Coletta, Massimiliano; Biocca, Silvia

    2015-01-01

    The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MβCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding. PMID:26495844

  14. Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells

    PubMed Central

    Testa, Barbara; Raniolo, Sofia; Fasciglione, Giovanni Francesco; Coletta, Massimiliano; Biocca, Silvia

    2015-01-01

    The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MβCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding. PMID:26495844

  15. Anatomy of the red cell membrane skeleton: unanswered questions.

    PubMed

    Lux, Samuel E

    2016-01-14

    The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3-containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton's physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.

  16. High cell-surface density of HER2 deforms cell membranes

    PubMed Central

    Chung, Inhee; Reichelt, Mike; Shao, Lily; Akita, Robert W.; Koeppen, Hartmut; Rangell, Linda; Schaefer, Gabriele; Mellman, Ira; Sliwkowski, Mark X.

    2016-01-01

    Breast cancers (BC) with HER2 overexpression (referred to as HER2 positive) progress more aggressively than those with normal expression. Targeted therapies against HER2 can successfully delay the progression of HER2-positive BC, but details of how this overexpression drives the disease are not fully understood. Using single-molecule biophysical approaches, we discovered a new effect of HER2 overexpression on disease-relevant cell biological changes in these BC. We found HER2 overexpression causes deformation of the cell membranes, and this in turn disrupts epithelial features by perturbing cell–substrate and cell–cell contacts. This membrane deformation does not require receptor signalling activities, but results from the high levels of HER2 on the cell surface. Our finding suggests that early-stage morphological alterations of HER2-positive BC cells during cancer progression can occur in a physical and signalling-independent manner. PMID:27599456

  17. Effect of EMP fields on cell membrane potentials

    SciTech Connect

    Gailey, P.C.; Easterly, C.E.

    1993-06-01

    A simple model is presented for cell membrane potentials induced during exposure to electromagnetic pulse (EMP). Using calculated values of internal electric field strength induced during EMP exposure, the model predicts that cell membrane potentials of about 100 mV may be induced for time frames on the order of 10 ns. Possible biological effects of these potentials including electroporation area discussed.

  18. Alleviation of Cu and Pb rhizotoxicities in cowpea (Vigna unguiculata) as related to ion activities at root-cell plasma membrane surface.

    PubMed

    Kopittke, Peter M; Kinraide, Thomas B; Wang, Peng; Blamey, F Pax C; Reichman, Suzie M; Menzies, Neal W

    2011-06-01

    Cations, such as Ca and Mg, are generally thought to alleviate toxicities of trace metals through site-specific competition (as incorporated in the biotic ligand model, BLM). Short-term experiments were conducted with cowpea (Vigna unguiculata L. Walp.) seedlings in simple nutrient solutions to examine the alleviation of Cu and Pb toxicities by Al, Ca, H, Mg, and Na. For Cu, the cations depolarized the plasma membrane (PM) and reduced the negativity of ψ(0)(o) (electrical potential at the outer surface of the PM) and thereby decreased {Cu(2+)}(0)(o) (activity of Cu(2+) at the outer surface of the PM). For Pb, root elongation was generally better correlated to the activity of Pb(2+) in the bulk solution than to {Pb(2+)}(0)(o). However, we propose that the addition of cations resulted in a decrease in {Pb(2+)}(0)(o) but a simultaneous increase in the rate of Pb uptake (due to an increase in the negativity of E(m,surf), the difference in potential between the inner and outer surfaces of the PM) thus offsetting the decrease in {Pb(2+)}(0)(o). In addition, Ca was found to alleviate Pb toxicity through a specific effect. Although our data do not preclude site-specific competition (as incorporated in the BLM), we suggest that electrostatic effects have an important role.

  19. Membrane nanowaves in single and collective cell migration.

    PubMed

    Zouani, Omar F; Gocheva, Veronika; Durrieu, Marie-Christine

    2014-01-01

    We report the characterization of three-dimensional membrane waves for migrating single and collective cells and describe their propagation using wide-field optical profiling technique with nanometer resolution. We reveal the existence of small and large membrane waves the amplitudes of which are in the range of ∼ 3-7 nm to ∼ 16-25 nm respectively, through the cell. For migrating single-cells, the amplitude of these waves is about 30 nm near the cell edge. Two or more different directions of propagation of the membrane nanowaves inside the same cell can be observed. After increasing the migration velocity by BMP-2 treatment, only one wave direction of propagation exists with an increase in the average amplitude (more than 80 nm near the cell edge). Furthermore for collective-cell migration, these membrane nanowaves are attenuated on the leader cells and poor transmission of these nanowaves to follower cells was observed. After BMP-2 treatment, the membrane nanowaves are transmitted from the leader cell to several rows of follower cells. Surprisingly, the vast majority of the observed membrane nanowaves is shared between the adjacent cells. These results give a new view on how single and collective-cells modulate their motility. This work has significant implications for the therapeutic use of BMPs for the regeneration of skin tissue. PMID:24846182

  20. Detergent Induction of HEK 293A Cell Membrane Permeability Measured under Quiescent and Superfusion Conditions Using Whole Cell Patch Clamp

    PubMed Central

    2015-01-01

    Detergents have several biological applications but present cytotoxicity concerns, since they can solubilize cell membranes. Using the IonFlux 16, an ensemble whole cell planar patch clamp, we observed that anionic sodium dodecyl sulfate (SDS), cationic cetyltrimethylammonium bromide (CTAB), and cationic, fluorescent octadecyl rhodamine B (ORB) increased the membrane permeability of cells substantially within a second of exposure, under superfusion conditions. Increased permeability was irreversible for 15 min. At subsolubilizing detergent concentrations, patched cells showed increased membrane currents that reached a steady state and were intact when imaged using fluorescence microscopy. SDS solubilized cells at concentrations of 2 mM (2× CMC), while CTAB did not solubilize cells even at concentrations of 10 mM (1000× CMC). The relative activity for plasma membrane current induction was 1:20:14 for SDS, CTAB, and ORB, respectively. Under quiescent conditions, the relative ratio of lipid to detergent in cell membranes at the onset of membrane permeability was 1:7:5 for SDS, CTAB, and ORB, respectively. The partition constants (K) for SDS, CTAB, and ORB were 23000, 55000, and 39000 M–1, respectively. Combining the whole cell patch clamp data and XTT viability data, SDS ≤ 0.2 mM and CTAB and ORB ≤ 1 mM induced cell membrane permeability without causing acute toxicity. PMID:24548291

  1. Detergent induction of HEK 293A cell membrane permeability measured under quiescent and superfusion conditions using whole cell patch clamp.

    PubMed

    Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M

    2014-02-27

    Detergents have several biological applications but present cytotoxicity concerns, since they can solubilize cell membranes. Using the IonFlux 16, an ensemble whole cell planar patch clamp, we observed that anionic sodium dodecyl sulfate (SDS), cationic cetyltrimethylammonium bromide (CTAB), and cationic, fluorescent octadecyl rhodamine B (ORB) increased the membrane permeability of cells substantially within a second of exposure, under superfusion conditions. Increased permeability was irreversible for 15 min. At subsolubilizing detergent concentrations, patched cells showed increased membrane currents that reached a steady state and were intact when imaged using fluorescence microscopy. SDS solubilized cells at concentrations of 2 mM (2× CMC), while CTAB did not solubilize cells even at concentrations of 10 mM (1000× CMC). The relative activity for plasma membrane current induction was 1:20:14 for SDS, CTAB, and ORB, respectively. Under quiescent conditions, the relative ratio of lipid to detergent in cell membranes at the onset of membrane permeability was 1:7:5 for SDS, CTAB, and ORB, respectively. The partition constants (K) for SDS, CTAB, and ORB were 23000, 55000, and 39000 M(-1), respectively. Combining the whole cell patch clamp data and XTT viability data, SDS ≤ 0.2 mM and CTAB and ORB ≤ 1 mM induced cell membrane permeability without causing acute toxicity.

  2. Killing of Staphylococci by θ-Defensins Involves Membrane Impairment and Activation of Autolytic Enzymes

    PubMed Central

    Wilmes, Miriam; Stockem, Marina; Bierbaum, Gabriele; Schlag, Martin; Götz, Friedrich; Tran, Dat Q.; Schaal, Justin B.; Ouellette, André J.; Selsted, Michael E.; Sahl, Hans-Georg

    2014-01-01

    θ-Defensins are cyclic antimicrobial peptides expressed in leukocytes of Old world monkeys. To get insight into their antibacterial mode of action, we studied the activity of RTDs (rhesus macaque θ-defensins) against staphylococci. We found that in contrast to other defensins, RTDs do not interfere with peptidoglycan biosynthesis, but rather induce bacterial lysis in staphylococci by interaction with the bacterial membrane and/or release of cell wall lytic enzymes. Potassium efflux experiments and membrane potential measurements revealed that the membrane impairment by RTDs strongly depends on the energization of the membrane. In addition, RTD treatment caused the release of Atl-derived cell wall lytic enzymes probably by interaction with membrane-bound lipoteichoic acid. Thus, the premature and uncontrolled activity of these enzymes contributes strongly to the overall killing by θ-defensins. Interestingly, a similar mode of action has been described for Pep5, an antimicrobial peptide of bacterial origin. PMID:25632351

  3. 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

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

    PubMed

    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 b 5 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

