Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

USGS Publications Warehouse

Stolz, J.F.; Gugliuzza, T.; Switzer, Blum J.; Oremland, R.; Martinez, Murillo F.

1997-01-01

The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.

Phospholipase A2 activity-dependent and -independent fusogenic activity of Naja nigricollis CMS-9 on zwitterionic and anionic phospholipid vesicles.

PubMed

Chiou, Yi-Ling; Chen, Ying-Jung; Lin, Shinne-Ren; Chang, Long-Sen

2011-11-01

CMS-9, a phospholipase A(2) (PLA(2)) from Naja nigricollis venom, induced the death of human breast cancer MCF-7 cells accompanied with the formation of cell clumps without clear boundaries between cells. Annexin V-FITC staining indicated that abundant phosphatidylserine appeared on the outer membrane of MCF-7 cell clumps, implying the possibility that CMS-9 may promote membrane fusion via anionic phospholipids. To validate this proposition, fusogenic activity of CMS-9 on vesicles composed of zwitterionic phospholipid alone or a combination of zwitterionic and anionic phospholipids was examined. Although CMS-9-induced fusion of zwitterionic phospholipid vesicles depended on PLA(2) activity, CMS-9-induced fusion of vesicles containing anionic phospholipids could occur without the involvement of PLA(2) activity. Membrane-damaging activity of CMS-9 was associated with its fusogenicity. Moreover, CMS-9 induced differently membrane leakage and membrane fusion of vesicles with different compositions. Membrane fluidity and binding capability with phospholipid vesicles were not related to the fusogenicity of CMS-9. However, membrane-bound conformation and mode of CMS-9 depended on phospholipid compositions. Collectively, our data suggest that PLA(2) activity-dependent and -independent fusogenicity of CMS-9 are closely related to its membrane-bound modes and targeted membrane compositions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Requirement for Coenzyme Q in Plasma Membrane Electron Transport

NASA Astrophysics Data System (ADS)

Sun, I. L.; Sun, E. E.; Crane, F. L.; Morre, D. J.; Lindgren, A.; Low, H.

1992-12-01

Coenzyme Q is required in the electron transport system of rat hepatocyte and human erythrocyte plasma membranes. Extraction of coenzyme Q from the membrane decreases NADH dehydrogenase and NADH:oxygen oxidoreductase activity. Addition of coenzyme Q to the extracted membrane restores the activity. Partial restoration of activity is also found with α-tocopherylquinone, but not with vitamin K_1. Analogs of coenzyme Q inhibit NADH dehydrogenase and oxidase activity and the inhibition is reversed by added coenzyme Q. Ferricyanide reduction by transmembrane electron transport from HeLa cells is inhibited by coenzyme Q analogs and restored with added coenzyme Q10. Reduction of external ferricyanide and diferric transferrin by HeLa cells is accompanied by proton release from the cells. Inhibition of the reduction by coenzyme Q analogs also inhibits the proton release, and coenzyme Q10 restores the proton release activity. Trans-plasma membrane electron transport stimulates growth of serum-deficient cells, and added coenzyme Q10 increases growth of HeLa (human adenocarcinoma) and BALB/3T3 (mouse fibroblast) cells. The evidence is consistent with a function for coenzyme Q in a trans-plasma membrane electron transport system which influences cell growth.

Propagating Cell-Membrane Waves Driven by Curved Activators of Actin Polymerization

PubMed Central

Peleg, Barak; Disanza, Andrea; Scita, Giorgio; Gov, Nir

2011-01-01

Cells exhibit propagating membrane waves which involve the actin cytoskeleton. One type of such membranal waves are Circular Dorsal Ruffles (CDR) which are related to endocytosis and receptor internalization. Experimentally, CDRs have been associated with membrane bound activators of actin polymerization of concave shape. We present experimental evidence for the localization of convex membrane proteins in these structures, and their insensitivity to inhibition of myosin II contractility in immortalized mouse embryo fibroblasts cell cultures. These observations lead us to propose a theoretical model which explains the formation of these waves due to the interplay between complexes that contain activators of actin polymerization and membrane-bound curved proteins of both types of curvature (concave and convex). Our model predicts that the activity of both types of curved proteins is essential for sustaining propagating waves, which are abolished when one type of curved activator is removed. Within this model waves are initiated when the level of actin polymerization induced by the curved activators is higher than some threshold value, which allows the cell to control CDR formation. We demonstrate that the model can explain many features of CDRs, and give several testable predictions. This work demonstrates the importance of curved membrane proteins in organizing the actin cytoskeleton and cell shape. PMID:21533032

Phytochemicals prevent mitochondrial membrane permeabilization and protect SH-SY5Y cells against apoptosis induced by PK11195, a ligand for outer membrane translocator protein.

PubMed

Wu, Yuqiu; Shamoto-Nagai, Masayo; Maruyama, Wakako; Osawa, Toshihiko; Naoi, Makoto

2017-01-01

Epidemiological studies present the beneficial effects of dietary habits on prevention of aging-associated decline of brain function. Phytochemicals, the second metabolites of food, protect neuronal cells from cell death in cellular models of neurodegenerative disorders, and the neuroprotective activity has been ascribed to the anti-oxidant and anti-inflammatory functions. In this paper, the cellular mechanism of neuroprotection by phytochemicals was investigated, using the cellular model of mitochondrial apoptosis induced by PK11195, a ligand of outer membrane translocator protein, in SH-SY5Y cells. PK11195 induced mitochondrial membrane permeabilization with rapid transit production of superoxide (superoxide flashes) and calcium release from mitochondria, and activated apoptosis signal pathway. Study on the structure-activity relationship of astaxanthin, ferulic acid derivatives, and sesame lignans revealed that these phytochemicals inhibited mitochondrial membrane permeabilization and protected cells from apoptosis. Ferulic acid derivatives and sesame lignans inhibited or enhanced the mitochondrial pore formation and cell death by PK11195 according to their amphiphilic properties, not directly depending on the antioxidant activity. Regulation of pore formation at mitochondrial membrane is discussed as a novel mechanism behind neuroprotective activity of phytochemicals in aging and age-associated neurodegenerative disorders, and also behind dual functions of phytochemicals in neuronal and cancer cells.

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex

PubMed Central

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; James, Ho C. S.; Rydström, Anna; Ngassam, Viviane N.; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N.; Svanborg, Catharina

2015-01-01

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death. PMID:26561036

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

PubMed

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

2015-11-12

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

Extracellular localization of catalase is associated with the transformed state of malignant cells.

PubMed

Böhm, Britta; Heinzelmann, Sonja; Motz, Manfred; Bauer, Georg

2015-12-01

Oncogenic transformation is dependent on activated membrane-associated NADPH oxidase (NOX). However, the resultant extracellular superoxide anions are also driving the NO/peroxynitrite and the HOCl pathway, which eliminates NOX-expressing transformed cells through selective apoptosis induction. Tumor progression is dependent on dominant interference with intercellular apoptosis-inducing ROS signaling through membrane-associated catalase, which decomposes H2O2 and peroxynitrite and oxidizes NO. Particularly, the decomposition of extracellular peroxynitrite strictly requires membrane-associated catalase. We utilized small interfering RNA (siRNA)-mediated knockdown of catalase and neutralizing antibodies directed against the enzyme in combination with challenging H2O2 or peroxynitrite to determine activity and localization of catalase in cells from three distinct steps of multistage oncogenesis. Nontransformed cells did not generate extracellular superoxide anions and only showed intracellular catalase activity. Transformed cells showed superoxide anion-dependent intercellular apoptosis-inducing ROS signaling in the presence of suboptimal catalase activity in their membrane. Tumor cells exhibited tight control of intercellular apoptosis-inducing ROS signaling through a high local concentration of membrane-associated catalase. These data demonstrate that translocation of catalase to the outside of the cell membrane is already associated with the transformation step. A strong local increase in the concentration of membrane-associated catalase is achieved during tumor progression and is controlled by tumor cell-derived H2O2 and by transglutaminase.

Altered Regulation of Airway Epithelial Cell Chloride Channels in Cystic Fibrosis

NASA Astrophysics Data System (ADS)

Frizzell, Raymond A.; Rechkemmer, Gerhard; Shoemaker, Richard L.

1986-08-01

In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that β -adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains.

PubMed

Corcoran, Jennifer A; Salsman, Jayme; de Antueno, Roberto; Touhami, Ahmed; Jericho, Manfred H; Clancy, Eileen K; Duncan, Roy

2006-10-20

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling.

PubMed

Parag-Sharma, Kshitij; Leyme, Anthony; DiGiacomo, Vincent; Marivin, Arthur; Broselid, Stefan; Garcia-Marcos, Mikel

2016-12-30

GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gα i3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis.

PubMed

Oropesa-Ávila, M; Fernández-Vega, A; de la Mata, M; Maraver, J G; Cordero, M D; Cotán, D; de Miguel, M; Calero, C P; Paz, M V; Pavón, A D; Sánchez, M A; Zaderenko, A P; Ybot-González, P; Sánchez-Alcázar, J A

2013-03-07

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na(+)/Ca(2+) exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na(+)/K(+) pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis.

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis

PubMed Central

Oropesa-Ávila, M; Fernández-Vega, A; de la Mata, M; Maraver, J G; Cordero, M D; Cotán, D; de Miguel, M; Calero, C P; Paz, M V; Pavón, A D; Sánchez, M A; Zaderenko, A P; Ybot-González, P; Sánchez-Alcázar, J A

2013-01-01

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na+/Ca2+ exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na+/K+ pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis. PMID:23470534

Lytic agents, cell permeability, and monolayer penetrability.

PubMed

Salton, M R

1968-07-01

Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20-30 % lipid and 50-75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2-3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.

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. Copyright © 2015 the American Physiological Society.

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

PubMed Central

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

2015-01-01

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. PMID:26201952

Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium.

PubMed

Amsler, K; Kuwada, S K

1999-01-01

Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

PubMed

Nervi, Pierluigi; Li-Blatter, Xiaochun; Aänismaa, Päivi; Seelig, Anna

2010-03-01

We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux. Copyright 2009 Elsevier B.V. All rights reserved.

Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes.

PubMed

Schröter, W; Neuvians, M

1970-12-01

Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.

Altering calcium influx for selective destruction of breast tumor.

PubMed

Yu, Han-Gang; McLaughlin, Sarah; Newman, Mackenzie; Brundage, Kathleen; Ammer, Amanda; Martin, Karen; Coad, James

2017-03-04

Human triple-negative breast cancer has limited therapeutic choices. Breast tumor cells have depolarized plasma membrane potential. Using this unique electrical property, we aim to develop an effective selective killing of triple-negative breast cancer. We used an engineered L-type voltage-gated calcium channel (Cec), activated by membrane depolarization without inactivation, to induce excessive calcium influx in breast tumor cells. Patch clamp and flow cytometry were used in testing the killing selectivity and efficiency of human breast tumor cells in vitro. Bioluminescence and ultrasound imaging were used in studies of human triple-negative breast cancer cell MDA-MB-231 xenograft in mice. Histological staining, immunoblotting and immunohistochemistry were used to investigate mechanism that mediates Cec-induced cell death. Activating Cec channels expressed in human breast cancer MCF7 cells produced enormous calcium influx at depolarized membrane. Activating the wild-type Cav1.2 channels expressed in MCF7 cells also produced a large calcium influx at depolarized membrane, but this calcium influx was diminished at the sustained membrane depolarization due to channel inactivation. MCF7 cells expressing Cec died when the membrane potential was held at -10 mV for 1 hr, while non-Cec-expressing MCF7 cells were alive. MCF7 cell death was 8-fold higher in Cec-expressing cells than in non-Cec-expressing cells. Direct injection of lentivirus containing Cec into MDA-MB-231 xenograft in mice inhibited tumor growth. Activated caspase-3 protein was detected only in MDA-MB-231 cells expressing Cec, along with a significantly increased expression of activated caspase-3 in xenograft tumor treated with Cec. We demonstrated a novel strategy to induce constant calcium influx that selectively kills human triple-negative breast tumor cells.

Antifungal mechanism of an antimicrobial peptide, HP (2--20), derived from N-terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans.

PubMed

Lee, Dong Gun; Park, Yoonkyung; Kim, Hee Nam; Kim, Hyung Keun; Kim, Pyoung Il; Choi, Bo Hwa; Hahm, Kyung-Soo

2002-03-08

The antifungal activity and mechanism of HP (2-20), a peptide derived from the N-terminus sequence of Helicobacter pylori Ribosomal Protein L1 were investigated. HP (2--20) displayed a strong antifungal activity against various fungi, and the antifungal activity was inhibited by Ca(2+) and Mg(2+) ions. In order to investigate the antifungal mechanism(s) of HP (2-20), fluorescence activated flow cytometry was performed. As determined by propidium iodide staining, Candida albicans treated with HP (2-20) showed a higher fluorescence intensity than untreated cells and was similar to melittin-treated cells. The effect on fungal cell membranes was examined by investigating the change in membrane dynamics of C. albicans using 1,6-diphenyl-1,3,5-hexatriene as a membrane probe and by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w) and by treating protoplasts of C. albicans with the peptide. The action of peptide against fungal cell membrane was further examined by the potassium-release test, and HP (2-20) was able to increase the amount of K(+) released from the cells. The result suggests that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membrane via pore formation or directly interacts with the lipid bilayers in a salt-dependent manner.

Functional dynamics of cell surface membrane proteins

NASA Astrophysics Data System (ADS)

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

Functional dynamics of cell surface membrane proteins.

PubMed

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

Protein receptor-independent plasma membrane remodeling by HAMLET: A tumoricidal protein-lipid complex

DOE PAGES

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

2015-11-12

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. Wemore » identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. In conclusion, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.« less

Phospholipase D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane.

PubMed

Mor, Adam; Wynne, Joseph P; Ahearn, Ian M; Dustin, Michael L; Du, Guangwei; Philips, Mark R

2009-06-01

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.

The anti-cancer peptide, PNC-27, induces tumor cell necrosis of a poorly differentiated non-solid tissue human leukemia cell line that depends on expression of HDM-2 in the plasma membrane of these cells.

PubMed

Davitt, Katlin; Babcock, Blake D; Fenelus, Maly; Poon, Chi Kong; Sarkar, Abhishek; Trivigno, Vincent; Zolkind, Paul A; Matthew, Sheena M; Grin'kina, Natalia; Orynbayeva, Zulfiya; Shaikh, Mohammad F; Adler, Victor; Michl, Josef; Sarafraz-Yazdi, Ehsan; Pincus, Matthew R; Bowne, Wilbur B

2014-01-01

We have developed the anti-cancer peptide, PNC-27, which is a membrane-active peptide that binds to the HDM-2 protein expressed in the cancer cell membranes of solid tissue tumor cells and induces transmembrane pore formation in cancer, but not in normal cells, resulting in tumor cell necrosis that is independent of p53 activity in these cells. We now extend our study to non-solid tissue tumor cells, in this case, a primitive, possible stem cell human leukemia cell line (K562) that is also p53-homozygously deleted. Our purpose was twofold: to investigate if these cells likewise express HDM-2 in their plasma membranes and to determine if our anti-cancer peptide induces tumor cell necrosis in these non-solid tissue tumor cells in a manner that depends on the interaction between the peptide and membrane-bound HDM-2. The anti-cancer activity and mechanism of PNC-27, which carries a p53 aa12-26-leader sequence connected on its carboxyl terminal end to a trans-membrane-penetrating sequence or membrane residency peptide (MRP), was studied against p53-null K562 leukemia cells. Murine leukocytes were used as a non-cancer cell control. Necrosis was determined by measuring the lactate dehydrogenase (LDH) release and apoptosis was determined by the detection of Caspases 3 and 7. Membrane colocalization of PNC-27 with HDM-2 was analyzed microscopically using fluorescently labeled antibodies against HDM-2 and PNC-27 peptides. We found that K562 cells strongly express HDM-2 protein in their membranes and that PNC-27 co-localizes with this protein in the membranes of these cells. PNC-27, but not the negative control peptide PNC-29, is selectively cytotoxic to K562 cells, inducing nearly 100 percent cell killing with LDH release. In contrast, this peptide had no effect on the lymphocyte control cells. The results suggest that HDM-2 is expressed in the membranes of non-solid tissue tumor cells in addition to the membranes of solid tissue tumor cells. Since K-562 cells appear to be in the stem cell family, the results suggest that early developing tumor cells also express HDM-2 protein in their membranes. Since PNC-27 induces necrosis of K-562 leukemia cells and co-localizes with HDM-2 in the tumor cell membrane as an early event, we conclude that the association of PNC-27 with HDM-2 in the cancer cell membrane results in trans-membrane pore formation which results in cancer cell death, as previously discovered in a number of different solid tissue tumor cells. Since K562 cells lack p53 expression, these effects of PNC-27 on this leukemia cell line occur by a p53-independent pathway. © 2014 by the Association of Clinical Scientists, Inc.