  5. Dynamics of a membrane interacting with an active wall.

    PubMed

    Yasuda, Kento; Komura, Shigeyuki; Okamoto, Ryuichi

    2016-05-01

    Active motions of a biological membrane can be induced by nonthermal fluctuations that occur in the outer environment of the membrane. We discuss the dynamics of a membrane interacting hydrodynamically with an active wall that exerts random velocities on the ambient fluid. Solving the hydrodynamic equations of a bound membrane, we first derive a dynamic equation for the membrane fluctuation amplitude in the presence of different types of walls. Membrane two-point correlation functions are calculated for three different cases: (i) a static wall, (ii) an active wall, and (iii) an active wall with an intrinsic time scale. We focus on the mean squared displacement (MSD) of a tagged membrane describing the Brownian motion of a membrane segment. For the static wall case, there are two asymptotic regimes of MSD (∼t^{2/3} and ∼t^{1/3}) when the hydrodynamic decay rate changes monotonically. In the case of an active wall, the MSD grows linearly in time (∼t) in the early stage, which is unusual for a membrane segment. This linear-growth region of the MSD is further extended when the active wall has a finite intrinsic time scale. PMID:27300924

  6. Cell membrane thermal gradients induced by electromagnetic fields

    NASA Astrophysics Data System (ADS)

    Garner, Allen L.; Deminsky, Maxim; Bogdan Neculaes, V.; Chashihin, V.; Knizhnik, Andrey; Potapkin, Boris

    2013-06-01

    While electromagnetic fields induce structural changes in cell membranes, particularly electroporation, much remains to be understood about membrane level temperature gradients. For instance, microwaves induce cell membrane temperature gradients (∇T) and bioeffects with little bulk temperature change. Recent calculations suggest that nanosecond pulsed electric fields (nsPEFs) may also induce such gradients that may additionally impact the electroporation threshold. Here, we analytically and numerically calculate the induced ∇T as a function of pulse duration and pulse repetition rate. We relate ∇T to the thermally induced cell membrane electric field (Em) by assuming the membrane behaves as a thermoelectric such that Em ˜ ∇T. Focusing initially on applying nsPEFs to a uniform membrane, we show that reducing pulse duration and increasing pulse repetition rate (or using higher frequency for alternating current (AC) fields) maximizes the magnitude and duration of ∇T and, concomitantly, Em. The maximum ∇T initially occurs at the interface between the cell membrane and extracellular fluid before becoming uniform across the membrane, potentially enabling initial molecular penetration and subsequent transport across the membrane. These results, which are equally applicable to AC fields, motivate further studies to elucidate thermoelectric behavior in a model membrane system and the coupling of the Em induced by ∇T with that created directly by the applied field.

  7. Fisetin inhibits growth, induces G₂ /M arrest and apoptosis of human epidermoid carcinoma A431 cells: role of mitochondrial membrane potential disruption and consequent caspases activation.

    PubMed

    Pal, Harish C; Sharma, Samriti; Elmets, Craig A; Athar, Mohammad; Afaq, Farrukh

    2013-07-01

    Non-melanoma skin cancers (NMSCs), one of the most common neoplasms, cause serious morbidity and mortality. Therefore, identification of non-toxic phytochemicals for prevention/treatment of NMSCs is highly desirable. Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid, present in fruits and vegetables possesses anti-oxidant and antiproliferative properties. The aim of this study was to investigate the chemotherapeutic potential of fisetin in cultured human epidermoid carcinoma A431 cells. Treatment of A431 cells with fisetin (5-80 μm) resulted in a significant decrease in cell viability in a dose- and time-dependent manner. Employing clonogenic assay, we found that fisetin treatment significantly reduced colony formation in A431 cells. Fisetin treatment of A431 cells resulted in G₂ /M arrest and induction of apoptosis. Furthermore, treatment of A431 cells with fisetin resulted in (i) decreased expression of anti-apoptotic proteins (Bcl2; Bcl-xL and Mcl-1); (ii) increased expression of pro-apoptotic proteins (Bax, Bak and Bad); (iii) disruption of mitochondrial potential; (iv) release of cytochrome c and Smac/DIABLO from mitochondria; (v) activation of caspases; and (vi) cleavage of Poly(ADP-ribose) polymerase (PARP) protein. Pretreatment of A431 cells with the pan-caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced cleavage of caspases and PARP. Taken together, these data provide evidence that fisetin possesses chemotherapeutic potential against human epidermoid carcinoma A431 cells. Overall, these results suggest that fisetin could be developed as a novel therapeutic agent for the management of NMSCs.

  8. Multiplex lithography for multilevel multiscale architectures and its application to polymer electrolyte membrane fuel cell

    PubMed Central

    Cho, Hyesung; Moon Kim, Sang; Sik Kang, Yun; Kim, Junsoo; Jang, Segeun; Kim, Minhyoung; Park, Hyunchul; Won Bang, Jung; Seo, Soonmin; Suh, Kahp-Yang; Sung, Yung-Eun; Choi, Mansoo

    2015-01-01

    The production of multiscale architectures is of significant interest in materials science, and the integration of those structures could provide a breakthrough for various applications. Here we report a simple yet versatile strategy that allows for the LEGO-like integrations of microscale membranes by quantitatively controlling the oxygen inhibition effects of ultraviolet-curable materials, leading to multilevel multiscale architectures. The spatial control of oxygen concentration induces different curing contrasts in a resin allowing the selective imprinting and bonding at different sides of a membrane, which enables LEGO-like integration together with the multiscale pattern formation. Utilizing the method, the multilevel multiscale Nafion membranes are prepared and applied to polymer electrolyte membrane fuel cell. Our multiscale membrane fuel cell demonstrates significant enhancement of performance while ensuring mechanical robustness. The performance enhancement is caused by the combined effect of the decrease of membrane resistance and the increase of the electrochemical active surface area. PMID:26412619

  9. Effects of an antibacterial membrane on osteoblast-like cells in vitro

    PubMed Central

    Ye, Jun; Yao, Qianqian; Mo, Anchun; Nie, Jing; Liu, Wenwen; Ye, Cui; Chen, Xianji

    2011-01-01

    Infection around membranes is often found in guided bone regeneration (GBR). The excellent antibacterial properties of Ag-nHA-nTiO2/polyamide-66 (PA66) nanocomposite membranes have been demonstrated previously. The aim of this study was to observe the microstructure of an Ag-nHA-nTiO2/PA66 membrane and its effects on osteoblast-like cells in vitro. An Ag-nHA-nTiO2/PA66 membrane was used in the experimental group, and both nHA/PA66 and expanded poly tetrafluroethylene (e-PTFE) membranes were set as control. MG63 osteoblast-like cells were cultured on the three kinds of membrane and tissue culture polystyrene (TCP). The microstructure of the above membranes and the cells adhered on them were detected by scanning electronic microscope (SEM). Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell viability with a cell viability analyzer, and alkaline phosphatase (ALP) activity and Ca2+ concentration of osteoblast-like cell matrix by enzyme-linked immunosorbent assay. SEM showed that both Ag-nHA-nTiO2/PA66 membranes and nHA/PA66 membranes were composed of porous obverse face and smooth opposite face. The e-PTFE membranes showed elliptic surface structure with many tiny lined cracks. The MG63 cells adhered and proliferated well on all three kinds of membranes. Though cell viability on Ag-nHA-nTiO2/PA66 membranes was significantly lower than that of the control groups (P < 0.05), MTT values, ALP activity, and Ca2+ concentration did not differ significantly among the three kinds of membranes (P > 0.05). From these findings, it can be concluded that Ag-nHA-nTiO2/PA66 membranes are as biocompatible as nHA/ PA66 membranes and TCP, thus may be applied safely in GBR. PMID:21931481

  10. Cell-free methods to produce structurally intact mammalian membrane proteins

    PubMed Central

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ishizuka-Katsura, Yoshiko; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Tomita, Taisuke; Ishibashi, Yohei; Hirabayashi, Yoshio; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2016-01-01

    The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1–1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin. PMID:27465719

  11. Cell-free methods to produce structurally intact mammalian membrane proteins.

    PubMed

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ishizuka-Katsura, Yoshiko; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Tomita, Taisuke; Ishibashi, Yohei; Hirabayashi, Yoshio; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2016-01-01

    The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1-1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin. PMID:27465719

  12. Studying the Nucleated Mammalian Cell Membrane by Single Molecule Approaches

    PubMed Central

    Wang, Feng; Wu, Jiazhen; Gao, Jing; Liu, Shuheng; Jiang, Junguang; Jiang, Shibo; Wang, Hongda

    2014-01-01

    The cell membrane plays a key role in compartmentalization, nutrient transportation and signal transduction, while the pattern of protein distribution at both cytoplasmic and ectoplasmic sides of the cell membrane remains elusive. Using a combination of single-molecule techniques, including atomic force microscopy (AFM), single molecule force spectroscopy (SMFS) and stochastic optical reconstruction microscopy (STORM), to study the structure of nucleated cell membranes, we found that (1) proteins at the ectoplasmic side of the cell membrane form a dense protein layer (4 nm) on top of a lipid bilayer; (2) proteins aggregate to form islands evenly dispersed at the cytoplasmic side of the cell membrane with a height of about 10–12 nm; (3) cholesterol-enriched domains exist within the cell membrane; (4) carbohydrates stay in microdomains at the ectoplasmic side; and (5) exposed amino groups are asymmetrically distributed on both sides. Based on these observations, we proposed a Protein Layer-Lipid-Protein Island (PLLPI) model, to provide a better understanding of cell membrane structure, membrane trafficking and viral fusion mechanisms. PMID:24806512

  13. Lactobacillus casei combats acid stress by maintaining cell membrane functionality.

    PubMed

    Wu, Chongde; Zhang, Juan; Wang, Miao; Du, Guocheng; Chen, Jian

    2012-07-01

    Lactobacillus casei strains have traditionally been recognized as probiotics and frequently used as adjunct culture in fermented dairy products where lactic acid stress is a frequently encountered environmental condition. We have investigated the effect of lactic acid stress on the cell membrane of L. casei Zhang [wild type (WT)] and its acid-resistant mutant Lbz-2. Both strains were grown under glucose-limiting conditions in chemostats; following challenge by low pH, the cell membrane stress responses were investigated. In response to acid stress, cell membrane fluidity decreased and its fatty acid composition changed to reduce the damage caused by lactic acid. Compared with the WT, the acid-resistant mutant exhibited numerous survival advantages, such as higher membrane fluidity, higher proportions of unsaturated fatty acids, and higher mean chain length. In addition, cell integrity analysis showed that the mutant maintained a more intact cellular structure and lower membrane permeability after environmental acidification. These results indicate that alteration in membrane fluidity, fatty acid distribution, and cell integrity are common mechanisms utilized by L. casei to withstand severe acidification and to reduce the deleterious effect of lactic acid on the cell membrane. This detailed comparison of cell membrane responses between the WT and mutant add to our knowledge of the acid stress adaptation and thus enable new strategies to be developed aimed at improving the industrial performance of this species under acid stress. PMID:22366811

  14. Durability of symmetrically and asymmetrically porous polybenzimidazole membranes for high temperature proton exchange membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Jheng, Li-Cheng; Chang, Wesley Jen-Yang; Hsu, Steve Lien-Chung; Cheng, Po-Yang

    2016-08-01

    Two types of porous polybenzimidazole (PBI) membranes with symmetric and asymmetric morphologies were fabricated by the template-leaching method and characterized by scanning electron microscope (SEM). Their physicochemical properties were compared in terms of acid-doping level, proton conductivity, mechanical strength, and oxidative stability. The durability of fuel cell operation is one of the most challenging for the PBI based membrane electrode assembly (MEA) used in high-temperature proton exchange membrane fuel cells (HT-PEMFCs). In the present work, we carried out a long-term steady-state fuel cell test to compare the effect of membrane structure on the cell voltage degradation. It has also been demonstrated that the asymmetrically porous PBI could bring some notable improvements on the durability of fuel cell operation, the fuel crossover problem, and the phosphoric acid leakage.

  15. Isolation of mitochondria by gentle cell membrane disruption, and their subsequent characterization.

    PubMed

    Shibata, Takahiro; Yamashita, Saki; Hirusaki, Kotoe; Katoh, Kaoru; Ohta, Yoshihiro

    2015-08-01

    Mitochondria play a key role in several physiological processes as in integrating signals in the cell. However, understanding of the mechanism by which mitochondria sense and respond to signals has been limited due to the lack of an appropriate model system. In this study, we developed a method to isolate and characterize mitochondria without cell homogenization. By gently pipetting cells treated with streptolysin-O, a pore-forming membrane protein, we disrupted the cell membrane and were able to isolate both elongated and spherical mitochondria. Fluorescence imaging combined with super resolution microscopy showed that both the outer and inner membranes of the elongated mitochondria isolated using the newly developed method were intact. In addition, a FRET-based ATP sensor expressed in the mitochondrial matrix demonstrated that ATP generation by FoF1-ATPase in the isolated elongated mitochondria was as high as that in intracellular mitochondria. On the other hand, some of the spherical mitochondria isolated with this method had the outer membrane that no longer encapsulated the inner membrane. In addition, all mitochondria isolated using conventional procedures involving homogenization were spherical, many of them had damaged membranes, and low levels of ATP generation. Our results suggest that elongated mitochondria isolated from cells through gentle cell membrane disruption using a pore-forming protein tend to be more similar to intracellular mitochondria, having an intact membrane system and higher activity than spherical mitochondria.

  16. Radiation-Grafted Polymer Electrolyte Membranes for Water Electrolysis Cells: Evaluation of Key Membrane Properties.

    PubMed

    Albert, Albert; Barnett, Alejandro O; Thomassen, Magnus S; Schmidt, Thomas J; Gubler, Lorenz

    2015-10-14

    Radiation-grafted membranes can be considered an alternative to perfluorosulfonic acid (PFSA) membranes, such as Nafion, in a solid polymer electrolyte electrolyzer. Styrene, acrylonitrile, and 1,3-diisopropenylbenzene monomers are cografted into preirradiated 50 μm ethylene tetrafluoroethylene (ETFE) base film, followed by sulfonation to introduce proton exchange sites to the obtained grafted films. The incorporation of grafts throughout the thickness is demonstrated by scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDX) analysis of the membrane cross-sections. The membranes are analyzed in terms of grafting kinetics, ion-exchange capacity (IEC), and water uptake. The key properties of radiation-grafted membranes and Nafion, such as gas crossover, area resistance, and mechanical properties, are evaluated and compared. The plot of hydrogen crossover versus area resistance of the membranes results in a property map that indicates the target areas for membrane development for electrolyzer applications. Tensile tests are performed to assess the mechanical properties of the membranes. Finally, these three properties are combined to establish a figure of merit, which indicates that radiation-grafted membranes obtained in the present study are promising candidates with properties superior to those of Nafion membranes. A water electrolysis cell test is performed as proof of principle, including a comparison to a commercial membrane electrode assembly (MEA).

  17. Vascular endothelial cell membranes differentiate between stretch and shear stress through transitions in their lipid phases.

    PubMed

    Yamamoto, Kimiko; Ando, Joji

    2015-10-01

    Vascular endothelial cells (ECs) respond to the hemodynamic forces stretch and shear stress by altering their morphology, functions, and gene expression. However, how they sense and differentiate between these two forces has remained unknown. Here we report that the plasma membrane itself differentiates between stretch and shear stress by undergoing transitions in its lipid phases. Uniaxial stretching and hypotonic swelling increased the lipid order of human pulmonary artery EC plasma membranes, thereby causing a transition from the liquid-disordered phase to the liquid-ordered phase in some areas, along with a decrease in membrane fluidity. In contrast, shear stress decreased the membrane lipid order and increased membrane fluidity. A similar increase in lipid order occurred when the artificial lipid bilayer membranes of giant unilamellar vesicles were stretched by hypotonic swelling, indicating that this is a physical phenomenon. The cholesterol content of EC plasma membranes significantly increased in response to stretch but clearly decreased in response to shear stress. Blocking these changes in the membrane lipid order by depleting membrane cholesterol with methyl-β-cyclodextrin or by adding cholesterol resulted in a marked inhibition of the EC response specific to stretch and shear stress, i.e., phosphorylation of PDGF receptors and phosphorylation of VEGF receptors, respectively. These findings indicate that EC plasma membranes differently respond to stretch and shear stress by changing their lipid order, fluidity, and cholesterol content in opposite directions and that these changes in membrane physical properties are involved in the mechanotransduction that activates membrane receptors specific to each force.

  18. The Structural Basis of Cholesterol Activity in Membranes

    SciTech Connect

    Olsen, Brett N.; Bielska, Agata; Lee, Tiffany; Daily, Michael D.; Covey, Douglas F.; Schlesinger, Paul H.; Baker, Nathan A.; Ory, Daniel S.

    2013-10-15

    Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.

  19. On-line comprehensive two-dimensional HepG2 cell membrane chromatographic analysis system for charactering anti-hepatoma components from rat serum after oral administration of Radix scutellariae: A strategy for rapid screening active compounds in vivo.

    PubMed

    Jia, Dan; Chen, Xiaofei; Cao, Yan; Wu, Xunxun; Ding, Xuan; Zhang, Hai; Zhang, Chuan; Chai, Yifeng; Zhu, Zhenyu

    2016-01-25

    Cell membrane chromatography (CMC) is a bioaffinity chromatography technique for characterizing interactions between drugs and membrane receptors and has been widely used to screen active components from complex samples such as herbal medicines (HMs). However, it has never been applied in vivo due to its relatively high limit of detection (LOD) and the matrix interferences. In this study, a novel on-line comprehensive two-dimensional HepG2/CMC/enrich columns/high performance liquid chromatography/time-of-flight mass spectrometry system was developed to rapidly screen potential anti-hepatoma components from drug-containing serum of rats after oral administration of Radix scutellariae. A matrix interference deduction method with a home-written program in MATLAB was developed, which could successfully eliminate the interference of endogenous substances in serum. Baicalein, wogonin, chrysin, oroxylin A, neobaicalein and rivularin from Radix scutellariae extraction were significantly retained in the HepG2/CMC column. Three potential active components, wogonin, oroxylin A and neobaicalein were firstly screened from the drug-containing serum as well. The cell counting kit-8 assay demonstrated that wogonin, oroxylin A and chrysin showed high inhibitory activities in a dose-dependent manner on HepG2 cells at the concentration of 12.5-200 μM (p<0.05) and the IC50 values were 69.83, 16.66 and 51.6 μM, respectively. Wogonin and oroxylin A, which were screened both from Radix scutellariae extraction and the drug-containing serum, could be selected as lead compounds to obtain good anti-hepatoma effects. The proposed comprehensive 2D CMC system and matrix interference elimination strategy have significant advantages for in vivo screening of active components from complex biological samples and could be applied to other biochromatography models.