Complement activation on B lymphocytes opsonized with rituximab or ofatumumab produces substantial changes in membrane structure preceding cell lysis.

PubMed

Beum, Paul V; Lindorfer, Margaret A; Beurskens, Frank; Stukenberg, P Todd; Lokhorst, Henk M; Pawluczkowycz, Andrew W; Parren, Paul W H I; van de Winkel, Jan G J; Taylor, Ronald P

2008-07-01

Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death.

Histochemical and biochemical studies of carbonic anhydrase activity in the opercular epithelium of the euryhaline teleost, Fundulus heteroclitus.

PubMed

Lacy, E R

1983-01-01

Carbonic anhydrase (CAH) activity was biochemically measured and histochemically localized (at both the light and electron microscope levels) in isolated opercular membranes from teleost fish, Fundulus heteroclitus, adapted to freshwater (FW), seawater (SW), and double-strength seawater (2 x SW). The normal morphology of this membrane showed that its epithelial portion consisted of five cell types: (1) chloride cells, which have been previously implicated as responsible for the active chloride transport across the epithelium; (2) mucous cells; (3) pavement cells, which formed the major portion of the free epithelial surface; (4) supportive cells, which had an abundance of intermediate (10 nm)-type filaments suggesting a structural role for these cells; and (5) vesicular cells, which were characterized by various types of membrane-bound vesicles, including lysosomes, and numerous free ribosomes. Vesicular cells may be stem cells and/or endocrine cells. Hansson's histochemical method for CAH revealed cobalt sulfide reaction product confined to the following structures in fish from each environment: (1) chloride cells: throughout the cytoplasm and some nuclear staining; (2) mucous cells: throughout the cytoplasm, some nuclear staining, and some in mucous granules; (3) vesicular cells: confined to lysosomes, some of the vesicles, and nucleoli; (4) a small portion of the intracellular space between adjacent vesicular cells and supportive cells; and (5) supportive cells: in nucleoli and occasionally in larger membrane-bound lysosomelike structures. Acetazolamide (10(-5) M) and potassium cyanate (KCNO) (10(-1) M) in Hansson's incubation medium completely inhibited the formation of reaction product. Biochemical determination of CAH activity on vascularly perfused, isolated opercular membranes showed no statistically significant difference in enzyme activity between environmental groups. The following units of activity/mg opercular membrane protein were measured: FW: 0.63 +/- 0.02; SW: 0.43 +/- 0.08; 2 x SW: 0.64 +/- 0.09.

Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

PubMed

Niczyj, Marta; Champagne, Antoine; Alam, Iftekhar; Nader, Joseph; Boutry, Marc

2016-11-01

Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H + -ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H + -ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H + -ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H + -ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

A nonlinear cochlear model with the outer hair cell piezoelectric activity

NASA Astrophysics Data System (ADS)

Jiang, Xiaoai; Grosh, Karl

2003-10-01

In this paper we present a simple cochlear model which captures the most important aspect of nonlinearity in the cochlea-the nonlinearity caused by the piezoelectric-like activity of outer hair cells and the variable conductance of the outer hair cell stereocilia. A one-dimensional long-wave model is built to simulate the dynamic response of the fluid-loaded basilar membrane. The basilar membrane is simulated as isolated linear oscillators along the cochlear length, and its motion is coupled with the fluid pressure and the nonlinear force produced by the outer hair cells. As the basilar membrane moves, the fluid shears stereocilia, and the resulting ion flow changes the transmembrane potential of the outer hair cells and subsequently their length, leading to further movement of the basilar membrane. The piezoelectric-like activity of the outer hair cell is simulated by a current source, and stereocilia motion is modeled as a varying conductance that changes as the basilar membrane moves. A solution in the time domain will be presented. [Work supported by NIH.

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.

Profiling the outer membrane proteome during growth and development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles.

PubMed

Kahnt, Jörg; Aguiluz, Kryssia; Koch, Jürgen; Treuner-Lange, Anke; Konovalova, Anna; Huntley, Stuart; Hoppert, Michael; Søgaard-Andersen, Lotte; Hedderich, Reiner

2010-10-01

Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.

Dynamic activation of basilar membrane macrophages in response to chronic sensory cell degeneration in aging mouse cochleae

PubMed Central

Frye, Mitchell D.; Yang, Weiping; Zhang, Celia; Xiong, Binbin; Hu, Bo Hua

2016-01-01

In the sensory epithelium, macrophages have been identified on the scala tympani side of the basilar membrane. These basilar membrane macrophages are the spatially closest immune cells to sensory cells and are able to directly respond to and influence sensory cell pathogenesis. While basilar membrane macrophages have been studied in acute cochlear stresses, their behavior in response to chronic sensory cell degeneration is largely unknown. Here we report a systematic observation of the variance in phenotypes, the changes in morphology and distribution of basilar membrane tissue macrophages in different age groups of C57BL/6J mice, a mouse model of age-related sensory cell degeneration. This study reveals that mature, fully differentiated tissue macrophages, not recently infiltrated monocytes, are the major macrophage population for immune responses to chronic sensory cell death. These macrophages display dynamic changes in their numbers and morphologies as age increases, and the changes are related to the phases of sensory cell degeneration. Notably, macrophage activation precedes sensory cell pathogenesis, and strong macrophage activity is maintained until sensory cell degradation is complete. Collectively, these findings suggest that mature tissue macrophages on the basilar membrane are a dynamic group of cells that are capable of vigorous adaptation to changes in the local sensory epithelium environment influenced by sensory cell status. PMID:27837652

Dynamic activation of basilar membrane macrophages in response to chronic sensory cell degeneration in aging mouse cochleae.

PubMed

Frye, Mitchell D; Yang, Weiping; Zhang, Celia; Xiong, Binbin; Hu, Bo Hua

2017-02-01

In the sensory epithelium, macrophages have been identified on the scala tympani side of the basilar membrane. These basilar membrane macrophages are the spatially closest immune cells to sensory cells and are able to directly respond to and influence sensory cell pathogenesis. While basilar membrane macrophages have been studied in acute cochlear stresses, their behavior in response to chronic sensory cell degeneration is largely unknown. Here we report a systematic observation of the variance in phenotypes, the changes in morphology and distribution of basilar membrane tissue macrophages in different age groups of C57BL/6J mice, a mouse model of age-related sensory cell degeneration. This study reveals that mature, fully differentiated tissue macrophages, not recently infiltrated monocytes, are the major macrophage population for immune responses to chronic sensory cell death. These macrophages display dynamic changes in their numbers and morphologies as age increases, and the changes are related to the phases of sensory cell degeneration. Notably, macrophage activation precedes sensory cell pathogenesis, and strong macrophage activity is maintained until sensory cell degradation is complete. Collectively, these findings suggest that mature tissue macrophages on the basilar membrane are a dynamic group of cells that are capable of vigorous adaptation to changes in the local sensory epithelium environment influenced by sensory cell status. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

PubMed Central

Salamone, Monica; Carfì Pavia, Francesco

2016-01-01

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

Modulation of the interface between polyester and spent coffee grounds in polysaccharide membranes: Preparation, cell proliferation, antioxidant activity and tyrosinase activity.

PubMed

Wu, Chin-San

2017-09-01

The structural, antioxidant and cytocompatibility properties of membranes prepared from polyhydroxyalkanoate (PHA) and spent coffee ground (SCG) blends (PHA/SCG) were studied. Acrylic acid-grafted PHA (PHA-g-AA) was used to enhance the desirable characteristics of these membranes, which had better tensile properties than the corresponding PHA/SCG membranes. The water resistance of the PHA-g-AA/SCG membranes was greater than that of the PHA/SCG membranes, and a cytocompatibility evaluation with mouse normal tail fibroblasts (FBs) indicated that both materials were nontoxic. Cell cycle assays of FBs on PHA/SCG and PHA-g-AA/SCG membrane samples were not affected by the DNA content related to damage. Moreover, SCG enhanced the saccharide and polyphenol contents, and antioxidant properties, of the PHA-g-AA/SCG and PHA/SCG membranes. Therefore, we analysed the effects of these compounds' membranes on melanogenesis in B16-F10 melanoma cells. The results demonstrated that PHA/SCG and PHA-g-AA/SCG membranes reduced cellular tyrosinase activities in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

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 mechanism by which these membrane-active peptides lyse membranes. The last class of membrane-active peptides discussed are the CPPs, which translocate across the lipid bilayer without inducing severe disruption and have potential as drug vehicles. CPPs are typically highly charged and can show antimicrobial activity by targeting an intracellular target rather than via a direct membrane lytic mechanism. A critical aspect in the structure-function relationship of membrane-active peptides is their specific activity relative to the lipid membrane composition of the cell target. Cell membranes have a wide diversity of lipids, and those of eukaryotic and prokaryotic species differ greatly in composition and structure. The activity of AMPs from Australian tree frogs, toxins, and CPPs has been investigated within various lipid systems to assess whether a relationship between peptide and membrane composition could be identified. NMR spectroscopy techniques are being used to gain atomistic details of how these membrane-active peptides interact with model membranes and cells, and in particular, competitive assays demonstrate the difference between affinity and activity for a specific lipid environment. Overall, the interactions between these relatively small sized peptides and various lipid bilayers give insight into how these peptides function at the membrane interface.

Giga-ohm seals on intracellular membranes: a technique for studying intracellular ion channels in intact cells.

PubMed

Jonas, E A; Knox, R J; Kaczmarek, L K

1997-07-01

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling.

PubMed

Lou, Hsin-Ya; Zhao, Wenting; Zeng, Yongpeng; Cui, Bianxiao

2018-05-15

Over the past decade, there has been growing interest in developing biosensors and devices with nanoscale and vertical topography. Vertical nanostructures induce spontaneous cell engulfment, which enhances the cell-probe coupling efficiency and the sensitivity of biosensors. Although local membranes in contact with the nanostructures are found to be fully fluidic for lipid and membrane protein diffusions, cells appear to actively sense and respond to the surface topography presented by vertical nanostructures. For future development of biodevices, it is important to understand how cells interact with these nanostructures and how their presence modulates cellular function and activities. How cells recognize nanoscale surface topography has been an area of active research for two decades before the recent biosensor works. Extensive studies show that surface topographies in the range of tens to hundreds of nanometers can significantly affect cell functions, behaviors, and ultimately the cell fate. For example, titanium implants having rough surfaces are better for osteoblast attachment and host-implant integration than those with smooth surfaces. At the cellular level, nanoscale surface topography has been shown by a large number of studies to modulate cell attachment, activity, and differentiation. However, a mechanistic understanding of how cells interact and respond to nanoscale topographic features is still lacking. In this Account, we focus on some recent studies that support a new mechanism that local membrane curvature induced by nanoscale topography directly acts as a biochemical signal to induce intracellular signaling, which we refer to as the curvature hypothesis. The curvature hypothesis proposes that some intracellular proteins can recognize membrane curvatures of a certain range at the cell-to-material interface. These proteins then recruit and activate downstream components to modulate cell signaling and behavior. We discuss current technologies allowing the visualization of membrane deformation at the cell membrane-to-substrate interface with nanometer precision and demonstrate that vertical nanostructures induce local curvatures on the plasma membrane. These local curvatures enhance the process of clathrin-mediated endocytosis and affect actin dynamics. We also present evidence that vertical nanostructures can induce significant deformation of the nuclear membrane, which can affect chromatin distribution and gene expression. Finally, we provide a brief perspective on the curvature hypothesis and the challenges and opportunities for the design of nanotopography for manipulating cell behavior.

Dual patch voltage clamp study of low membrane resistance astrocytes in situ.

PubMed

Ma, Baofeng; Xu, Guangjin; Wang, Wei; Enyeart, John J; Zhou, Min

2014-03-17

Whole-cell patch clamp recording has been successfully used in identifying the voltage-dependent gating and conductance properties of ion channels in a variety of cells. However, this powerful technique is of limited value in studying low membrane resistance cells, such as astrocytes in situ, because of the inability to control or accurately measure the real amplitude of command voltages. To facilitate the study of ionic conductances of astrocytes, we have developed a dual patch recording method which permits membrane current and membrane potential to be simultaneously recorded from astrocytes in spite of their extraordinarily low membrane resistance. The utility of this technique is demonstrated by measuring the voltage-dependent activation of the inwardly rectifying K+ current abundantly expressed in astrocytes and multiple ionic events associated with astrocytic GABAA receptor activation. This protocol can be performed routinely in the study of astrocytes. This method will be valuable for identifying and characterizing the individual ion channels that orchestrate the electrical activity of low membrane resistance cells.

Mechanism of Azalomycin F5a against Methicillin-Resistant Staphylococcus aureus

PubMed Central

Xu, Li; Xu, Xuejie; Wang, Yimin; Liu, Erxiao

2018-01-01

To investigate the mechanism of azalomycin F5a against methicillin-resistant Staphylococcus aureus (MRSA), the conductivity of MRSA suspension and the adenylate kinase activity of MRSA culture were determined with the intervention of azalomycin F5a, which were significantly increased compared to those of blank controls. This inferred that azalomycin F5a could lead to the leakage of cellular substances possibly by increasing permeability to kill MRSA. As phospholipid bilayer was mainly responsible for cell-membrane permeability, the interaction between azalomycin F5a and cell-membrane lipids was further researched by determining the anti-MRSA activities of azalomycin F5a combined with cell-membrane lipids extracted from test MRSA or with 1,2-dipalmitoyl-sn-glycero-3-phospho-glycerol (DPPG) for possible molecular targets lying in MRSA cell-membrane. The results indicated that the anti-MRSA activity of azalomycin F5a remarkably decreased when it combined with membrane lipids or DPPG. This indicated that cell-membrane lipids especially DPPG might be important targets of azalomycin F5a against MRSA. PMID:29607325

An activating mutant of Rac1 that fails to interact with Rho GDP-dissociation inhibitor stimulates membrane ruffling in mammalian cells.

PubMed Central

Gandhi, Payal N; Gibson, Richard M; Tong, Xiaofeng; Miyoshi, Jun; Takai, Yoshimi; Konieczkowski, Martha; Sedor, John R; Wilson-Delfosse, Amy L

2004-01-01

Rac1, a member of the Rho family of small GTP-binding proteins, is involved in the regulation of the actin cytoskeleton via activation of lamellipodia and membrane ruffle formation. RhoGDI (Rho-family-specific GDP-dissociation inhibitor) forms a complex with Rho proteins in the cytosol of mammalian cells. It not only regulates guanine nucleotide binding to Rho proteins, but may also function as a molecular shuttle to carry Rho proteins from an inactive cytosolic pool to the membrane for activation. These studies tested if RhoGDI is necessary for the translocation of Rac1 from the cytosol to the plasma membrane for the formation of membrane ruffles. We describe a novel mutant of Rac1, R66E (Arg66-->Glu), that fails to bind RhoGDI. This RhoGDI-binding-defective mutation is combined with a Rac1-activating mutation G12V, resulting in a double-mutant [Rac1(G12V/R66E)] that fails to interact with RhoGDI in COS-7 cells, but remains constitutively activated. This double mutant stimulates membrane ruffling to a similar extent as that observed after epidermal growth factor treatment of non-transfected cells. To confirm that Rac1 can signal ruffle formation in the absence of interaction with RhoGDI, Rac1(G12V) was overexpressed in cultured mesangial cells derived from a RhoGDI knockout mouse. Rac1-mediated membrane ruffling was indistinguishable between the RhoGDI(-/-) and RhoGDI(+/+) cell lines. In both the COS-7 and cultured mesangial cells, Rac1(G12V) and Rac1(G12V/R66E) co-localize with membrane ruffles. These findings suggest that interaction with RhoGDI is not essential in the mechanism by which Rac1 translocates to the plasma membrane to stimulate ruffle formation. PMID:14629200

Evaluation of the damage of cell wall and cell membrane for various extracellular polymeric substance extractions of activated sludge.