  20. Cell membrane potentials induced during exposure to EMP fields

    SciTech Connect

    Gailey, P.C.; Easterly, C.E.

    1994-09-01

    Internal current densities and electric fields induced in the human body during exposure to EMP fields are reviewed and used to predict resulting cell membrane potentials. Using several different approaches, membrane potentials of about 100 mV are predicted. These values are comparable to the static membrane potentials maintained by cells as a part of normal physiological function, but the EMP-induced potentials persist for only about 10 ns. Possible biological implications of EMP-induced membrane potentials including conformational changes and electroporation are discussed.

  1. [Research progress of corneal epithelial basal cells and basement membrane].

    PubMed

    Qu, J H; Sun, X G

    2016-09-11

    The cylinder cells at the bottom of corneal epithelial cells are basal cells. Their cytoplasm contains keratin intermediate filament which is important in secretion of basement membrane. Corneal epithelial dysfunction due to diabetes or ocular surgery is intimately related with basal cell abnormality. Corneal epithelial basement membrane is a highly specific extracellular matrix which is made up of lamina lucida and lamina densa. It plays an extremely important role in renewal and restoration. Many ocular abnormalities and diseases have been described to relate to the corneal epithelial basement membrane, such as traumatic recurrent corneal erosion, corneal dystrophy and keratoconus. (Chin J Ophthalmol, 2016, 52: 703-707). PMID:27647251

  2. Initial conditioning of polymer eelectrolyte membrane fuel cell by temperature and potential cycling.

    PubMed

    Bezmalinović, Dario; Radošević, Jagoda; Barbir, Frano

    2015-01-01

    Polymer electrolyte membrane fuel cells need initial conditioning, activation or break-in the first time they are operated after being assembled. During this period performance of the fuel cell improves until it reaches its nominal performance. The exact mechanism of this initial conditioning is not completely understood, but it is assumed that during the conditioning process the polymer membrane, as well as the polymer in the catalyst layer, get humidified, and the number of active catalyst sites increases. Activation procedure proposed here consists of temperature and potential cycling. Temperature cycling is a new approach for the conditioning and the idea is to rapidly cool the running cell at some point to allow the membrane to equilibrate with condensed water which should result in higher intake of water within the membrane. The results show that proposed procedure is better or at least comparable to some conventional procedures for the initial conditioning. PMID:25830963

  3. HGAL localization to cell membrane regulates B-cell receptor signaling

    PubMed Central

    Lu, Xiaoqing; Sicard, Renaud; Jiang, Xiaoyu; Stockus, Jessica N.; McNamara, George; Abdulreda, Midhat; Moy, Vincent T.; Landgraf, Ralf

    2015-01-01

    Human germinal center–associated lymphoma (HGAL) is specifically expressed only in germinal center (GC) B lymphocytes and GC-derived lymphomas. HGAL protein decreases lymphocyte motility by inhibiting the ability of myosin to translocate actin via direct interaction with F-actin and myosin II and by activating RhoA signaling via direct interactions with RhoA-specific guanine nucleotide exchange factors. HGAL protein also regulates B-cell receptor (BCR) signaling by directly binding to and enhancing Syk kinase activity and activation of its downstream effectors. Herein we demonstrate that HGAL protein can be myristoylated and palmitoylated and that these modifications localize HGAL to cellular membrane raft microdomains with distinct consequences for BCR signaling and chemoattractant-induced cell mobility. In BCR signaling, raft localization of HGAL facilitates interaction with Syk and modulation of the BCR activation and signaling, which induces HGAL phosphorylation and redistribution from lipid raft to bulk membrane and cytoplasm, followed by degradation. In contrast, HGAL myristoylation and palmitoylation avert its inhibitory effects on chemoattractant-induced cell motility. These findings further elucidate the growing and complex role of HGAL in B-cell biology and suggest that membrane-bound and cytoplasmic HGAL protein differently regulates distinct biological processes. PMID:25381061

  4. Proteomics and Phosphoproteomics Analysis of Human Lens Fiber Cell Membranes

    PubMed Central

    Wang, Zhen; Han, Jun; David, Larry L.; Schey, Kevin L.

    2013-01-01

    Purpose. The human lens fiber cell insoluble membrane fraction contains important membrane proteins, cytoskeletal proteins, and cytosolic proteins that are strongly associated with the membrane. The purpose of this study was to characterize the lens fiber cell membrane proteome and phosphoproteome from human lenses. Methods. HPLC-mass spectrometry–based multidimensional protein identification technology (MudPIT), without or with phosphopeptide enrichment, was applied to study the proteome and phosphoproteome of lens fiber cell membranes, respectively. Results. In total, 951 proteins were identified, including 379 integral membrane and membrane-associated proteins. Enriched gene categories and pathways based on the proteomic analysis include carbohydrate metabolism (glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism), proteasome, cell-cell signaling and communication (GTP binding, gap junction, focal adhesion), glutathione metabolism, and actin regulation. The combination of TiO2 phosphopeptide enrichment and MudPIT analysis revealed 855 phosphorylation sites on 271 proteins, including 455 phosphorylation sites that have not been previously identified. PKA, PKC, CKII, p38MAPK, and RSK are predicted as the major kinases for phosphorylation on the sites identified in the human lens membrane fraction. Conclusions. The results presented herein significantly expand the characterized proteome and phosphoproteome of the human lens fiber cell and provide a valuable reference for future research in studies of lens development and disease. PMID:23349431

  5. Membrane Bound GSK-3 Activates Wnt Signaling through Disheveled and Arrow

    PubMed Central

    Mannava, Anirudh G.; Tolwinski, Nicholas S.

    2015-01-01

    Wnt ligands and their downstream pathway components coordinate many developmental and cellular processes. In adults, they regulate tissue homeostasis through regulation of stem cells. Mechanistically, signal transduction through this pathway is complicated by pathway components having both positive and negative roles in signal propagation. Here we examine the positive role of GSK-3/Zw3 in promoting signal transduction at the plasma membrane. We find that targeting GSK-3 to the plasma membrane activates signaling in Drosophila embryos. This activation requires the presence of the co-receptor Arrow-LRP5/6 and the pathway activating protein Disheveled. Our results provide genetic evidence for evolutionarily conserved, separable roles for GSK-3 at the membrane and in the cytosol, and are consistent with a model where the complex cycles from cytosol to membrane in order to promote signaling at the membrane and to prevent it in the cytosol. PMID:25848770

  6. 160 C PROTON EXCHANGE MEMBRANE (PEM) FUEL CELL SYSTEM DEVELOPMENT

    SciTech Connect

    L.G. Marianowski

    2001-12-21

    The objectives of this program were: (a) to develop and demonstrate a new polymer electrolyte membrane fuel cell (PEMFC) system that operates up to 160 C temperatures and at ambient pressures for stationary power applications, and (b) to determine if the GTI-molded composite graphite bipolar separator plate could provide long term operational stability at 160 C or higher. There are many reasons that fuel cell research has been receiving much attention. Fuel cells represent environmentally friendly and efficient sources of electrical power generation that could use a variety of fuel sources. The Gas Technology Institute (GTI), formerly Institute of Gas Technology (IGT), is focused on distributed energy stationary power generation systems. Currently the preferred method for hydrogen production for stationary power systems is conversion of natural gas, which has a vast distribution system in place. However, in the conversion of natural gas into a hydrogen-rich fuel, traces of carbon monoxide are produced. Carbon monoxide present in the fuel gas will in time cumulatively poison, or passivate the active platinum catalysts used in the anodes of PEMFC's operating at temperatures of 60 to 80 C. Various fuel processors have incorporated systems to reduce the carbon monoxide to levels below 10 ppm, but these require additional catalytic section(s) with sensors and controls for effective carbon monoxide control. These CO cleanup systems must also function especially well during transient load operation where CO can spike 300% or more. One way to circumvent the carbon monoxide problem is to operate the fuel cell at a higher temperature where carbon monoxide cannot easily adsorb onto the catalyst and poison it. Commercially available polymer membranes such as Nafion{trademark} are not capable of operation at temperatures sufficiently high to prevent this. Hence this project investigated a new polymer membrane alternative to Nafion{trademark} that is capable of operation at

  7. Ionic Liquids and New Proton Exchange Membranes for Fuel Cells

    NASA Technical Reports Server (NTRS)

    Belieres, Jean-Philippe

    2004-01-01

    There is currently a great surge of activity in fuel cell research as laboratories across the world seek to take advantage of the high energy capacity provided by &el cells relative to those of other portable electrochemical power systems. Much of this activity is aimed at high temperature fie1 cells, and a vital component of such &el cells must be the availability of a high temperature stable proton-permeable membrane. NASA Glenn Research Center is greatly involved in developing this technology. Other approaches to the high temperature fuel cell involve the use of single- component or almost-single-component electrolytes that provide a path for protons through the cell. A heavily researched case is the phosphoric acid fuel cell, in which the electrolyte is almost pure phosphoric acid and the cathode reaction produces water directly. The phosphoric acid fie1 cell delivers an open circuit voltage of 0.9 V falling to about 0.7 V under operating conditions at 170 C. The proton transport mechanism is mainly vehicular in character according to the viscosity/conductance relation. Here we describe some Proton Transfer Ionic Liquids (PTILs) with low vapor pressure and high temperature stability that have conductivities of unprecedented magnitude for non-aqueous systems. The first requirement of an ionic liquid is that, contrary to experience with most liquids consisting of ions, it must have a melting point that is not much above room temperature. The limit commonly suggested is 100 C. PTILs constitute an interesting class of non-corrosive proton-exchange electrolyte, which can serve well in high temperature (T = 100 - 250 C) fuel cell applications. We will present cell performance data showing that the open circuit voltage output, and the performance of a simple H2(g)Pt/PTIL/Pt/O2(g) fuel cell may be superior to those of the equivalent phosphoric acid electrolyte fuel cell both at ambient temperature and temperatures up to and above 200 C. My work at NASA Glenn Research

  8. Cell Membranes Under Hydrostatic Pressure Subjected to Micro-Injection

    NASA Astrophysics Data System (ADS)

    Vassilev, Vassil M.; Kostadinov, Kostadin G.; Mladenov, Ivaïlo M.; Shulev, Assen A.; Stoilov, Georgi I.; Djondjorov, Peter A.

    2011-04-01

    The work is concerned with the determination of the mechanical behaviour of cell membranes under uniform hydrostatic pressure subject to micro-injections. For that purpose, assuming that the shape of the deformed cell membrane is axisymmetric a variational statement of the problem is developed on the ground of the so-called spontaneous curvature model. In this setting, the cell membrane is regarded as an axisymmetric surface in the three-dimensional Euclidean space providing a stationary value of the shape energy functional under the constraint of fixed total area and fixed enclosed volume. The corresponding Euler-Lagrange equations and natural boundary conditions are derived, analyzed and used to express the forces and moments in the membrane. Several examples of such surfaces representing possible shapes of cell membranes under pressure subjected to micro injection are determined numerically.

  9. Selective blockade of CaMKII-alpha inhibits NMDA-induced caspase-3-dependent cell death but does not arrest PARP-1 activation or loss of plasma membrane selectivity in rat retinal neurons.

    PubMed

    Goebel, Dennis J

    2009-02-23

    Calcium/calmodulin-dependent protein kinase II-alpha (CaMKII-alpha) has been implicated in a number of receptor mediated events in neurons. Pharmacological blockade of CaMKII-alpha has been shown to prevent phosphorylation of NMDA-R2A and R2B receptor subunits, suggesting that this enzyme may be linked to receptor trafficking of glutamate receptors and serve as a regulatory protein for neuronal cell death. In the retina, inhibition of CaMKII-alpha has been reported to be neuroprotective against NMDA-induced cell death by preventing the activation of the caspase-3 dependent pathway. However, the effects of CaMKII-alpha blockade on the caspase-3 independent, PARP-1 dependent and the non-programmed cell death pathways have not previously been investigated. In the present study, blockade of CaMKII-alpha with the highly specific antagonist myristoylated autocamtide-2-related inhibitory peptide (AIP) was used in a rat in vivo model of retinal toxicity to compare the effects of on NMDA-induced caspase-3-dependent, PARP-1 dependent and the non-programmed (necrosis) cell death pathways. Results confirmed that AIP fully attenuates caspase-3 activation for at least 8 h following NMDA insult and also significantly improves retinal ganglion cell survival. However, this blockade had little effect on reducing the loss of plasma membrane selectivity (LPMS, e.g. necrosis) in cells located in the ganglion cell and inner nuclear layers and did not alter NMDA-induced PARP-1 hyperactivation, or prevent TUNEL labeling following a moderate NMDA-insult. These findings support a specific role for CaMKII-alpha in mediating the caspase-3 dependent cell death pathway and provide evidence that it is not directly linked to the signaling of either the PARP-1 dependent or the non-programmed cell death pathways.

  10. Development of active-transport membrane devices

    SciTech Connect

    Laciak, D.V.

    1994-07-01

    This report introduces the concept of Air Products` AT membranes for the separation of NH{sub 3} and CO{sub 2} from process gas streams and presents results from the first year fabrication concept development studies.

  11. Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

    PubMed Central

    Alonso, Jose L.; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A.; Hernández, Javier

    2002-01-01

    We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells. PMID:12324366

  12. Double-staining method for differentiation of morphological changes and membrane integrity of Campylobacter coli cells.

    PubMed

    Alonso, Jose L; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A; Hernández, Javier

    2002-10-01

    We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.

  13. How the antimicrobial peptides destroy bacteria cell membrane: Translocations vs. membrane buckling

    NASA Astrophysics Data System (ADS)

    Golubovic, Leonardo; Gao, Lianghui; Chen, Licui; Fang, Weihai

    2012-02-01

    In this study, coarse grained Dissipative Particle Dynamics simulation with implementation of electrostatic interactions is developed in constant pressure and surface tension ensemble to elucidate how the antimicrobial peptide molecules affect bilayer cell membrane structure and kill bacteria. We find that peptides with different chemical-physical properties exhibit different membrane obstructing mechanisms. Peptide molecules can destroy vital functions of the affected bacteria by translocating across their membranes via worm-holes, or by associating with membrane lipids to form hydrophilic cores trapped inside the hydrophobic domain of the membranes. In the latter scenario, the affected membranes are strongly corrugated (buckled) in accord with very recent experimental observations [G. E. Fantner et al., Nat. Nanotech., 5 (2010), pp. 280-285].

  14. Dynamic maintenance of stochastic molecular clusters on cell membranes

    NASA Astrophysics Data System (ADS)

    Mugler, Andrew; Wehrens, Martijn; Ten Wolde, Pieter Rein

    2015-03-01

    Clustering of molecules on cell membranes is a widely observed phenomenon. A key example is the oncoprotein Ras. Maintenance of Ras clusters has been linked to proper Ras signaling. Yet, the mechanism by which Ras clusters are maintained remains unclear. Recently it was discovered that activated Ras promotes further Ras activation. We show using particle-based simulation that this positive feedback link is sufficient to produce persistent clusters of active Ras molecules via a dynamic nucleation mechanism. The cluster statistics are consistent with experimental observations. Interestingly, our model does not support a Turing regime of macroscopic reaction-diffusion patterning. This means that the clustering we observe is a purely stochastic effect, arising from the coupling of the positive feedback network with the discrete nature of individual molecules. These findings underscore the importance of stochastic and dynamic properties of reaction diffusion systems for biological behavior.

  15. Capillary electrophoresis-based nanoscale assays for monitoring ecto-5'-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes.

    PubMed

    Iqbal, Jamshed; Jirovsky, David; Lee, Sang-Yong; Zimmermann, Herbert; Müller, Christa E

    2008-02-01

    Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5'-nucleotidase (ecto-5'-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5'-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 microM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10mM Hepes [pH 7.4], 2mM MgCl2, and 1mM CaCl2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 microM i.d.), followed by enzyme (recombinant rat ecto-5'-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5'-NT.

  16. Bioengineering of living renal membranes consisting of hierarchical, bioactive supramolecular meshes and human tubular cells.

    PubMed

    Dankers, Patricia Y W; Boomker, Jasper M; Huizinga-van der Vlag, Ali; Wisse, Eva; Appel, Wilco P J; Smedts, Frank M M; Harmsen, Martin C; Bosman, Anton W; Meijer, W; van Luyn, Marja J A

    2011-01-01

    Maintenance of polarisation of epithelial cells and preservation of their specialized phenotype are great challenges for bioengineering of epithelial tissues. Mimicking the basement membrane and underlying extracellular matrix (ECM) with respect to its hierarchical fiber-like morphology and display of bioactive signals is prerequisite for optimal epithelial cell function in vitro. We report here on a bottom-up approach based on hydrogen-bonded supramolecular polymers and ECM-peptides to make an electro-spun, bioactive supramolecular mesh which can be applied as synthetic basement membrane. The supramolecular polymers used, self-assembled into nano-meter scale fibers, while at micro-meter scale fibers were formed by electro-spinning. We introduced bioactivity into these nano-fibers by intercalation of different ECM-peptides designed for stable binding. Living kidney membranes were shown to be bioengineered through culture of primary human renal tubular epithelial cells on these bioactive meshes. Even after a long-term culturing period of 19 days, we found that the cells on bioactive membranes formed tight monolayers, while cells on non-active membranes lost their monolayer integrity. Furthermore, the bioactive membranes helped to support and maintain renal epithelial phenotype and function. Thus, incorporation of ECM-peptides into electro-spun meshes via a hierarchical, supramolecular method is a promising approach to engineer bioactive synthetic membranes with an unprecedented structure. This approach may in future be applied to produce living bioactive membranes for a bio-artificial kidney.

  17. Time-resolved molecular transport across living cell membranes.

    PubMed

    Zeng, Jia; Eckenrode, Heather M; Dounce, Susan M; Dai, Hai-Lung

    2013-01-01

    It is shown that the nonlinear optical phenomenon known as second-harmonic generation can be used for label-free, time-resolved study of the transport of molecules through living cell membranes. The adsorption and transport of a 300-Da molecular-mass hydrophobic ion at the Escherichia coli membrane is observed. Remarkably, at low ion concentrations, the second-harmonic generation technique clearly exposes a multistep molecular transport process: Transport of the molecular ion across the outer and cytoplasmic membranes of the Gram-negative bacteria is recorded, in sequence, in time. Fitting of the data to a multiprocess kinematic model reveals that the transport of this hydrophobic ion through the outer membrane is much faster than through the cytoplasmic membrane, likely reflecting the effectiveness of ion transport porins. The observations illustrate an experimental means for studying the interactions of small molecules with cell membranes.