PubMed

Guo, Xuesong; Liu, Junxin; Xiao, Benyi

2014-10-20

Extracellular polymeric substances (EPS) are susceptible to contamination by intracellular substances released during the extraction of EPS owing to the damage caused to microbial cell structures. The damage to cell walls and cell membranes in nine EPS extraction processes of activated sludge was evaluated in this study. The extraction of EPS (including proteins, carbohydrates and DNA) was the highest using the NaOH extraction method and the lowest using formaldehyde extraction. All nine EPS extraction methods in this study resulted in cell wall and membrane damage. The damage to cell walls, evaluated by 2-keto-3-deoxyoctonate (KDO) and N-acetylglucosamine content changes in extracted EPS, was the most significant in the NaOH extraction process. Formaldehyde extraction showed a similar extent of damage to cell walls to those detected in the control method (centrifugation), while those in the formaldehyde-NaOH and cation exchange resin extractions were slightly higher than those detected in the control. N-acetylglucosamine was more suitable than KDO for the evaluation of cell wall damage in the EPS extraction of activated sludge. The damage to cell membranes was characterized by two fluorochromes (propidium iodide and FITC Annexin V) with flow cytometry (FCM) measurement. The highest proportion of membrane-damaged cells was detected in NaOH extraction (26.54% of total cells) while membrane-damaged cells comprised 8.19% of total cells in the control. Copyright © 2014 Elsevier B.V. All rights reserved.

Calcium-activated potassium channels in basolateral membranes of colon epithelial cells; reconstitution and functional properties.

PubMed

Wiener, H; Turnheim, K

1990-10-26

Using differential sedimentation, isopycnic and Ficoll-400 barrier centrifugation, basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon were enriched 34- and 9-fold, respectively. 86Rb(+)-uptake into these vesicles, driven by an electrical potential difference, was stimulated by submicromolar Ca2+ activities and inhibited by Ba2+. These findings indicate the presence of Ca2(+)-activated K+ channels. The K+ channels in surface and crypt cell membranes differed with respect to inhibition by the bee venom apamin, the scorpion venom charybdotoxin and tetraethylammonium and exhibited a different pH dependence. Fusion of basolateral membrane vesicles with planar phospholipid bilayers revealed the presence of high-conductance Ba2(+)-sensitive K+ channels which were activated by micromolar Ca2+ and inhibited by crude scorpion venom and trifluoperazine. These K+ channels may be involved in the coupling of apical and basolateral membrane conductances during Na+ absorption and Cl- secretion, but they may also play a role in cell volume regulation.

Membrane cholesterol plays an important role in enteropathogen adhesion and the activation of innate immunity via flagellin-TLR5 signaling.

PubMed

Zhou, Mingxu; Duan, Qiangde; Li, Yinchau; Yang, Yang; Hardwidge, Philip R; Zhu, Guoqiang

2015-08-01

Lipid rafts are cholesterol- and sphingolipid-rich ordered microdomains distributed in the plasma membrane that participates in mammalian signal transduction pathways. To determine the role of lipid rafts in mediating interactions between enteropathogens and intestinal epithelial cells, membrane cholesterol was depleted from Caco-2 and IPEC-J2 cells using methyl-β-cyclodextrin. Cholesterol depletion significantly reduced Escherichia coli and Salmonella enteritidis adhesion and invasion into intestinal epithelial cells. Complementation with exogenous cholesterol restored bacterial adhesion to basal levels. We also evaluated the role of lipid rafts in the activation of Toll-like receptor 5 signaling by bacterial flagellin. Depleting membrane cholesterol reduced the ability of purified recombinant E. coli flagellin to activate TLR5 signaling in intestinal cells. These data suggest that both membrane cholesterol and lipid rafts play important roles in enteropathogen adhesion and contribute to the activation of innate immunity via flagellin-TLR5 signaling.

Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

PubMed Central

Delorme-Walker, Violaine; Seo, Ji-Yeon; Gohla, Antje; Fowler, Bruce; Bohl, Ben; DerMardirossian, Céline

2015-01-01

Cofilin, a critical player of actin dynamics, is spatially and temporally regulated to control the direction and force of membrane extension required for cell locomotion. In carcinoma cells, although the signaling pathways regulating cofilin activity to control cell direction have been established, the molecular machinery required to generate the force of the protrusion remains unclear. We show that the cofilin phosphatase chronophin (CIN) spatiotemporally regulates cofilin activity at the cell edge to generate persistent membrane extension. We show that CIN translocates to the leading edge in a PI3-kinase–, Rac1-, and cofilin-dependent manner after EGF stimulation to activate cofilin, promotes actin free barbed end formation, accelerates actin turnover, and enhances membrane protrusion. In addition, we establish that CIN is crucial for the balance of protrusion/retraction events during cell migration. Thus, CIN coordinates the leading edge dynamics by controlling active cofilin levels to promote MTLn3 cell protrusion. PMID:26324884

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

PubMed

Smith, Alyson S; Nowak, Roberta B; Zhou, Sitong; Giannetto, Michael; Gokhin, David S; Papoin, Julien; Ghiran, Ionita C; Blanc, Lionel; Wan, Jiandi; Fowler, Velia M

2018-05-08

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

Thermodynamic limitations on the temperature sensitivity of cell-membrane ion channels: Trouble with enthalpy uncertainty

NASA Astrophysics Data System (ADS)

Zheltikov, A. M.

2018-06-01

Energy exchange between a thermodynamic ensemble of heat- and cold-activated cell-membrane ion channels and the surrounding heat reservoir is shown to impose fundamental limitations on the performance of such channels as temperature-controlled gates for thermal cell activation. Analysis of unavoidable thermodynamic internal-energy fluctuations caused by energy exchange between the ion channels and the heat bath suggests that the resulting enthalpy uncertainty is too high for a robust ion-current gating by a single ion channel, implying that large ensembles of ion channels are needed for thermal cell activation. We argue, based on this thermodynamic analysis, that, had thermosensitive cell-membrane ion channels operated individually, rather than as large ensembles, robust thermal cell activation would have been impossible because of thermodynamic fluctuations.

A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells.

PubMed

Chen, Lihong; Meng, Qingli; Jing, Xian; Xu, Pingxiang; Luo, Dali

2011-02-01

Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²(+) signalling. In HEK293 and Jurkat cells, the Ca²(+) release and Ca²(+) uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²(+) responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²(+) concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²(+) flux. Copyright © 2010 Elsevier Inc. All rights reserved.

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.

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

PubMed

Zhang, S X; Kobayashi, T; Okada, T; García del Saz, E; Seguchi, H

1991-07-01

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.

PTK6 activation at the membrane regulates epithelial-mesenchymal transition in prostate cancer.

PubMed

Zheng, Yu; Wang, Zebin; Bie, Wenjun; Brauer, Patrick M; Perez White, Bethany E; Li, Jing; Nogueira, Veronique; Raychaudhuri, Pradip; Hay, Nissim; Tonetti, Debra A; Macias, Virgilia; Kajdacsy-Balla, André; Tyner, Angela L

2013-09-01

The intracellular tyrosine kinase protein tyrosine kinase 6 (PTK6) lacks a membrane-targeting SH4 domain and localizes to the nuclei of normal prostate epithelial cells. However, PTK6 translocates from the nucleus to the cytoplasm in human prostate tumor cells. Here, we show that while PTK6 is located primarily within the cytoplasm, the pool of active PTK6 in prostate cancer cells localizes to membranes. Ectopic expression of membrane-targeted active PTK6 promoted epithelial-mesenchymal transition in part by enhancing activation of AKT, thereby stimulating cancer cell migration and metastases in xenograft models of prostate cancer. Conversely, siRNA-mediated silencing of endogenous PTK6 promoted an epithelial phenotype and impaired tumor xenograft growth. In mice, PTEN deficiency caused endogenous active PTK6 to localize at membranes in association with decreased E-cadherin expression. Active PTK6 was detected at membranes in some high-grade human prostate tumors, and PTK6 and E-cadherin expression levels were inversely correlated in human prostate cancers. In addition, high levels of PTK6 expression predicted poor prognosis in patients with prostate cancer. Our findings reveal novel functions for PTK6 in the pathophysiology of prostate cancer, and they define this kinase as a candidate therapeutic target. Cancer Res; 73(17); 5426-37. ©2013 AACR.

Cryptic antifungal compounds active by synergism with polyene antibiotics.

PubMed

Kinoshita, Hiroshi; Yoshioka, Mariko; Ihara, Fumio; Nihira, Takuya

2016-04-01

The majority of antifungal compounds reported so far target the cell wall or cell membrane of fungi, suggesting that other types of antibiotics cannot exert their activity because they cannot penetrate into the cells. Therefore, if the permeability of the cell membrane could be enhanced, many antibiotics might be found to have antifungal activity. We here used the polyene antibiotic nystatin, which binds to ergosterol and forms pores at the cell membrane, to enhance the cellular permeability. In the presence of nystatin, many culture extracts from entomopathogenic fungi displayed antifungal activity. Among all the active extracts, two active components were purified and identified as helvolic acid and terramide A. Because the minimum inhibitory concentration of either compound was reduced four-fold in the presence of nystatin, it can be concluded that this screening method is useful for detecting novel antifungal activity. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase.

PubMed

Morales-Cedillo, Francisco; González-Solís, Ariadna; Gutiérrez-Angoa, Lizbeth; Cano-Ramírez, Dora Luz; Gavilanes-Ruiz, Marina

2015-04-01

Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.

Sirtuin activation: a role for plasma membrane in the cell growth puzzle.

PubMed

Crane, Frederick L; Navas, Plácido; Low, Hans; Sun, Iris L; de Cabo, Rafael

2013-04-01

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.

Nanoscale manipulation of membrane curvature for probing endocytosis in live cells.

PubMed

Zhao, Wenting; Hanson, Lindsey; Lou, Hsin-Ya; Akamatsu, Matthew; Chowdary, Praveen D; Santoro, Francesca; Marks, Jessica R; Grassart, Alexandre; Drubin, David G; Cui, Yi; Cui, Bianxiao

2017-08-01

Clathrin-mediated endocytosis (CME) involves nanoscale bending and inward budding of the plasma membrane, by which cells regulate both the distribution of membrane proteins and the entry of extracellular species. Extensive studies have shown that CME proteins actively modulate the plasma membrane curvature. However, the reciprocal regulation of how the plasma membrane curvature affects the activities of endocytic proteins is much less explored, despite studies suggesting that membrane curvature itself can trigger biochemical reactions. This gap in our understanding is largely due to technical challenges in precisely controlling the membrane curvature in live cells. In this work, we use patterned nanostructures to generate well-defined membrane curvatures ranging from +50 nm to -500 nm radius of curvature. We find that the positively curved membranes are CME hotspots, and that key CME proteins, clathrin and dynamin, show a strong preference towards positive membrane curvatures with a radius <200 nm. Of ten CME-related proteins we examined, all show preferences for positively curved membrane. In contrast, other membrane-associated proteins and non-CME endocytic protein caveolin1 show no such curvature preference. Therefore, nanostructured substrates constitute a novel tool for investigating curvature-dependent processes in live cells.

Membrane perturbation activity of cationic phenylene ethynylene oligomers and polymers: selectivity against model bacterial and mammalian membranes.

PubMed

Wang, Ying; Tang, Yanli; Zhou, Zhijun; Ji, Eunkyung; Lopez, Gabriel P; Chi, Eva Y; Schanze, Kirk S; Whitten, David G

2010-08-03

Poly(phenylene ethyneylene) (PPE)-based cationic conjugated polyelectrolytes (CPEs) and cationic phenylene ethynylene oligomers (OPEs) exhibit broad-spectrum antimicrobial activity, and their main target is believed to be the cell membrane. To understand better how these antimicrobial molecules interact with membranes, a series of PPE-based CPEs and OPEs with different side chains were studied. Large unilamellar vesicles with lipid compositions mimicking those of mammalian or bacterial membranes were used as model membranes. Among the CPEs and OPEs tested, the anionic CPE, PPE-SO(3)(2-) and the smallest cationic OPE-1 are inactive against all vesicles. Other cationic CPEs and OPEs show significant membrane perturbation ability against bacterial membrane mimics but are inactive against a mammalian cell membrane mimic with the exception of PPE-DABCO and two end-only-functionalized OPEs, which also disrupted a mammalian cell membrane mimic. The results suggest that the phospholipid composition of vesicles dominates the interaction of CPE and OPE with lipid membranes.

Phosphatidylserine metabolism modification precedes manganese-induced apoptosis and phosphatidylserine exposure in PC12 cells.

PubMed

Ferrara, G; Gambelunghe, A; Mozzi, R; Marchetti, M C; Migliorati, G; Muzi, G; Buratta, S

2013-12-01

Long-term exposure to high manganese (Mn) levels can lead to Parkinson-like neurological disorders. Molecular mechanisms underlying Mn cytotoxicity have been not defined. It is known that Mn induces apoptosis in PC12 cells and that this involves the activation of some signal transduction pathways. Although the role of phospholipids in apoptosis and signal transduction is well-known, the membrane phospholipid component in Mn-related damage has not yet been investigated. Phosphatidylserine (PS) facilitates protein translocation from cytosol to plasma membrane and PS exposure on the cell surface allows macrophage recognition of apoptotic cells. This study investigates the effects of MnCl2 on PS metabolism in PC12 cells, relating them to those on cell apoptosis. Apoptosis induction decreased PS radioactivity of PC12 cells incubated with radioactive serine. MnCl2 reduced PS radioactivity even under conditions that did not affect cell viability or PS exposure, suggesting that the effects on PS metabolism may represent an early event in cell apoptosis. Thus the latter conditions that also induced a greater PS decarboxylation were utilized for further investigating on the effects on PS synthesis, by measuring the activity and expression of PS-synthesizing enzymes, in cell lysates and in total cellular membranes (TM). Compared with corresponding controls, enzyme activity of MnCl2-treated cells was lower in cell lysates and greater in TM. Evaluating the expression of two isoforms of PS-synthesizing enzyme (PSS), PSSII was increased both in cell lysate and TM, while PSSI was unchanged. MnCl2 addition to control cell lysate reduced enzyme activity. These results suggest Mn plays a dual role on PS synthesis. Once inside the cell, Mn inhibits the enzyme/s, thus accounting for reduced PS synthesis in lysates and intact cells. On the other hand, it increases PSSII expression in cell membranes. The possibility that this occurs to counteract the direct effects of Mn ions on enzyme activity cannot be excluded. The effects on membrane enzyme activity and expression may also participate to PS exposure, observed at longer periods of treatment, by increasing membrane PS content. Copyright © 2013 Elsevier Inc. All rights reserved.

Ammonium-sensitive protein kinase activity in plasma membranes of the cyanobacterium Anacystis nidulans.

PubMed

Rodríguez, R; García-González, M; Guerrero, M G; Lara, C

1994-08-15

Cytoplasmic membranes prepared from nitrate-grown Anacystis nidulans cells exhibit a Mg(2+)-dependent protein kinase activity able to phosphorylate in vitro plasma membrane polypeptides with molecular masses of 98, 93, 83, 47, 44 and 31 kDa. The protein kinase activity was inhibited in cytoplasmic membrane preparations from nitrate-grown cells which had been exposed to ammonium for 5 min. Parallely, ammonium exposure also resulted in a more than two-fold activation of an alkaline phosphatase activity present in the soluble fraction. These results are discussed in relation to the well-known inhibition by ammonium of nitrate transport activity, and a hypothesis for the regulatory mechanism involved is presented.

A Novel Plasma Membrane-Anchored Protein Regulates Xylem Cell-Wall Deposition through Microtubule-Dependent Lateral Inhibition of Rho GTPase Domains.

PubMed

Sugiyama, Yuki; Wakazaki, Mayumi; Toyooka, Kiminori; Fukuda, Hiroo; Oda, Yoshihisa

2017-08-21

Spatial control of cell-wall deposition is essential for determining plant cell shape [1]. Rho-type GTPases, together with the cortical cytoskeleton, play central roles in regulating cell-wall patterning [2]. In metaxylem vessel cells, which are the major components of xylem tissues, active ROP11 Rho GTPases form oval plasma membrane domains that locally disrupt cortical microtubules, thereby directing the formation of oval pits in secondary cell walls [3-5]. However, the regulatory mechanism that determines the planar shape of active Rho of Plants (ROP) domains is still unknown. Here we show that IQD13 associates with cortical microtubules and the plasma membrane to laterally restrict the localization of ROP GTPase domains, thereby directing the formation of oval secondary cell-wall pits. Loss and overexpression of IQD13 led to the formation of abnormally round and narrow secondary cell-wall pits, respectively. Ectopically expressed IQD13 increased the presence of parallel cortical microtubules by promoting microtubule rescue. A reconstructive approach revealed that IQD13 confines the area of active ROP domains within the lattice of the cortical microtubules, causing narrow ROP domains to form. This activity required the interaction of IQD13 with the plasma membrane. These findings suggest that IQD13 positively regulates microtubule dynamics as well as their linkage to the plasma membrane, which synergistically confines the area of active ROP domains, leading to the formation of oval secondary cell-wall pits. This finding sheds light on the role of microtubule-plasma membrane linkage as a lateral fence that determines the planar shape of Rho GTPase domains. Copyright © 2017 Elsevier Ltd. All rights reserved.

ω-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis

PubMed Central

Levental, Kandice R.; Surma, Michal A.; Skinkle, Allison D.; Lorent, Joseph H.; Zhou, Yong; Klose, Christian; Chang, Jeffrey T.; Hancock, John F.; Levental, Ilya

2017-01-01

Mammalian cells produce hundreds of dynamically regulated lipid species that are actively turned over and trafficked to produce functional membranes. These lipid repertoires are susceptible to perturbations from dietary sources, with potentially profound physiological consequences. However, neither the lipid repertoires of various cellular membranes, their modulation by dietary fats, nor their effects on cellular phenotypes have been widely explored. We report that differentiation of human mesenchymal stem cells (MSCs) into osteoblasts or adipocytes results in extensive remodeling of the plasma membrane (PM), producing cell-specific membrane compositions and biophysical properties. The distinct features of osteoblast PMs enabled rational engineering of membrane phenotypes to modulate differentiation in MSCs. Specifically, supplementation with docosahexaenoic acid (DHA), a lipid component characteristic of osteoblast membranes, induced broad lipidomic remodeling in MSCs that reproduced compositional and structural aspects of the osteoblastic PM phenotype. The PM changes induced by DHA supplementation potentiated osteogenic differentiation of MSCs concurrent with enhanced Akt activation at the PM. These observations prompt a model wherein the DHA-induced lipidome leads to more stable membrane microdomains, which serve to increase Akt activity and thereby enhance osteogenic differentiation. More broadly, our investigations suggest a general mechanism by which dietary fats affect cellular physiology through remodeling of membrane lipidomes, biophysical properties, and signaling. PMID:29134198

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

PubMed

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

2008-12-01

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

Increased Long Chain acyl-Coa Synthetase Activity and Fatty Acid Import Is Linked to Membrane Synthesis for Development of Picornavirus Replication Organelles

PubMed Central

Scott, Alison J.; Ford, Lauren A.; Pei, Zhengtong; Watkins, Paul A.; Ernst, Robert K.; Belov, George A.

2013-01-01

All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be relevant for other (+)RNA viruses as well. PMID:23762027

Spectrophotometric and cytochemical analyses of phosphatase activity in Beta vulgaris L.

PubMed

Pesacreta, T C; Bennett, A B; Lucas, W J

1986-03-01

Spectrophotometric and cytochemical methods were used to investigate the localization and/or the sensitivity of phosphatase activities in aldehyde-fixed beet leaves and membrane fractions. The nonspecific acid phosphatase substrates, p-nitrophenyl phosphate and beta-glycerol phosphate, each exhibited unique spectrophotometric patterns of hydrolysis as a function of pH. Additionally, beta-glycerol phosphatase activity was primarily present on the tonoplast, whereas p-nitrophenyl phosphatase was present on the plasma membrane. Because of the unique pH response of each enzyme and their different localization, we conclude that they cannot be entirely "nonspecific." The spectrophotometric pattern of ATP hydrolysis differed from that of p-nitrophenol phosphate in that it decreased at pH 5.0-5.5 and was greatly inhibited by 10 mM sodium fluoride; however, both activities were on the plasma membrane. Therefore, we conclude that these activities represent either two enzymes or only one enzyme that differs in its ability to hydrolyze these two substrates. Generally, enzymatically produced lead deposits on the plasma membrane of non-vascular cells were as frequent and large as those on phloem cells; frequently, deposits on sieve element plasma membranes were relatively small. We therefore conclude that there is no evidence for the presence of relatively intense ATPase activity on the plasma membrane of phloem cells in beet leaf, in contrast to other species. Studies with membrane fractions indicated that formaldehyde could completely inhibit the inhibitor-sensitive phosphatase activities in mitochondrial and vacuolar fractions while preserving significant activity in the plasma membrane fraction.

Withaferin-A Induces Apoptosis in Osteosarcoma U2OS Cell Line via Generation of ROS and Disruption of Mitochondrial Membrane Potential.

PubMed

Zhang, Hui-Liang; Zhang, Hong

2017-01-01

Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania sominifera . It has tremendous pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti-cancer therapeutic development is to identify apoptosis inducing drug-like molecules. Osteosarcoma is a rare type of osteocancer, affecting human. The present study therefore focused on the evaluation of antitumor potential of WF-A against several osteosarcoma cell lines. MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC 50 . DAPI staining was used to confirm the apoptosis inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis. The results revealed that that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC 50 ranging from 0.32 to 7.6 μM. The lowest IC 50 (0.32 μM) was observed against U2OS cell line and therefore it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced ROS-mediated reduction in mitochondrial membrane potential ΔΨm) in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells. We propose that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential. WF-A exhibits strong anticancer activity against osteosarcoma cell linesAntiproliferative activity of WF-A is via induction of apoptosisWF-A induced ROS-mediated reduction in mitochondrial membrane potentialWF-A induced expression of caspase-3 in osteosarcoma cells. Abbreviations used: WA: Withaferin A; ROS: Reactive oxygen species; OS: Osteosarcoma; MMP: Mitochondrial membrane potential.

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. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Capacitance measurements of regulated exocytosis in mouse taste cells.

PubMed

Vandenbeuch, Aurelie; Zorec, Robert; Kinnamon, Sue C

2010-11-03

Exocytosis, consisting of the merger of vesicle and plasma membrane, is a common mechanism used by different types of nucleated cells to release their vesicular contents. Taste cells possess vesicles containing various neurotransmitters to communicate with adjacent taste cells and afferent nerve fibers. However, whether these vesicles engage in exocytosis on a stimulus is not known. Since vesicle membrane merger with the plasma membrane is reflected in plasma membrane area fluctuations, we measured membrane capacitance (C(m)), a parameter linearly related to membrane surface area. To investigate whether taste cells undergo regulated exocytosis, we used the compensated tight-seal whole-cell recording technique to monitor depolarization-induced changes in C(m) in the different types of taste cells. To identify taste cell types, mice expressing green fluorescent protein from the TRPM5 promoter or from the GAD67 promoter were used to discriminate type II and type III taste cells, respectively. Moreover, the cell types were also identified by monitoring their voltage-current properties. The results demonstrate that only type III taste cells show significant depolarization-induced increases in C(m), which were correlated to the voltage-activated calcium currents. The results suggest that type III, but neither type II nor type I cells exhibit depolarization-induced regulated exocytosis to release transmitter and activate gustatory afferent nerve fibers.

Modification of erythrocyte membrane proteins, enzymes and transport mechanisms in chronic alcoholics: an in vivo and in vitro study.

PubMed

Maturu, Paramahamsa; Vaddi, Damodara Reddy; Pannuru, Padmavathi; Nallanchakravarthula, Varadacharyulu

2013-01-01

The aim of the study was to elucidate the molecular mechanisms underlying the alcohol perturbation leading to deleterious effects on erythrocyte membrane transport in chronic alcoholics. Membrane bound enzyme activities such as Na(+), K(+)-ATPase, Ca(2+),Mg(2+)-ATPase and acetylcholine esterase and membrane transport analysis by in vitro and erythrocyte membrane profile analysis in controls and chronic alcoholic red cells were analyzed. It was observed that decreased Na(+), K(+)-ATPase enzyme activity and increased activities of Ca(2+),Mg(2+)-ATPase and acetylcholine esterase in chronic alcoholics compared to controls. The in vitro studies of erythrocytes suggested that there is an increased uptake of glucose through chronic alcoholic red cells. However, glucose utilization by chronic alcoholic red cells was decreased. An increased sensitivity of ouabain for its binding site on Na(+), K(+)-ATPase in chronic alcoholic erythrocyte membrane was evident from this study. Though there appears to be an increased Na(+) influx in chronic alcoholic cells, the status of Na(+) transport is not altered much. However, ouabain caused slight disturbances in the transport of sodium, similar disturbances in the potassium transport resulting in much accumulation of potassium in red cells. It was concluded that chronic alcohol consumption modified certain membrane bound proteins, enzymes and transport mechanisms in chronic alcoholics.

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden

PubMed Central

Martin, Erik W.; Buzza, Marguerite S.; Driesbaugh, Kathryn H.; Liu, Shihui; Fortenberry, Yolanda M.; Leppla, Stephen H.; Antalis, Toni M.

2015-01-01

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent. PMID:26392335

Cell activation and cellular-cellular interactions during hemodialysis: effect of dialyzer membrane.

PubMed

Sirolli, V; Ballone, E; Di Stante, S; Amoroso, L; Bonomini, M

2002-06-01

During hemodialysis (HD), circulating blood cells can be activated and also engage in dynamic interplay. These phenomena may be important factors behind dialysis membrane bio(in)compatibility. In the present prospective cross-over study, we have used flow cytometry to evaluate the influence of different dialysis membranes on the activation of circulating blood cells (leukocytes, platelets) and their dynamic interactions (formation of circulating platelet-leukocyte and platelet-erythrocyte aggregates) during in vivo HD. Each patient (n = 10) was treated with dialyzers containing membranes of cellulose diacetate, polysulfone and ethylenevinylalcohol (EVAL) in a randomized order. Upregulation of adhesion receptor expression (CD15s, CD11b/CD18) occurred mainly with the cellulosic membrane, though an increase in CD11b/CD18 circulating on neutrophils was also found with both synthetic membranes. Circulating activated platelets (P-selectin/CD63-positive platelets) increased during HD sessions with cellulose diacetate and polysulfone. An increased formation of platelet-neutrophil aggregates was found at 15 and 30 min during dialysis with cellulose diacetate and polysulfone but not with EVAL. Platelet-erythrocyte aggregates also increased with cellulose diacetate and at 15 min with polysulfone as well. Generally in concomitance with the increase in platelet-neutrophil coaggregates, there was an increased hydrogen peroxide production by neutrophils. The results of this study indicate that cellular mechanisms can be activated during HD largely depending on the membrane material, EVAL causing less reactivity than the other two membranes. It appears that each dialysis membrane has multiple and different characteristics that may contribute to interactions with blood components. Our results also indicate that derivatizing cellulose (cellulose diacetate) may be a useful way to improve the biocompatibility of the cellulose polymer and that there may be great variability in the biocompatibility profile of synthetic membranes, dialysis with polysulfone being in general associated with a higher degree of cell activation than EVAL membrane.

Separation of metabolic supply and demand: aerobic glycolysis as a normal physiological response to fluctuating energetic demands in the membrane.

PubMed

Epstein, Tamir; Xu, Liping; Gillies, Robert J; Gatenby, Robert A

2014-01-01

Cancer cells, and a variety of normal cells, exhibit aerobic glycolysis, high rates of glucose fermentation in the presence of normal oxygen concentrations, also known as the Warburg effect. This metabolism is considered abnormal because it violates the standard model of cellular energy production that assumes glucose metabolism is predominantly governed by oxygen concentrations and, therefore, fermentative glycolysis is an emergency back-up for periods of hypoxia. Though several hypotheses have been proposed for the origin of aerobic glycolysis, its biological basis in cancer and normal cells is still not well understood. We examined changes in glucose metabolism following perturbations in membrane activity in different normal and tumor cell lines and found that inhibition or activation of pumps on the cell membrane led to reduction or increase in glycolysis, respectively, while oxidative phosphorylation remained unchanged. Computational simulations demonstrated that these findings are consistent with a new model of normal physiological cellular metabolism in which efficient mitochondrial oxidative phosphorylation supplies chronic energy demand primarily for macromolecule synthesis and glycolysis is necessary to supply rapid energy demands primarily to support membrane pumps. A specific model prediction was that the spatial distribution of ATP-producing enzymes in the glycolytic pathway must be primarily localized adjacent to the cell membrane, while mitochondria should be predominantly peri-nuclear. The predictions were confirmed experimentally. Our results show that glycolytic metabolism serves a critical physiological function under normoxic conditions by responding to rapid energetic demand, mainly from membrane transport activities, even in the presence of oxygen. This supports a new model for glucose metabolism in which glycolysis and oxidative phosphorylation supply different types of energy demand. Cells use efficient but slow-responding aerobic metabolism to meet baseline, steady energy demand and glycolytic metabolism, which is inefficient but can rapidly increase adenosine triphosphate (ATP) production, to meet short-timescale energy demands, mainly from membrane transport activities. In this model, the origin of the Warburg effect in cancer cells and aerobic glycolysis in general represents a normal physiological function due to enhanced energy demand for membrane transporters activity required for cell division, growth, and migration.

Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

PubMed Central

Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

1996-01-01

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

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.

Polycation-induced Cell Membrane Permeability Does Not Enhance Cellular Uptake or Expression Efficiency of Delivered DNA

PubMed Central

Prevette, Lisa E.; Mullen, Douglas G.; Banaszak Holl, Mark M.

2010-01-01

Polycationic materials commonly used to delivery DNA to cells are known to induce cell membrane porosity in a charge-density dependent manner. It has been suggested that these pores may provide a mode of entry of the polymer-DNA complexes (polyplexes) into cells. To examine the correlation between membrane permeability and biological activity, we used two-color flow cytometry on two mammalian cell lines to simultaneously measure gene expression of a plasmid DNA delivered with four common nonviral vectors and cellular uptake of normally excluded fluorescent dye molecules of two different sizes, 668 Da and 2 MDa. We also followed gene expression in cells sorted based on the retention of endogenous fluorescein. We have found that cell membrane porosity caused by polycationic vectors does not enhance internalization or gene expression. Based on this single-cell study, membrane permeability is found to be an unwanted side effect that limits transfection efficiency, possibly through leakage of the delivered nucleic acid through the pores prior to transcription and translation and/or activation of cell defense mechanisms that restrict transgene expression. PMID:20349965

In vitro regulation of pericellular proteolysis in prostatic tumor cells treated with bombesin.