  18. Membrane curvature in cell biology: An integration of molecular mechanisms.

    PubMed

    Jarsch, Iris K; Daste, Frederic; Gallop, Jennifer L

    2016-08-15

    Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists.

  19. Membrane curvature in cell biology: An integration of molecular mechanisms.

    PubMed

    Jarsch, Iris K; Daste, Frederic; Gallop, Jennifer L

    2016-08-15

    Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists. PMID:27528656

  20. Cholesterol depletion increases membrane stiffness of aortic endothelial cells.

    PubMed

    Byfield, Fitzroy J; Aranda-Espinoza, Helim; Romanenko, Victor G; Rothblat, George H; Levitan, Irena

    2004-11-01

    This study has investigated the effect of cellular cholesterol on membrane deformability of bovine aortic endothelial cells. Cellular cholesterol content was depleted by exposing the cells to methyl-beta-cyclodextrin or enriched by exposing the cells to methyl-beta-cyclodextrin saturated with cholesterol. Control cells were treated with methyl-beta-cyclodextrin-cholesterol at a molar ratio that had no effect on the level of cellular cholesterol. Mechanical properties of the cells with different cholesterol contents were compared by measuring the degree of membrane deformation in response to a step in negative pressure applied to the membrane by a micropipette. The experiments were performed on substrate-attached cells that maintained normal morphology. The data were analyzed using a standard linear elastic half-space model to calculate Young elastic modulus. Our observations show that, in contrast to the known effect of cholesterol on membrane stiffness of lipid bilayers, cholesterol depletion of bovine aortic endothelial cells resulted in a significant decrease in membrane deformability and a corresponding increase in the value of the elastic coefficient of the membrane, indicating that cholesterol-depleted cells are stiffer than control cells. Repleting the cells with cholesterol reversed the effect. An increase in cellular cholesterol to a level higher than that of normal cells, however, had no effect on the elastic properties of bovine aortic endothelial cells. We also show that although cholesterol depletion had no apparent effect on the intensity of F-actin-specific fluorescence, disrupting F-actin with latrunculin A abrogated the stiffening effect. We suggest that cholesterol depletion increases the stiffness of the membrane by altering the properties of the submembrane F-actin and/or its attachment to the membrane.

  1. Human hepatocytes and endothelial cells in organotypic membrane systems.

    PubMed

    Salerno, Simona; Campana, Carla; Morelli, Sabrina; Drioli, Enrico; De Bartolo, Loredana

    2011-12-01

    The realization of organotypic liver model that exhibits stable phenotype is a major challenge in the field of liver tissue engineering. In this study we developed liver organotypic co-culture systems by using synthetic and biodegradable membranes with primary human hepatocytes and human umbilical vein endothelial cells (HUVEC). Synthetic membranes prepared by a polymeric blend constituted of modified polyetheretherketone (PEEK-WC) and polyurethane (PU) and biodegradable chitosan membranes were developed by phase inversion technique and used in homotypic and organotypic culture systems. The morphological and functional characteristics of cells in the organotypic co-culture membrane systems were evaluated in comparison with homotypic cultures and traditional systems. Hepatocytes in the organotypic co-culture systems exhibit compact polyhedral cells with round nuclei and well demarcated cell-cell borders like in vivo, as a result of heterotypic interaction with HUVECs. In addition HUVECs formed tube-like structures directly through the interactions with the membranes and hepatocytes and indirectly through the secretion of ECM proteins which secretion improved in the organotypic co-culture membrane systems. The heterotypic cell-cell contacts have beneficial effect on the hepatocyte albumin production, urea synthesis and drug biotransformation. The developed organotypic co-culture membrane systems elicit liver specific functions in vitro and could be applied for the realization of engineered liver tissues to be used in tissue engineering, drug metabolism studies and bioartificial liver devices. PMID:21871658

  2. Coating nanoparticles with cell membranes for targeted drug delivery.

    PubMed

    Gao, Weiwei; Zhang, Liangfang

    2015-01-01

    Targeted delivery allows drug molecules to preferentially accumulate at the sites of action and thus holds great promise to improve therapeutic index. Among various drug-targeting approaches, nanoparticle-based delivery systems offer some unique strengths and have achieved exciting preclinical and clinical results. Herein, we aim to provide a review on the recent development of cell membrane-coated nanoparticle system, a new class of biomimetic nanoparticles that combine both the functionalities of cellular membranes and the engineering flexibility of synthetic nanomaterials for effective drug delivery and novel therapeutics. This review is particularly focused on novel designs of cell membrane-coated nanoparticles as well as their underlying principles that facilitate the purpose of drug targeting. Three specific areas are highlighted, including: (i) cell membrane coating to prolong nanoparticle circulation, (ii) cell membrane coating to achieve cell-specific targeting and (iii) cell membrane coating for immune system targeting. Overall, cell membrane-coated nanoparticles have emerged as a novel class of targeted nanotherapeutics with strong potentials to improve on drug delivery and therapeutic efficacy for treatment of various diseases.

  3. Scanning Ion Conductance Microscopy for living cell membrane potential measurement

    NASA Astrophysics Data System (ADS)

    Panday, Namuna

    Recently, the existence of multiple micro-domains of extracellular potential around individual cells have been revealed by voltage reporter dye using fluorescence microscopy. One hypothesis is that these long lasting potential patterns play a vital role in regulating important cell activities such as embryonic patterning, regenerative repair and reduction of cancerous disorganization. We used multifunctional Scanning Ion Conductance Microscopy (SICM) to study these extracellular potential patterns of single cell with higher spatial resolution. To validate this novel technique, we compared the extracellular potential distribution on the fixed HeLa cell surface and Polydimethylsiloxane (PDMS) surface and found significant difference. We then measured the extracellular potential distributions of living melanocytes and melanoma cells and found both the mean magnitude and spatial variation of extracellular potential of the melanoma cells are bigger than those of melanocytes. As compared to the voltage reporter dye based fluorescence microscope method, SICM can achieve quantitative potential measurements of non-labeled living cell membranes with higher spatial resolution.

  4. Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides.

    PubMed

    Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

    2015-02-10

    Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin.

  5. T Lymphocyte Activation Threshold and Membrane Reorganization Perturbations in Unique Culture Model

    NASA Technical Reports Server (NTRS)

    Adams, C. L.; Sams, C. F.

    2000-01-01

    Quantitative activation thresholds and cellular membrane reorganization are mechanisms by which resting T cells modulate their response to activating stimuli. Here we demonstrate perturbations of these cellular processes in a unique culture system that non-invasively inhibits T lymphocyte activation. During clinorotation, the T cell activation threshold is increased 5-fold. This increased threshold involves a mechanism independent of TCR triggering. Recruitment of lipid rafts to the activation site is impaired during clinorotation but does occur with increased stimulation. This study describes a situation in which an individual cell senses a change in its physical environment and alters its cell biological behavior.

  6. Alkaline polymer electrolyte membranes for fuel cell applications.

    PubMed

    Wang, Yan-Jie; Qiao, Jinli; Baker, Ryan; Zhang, Jiujun

    2013-07-01

    In this review, we examine the most recent progress and research trends in the area of alkaline polymer electrolyte membrane (PEM) development in terms of material selection, synthesis, characterization, and theoretical approach, as well as their fabrication into alkaline PEM-based membrane electrode assemblies (MEAs) and the corresponding performance/durability in alkaline polymer electrolyte membrane fuel cells (PEMFCs). Respective advantages and challenges are also reviewed. To overcome challenges hindering alkaline PEM technology advancement and commercialization, several research directions are then proposed.

  7. Improved Membrane Materials for PEM Fuel Cell Application

    SciTech Connect

    Kenneth A. Mauritz; Robert B. Moore

    2008-06-30

    The overall goal of this project is to collect and integrate critical structure/property information in order to develop methods that lead to significant improvements in the durability and performance of polymer electrolyte membrane fuel cell (PEMFC) materials. This project is focused on the fundamental improvement of PEMFC membrane materials with respect to chemical, mechanical and morphological durability as well as the development of new inorganically-modified membranes.

  8. Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates

    PubMed Central

    Tharad, Sudarat; Toca-Herrera, José L.; Promdonkoy, Boonhiang; Krittanai, Chartchai

    2016-01-01

    Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and in vitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane. PMID:27612497

  9. Radiation Interaction with Therapeutic Drugs and Cell Membranes

    NASA Astrophysics Data System (ADS)

    Martin, Diana I.; Manaila, Elena N.; Moisescu, Mihaela I.; Savopol, Tudor D.; Kovacs, Eugenia A.; Cinca, Sabin A.; Matei, Constantin I.; Margaritescu, Irina D.; Iacob, Nicusor I.; Ighigeanu, Daniel I.; Craciun, Gabriela D.

    2007-04-01

    This transient permeabilized state of the cell membrane, named the ``cell electroporation'' (CE) can be used to increase cells uptake of drugs that do not readily pass cell membrane, thus enabling their cytotoxicity. The anticancer drugs, such as bleomycin (BL) and cisplatin, are the most candidates for the combined use with ionizing and non-ionizing radiation fields. The methods and installations for the cell electroporation by electron beam (EB) and microwave (MW) irradiation are presented. The viability tests of the human leukocytes under EB and MW exposure with/without the BL in the cell cultures are discussed.