PubMed

Festuccia, C; Guerra, F; D'Ascenzo, S; Giunciuglio, D; Albini, A; Bologna, M

1998-01-30

Bombesin is a potent inducer of signal trasduction pathways involved in the proliferation and invasion of androgen-insensitive prostatic tumor cells. This study examines the bombesin-mediated modulation of pericellular proteolysis, monitoring cell capability to migrate and invade basement membranes, using a chemo-invasion assay and analyzing protease production. The results suggest that bombesin could modulate the invasive potential of prostatic cell lines regulating secretion and cell-surface uptake of uPA and MMP-9 activation. In fact, in PC3 and DU145 cells but not in LNCaP cells, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are induced by bombesin treatment. Bombesin also stimulates cell proliferation and this effect can be inhibited blocking uPA by antibodies and/or uPA inhibitor p-aminobenzamidine. Moreover, HMW-uPA induces cell proliferation in LNCaP cells, which do not produce uPA in the basal conditions, while PC3 and DU145 cell growth is supported by autocrine production of uPA. The increment of uPA activity on the external plasma membrane causes an increased pericellular plasmin activation. This effect is inhibited by antibodies against uPA and by p-aminobenzamidine. Similarly to EGF, bombesin stimulates secretion and activation of MMP-9 and TIMP-1 production. MMP-9 activation can be also obtained by HMW-uPA treatment, suggesting that plasma-membrane-bound uPA can start a proteolytic cascade involving MMP-9. Therefore, in in vitro assays, bombesin is able to modulate pericellular proteolysis and cell proliferation, differently distributing and activating proteolytic activities. This effect can be related to the "non-random" degradation of the extracellular matrix in which membrane uPA-uPAreceptor complexes could start bombesin-induced directional protein degradation during metastatic spread.

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

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.

A Mechanistic Understanding of Pyroptosis: The Fiery Death Triggered by Invasive Infection.

PubMed

Liu, Xing; Lieberman, Judy

2017-01-01

Immune cells and skin and mucosal epithelial cells recognize invasive microbes and other signs of danger to sound alarms that recruit responder cells and initiate an immediate "innate" immune response. An especially powerful alarm is triggered by cytosolic sensors of invasive infection that assemble into multimolecular complexes, called inflammasomes, that activate the inflammatory caspases, leading to maturation and secretion of proinflammatory cytokines and pyroptosis, an inflammatory death of the infected cell. Work in the past year has defined the molecular basis of pyroptosis. Activated inflammatory caspases cleave Gasdermin D (GSDMD), a cytosolic protein in immune antigen-presenting cells and epithelia. Cleavage separates the autoinhibitory C-terminal fragment from the active N-terminal fragment, which moves to the cell membrane, binds to lipids on the inside of the cell membrane, and oligomerizes to form membrane pores that disrupt cell membrane integrity, causing death and leakage of small molecules, including the proinflammatory cytokines and GSDMD itself. GSDMD also binds to cardiolipin on bacterial membranes and kills the very bacteria that activate the inflammasome. GSDMD belongs to a family of poorly studied gasdermins, expressed in the skin and mucosa, which can also form membrane pores. Spontaneous mutations that disrupt the binding of the N- and C-terminal domains of other gasdermins are associated with alopecia and asthma. Here, we review recent studies that identified the roles of the inflammasome, inflammatory caspases, and GSDMD in pyroptosis and highlight some of the outstanding questions about their roles in innate immunity, control of infection, and sepsis. © 2017 Elsevier Inc. All rights reserved.

Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.

2006-07-05

The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1{sup V12} or Cdc42{sup V12} could increase cell-cell fusion promoted by the Hendra ormore » SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA{sup L63} decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia.« less

Inflammasome - activated gasdermin D causes pyroptosis by forming membrane pores

PubMed Central

Liu, Xing; Zhang, Zhibin; Ruan, Jianbin; Pan, Youdong; Magupalli, Venkat Giri; Wu, Hao; Lieberman, Judy

2017-01-01

Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β1,2. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined. PMID:27383986

Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.

PubMed

Liu, Xing; Zhang, Zhibin; Ruan, Jianbin; Pan, Youdong; Magupalli, Venkat Giri; Wu, Hao; Lieberman, Judy

2016-07-07

Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined.

[The effect of nitrofurans on the membrane apparatus of coccal bacteria].

PubMed

Kutsmako, R T; Kuliash, Iu V; Rudzit, E A; Ermachenko, V A; Simakova, I M

1984-01-01

Secondary membrane effects on the membrane apparatus of coccus bacteria were being studied. Cultivation of Micrococcus lysodeikticus and Staphylococcus aureus cells on subbacteriostatic concentrations of nitrofurans results in a lower biosynthesis of many membrane proteins, as well as in inhibiting the activity of respiratory enzymes, i. e. the specific concentration of cytochromes and specific activity of NADH-, malate-, lactate oxidases and some reductases drop. Some cytological changes were revealed, when cells were grown on solafur, furazolidone, and furacriline.

The effect of boron on plasma membrane electron transport and associated proton secretion by cultured carrot cells.

PubMed

Barr, R; Böttger, M; Crane, F L

1993-09-01

Plasma membrane electron transport reactions and associated proton secretion were studied in boron-deficient carrot cells. It was found that the hormone-sensitive plasma membrane NADH oxidase was inhibited by boron deficiency and that under such conditions activity could be restored by exogenous boric acid with or without 2,4-dichlorophenoxy acetic acid. Gramicidin, a channel-forming protonophore, further stimulated NADH oxidase by carrot cells. Proton secretion, associated with plasma membrane H(+)-ATPase, was also affected by boron deficiency, but not as severely as ferricyanide-generated proton secretion, reflecting plasma membrane electron transport. The addition of 1 mM boric acid and 1 microM 2,4-dichlorophenoxy acetic acid to carrot cells fully restored the H+ secretion in presence of ferricyanide. The effect of boron deficiency in cultured carrot cells can, therefore, be directly associated with cell growth through its effect on the plasma membrane NADH oxidase and H+ secretion. Ferricyanide provides a probe which activates transmembrane electron transport that is only coupled to proton release when boron is present.

Opposing effects of estradiol- and testosterone-membrane binding sites on T47D breast cancer cell apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kampa, Marilena; Nifli, Artemissia-Phoebe; Charalampopoulos, Ioannis

Classical steroid mode of action involves binding to intracellular receptors, the later acting as ligand-activated nuclear transcription factors. Recently, membrane sites for different steroids have been also identified, mediating rapid, non-genomic, steroid actions. Membrane sites for estrogen and androgen have been found in a number of different cell types, bearing or not classical intracellular receptors. In the present study, with the use of radioligand binding, flow cytometry and confocal laser microscopy, we report that T47D human breast cancer cells express specific and saturable membrane receptors for both estrogen (K {sub D} 4.06 {+-} 3.31 nM) and androgen (K {sub D}more » 7.64 {+-} 3.15 nM). Upon activation with BSA-conjugated, non-permeable ligands (E{sub 2}-BSA and testosterone-BSA), membrane estrogen receptors protect cells from serum-deprivation-induced apoptosis, while androgen receptors induce apoptosis in serum-supplemented T47D cells. In addition, co-incubation of cells with a fixed concentration of one steroid and varying concentrations of the other reversed the abovementioned effect (apoptosis for androgen, and anti-apoptosis for E{sub 2}), suggesting that the fate of the cell depends on the relative concentration of either steroid in the culture medium. We also report the identification of membrane receptors for E{sub 2} and androgen in biopsy slides from breast cancer patients. Both sites are expressed, with the staining for membrane E{sub 2} being strongly present in ER-negative, less differentiated, more aggressive tumors. These findings suggest that aromatase inhibitors may exert their beneficial effects on breast cancer by also propagating the metabolism of local steroids towards androgen, inducing thus cell apoptosis through membrane androgen receptor activation.« less

Monitoring Ion Activities In and Around Cells Using Ion-Selective Liquid-Membrane Microelectrodes

PubMed Central

Lee, Seong-Ki; Boron, Walter F.; Parker, Mark D.

2013-01-01

Determining the effective concentration (i.e., activity) of ions in and around living cells is important to our understanding of the contribution of those ions to cellular function. Moreover, monitoring changes in ion activities in and around cells is informative about the actions of the transporters and/or channels operating in the cell membrane. The activity of an ion can be measured using a glass microelectrode that includes in its tip a liquid-membrane doped with an ion-selective ionophore. Because these electrodes can be fabricated with tip diameters that are less than 1 μm, they can be used to impale single cells in order to monitor the activities of intracellular ions. This review summarizes the history, theory, and practice of ion-selective microelectrode use and brings together a number of classic and recent examples of their usefulness in the realm of physiological study. PMID:23322102

Beyond the hydrophobic effect: Critical function of water at biological phase boundaries--A hypothesis.

PubMed

Damodaran, Srinivasan

2015-07-01

Many life-sustaining processes in living cells occur at the membrane-water interface. The pertinent questions that need to be asked are what is the evolutionary reason for biology to choose the membrane-water interface as the site for performing and/or controlling crucial biological reactions and what is the key physical principle that is singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this review, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes and receptor activated processes via manipulating the thermodynamic activity of water at the membrane-water interfacial region. In support of this hypothesis, first we establish that the surface pressure of a lipid monolayer is a direct measure of a reduction in the thermodynamic activity of interfacial water. Second, we show that the surface pressure-dependent activation/inactivation of interfacial enzymes is fundamentally related to their dependence on interfacial water activity. We extend this argument to infer that cells might manipulate activities of membrane-associated biological processes via manipulating the activity of interfacial water via localized compression or expansion of the interface. In this paper, we critically analyze literature data on mechano-activation of large pore ion channels in Escherichia coli spheroplasts and G-proteins in reconstituted lipid vesicles, and show that these pressure-induced activation processes are fundamentally and quantitatively related to changes in the thermodynamic state of interfacial water, caused by mechanical stretching of the bilayer. Copyright © 2015 Elsevier B.V. All rights reserved.

Content of endoplasmic reticulum and Golgi complex membranes positively correlates with the proliferative status of brain cells.

PubMed

Silvestre, David C; Maccioni, Hugo J F; Caputto, Beatriz L

2009-03-01

Although the molecular and cellular basis of particular events that lead to the biogenesis of membranes in eukaryotic cells has been described in detail, understanding of the intrinsic complexity of the pleiotropic response by which a cell adjusts the overall activity of its endomembrane system to accomplish these requirements is limited. Here we carried out an immunocytochemical and biochemical examination of the content and quality of the endoplasmic reticulum (ER) and Golgi apparatus membranes in two in vivo situations characterized by a phase of active cell proliferation followed by a phase of declination in proliferation (rat brain tissue at early and late developmental stages) or by permanent active proliferation (gliomas and their most malignant manifestation, glioblastomas multiforme). It was found that, in highly proliferative phases of brain development (early embryo brain cells), the content of ER and Golgi apparatus membranes, measured as total lipid phosphorous content, is higher than in adult brain cells. In addition, the concentration of protein markers of ER and Golgi is also higher in early embryo brain cells and in human glioblastoma multiforme cells than in adult rat brain or in nonpathological human brain cells. Results suggest that the amount of endomembranes and the concentration of constituent functional proteins diminish as cells decline in their proliferative activity.

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. © 2011 Macmillan Publishers Limited. All rights reserved.

PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

PubMed Central

Su, Tizhi; Bao, Liwei; Xie, Xiujie; Lehner, Caryn L.; Cavey, Greg S.; Teknos, Theodoros N.

2013-01-01

Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA. PMID:24191200

Linoleic Acid Permeabilizes Gastric Epithelial Cells by Increasing Connexin43 Levels in the Cell Membrane Via a GPR40- and Akt-Dependent Mechanism

PubMed Central

Puebla, Carlos; Cisterna, Bruno A.; Salas, Daniela P.; Delgado-López, Fernando; Lampe, Paul D.; Sáez, Juan C.

2016-01-01

Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintain the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt. PMID:26869446

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

PubMed

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

2008-08-01

Plant cell wall production is a membrane-bound process. Cell walls are composed of cellulose microfibrils, embedded inside a matrix of other polysaccharides and glycoproteins. The cell wall matrix is extruded into the existing cell wall by exocytosis. This same process also inserts the cellulose synthase complexes into the plasma membrane. These complexes, the nanomachines that produce the cellulose microfibrils, move inside the plasma membrane leaving the cellulose microfibrils in their wake. Cellulose microfibril angle is an important determinant of cell development and of tissue properties and as such relevant for the industrial use of plant material. Here, we provide an integrated view of the events taking place in the not more than 100 nm deep area in and around the plasma membrane, correlating recent results provided by the distinct field of plant cell biology. We discuss the coordinated activities of exocytosis, endocytosis, and movement of cellulose synthase complexes while producing cellulose microfibrils and the link of these processes to the cortical microtubules.

Diatomite reinforced chitosan composite membrane as potential scaffold for guided bone regeneration.

PubMed

Tamburaci, Sedef; Tihminlioglu, Funda

2017-11-01

In this study, natural silica source, diatomite, incorporated novel chitosan based composite membranes were fabricated and characterized for bone tissue engineering applications as possible bone regeneration membrane. The effect of diatomite loading on the mechanical, morphological, chemical, thermal and surface properties, wettability and in vitro cytotoxicity and cell proliferation on of composite membranes were investigated and observed by tensile test, atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), protein adsorption assay, air/water contact angle analysis and WST-1 respectively. Swelling studies were also performed by water absorption capacity determination. Results showed that incorporation of diatomite to the chitosan matrix increased the surface roughness, swelling capacity and tensile modulus of membranes. An increase of about 52% in Young's modulus was achieved for 10wt% diatomite composite membranes compared with chitosan membranes. High cell viability results were obtained with indirect extraction method. Besides, in vitro cell proliferation and ALP activity results showed that diatom incorporation significantly increased the ALP activity of Saos-2 cells cultured on chitosan membranes. The novel composite membranes prepared in the present study with tunable properties can be considered as a potential candidate as a scaffold in view of its enhanced physical & chemical properties as well as biological activities for bone tissue engineering applications. Copyright © 2017 Elsevier B.V. All rights reserved.

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

PubMed Central

Alvares, Stacy M.; Dunn, Clarence A.; Brown, Tod A.; Wayner, Elizabeth E.; Carter, William G.

2008-01-01

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals- possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior. PMID:18269919

WAVE2 forms a complex with PKA and is involved in PKA enhancement of membrane protrusions.

PubMed

Yamashita, Hiroshi; Ueda, Kazumitsu; Kioka, Noriyuki

2011-02-04

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation.

High yield cell-free production of integral membrane proteins without refolding or detergents.

PubMed

Wuu, Jessica J; Swartz, James R

2008-05-01

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

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

NASA Astrophysics Data System (ADS)

Belyavskaya, N. A.

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

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

Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.

PubMed

Stone, Matthew B; Shelby, Sarah A; Núñez, Marcos F; Wisser, Kathleen; Veatch, Sarah L

2017-02-01

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

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

PubMed

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

2018-06-01

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

Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

PubMed Central

Annaba, Fadi; Sarwar, Zaheer; Kumar, Pradeep; Saksena, Seema; Turner, Jerrold R.; Dudeja, Pradeep K.; Gill, Ravinder K.; Alrefai, Waddah A.

2016-01-01

Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MβCD. The inhibition in ASBT activity by MβCD was blocked in the cells treated with MβCD-cholesterol complexes. Kinetic analysis revealed that MβCD treatment decreased the Vmax of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis. PMID:18063707

Prolonged Intake of Dietary Lipids Alters Membrane Structure and T Cell Responses in LDLr-/- Mice.

PubMed

Pollock, Abigail H; Tedla, Nicodemus; Hancock, Sarah E; Cornely, Rhea; Mitchell, Todd W; Yang, Zhengmin; Kockx, Maaike; Parton, Robert G; Rossy, Jérémie; Gaus, Katharina

2016-05-15

Although it is recognized that lipids and membrane organization in T cells affect signaling and T cell activation, to what extent dietary lipids alter T cell responsiveness in the absence of obesity and inflammation is not known. In this study, we fed low-density lipoprotein receptor knockout mice a Western high-fat diet for 1 or 9 wk and examined T cell responses in vivo along with T cell lipid composition, membrane order, and activation ex vivo. Our data showed that high levels of circulating lipids for a prolonged period elevated CD4(+) and CD8(+) T cell proliferation and resulted in an increased proportion of CD4(+) central-memory T cells within the draining lymph nodes following induction of contact hypersensitivity. In addition, the 9-wk Western high-fat diet elevated the total phospholipid content and monounsaturated fatty acid level, but decreased saturated phosphatidylcholine and sphingomyelin within the T cells. The altered lipid composition in the circulation, and of T cells, was also reflected by enhanced membrane order at the activation site of ex vivo activated T cells that corresponded to increased IL-2 mRNA levels. In conclusion, dietary lipids can modulate T cell lipid composition and responses in lipoprotein receptor knockout mice even in the absence of excess weight gain and a proinflammatory environment. Copyright © 2016 by The American Association of Immunologists, Inc.