  10. Radiation Interaction with Therapeutic Drugs and Cell Membranes

    SciTech Connect

    Martin, Diana I.; Manaila, Elena N.; Matei, Constantin I.; Iacob, Nicusor I.; Ighigeanu, Daniel I.; Craciun, Gabriela D.; Moisescu, Mihaela I.; Savopol, Tudor D.; Kovacs, Eugenia A.; Cinca, Sabin A.; Margaritescu, Irina D.

    2007-04-23

    This transient permeabilized state of the cell membrane, named the 'cell electroporation' (CE) can be used to increase cells uptake of drugs that do not readily pass cell membrane, thus enabling their cytotoxicity. The anticancer drugs, such as bleomycin (BL) and cisplatin, are the most candidates for the combined use with ionizing and non-ionizing radiation fields. The methods and installations for the cell electroporation by electron beam (EB) and microwave (MW) irradiation are presented. The viability tests of the human leukocytes under EB and MW exposure with/without the BL in the cell cultures are discussed.

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

    PubMed

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

    2014-07-24

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

  12. Investigating cell membrane structure and dynamics with TCSPC-FLIM

    NASA Astrophysics Data System (ADS)

    Le Marois, Alix; Owen, Dylan M.; Suhling, Klaus

    2015-03-01

    We report the use of Time-Correlated Single Photon Counting (TCSPC) in a polarization-resolved Fluorescence Lifetime Imaging (FLIM) setup for the investigation of cell membrane structural and dynamic properties. This technique allows us to study the orientation and mobility of fluorescent membrane dyes, namely di-4-ANEPPDHQ and DiO, in model bilayers of different lipid compositions. Dipole alignment and extent of rotational motion can be linked to membrane order and fluidity. Comparison of the time-resolved anisotropy decays of the two fluorescent dyes suggests that rotational motion of membrane constituents is restricted in liquid-ordered phases, and appears to be limited to the region of aliphatic tails in liquid-disordered phases. In living cells, understanding the membrane structure provides crucial information on its functional properties, such as exo- and endocytosis, cell mobility and signal transduction.

  13. Automated membrane test cell apparatus and method for so using

    SciTech Connect

    Yeager, H.L.; Malinsky, J.D.

    1984-11-20

    An automated electrolytic membrane test cell apparatus adaptable for the purpose of accurately measuring cationic transport and water transport numbers for membranes used in chlor-alkali cells under operating conditions similar to those used in such cells is disclosed. The apparatus comprises a test cell, said test cell being adapted to hold a permselective membrane sealingly supported therein so as to create separate anode and cathode compartments, each of said compartments having a suitable electrode, and heating electrolyte inlet and outlet means attached thereto. The apparatus further comprises means to select one of a plurality of anolyte and catholyte test solutions and control means adapted to control the electrolysis, circulation and heating of said solutions and the generation of all test samples needed to perform the measurements necessary to calculate said transport numbers. When used in conjunction with radioactive tracer techniques, considerably improvements are possible in the accuracy and ease with which transport phenomena in said membrane can be studied.

  14. Unraveling the toxicity mechanisms of the herbicide diclofop-methyl in rice: modulation of the activity of key enzymes involved in citrate metabolism and induction of cell membrane anion channels.

    PubMed

    Ding, Haiyan; Lu, Haiping; Lavoie, Michel; Xie, Jun; Li, Yali; Lv, Xiaolu; Fu, Zhengwei; Qian, Haifeng

    2014-11-01

    Residual soil concentrations of the herbicide diclofop-methyl (DM) can be toxic to other nontarget plant species, but the toxicity mechanisms at play are not fully understood. In the present study, we analyzed the toxic effect of DM on root growth and metabolism in the rice species Oryza sativa. The results show that a 48-h exposure to a trace level (5 μg/L) of DM inhibits rice root growth by almost 70%. A 48-h exposure to 5 μg/L DM also leads to an ≈2.5-fold increase in citrate synthase (CS) activity (and CS gene transcription) and an ≈2-fold decrease in the citrate lyase gene transcripts, which lead to an increase in the intracellular concentration of citrate and in citrate exudation rate. Addition of a specific inhibitor of cell membrane anion channel, anthracene-9-carboxylic acid, decreased citrate release in the culture, suggesting that DM-induced citrate loss from the cells is mediated by a specific membrane-bound channel protein. This study brings new insights into the key biochemical mechanisms leading to DM toxicity in rice.

  15. Purinergically induced membrane fluidization in ciliary cells: characterization and control by calcium and membrane potential.

    PubMed Central

    Alfahel, E; Korngreen, A; Parola, A H; Priel, Z

    1996-01-01

    To examine the role of membrane dynamics in transmembrane signal transduction, we studied changes in membrane fluidity in mucociliary tissues from frog palate and esophagus epithelia stimulated by extracellular ATP. Micromolar concentrations of ATP induced strong changes in fluorescence polarization, possibly indicating membrane fluidization. This effect was dosage dependent, reaching a maximum at 10-microM ATP. It was dependent on the presence of extracellular Ca2+ (or Mg2+), though it was insensitive to inhibitors of voltage-gated calcium channels. It was inhibited by thapsigargin and by ionomycin (at low extracellular Ca2+ concentration), both of which deplete Ca2+ stores. It was inhibited by the calcium-activated potassium channel inhibitors quinidine, charybdotoxin, and apamine and was reduced considerably by replacement of extracellular Na+ with K+. Hyperpolarization, or depolarization, of the mucociliary membrane induced membrane fluidization. The degree of membrane fluidization depended on the degree of hyperpolarization or depolarization of the ciliary membrane potential and was considerably lower than the effect induced by extracellular ATP. These results indicate that appreciable membrane fluidization induced by extracellular ATP depends both on an increase in intracellular Ca2+, mainly from its internal stores, and on hyperpolarization of the membrane. Calcium-dependent potassium channels couple the two effects. In light of recent results on the enhancement of ciliary beat frequency, it would appear that extracellular ATP-induced changes both in ciliary beat frequency and in membrane fluidity are triggered by similar signal transduction pathways. PMID:8789123

  16. New modified polyetheretherketone membrane for liver cell culture in biohybrid systems: adhesion and specific functions of isolated hepatocytes.

    PubMed

    De Bartolo, L; Morelli, S; Rende, M; Gordano, A; Drioli, E

    2004-08-01

    There has been growing interest in innovative materials with physico-chemical properties that provide improved blood/cell compatibility. We propose new polymeric membranes made of modified polyetheretherketone (PEEK-WC) as materials with potential for use in biohybrid devices. PEEK-WC exhibits high chemical, thermal stability and mechanical resistance. Owing to its lack of crystallinity this polymer can be used for preparing membranes with cheap and flexible methods. We compared the properties of PEEK-WC membranes to polyurethane membranes prepared using the same phase inverse technique and commercial membranes. The physico-chemical properties of the membranes were characterised by contact angle measurements. The different parameters acid (gamma+), base (gamma-) and Lifshitz-van der Waals (gammaLW) of the surface free energy were calculated according to Good-van Oss's model. We evaluated the cytocompatibility of PEEK-WC membranes by culturing hepatocytes isolated from rat liver. Cell adhesion and metabolic behaviour in terms of ammonia elimination, urea synthesis and protein synthesis were evaluated during the first days of culture. Liver cells adhered and formed three-dimensional aggregates on the most tested membranes. PEEK-WC membranes promoted hepatocyte adhesion most effectively. Urea synthesis, ammonia elimination and protein synthesis improved significantly when cells adhered to PEEK-WC membrane. The considerable metabolic activities of cells cultured on this membrane confirmed the good structural and physico-chemical properties of the PEEK-WC membrane that could be a promising biomaterial for cell culture in biohybrid devices. PMID:15020136

  17. Membrane-electrode assemblies for electrochemical cells

    DOEpatents

    Swathirajan, Sundararajan; Mikhail, Youssef M.

    1993-01-01

    A combination, unitary, membrane and electrode assembly with a solid polymer electrolyte membrane, and first and second electrodes at least partially embedded in opposed surfaces of the membrane. The electrodes each comprise a respective group of finely divided carbon particles, very finely divided catalytic particles supported on internal and external surfaces of the carbon particles and a proton conductive material intermingled with the catalytic and carbon particles. A first group of finely divided carbon particles forming the first electrode has greater water attraction and retention properties, and is more hydrophilic than a second group of carbon particles forming the second electrode. In a preferred method, the membrane electrode assembly of the invention is prepared by forming a slurry of proton conductive material and at least one group of the carbon and catalyst particles. The slurry is applied to the opposed surfaces of the membrane and heated while being pressed to the membrane for a time and at a temperature and compressive load sufficient to embed at least a portion of the particles into the membrane.