Modeling 3-D deformation of outer hair cells and their production of the active force in the cochlea.

PubMed

Spector, A A; Ameen, M; Schmiedt, R A

2002-10-01

We analyze the deformation of the outer hair cell and its production of active force under physiological conditions. The active force has two components. One results from the strain caused by loading in the organ of Corti in the cochlea and depends on the level of the acoustic signal; the other is related to the intrinsic active properties of the cell membrane. We demonstrate our approach by considering, as a basic model of an outer hair cell in the organ of Corti, a cylindrical shell that is filled with an incompressible fluid and located between two planes that move relative to each other. These planes represent the basilar membrane and tectorial membrane complexes. We show that the deformed state of the cell has a 3-D nature, including bending and twisting components. This is different from the experimental conditions in which the active force is usually measured. We estimate the active force as a function of the relative position of the planes, angle of the cell's inclination, and the cell length.

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells.

PubMed

Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José; Muriaux, Delphine

2015-08-01

During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

PubMed Central

Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José

2015-01-01

ABSTRACT During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. IMPORTANCE During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes. PMID:26018170

Quantitative transporter proteomics by liquid chromatography with tandem mass spectrometry: addressing methodologic issues of plasma membrane isolation and expression-activity relationship.

PubMed

Kumar, Vineet; Prasad, Bhagwat; Patilea, Gabriela; Gupta, Anshul; Salphati, Laurent; Evers, Raymond; Hop, Cornelis E C A; Unadkat, Jashvant D

2015-02-01

To predict transporter-mediated drug disposition using physiologically based pharmacokinetic models, one approach is to measure transport activity and relate it to protein expression levels in cell lines (overexpressing the transporter) and then scale these to via in vitro to in vivo extrapolation (IVIVE). This approach makes two major assumptions. First, that the expression of the transporter is predominantly in the plasma membrane. Second, that there is a linear correlation between expression level and activity of the transporter protein. The present study was conducted to test these two assumptions. We evaluated two commercially available kits that claimed to separate plasma membrane from other cell membranes. The Qiagen Qproteome kit yielded very little protein in the fraction purported to be the plasma membrane. The Abcam Phase Separation kit enriched the plasma membrane but did not separate it from other intracellular membranes. For the Abcam method, the expression level of organic anion-transporting polypeptides (OATP) 1B1/2B1 and breast cancer resistance protein (BCRP) proteins in all subcellular fractions isolated from cells or human liver tissue tracked that of Na⁺-K⁺ ATPase. Assuming that Na⁺-K⁺ ATPase is predominantly located in the plasma membrane, these data suggest that the transporters measured are also primarily located in the plasma membrane. Using short hairpin RNA, we created clones of cell lines with varying degrees of OATP1B1 or BCRP expression level. In these clones, transport activity of OATP1B1 or BCRP was highly correlated with protein expression level (r² > 0.9). These data support the use of transporter expression level data and activity data from transporter overexpressing cell lines for IVIVE of transporter-mediated disposition of drugs. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Extracts of Edible and Medicinal Plants Damage Membranes of Vibrio cholerae▿

PubMed Central

Sánchez, Eduardo; García, Santos; Heredia, Norma

2010-01-01

The use of natural compounds from plants can provide an alternative approach against food-borne pathogens. The mechanisms of action of most plant extracts with antimicrobial activity have been poorly studied. In this work, changes in membrane integrity, membrane potential, internal pH (pHin), and ATP synthesis were measured in Vibrio cholerae cells after exposure to extracts of edible and medicinal plants. A preliminary screen of methanolic, ethanolic, and aqueous extracts of medicinal and edible plants was performed. Minimal bactericidal concentrations (MBCs) were measured for extracts showing high antimicrobial activity. Our results indicate that methanolic extracts of basil (Ocimum basilicum L.), nopal cactus (Opuntia ficus-indica var. Villanueva L.), sweet acacia (Acacia farnesiana L.), and white sagebrush (Artemisia ludoviciana Nutt.) are the most active against V. cholera, with MBCs ranging from 0.5 to 3.0 mg/ml. Using four fluorogenic techniques, we studied the membrane integrity of V. cholerae cells after exposure to these four extracts. Extracts from these plants were able to disrupt the cell membranes of V. cholerae cells, causing increased membrane permeability, a clear decrease in cytoplasmic pH, cell membrane hyperpolarization, and a decrease in cellular ATP concentration in all strains tested. These four plant extracts could be studied as future alternatives to control V. cholerae contamination in foods and the diseases associated with this microorganism. PMID:20802077

Extracts of edible and medicinal plants damage membranes of Vibrio cholerae.

PubMed

Sánchez, Eduardo; García, Santos; Heredia, Norma

2010-10-01

The use of natural compounds from plants can provide an alternative approach against food-borne pathogens. The mechanisms of action of most plant extracts with antimicrobial activity have been poorly studied. In this work, changes in membrane integrity, membrane potential, internal pH (pH(in)), and ATP synthesis were measured in Vibrio cholerae cells after exposure to extracts of edible and medicinal plants. A preliminary screen of methanolic, ethanolic, and aqueous extracts of medicinal and edible plants was performed. Minimal bactericidal concentrations (MBCs) were measured for extracts showing high antimicrobial activity. Our results indicate that methanolic extracts of basil (Ocimum basilicum L.), nopal cactus (Opuntia ficus-indica var. Villanueva L.), sweet acacia (Acacia farnesiana L.), and white sagebrush (Artemisia ludoviciana Nutt.) are the most active against V. cholera, with MBCs ranging from 0.5 to 3.0 mg/ml. Using four fluorogenic techniques, we studied the membrane integrity of V. cholerae cells after exposure to these four extracts. Extracts from these plants were able to disrupt the cell membranes of V. cholerae cells, causing increased membrane permeability, a clear decrease in cytoplasmic pH, cell membrane hyperpolarization, and a decrease in cellular ATP concentration in all strains tested. These four plant extracts could be studied as future alternatives to control V. cholerae contamination in foods and the diseases associated with this microorganism.

Milestones and recent discoveries on cell death mediated by mitochondria and their interactions with biologically active amines.

PubMed

Grancara, Silvia; Ohkubo, Shinji; Artico, Marco; Ciccariello, Mauro; Manente, Sabrina; Bragadin, Marcantonio; Toninello, Antonio; Agostinelli, Enzo

2016-10-01

Mitochondria represent cell "powerhouses," being involved in energy transduction from the electrochemical gradient to ATP synthesis. The morphology of their cell types may change, according to various metabolic processes or osmotic pressure. A new morphology of the inner membrane and mitochondrial cristae, significantly different from the previous one, has been proposed for the inner membrane and mitochondrial cristae, based on the technique of electron tomography. Mitochondrial Ca(2+) transport (the transporter has been isolated) generates reactive oxygen species and induces the mitochondrial permeability transition of both inner and outer mitochondrial membranes, leading to induction of necrosis and apoptosis. In the mitochondria of several cell types (liver, kidney, and heart), mitochondrial oxidative stress is an essential step in the induction of cell death, although not in brain, in which the phenomenon is caused by a different mechanism. Mitochondrial permeability transition drives both apoptosis and necrosis, whereas mitochondrial outer membrane permeability is characteristic of apoptosis. Adenine nucleotide translocase remains the most important component involved in membrane permeability, with the opening of the transition pore, although other proteins, such as ATP synthase or phosphate carriers, have been proposed. Intrinsic cell death is triggered by the release from mitochondria of proteic factors, such as cytochrome c, apoptosis inducing factor, and Smac/DIABLO, with the activation of caspases upon mitochondrial permeability transition or mitochondrial outer membrane permeability induction. Mitochondrial permeability transition induces the permeability of the inner membrane in sites in contact with the outer membrane; mitochondrial outer membrane permeability forms channels on the outer membrane by means of various stimuli involving Bcl-2 family proteins. The biologically active amines, spermine, and agmatine, have specific functions on mitochondria which distinguish them from other amines. Enzymatic oxidative deamination of spermine by amine oxidases in tumor cells may produce reactive oxygen species, leading to transition pore opening and apoptosis. This process could be exploited as a new therapeutic strategy to combat cancer.

AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2

PubMed Central

Cho, Kwang-jin; Casteel, Darren E.; Prakash, Priyanka; Tan, Lingxiao; van der Hoeven, Dharini; Salim, Angela A.; Kim, Choel; Capon, Robert J.; Lacey, Ernest; Cunha, Shane R.; Gorfe, Alemayehu A.

2016-01-01

K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function. PMID:27697864

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

PubMed

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.

Virus-Mimetic Fusogenic Exosomes for Direct Delivery of Integral Membrane Proteins to Target Cell Membranes.

PubMed

Yang, Yoosoo; Hong, Yeonsun; Nam, Gi-Hoon; Chung, Jin Hwa; Koh, Eunee; Kim, In-San

2017-04-01

An efficient system for direct delivery of integral membrane proteins is successfully developed using a new biocompatible exosome-based platform. Fusogenic exosomes harboring viral fusogen, vascular stomatitis virus (VSV)-G protein, can fuse with and modify plasma membranes in a process called "membrane editing." This can facilitate the transfer of biologically active membrane proteins into the target cell membranes both in vitro and in vivo. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation of Chloride Channels by Protein Kinase C in Normal and Cystic Fibrosis Airway Epithelia

NASA Astrophysics Data System (ADS)

Li, Ming; McCann, John D.; Anderson, Matthew P.; Clancy, John P.; Liedtke, Carole M.; Nairn, Angus C.; Greengard, Paul; Welsh, Michael J.

1989-06-01

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.

Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane.

PubMed

Cho, Gota; Bragiel, Aneta M; Wang, Di; Pieczonka, Tomasz D; Skowronski, Mariusz T; Shono, Masayuki; Nielsen, Søren; Ishikawa, Yasuko

2015-04-01

The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased. The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions. Copyright © 2015 Elsevier B.V. All rights reserved.

Phytol shows anti-angiogenic activity and induces apoptosis in A549 cells by depolarizing the mitochondrial membrane potential.

PubMed

Sakthivel, Ravi; Malar, Dicson Sheeja; Devi, Kasi Pandima

2018-06-13

In the present study, the antiproliferative activity of phytol and its mechanism of action against human lung adenocarcinoma cell line A549 were studied in detail. Results showed that phytol exhibited potent antiproliferative activity against A549 cells in a dose and time-dependent manner with an IC 50 value of 70.81 ± 0.32 μM and 60.7 ± 0.47 μM at 24 and 48 h, respectively. Phytol showed no adverse toxic effect in normal human lung cells (L-132), but mild toxic effect was observed when treated with maximum dose (67 and 84 μM). No membrane-damaging effect was evidenced by PI staining and SEM analysis. The results of mitochondrial membrane potential analysis, cell cycle analysis, FT-IR and Western blotting analysis clearly demonstrated the molecular mechanism of phytol as induction of apoptosis in A549 cells, as evidenced by formation of shrinked cell morphology with membrane blebbing, depolarization of mitochondrial membrane potential, increased cell population in the sub-G0 phase, band variation in the DNA and lipid region, downregulation of Bcl-2, upregulation of Bax and the activation of caspase-9 and -3. In addition, phytol inhibited the CAM vascular growth as evidenced by CAM assay, which positively suggests that phytol has anti-angiogenic potential. Taken together, these findings clearly demonstrate the mode of action by which phytol induces cell death in A549 lung adenocarcinoma cells. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

ELECTRON MICROSCOPIC STUDY OF THE ATPASE ACTIVITY OF THE BAI STRAIN A (MYELOBLASTOSIS) AVIAN TUMOR VIRUS

PubMed Central

Novikoff, Alex B.; de Thé, Guy; Beard, D.; Beard, J. W.

1962-01-01

Thymus glands of chicks with leukemia induced by BAI strain A (myeloblastosis) virus were fixed in cold 4 per cent formaldehyde-sucrose. Frozen sections were incubated in the ATPase medium of Wachstein and Meisel and studied by light microscopy and electron microscopy. The ATPase activity of the virus is localized to the outermost membrane of the virus. The membrane of the blast-like cells of the thymus cortex from which the virus emerges, by budding, also possesses such activity. It appears likely that the outermost membrane of the virus is derived from the plasma membrane of these cells. PMID:13939125

Expression of membrane progesterone receptors (mPRs) in rat peripheral glial cell membranes and their potential role in the modulation of cell migration and protein expression.

PubMed

Castelnovo, Luca F; Magnaghi, Valerio; Thomas, Peter

2017-09-28

The role played by progestogens in modulating Schwann cell pathophysiology is well established. Progestogens exert their effects in these cells through both classical genomic and non-genomic mechanisms, the latter mediated by the GABA-A receptor. However, there is evidence that other receptors may be involved. Membrane progesterone receptors (mPRs) are novel 7-transmembrane receptors coupled to G proteins that have been characterized in different tissues and cells, including the central nervous system (CNS). The mPRs were shown to mediate some of progestogens' neuroprotective effects in the CNS, and to be upregulated in glial cells after traumatic brain injury. Based on this evidence, this paper investigated the possible involvement of mPRs in mediating progestogen actions in S42 Schwann cells. All five mPR isoforms and progesterone receptor membrane component 1 (PGRMC1) were detected in Schwann cells, and were present on the cell membrane. Progesterone and the mPR-specific agonist, Org-OD-02-0 (02) bound to these membranes, indicating the presence of functional mPRs. The mPR agonist 02 rapidly increased cell migration in an in vitro assay, suggesting a putative role of mPRs in the nerve regeneration process. Treatment with pertussis toxin and 8-Br-cAMP blocked 02-induced cell migration, suggesting this progestogen action is mediated by activation of an inhibitory G protein, leading to a decrease in intracellular cAMP levels. In contrast, long-term mPR activation led to increased expression levels of myelin associated glycoprotein (MAG). Taken together, these findings show that mPRs are present and active in Schwann cells and have a role in modulating their physiological processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Lactobacillus plantarum 299v surface-bound GAPDH: a new insight into enzyme cell walls location.

PubMed

Saad, N; Urdaci, M; Vignoles, C; Chaignepain, S; Tallon, R; Schmitter, J M; Bressollier, P

2009-12-01

The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.

Microdomains in the membrane landscape shape antigen-presenting cell function.

PubMed

Zuidscherwoude, Malou; de Winde, Charlotte M; Cambi, Alessandra; van Spriel, Annemiek B

2014-02-01

The plasma membrane of immune cells is a highly organized cell structure that is key to the initiation and regulation of innate and adaptive immune responses. It is well-established that immunoreceptors embedded in the plasma membrane have a nonrandom spatial distribution that is important for coupling to components of intracellular signaling cascades. In the last two decades, specialized membrane microdomains, including lipid rafts and TEMs, have been identified. These domains are preformed structures ("physical entities") that compartmentalize proteins, lipids, and signaling molecules into multimolecular assemblies. In APCs, different microdomains containing immunoreceptors (MHC proteins, PRRs, integrins, among others) have been reported that are imperative for efficient pathogen recognition, the formation of the immunological synapse, and subsequent T cell activation. In addition, recent work has demonstrated that tetraspanin microdomains and lipid rafts are involved in BCR signaling and B cell activation. Research into the molecular mechanisms underlying membrane domain formation is fundamental to a comprehensive understanding of membrane-proximal signaling and APC function. This review will also discuss the advances in the microscopy field for the visualization of the plasma membrane, as well as the recent progress in targeting microdomains as novel, therapeutic approach for infectious and malignant diseases.

Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes.

PubMed

Moniruzzaman, Md; Islam, Md Zahidul; Sharmin, Sabrina; Dohra, Hideo; Yamazaki, Masahito

2017-08-22

Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

[The nature of pacemaker activity].

PubMed

Kabakov, A Iu

1991-01-01

A general equation of the membrane resting potential (RP) has been derived for closed cell membrane (CM) model. It is shown that Na,K-ATPase of cardiomyocytes is in the antielectrogenic phase. A hypothesis is proposed: a pacemaker cell is an excitable cell, which has RP corresponding to the given activity of Na,K-ATPase and non-activated cationic conductivities of CM higher than the activation threshold of Na-channels. The equation of the equipotential levels of the membrane RP on the surface of the cationic conductivities has been derived. It is shown that the substances (e. g. neuromediator) that change the membrane cation permeability are able to depolarize or to hyperpolarize CM. The direction of polarization is dependent on the state of the cell electrogenic system. The following factors promote the hyperpolarizing effect of the magnifying cation permeability substances: 1) high activity of Na,K-ATPase, 2) low background cation permeability of CM (among their number the integrity of CM) and 3) high ratio of the potassium permeability alteration in respect to that of sodium which is evoked by the substance (delta gK/delta gNa).

Molecular simulation aspects of amyloid peptides at membrane interface.

PubMed

Liu, Yonglan; Ren, Baiping; Zhang, Yanxian; Sun, Yan; Chang, Yung; Liang, Guizhao; Xu, Lijian; Zheng, Jie

2018-02-06

The interactions of amyloid peptides with cell membranes play an important role in maintaining the integrity and functionality of cell membrane. A thorough molecular-level understanding of the structure, dynamics, and interactions between amyloid peptides and cell membranes is critical to amyloid aggregation and toxicity mechanisms for the bench-to-bedside applications. Here we review the most recent computational studies of amyloid peptides at model cell membranes. Different mechanisms of action of amyloid peptides on/in cell membranes, targeted by different computational techniques at different lengthscales and timescales, are rationally discussed. Finally, we have proposed some new insights into the remaining challenges and perspectives for future studies to improve our understanding of the activity of amyloid peptides associated with protein-misfolding diseases. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy. Copyright © 2018 Elsevier B.V. All rights reserved.

Microbubble-Assisted Ultrasound-Induced Transient Phosphatidylserine Translocation.

PubMed

Escoffre, Jean-Michel; Derieppe, Marc; Lammertink, Bart; Bos, Clemens; Moonen, Chrit

2017-04-01

Microbubble-assisted ultrasound (sonopermeabilization) results in reversible permeabilization of the plasma membrane of cells. This method is increasingly used in vivo because of its potential to deliver therapeutic molecules with limited cell damage. Nevertheless, the effects of sonopermeabilization on the plasma membrane remain not fully understood. We investigated the influence of sonopermeabilization on the transverse mobility of phospholipids, especially on phosphatidylserine (PS) externalization. We performed studies using optical imaging with Annexin V and FM1-43 probes to monitor PS externalization of rat glioma C6 cells. Sonopermeabilization induced transient membrane permeabilization, which is positively correlated with reversible PS externalization. This membrane disorganization was temporary and not associated with loss of cell viability. Sonopermeabilization did not induce PS externalization via activation of the scramblase. We hypothesize that acoustically induced membrane pores may provide a new pathway for PS migration between both membrane leaflets. During the membrane-resealing phase, PS asymmetry may be re-established by amino-phospholipid flippase activity and/or endocytosis, along with exocytosis processes. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Light-induced modification of plant plasma membrane ion transport.

PubMed

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

2010-09-01

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

Do mechanical forces contribute to nanoscale membrane organisation in T cells?

PubMed

Klotzsch, Enrico; Stiegler, Johannes; Ben-Ishay, Eldad; Gaus, Katharina

2015-04-01

Mechanotransduction describes how a cell senses and interacts with its environment. The concept originated in adhesion biology where adhesion receptors, integrins, facilitate force transmission between the extracellular matrix and the intracellular actin cytoskeleton. Indeed, during any adhesive contacts, cells do exert mechanical force. Hence, the probing of the local environment by cells results in mechanical cues that contribute to cellular functions and cell fate decisions such as migration, proliferation, differentiation and apoptosis. On the molecular level, mechanical forces can rearrange proteins laterally within the membrane, regulate their activity by inducing conformational changes and probe the mechanical properties and bond strength of receptor-ligands. From this point of view, it appears surprising that molecular forces have been largely overlooked in membrane organisation and ligand discrimination processes in lymphocytes. During T cell activation, the T cell receptor recognises and distinguishes antigenic from benign endogenous peptides to initiate the reorganisation of membrane proteins into signalling clusters within the immunological synapse. In this review, we asked whether characteristics of fibroblast force sensing could be applied to immune cell antigen recognition and signalling, and outline state-of-the-art experimental strategies for studying forces in the context of membrane organisation. This article is part of a Special Issue entitled: Nanoscale membrane orgainisation and signalling. Copyright © 2014 Elsevier B.V. All rights reserved.

Vorinostat-eluting poly(DL-lactide-co-glycolide) nanofiber-coated stent for inhibition of cholangiocarcinoma cells

PubMed Central

Song, Yeon Hui; Kim, Chan; Kim, Jungsoo; Seo, Sol-Ji; Jeong, Young-Il; Kang, Dae Hwan

2017-01-01

Purpose The aim of this study was to fabricate a vorinostat (Zolinza™)-eluting nanofiber membrane-coated gastrointestinal (GI) stent and to study its antitumor activity against cholangiocarcinoma (CCA) cells in vitro and in vivo. Methods Vorinostat and poly(DL-lactide-co-glycolide) dissolved in an organic solvent was sprayed onto a GI stent to make a nanofiber-coated stent using an electro-spinning machine. Intact vorinostat and vorinostat released from nanofibers was used to assess anticancer activity in vitro against various CCA cells. The antitumor activity of the vorinostat-eluting nanofiber membrane-coated stent was evaluated using HuCC-T1 bearing mice. Results A vorinostat-incorporated polymer nanofiber membrane was formed on the surface of the GI stent. Vorinostat was continuously released from the nanofiber membrane over 10 days, and its release rate was higher in cell culture media than in phosphate-buffered saline. Released vorinostat showed similar anticancer activity against various CCA cells in vitro compared to that of vorinostat. Like vorinostat, vorinostat released from nanofibers induced acetylation of histone H4 and inhibited histone deacetylases 1⋅3⋅4/5/7 expression in vitro and in vivo. Furthermore, vorinostat nanofibers showed a higher tumor growth inhibition rate in HuCC-T1 bearing mice than vorinostat injections. Conclusion Vorinostat-eluting nanofiber membranes showed significant antitumor activity against CCA cells in vitro and in vivo. We suggest the vorinostat nanofiber-coated stent may be a promising candidate for CCA treatment. PMID:29089762

Recycling of used perfluorosulfonic acid membranes

DOEpatents

Grot, Stephen [Middletown, DE; Grot, Walther [Chadds Ford, PA

2007-08-14

A method for recovering and recycling catalyst coated fuel cell membranes includes dissolving the used membranes in water and solvent, heating the dissolved membranes under pressure and separating the components. Active membranes are produced from the recycled materials.

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells.

PubMed

Laohavisit, Anuphon; Shang, Zhonglin; Rubio, Lourdes; Cuin, Tracey A; Véry, Anne-Aliénor; Wang, Aihua; Mortimer, Jennifer C; Macpherson, Neil; Coxon, Katy M; Battey, Nicholas H; Brownlee, Colin; Park, Ohkmae K; Sentenac, Hervé; Shabala, Sergey; Webb, Alex A R; Davies, Julia M

2012-04-01

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH•. In root cells, extracellular OH• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH•. An OH•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.

Tetraspanins and Transmembrane Adaptor Proteins As Plasma Membrane Organizers-Mast Cell Case.

PubMed

Halova, Ivana; Draber, Petr

2016-01-01

The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs) and transmembrane adaptor protein (TRAP)-enriched domains. Recent biophysical, microscopic, and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD)9, CD53, CD63, CD81, CD151)] or TRAPs [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG)] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B.

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 ofmore » 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.« less

Mechanical injury and repair of cells

NASA Technical Reports Server (NTRS)

Miyake, Katsuya; McNeil, Paul L.

2003-01-01

OBJECTIVE: To concisely review the field of cell plasma membrane disruption (torn cell surface) and repair. MAIN POINTS: Plasma membrane disruption is a common form of cell injury under physiologic conditions, after trauma, in certain muscular dystrophies, and during certain forms of clinical intervention. Rapid repair of a disruption is essential to cell survival and involves a complex and active cell response that includes membrane fusion and cytoskeletal activation. Tissues, such as cardiac and skeletal muscle, adapt to a disruption injury by hypertrophying. Cells adapt by increasing the efficiency of their resealing response. CONCLUSION: Plasma membrane disruption is an important cellular event in both health and disease. The disruption repair mechanism is now well understood at the cellular level, but much remains to be learned at the molecular level. Cell and tissue level adaptational responses to the disruption either prevent its further occurrence or facilitate future repairs. Therapeutically useful drugs might result if, using this accumulating knowledge, chemical agents can be developed that can enhance repair or adaptive responses.

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.

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.

Cell Membrane Transport Mechanisms: Ion Channels and Electrical Properties of Cell Membranes.

PubMed

Kulbacka, Julita; Choromańska, Anna; Rossowska, Joanna; Weżgowiec, Joanna; Saczko, Jolanta; Rols, Marie-Pierre

2017-01-01

Cellular life strongly depends on the membrane ability to precisely control exchange of solutes between the internal and external (environmental) compartments. This barrier regulates which types of solutes can enter and leave the cell. Transmembrane transport involves complex mechanisms responsible for passive and active carriage of ions and small- and medium-size molecules. Transport mechanisms existing in the biological membranes highly determine proper cellular functions and contribute to drug transport. The present chapter deals with features and electrical properties of the cell membrane and addresses the questions how the cell membrane accomplishes transport functions and how transmembrane transport can be affected. Since dysfunctions of plasma membrane transporters very often are the cause of human diseases, we also report how specific transport mechanisms can be modulated or inhibited in order to enhance the therapeutic effect.

Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis

PubMed Central

Chen, Xin; He, Wan-ting; Hu, Lichen; Li, Jingxian; Fang, Yuan; Wang, Xin; Xu, Xiaozheng; Wang, Zhuo; Huang, Kai; Han, Jiahuai

2016-01-01

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis. PMID:27573174

Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis.

PubMed

Chen, Xin; He, Wan-Ting; Hu, Lichen; Li, Jingxian; Fang, Yuan; Wang, Xin; Xu, Xiaozheng; Wang, Zhuo; Huang, Kai; Han, Jiahuai

2016-09-01

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis.

A Cell Number Counting Factor Regulates Akt/Protein Kinase B To Regulate Dictyostelium discoideum Group Size

PubMed Central

Gao, Tong; Knecht, David; Tang, Lei; Hatton, R. Diane; Gomer, Richard H.

2004-01-01

Little is known about how individual cells can organize themselves to form structures of a given size. During development, Dictyostelium discoideum aggregates in dendritic streams and forms groups of ∼20,000 cells. D. discoideum regulates group size by secreting and simultaneously sensing a multiprotein complex called counting factor (CF). If there are too many cells in a stream, the associated high concentration of CF will decrease cell-cell adhesion and increase cell motility, causing aggregation streams to break up. The pulses of cyclic AMP (cAMP) that mediate aggregation cause a transient translocation of Akt/protein kinase B (Akt/PKB) to the leading edge of the plasma membrane and a concomitant activation of the kinase activity, which in turn stimulates motility. We found that countin− cells (which lack bioactive CF) and wild-type cells starved in the presence of anticountin antibodies (which block CF activity) showed a decreased level of cAMP-stimulated Akt/PKB membrane translocation and kinase activity compared to parental wild-type cells. Recombinant countin has the bioactivity of CF, and a 1-min treatment of cells with recombinant countin potentiated Akt/PKB translocation to membranes and Akt/PKB activity. Western blotting of total cell lysates indicated that countin does not affect the total level of Akt/PKB. Fluorescence microscopy of cells expressing an Akt/PKB pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion protein indicated that recombinant countin and anti-countin antibodies do not obviously alter the distribution of Akt/PKB PH-GFP when it translocates to the membrane. Our data indicate that CF increases motility by potentiating the cAMP-stimulated activation and translocation of Akt/PKB. PMID:15470246

Response of Ca2+-ATPase to clinorotaion of pea seedlings. O. M. Nedukha and E. L. Kordyum

NASA Astrophysics Data System (ADS)

Nedukha, Olena

2016-07-01

The present study was aimed to reveal of response of Ca2+-ATPase activity of cortex cells in distal elongation zone of Pisum sativum root to slow clinorotation. Pea seedlings were grown on a horizontal clinostat (2 rpm) and in the stationary control for 6 days. The electron-cytochemical method was used to examine the effects of imitated microgravity on the distribution of Ca2+-ATPase in outer layers of root cortex. The quantitative analysis of the density of cytochemical reaction products was measured using the Image J program. Electron microscopy showed the presence of electron-dense lead phosphate precipitated grains, the enzymatic activity reaction products on the plasma membrane, membranes of vesicular structures, endoplasmic reticulum (ER) and on organelles envelope in both of samples of the stationary control and clinorotated seedlings. We revealed the sensitivity of Ca2+-ATPase to clinorotation. The quantitative analysis of the area and density of enzymatic activity reaction products revealed that clinorotation led to the decrease of 3.4 times the density of reaction products on the plasma membrane and the increase of reaction products density on endomembranes and organelles membranes, in particular: in 2.2 times on mitochondria membranes; in 1.3 times - on membranes of ER; in 2.5 times - on tonoplast; by an order of magnitude greater - on contacting membranes of organelles with plasma membrane in comparison with such in cells of control samples. The data analysis can indicate an intensification of calcium pump on endomembranes, on envelopes of cytoplasmic organelles and nucleus. The obtained data suggest that the redistribution of Ca2+-ATPase activity in cells can be mediated by the activation of certain isoforms of enzyme or/and by an activation of Ca2+/H+ antiporter in plasma membrane that helps to maintain optimal calcium balance in plant cells under imitated microgravity.

Premature activation of the paramyxovirus fusion protein before target cell attachment with corruption of the viral fusion machinery.

PubMed

Farzan, Shohreh F; Palermo, Laura M; Yokoyama, Christine C; Orefice, Gianmarco; Fornabaio, Micaela; Sarkar, Aurijit; Kellogg, Glen E; Greengard, Olga; Porotto, Matteo; Moscona, Anne

2011-11-04

Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents.

Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.

PubMed

Etter, E F; Minta, A; Poenie, M; Fay, F S

1996-05-28

(Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.

Roles of specific membrane lipid domains in EGF receptor activation and cell adhesion molecule stabilization in a developing olfactory system.

PubMed

Gibson, Nicholas J; Tolbert, Leslie P; Oland, Lynne A

2009-09-29

Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-beta-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons.

The Relationship between Respiration-Related Membrane Potential Slow Oscillations and Discharge Patterns in Mitral/Tufted Cells: What Are the Rules?

PubMed Central

Briffaud, Virginie; Fourcaud-Trocmé, Nicolas; Messaoudi, Belkacem; Buonviso, Nathalie; Amat, Corine

2012-01-01

Background A slow respiration-related rhythm strongly shapes the activity of the olfactory bulb. This rhythm appears as a slow oscillation that is detectable in the membrane potential, the respiration-related spike discharge of the mitral/tufted cells and the bulbar local field potential. Here, we investigated the rules that govern the manifestation of membrane potential slow oscillations (MPSOs) and respiration-related discharge activities under various afferent input conditions and cellular excitability states. Methodology and Principal Findings We recorded the intracellular membrane potential signals in the mitral/tufted cells of freely breathing anesthetized rats. We first demonstrated the existence of multiple types of MPSOs, which were influenced by odor stimulation and discharge activity patterns. Complementary studies using changes in the intracellular excitability state and a computational model of the mitral cell demonstrated that slow oscillations in the mitral/tufted cell membrane potential were also modulated by the intracellular excitability state, whereas the respiration-related spike activity primarily reflected the afferent input. Based on our data regarding MPSOs and spike patterns, we found that cells exhibiting an unsynchronized discharge pattern never exhibited an MPSO. In contrast, cells with a respiration-synchronized discharge pattern always exhibited an MPSO. In addition, we demonstrated that the association between spike patterns and MPSO types appeared complex. Conclusion We propose that both the intracellular excitability state and input strength underlie specific MPSOs, which, in turn, constrain the types of spike patterns exhibited. PMID:22952828

Ruthenium complexes with phenylterpyridine derivatives target cell membrane and trigger death receptors-mediated apoptosis in cancer cells.