  18. Lysosome fusion to the cell membrane is mediated by the dysferlin C2A domain in coronary arterial endothelial cells

    PubMed Central

    Han, Wei-Qing; Xia, Min; Xu, Ming; Boini, Krishna M.; Ritter, Joseph K.; Li, Ning-Jun; Li, Pin-Lan

    2012-01-01

    Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered in membrane raft macrodomains after Fas Ligand (FasL) stimulation as detected by confocal microscopy and membrane fraction flotation. Small-interfering RNA targeted to dysferlin prevented membrane raft clustering. Furthermore, the translocation of acid sphingomyelinase (ASMase) to membrane raft clusters, whereby local ASMase activation and ceramide production – an important step that mediates membrane raft clustering – was attenuated. Functionally, silencing of the dysferlin gene reversed FasL-induced impairment of endothelium-dependent vasodilation in isolated small coronary arteries. By monitoring fluorescence quenching or dequenching, silencing of the dysferlin gene was found to almost completely block lysosome fusion to plasma membrane upon FasL stimulation. Further studies to block C2A binding and silencing of AHNAK (a dysferlin C2A domain binding partner), showed that the dysferlin C2A domain is required for FasL-induced lysosome fusion to the cell membrane, ASMase translocation and membrane raft clustering. We conclude that dysferlin determines lysosome fusion to the plasma membrane through its C2A domain and it is therefore implicated in membrane-raft-mediated signaling and regulation of endothelial function in coronary circulation. PMID:22349696

  19. Catalytic membranes for CO oxidation in fuel cells

    DOEpatents

    Sandi-Tapia, Giselle; Carrado Gregar, Kathleen; Kizilel, Riza

    2010-06-08

    A hydrogen permeable membrane, which includes a polymer stable at temperatures of about 200 C having clay impregnated with Pt or Au or Ru or Pd particles or mixtures thereof with average diameters of less than about 10 nanometers (nms) is disclosed. The membranes are useful in fuel cells or any device which requires hydrogen to be separated from carbon monoxide.

  20. Daptomycin exerts rapid bactericidal activity against Bacillus anthracis without disrupting membrane integrity

    PubMed Central

    Xing, Yu-hua; Wang, Wei; Dai, Su-qin; Liu, Ti-yan; Tan, Jun-jie; Qu, Guo-long; Li, Yu-xia; Ling, Yan; Liu, Gang; Fu, Xue-qi; Chen, Hui-peng

    2014-01-01

    Aim: To examine whether the novel cyclic lipopeptide antibiotic daptomycin could be used to treat anthrax and to study the mechanisms underlying its bactericidal action against Bacillus anthracis. Methods: Spore-forming B anthracis AP422 was tested. MIC values of antibiotics were determined. Cell membrane potential was measured using flow cytometric assays with membrane potential-sensitive fluorescent dyes. Cell membrane integrity was detected using To-Pro-3 iodide staining and transmission electron microscopy. K+ efflux and Na+ influx were measured using the fluorescent probes PBFI and SBFI-AM, respectively. Results: Daptomycin exhibited rapid bactericidal activity against vegetative B anthracis with a MIC value of 0.78 μg/mL, which was comparable to those of ciprofloxacin and penicillin G. Furthermore, daptomycin prevented the germinated spores from growing into vegetative bacteria. Daptomycin concentration-dependently dissipated the membrane potential of B anthracis and caused K+ efflux and Na+ influx without disrupting membrane integrity. In contrast, both ciprofloxacin and penicillin G did not change the membrane potential of vegetative bacteria or spores. Penicillin G disrupted membrane integrity of B anthracis, whereas ciprofloxacin had no such effect. Conclusion: Daptomycin exerts rapid bactericidal action against B anthracis via reducing membrane potential without disrupting membrane integrity. This antibiotic can be used as an alternate therapy for B anthracis infections. PMID:24362329

  1. Long-lived anomalous thermal diffusion induced by elastic cell membranes on nearby particles

    NASA Astrophysics Data System (ADS)

    Daddi-Moussa-Ider, Abdallah; Guckenberger, Achim; Gekle, Stephan

    2016-01-01

    The physical approach of a small particle (virus, medical drug) to the cell membrane represents the crucial first step before active internalization and is governed by thermal diffusion. Using a fully analytical theory we show that the stretching and bending of the elastic membrane by the approaching particle induces a memory in the system, which leads to anomalous diffusion, even though the particle is immersed in a purely Newtonian liquid. For typical cell membranes the transient subdiffusive regime extends beyond 10 ms and can enhance residence times and possibly binding rates up to 50%. Our analytical predictions are validated by numerical simulations.

  2. Polymorphic membrane protein (PMP) 20 and PMP 21 of Chlamydia pneumoniae induce proinflammatory mediators in human endothelial cells in vitro by activation of the nuclear factor-kappaB pathway.

    PubMed

    Niessner, Alexander; Kaun, Christoph; Zorn, Gerlinde; Speidl, Walter; Türel, Zeynep; Christiansen, Gunna; Pedersen, Anna-Sofie; Birkelund, Svend; Simon, Susan; Georgopoulos, Apostolos; Graninger, Wolfgang; de Martin, Rainer; Lipp, Joachim; Binder, Bernd R; Maurer, Gerald; Huber, Kurt; Wojta, Johann

    2003-07-01

    We tested whether polymorphic membrane proteins (PMPs) of Chlamydia pneumoniae might play a role in triggering an inflammatory response in human endothelial cells. Of 15 purified, recombinant chlamydial PMPs tested, 2 (PMP 20 and PMP 21) dose-dependently increased the production of the inflammatory mediators interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1), in cultured human endothelial cells; production of IL-8 was also increased. When endothelial cells were infected by live C. pneumoniae, an increase in the production of IL-6, IL-8, and MCP-1 was seen. We used adenovirus-induced overexpression of IkappaBalpha-an inhibitor of nuclear factor (NF)-kappaB-to demonstrate that PMP 20 and PMP 21 increase the production of IL-6 and MCP-1 in human endothelial cells by activation of the NF-kappaB pathway, because, in cells overexpressing IkappaBalpha, treatment with the respective PMP did not result in increased production of IL-6 and MCP-1. Thus, C. pneumoniae could, by interactions of its PMPs with the endothelium, contribute to the process of vascular injury during the development and progression of atherosclerotic lesions.

  3. Sulfated Titania-Silica Reinforced Nafion Nanocomposite Membranes for Proton Exchange Membrane Fuel Cells.

    PubMed

    Abu Sayeed, M D; Kim, Hee Jin; Gopalan, A I; Kim, Young Ho; Lee, Kwang-Pill; Choi, Sang-June

    2015-09-01

    Sulfated titania-silica (SO4(2-)-/TiO2-SiO2) composites were prepared by a sol-gel method with sulfate reaction and characterized by X-ray diffraction (XRD) and energy-dispersive X-ray spectroscopy (EDS). The nanometric diameter and geometry of the sulfated titania-silica (STS) was investigated by transmission electron microscopy (TEM). A small amount of the STS composite in the range of 0.5-3 wt% was then added as reinforcing into the Nafion membrane by water-assisted solution casting method to prepare STS reinforced Nafion nanocomposite membranes (STS-Nafion nanocomposite membranes). The additional functional groups, sulfate groups, of the nanocomposite membrane having more surface oxygenated groups enhanced the fuel cell membrane properties. The STS-Nafion nanocomposite membranes exhibited improved water uptake compared to that of neat Nafion membranes, whereas methanol uptake values were decreased dramatically improved thermal property of the prepared nanocomposite membranes were measured by thermogravimetric analysis (TGA). Furthermore, increased ion exchange capacity values were obtained by thermoacidic pretreatment of the nanocomposite membranes.

  4. CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels.

    PubMed

    Itel, Fabian; Al-Samir, Samer; Öberg, Fredrik; Chami, Mohamed; Kumar, Manish; Supuran, Claudiu T; Deen, Peter M T; Meier, Wolfgang; Hedfalk, Kristina; Gros, Gerolf; Endeward, Volker

    2012-12-01

    Recent observations that some membrane proteins act as gas channels seem surprising in view of the classical concept that membranes generally are highly permeable to gases. Here, we study the gas permeability of membranes for the case of CO(2), using a previously established mass spectrometric technique. We first show that biological membranes lacking protein gas channels but containing normal amounts of cholesterol (30-50 mol% of total lipid), e.g., MDCK and tsA201 cells, in fact possess an unexpectedly low CO(2) permeability (P(CO2)) of ∼0.01 cm/s, which is 2 orders of magnitude lower than the P(CO2) of pure planar phospholipid bilayers (∼1 cm/s). Phospholipid vesicles enriched with similar amounts of cholesterol also exhibit P(CO2) ≈ 0.01 cm/s, identifying cholesterol as the major determinant of membrane P(CO2). This is confirmed by the demonstration that MDCK cells depleted of or enriched with membrane cholesterol show dramatic increases or decreases in P(CO2), respectively. We demonstrate, furthermore, that reconstitution of human AQP-1 into cholesterol-containing vesicles, as well as expression of human AQP-1 in MDCK cells, leads to drastic increases in P(CO2), indicating that gas channels are of high functional significance for gas transfer across membranes of low intrinsic gas permeability.

  5. Polymer-supported membranes as models of the cell surface

    NASA Astrophysics Data System (ADS)

    Tanaka, Motomu; Sackmann, Erich

    2005-09-01

    Lipid-bilayer membranes supported on solid substrates are widely used as cell-surface models that connect biological and artificial materials. They can be placed either directly on solids or on ultrathin polymer supports that mimic the generic role of the extracellular matrix. The tools of modern genetic engineering and bioorganic chemistry make it possible to couple many types of biomolecule to supported membranes. This results in sophisticated interfaces that can be used to control, organize and study the properties and function of membranes and membrane-associated proteins. Particularly exciting opportunities arise when these systems are coupled with advanced semiconductor technology.