PubMed

Deng, Zhiqin; Gao, Pan; Yu, Lianling; Ma, Bin; You, Yuanyuan; Chan, Leung; Mei, Chaoming; Chen, Tianfeng

2017-06-01

Elucidation of the communication between metal complexes and cell membrane may provide useful information for rational design of metal-based anticancer drugs. Herein we synthesized a novel class of ruthenium (Ru) complexes containing phtpy derivatives (phtpy = phenylterpyridine), analyzed their structure-activity relationship and revealed their action mechanisms. The result showed that, the increase in the planarity of hydrophobic Ru complexes significantly enhanced their lipophilicity and cellular uptake. Meanwhile, the introduction of nitro group effectively improved their anticancer efficacy. Further mechanism studies revealed that, complex (2c), firstly accumulated on cell membrane and interacted with death receptors to activate extrinsic apoptosis signaling pathway. The complex was then transported into cell cytoplasm through transferrin receptor-mediated endocytosis. Most of the intracellular 2c accumulated in cell plasma, decreasing the level of cellular ROS, inducing the activation of caspase-9 and thus intensifying the apoptosis. At the same time, the residual 2c can translocate into cell nucleus to interact with DNA, induce DNA damage, activate p53 pathway and enhance apoptosis. Comparing with cisplatin, 2c possesses prolonged circulation time in blood, comparable antitumor ability and importantly, much lower toxicity in vivo. Taken together, this study uncovers the role of membrane receptors in the anticancer actions of Ru complexes, and provides fundamental information for rational design of membrane receptor targeting anticancer drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

The antifungal activity and membrane-disruptive action of dioscin extracted from Dioscorea nipponica.

PubMed

Cho, Jaeyong; Choi, Hyemin; Lee, Juneyoung; Kim, Mi-Sun; Sohn, Ho-Yong; Lee, Dong Gun

2013-03-01

Dioscin is a kind of steroidal saponin isolated from the root bark of wild yam Dioscorea nipponica. We investigated the antifungal effect of dioscin against different fungal strains and its antifungal mechanism(s) in Candida albicans cells. Using the propidium iodide assay and calcein-leakage measurement, we confirmed that dioscin caused fungal membrane damage. Furthermore, we evaluated the ability of dioscin to disrupt the plasma membrane potential, using 3,3'-dipropylthiadicarbocyanine iodide [DiSC(3)(5)] and bis-(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)]. Cells stained with the dyes had a significant increase in fluorescent intensity after exposure to dioscin, indicating that dioscin has an effect on the membrane potential. To visualize the effect of dioscin on the cell membrane, we synthesized rhodamine-labeled giant unilamellar vesicles (GUVs) mimicking the outer leaflet of the plasma membrane of C. albicans. As seen in the result, the membrane disruptive action of dioscin caused morphological change and rhodamine leakage of the GUVs. In three-dimensional contour-plot analysis using flow cytometry, we observed a decrease in cell size, which is in agreement with our result from the GUV assay. These results suggest that dioscin exerts a considerable antifungal activity by disrupting the structure in membrane after invading into the fungal membrane, resulting in fungal cell death. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Exact solutions to a spatially extended model of kinase-receptor interaction.

PubMed

Szopa, Piotr; Lipniacki, Tomasz; Kazmierczak, Bogdan

2011-10-01

B and Mast cells are activated by the aggregation of the immune receptors. Motivated by this phenomena we consider a simple spatially extended model of mutual interaction of kinases and membrane receptors. It is assumed that kinase activates membrane receptors and in turn the kinase molecules bound to the active receptors are activated by transphosphorylation. Such a type of interaction implies positive feedback and may lead to bistability. In this study we apply the Steklov eigenproblem theory to analyze the linearized model and find exact solutions in the case of non-uniformly distributed membrane receptors. This approach allows us to determine the critical value of receptor dephosphorylation rate at which cell activation (by arbitrary small perturbation of the inactive state) is possible. We found that cell sensitivity grows with decreasing kinase diffusion and increasing anisotropy of the receptor distribution. Moreover, these two effects are cooperating. We showed that the cell activity can be abruptly triggered by the formation of the receptor aggregate. Since the considered activation mechanism is not based on receptor crosslinking by polyvalent antigens, the proposed model can also explain B cell activation due to receptor aggregation following binding of monovalent antigens presented on the antigen presenting cell.

Matrix-specific protein kinase A signaling regulates p21 activated kinase activation by flow in endothelial cells

PubMed Central

Funk, Steven Daniel; Yurdagul, Arif; Green, Jonette M.; Jhaveri, Krishna A.; Schwartz, Martin Alexander; Orr, A. Wayne

2010-01-01

Rationale Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and NF-κB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-κB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-κB. Objective To elucidate the mechanisms regulating matrix-specific PAK activation. Methods and Results We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead basement membrane proteins enhance flow-induced protein kinase A (PKA) activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-κB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the PGI2 analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKI injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI. Conclusions Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK. PMID:20224042

Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress.

PubMed

Soares, Júlia Ribeiro; José Tenório de Melo, Edésio; da Cunha, Maura; Fernandes, Kátia Valevski Sales; Taveira, Gabriel Bonan; da Silva Pereira, Lidia; Pimenta, Samy; Trindade, Fernanda Gomes; Regente, Mariana; Pinedo, Marcela; de la Canal, Laura; Gomes, Valdirene Moreira; de Oliveira Carvalho, André

2017-01-01

Plant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef 1 -Saccharomyces cerevisiae interaction. ApDef 1 -S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy. ApDef 1 caused S. cerevisiae cell death and MIC was 7.8μM. Whole cell population died after 18h of ApDef 1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef 1 induced death. ApDef 1 -S. cerevisiae interaction resulted in membrane permeabilization, H 2 O 2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. ApDef 1 -S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via. We show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef 1 -S. cerevisiae interaction. Copyright © 2016 Elsevier B.V. All rights reserved.

Mechanism of antifungal activity of antimicrobial peptide APP, a cell-penetrating peptide derivative, against Candida albicans: intracellular DNA binding and cell cycle arrest.

PubMed

Li, Lirong; Sun, Jin; Xia, Shufang; Tian, Xu; Cheserek, Maureen Jepkorir; Le, Guowei

2016-04-01

We investigated the antifungal properties and anti-candidal mechanism of antimicrobial peptide APP. The minimum inhibitory concentration of APP was 8 μM against Candida albicans and Aspeogillus flavus, the concentration against Saccharomyces cerevisiae and Cryptococcus neoformans was 16 μM, while 32 μM inhibited Aspergilla niger and Trichopyton rubrum. APP caused slight depolarization (12.32 ± 0.87%) of the membrane potential of intact C. albicans cells when it exerted its anti-candidal activity and only caused 21.52 ± 0.48% C. albicans cell membrane damage. APP interacted with cell wall membrane, caused potassium efflux and nucleotide leakage. However, confocal fluorescence microscopy experiment and flow cytometry confirmed that FITC-labeled APP penetrated C. albicans cell membrane with 52.31 ± 1.88% cell-penetrating efficiency and accumulated in the cytoplasm. Then, APP interact with C. albicans genomic DNA and completely suppressed DNA migration above weight ratio (peptide/DNA) of 2, and significantly arrested cell cycles during the S-phase (S-phase cell population was 27.09 ± 0.73%, p < 0.05) after penetrating the cell membrane. Results indicated that APP kills C. albicans for efficient cell-penetrating efficiency, strong DNA-binding affinity and significant physiological changes inducing S-phase arrest in intracellular environment.

Membrane rafts in host-pathogen interactions.

PubMed

Riethmüller, Joachim; Riehle, Andrea; Grassmé, Heike; Gulbins, Erich

2006-12-01

Central elements in the infection of mammalian cells with viral, bacterial and parasitic pathogens include the adhesion of the pathogen to surface receptors of the cell, recruitment of additional receptor proteins to the infection-site, a re-organization of the membrane and, in particular, the intracellular signalosome. Internalization of the pathogen results in the formation of a phagosome that is supposed to fuse with lysosomes to form phagolysosomes, which serve the degradation of the pathogen, an event actively prevented by some pathogens. In summary, these changes in the infected cell permit pathogens to trigger apoptosis (for instance of macrophages paralysing the initial immune response), to invade the cell and/or to survive in the cell, but they also serve the mammalian cell to defeat the infection, for instance by activation of transcription factors and the release of cytokines. Distinct membrane domains in the plasma membrane and intracellular vesicles that are mainly composed of sphingolipids and cholesterol or enriched with the sphingolipid ceramide, are critically involved in all of these events occurring during the infection. These membrane structures are therefore very attractive targets for novel drugs to interfere with bacterial, viral and parasitic infections.

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells.

PubMed

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A; Multhoff, Gabriele

2005-06-15

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

Heat Shock Protein 70 Surface-Positive Tumor Exosomes Stimulate Migratory and Cytolytic Activity of Natural Killer Cells

PubMed Central

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A.; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A.

2006-01-01

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo−/CX exosomes were Hsp70 membrane derived positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes. PMID:15958569

Aluminum and temperature alteration of cell membrane permeability of Quercus rubra

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 lipidmore » 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.« less

Lateral Membrane Waves Constitute a Universal Dynamic Pattern of Motile Cells

NASA Astrophysics Data System (ADS)

Döbereiner, Hans-Günther; Dubin-Thaler, Benjamin J.; Hofman, Jake M.; Xenias, Harry S.; Sims, Tasha N.; Giannone, Grégory; Dustin, Michael L.; Wiggins, Chris H.; Sheetz, Michael P.

2006-07-01

We have monitored active movements of the cell circumference on specifically coated substrates for a variety of cells including mouse embryonic fibroblasts and T cells, as well as wing disk cells from fruit flies. Despite having different functions and being from multiple phyla, these cell types share a common spatiotemporal pattern in their normal membrane velocity; we show that protrusion and retraction events are organized in lateral waves along the cell membrane. These wave patterns indicate both spatial and temporal long-range periodic correlations of the actomyosin gel.

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties[S

PubMed Central

Sousa, Tânia; Castro, Rui E.; Pinto, Sandra N.; Coutinho, Ana; Lucas, Susana D.; Moreira, Rui; Rodrigues, Cecília M. P.; Prieto, Manuel; Fernandes, Fábio

2015-01-01

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure. PMID:26351365

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

PubMed

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

2017-09-01

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase.

PubMed

Kolesnick, R N; Clegg, S

1988-05-15

It has been suggested that sphingoid bases may serve as physiologic inhibitors of protein kinase C. Because 1,2-diacylglycerols, but not phorbol esters, enhance sphingomyelin degradation via a sphingomyelinase in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762), the effects of phorbol esters, 1,2-diacylglycerols, and sphingomyelinase on protein kinase C activation were assessed. Under basal conditions, the inactive cytosolic form of protein kinase C predominated. 1,2-Diacylglycerols stimulated transient protein kinase C redistribution to the membrane. 1,2-Dioctanoylglycerol (200 micrograms/ml) reduced cytosolic protein kinase C activity to 67% of control from 72 to 48 pmol.min-1.10(6) cells-1 and enhanced membrane-bound activity to 430% of control from 6 to 25 pmol.min-1.10(6) cells-1 after 4 min of stimulation. Thereafter, protein kinase C activity returned to the cytosol. In contrast, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated redistribution to the membrane without return to the cytosol. Exogenous sphingomyelinase reduced membrane-bound protein kinase C activity to 30% of control, yet did not alter cytosolic activity. Sphingomyelinase, added after phorbol ester-induced redistribution was completed, restored activity to the cytosol. In these studies, TPA (10(-8) M) reduced cytosolic activity to 62% of control and elevated membrane-bound protein kinase C activity to 650% of control. Sphingomyelinase restored cytosolic activity to 84% of control and reduced membrane-bound activity to 297% of control. Similarly, the free sphingoid bases, sphingosine, sphinganine, and phytosphingosine, reversed phorbol ester-induced protein kinase C redistribution. Since 1,2-diacylglycerols activate a sphingomyelinase and sphingomyelinase action can reverse protein kinase C activation, these studies suggest that a pathway involving a sphingomyelinase might comprise a physiologic negative effector system for protein kinase C. Further, the failure of phorbol esters to activate this system might account for some differences between these agents.

Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion.

PubMed

Holst, Mikkel Roland; Vidal-Quadras, Maite; Larsson, Elin; Song, Jie; Hubert, Madlen; Blomberg, Jeanette; Lundborg, Magnus; Landström, Maréne; Lundmark, Richard

2017-08-22

Cellular blebbing, caused by local alterations in cell-surface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrin-independent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension-mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol-interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans

PubMed Central

Alvarez, Francisco J.; Douglas, Lois M.; Rosebrock, Adam

2008-01-01

The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Δ mutant are similar to the effects of inhibiting β-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains. PMID:18799621

Enhanced Membrane Pore Formation through High-Affinity Targeted Antimicrobial Peptides

PubMed Central

Arnusch, Christopher J.; Pieters, Roland J.; Breukink, Eefjan

2012-01-01

Many cationic antimicrobial peptides (AMPs) target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted. PMID:22768121

WAVE2 Forms a Complex with PKA and Is Involved in PKA Enhancement of Membrane Protrusions*

PubMed Central

Yamashita, Hiroshi; Ueda, Kazumitsu; Kioka, Noriyuki

2011-01-01

PKA contributes to many physiological processes, including glucose homeostasis and cell migration. The substrate specificity of PKA is low compared with other kinases; thus, complex formation with A-kinase-anchoring proteins is important for the localization of PKA in specific subcellular regions and the phosphorylation of specific substrates. Here, we show that PKA forms a complex with WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) in MDA-MB-231 breast cancer cells and mouse brain extracts. Two separate regions of WAVE2 are involved in WAVE2-PKA complex formation. This complex localizes to the leading edge of MDA-MB-231 cells. PKA activation results in enlargement of the membrane protrusion. WAVE2 depletion impairs PKA localization at membrane protrusions and the enlargement of membrane protrusion induced by PKA activation. Together, these results suggest that WAVE2 works as an A-kinase-anchoring protein that recruits PKA at membrane protrusions and plays a role in the enlargement of membrane protrusions induced by PKA activation. PMID:21119216

Fractal avalanche ruptures in biological membranes

NASA Astrophysics Data System (ADS)

Gözen, Irep; Dommersnes, Paul; Czolkos, Ilja; Jesorka, Aldo; Lobovkina, Tatsiana; Orwar, Owe

2010-11-01

Bilayer membranes envelope cells as well as organelles, and constitute the most ubiquitous biological material found in all branches of the phylogenetic tree. Cell membrane rupture is an important biological process, and substantial rupture rates are found in skeletal and cardiac muscle cells under a mechanical load. Rupture can also be induced by processes such as cell death, and active cell membrane repair mechanisms are essential to preserve cell integrity. Pore formation in cell membranes is also at the heart of many biomedical applications such as in drug, gene and short interfering RNA delivery. Membrane rupture dynamics has been studied in bilayer vesicles under tensile stress, which consistently produce circular pores. We observed very different rupture mechanics in bilayer membranes spreading on solid supports: in one instance fingering instabilities were seen resulting in floral-like pores and in another, the rupture proceeded in a series of rapid avalanches causing fractal membrane fragmentation. The intermittent character of rupture evolution and the broad distribution in avalanche sizes is consistent with crackling-noise dynamics. Such noisy dynamics appear in fracture of solid disordered materials, in dislocation avalanches in plastic deformations and domain wall magnetization avalanches. We also observed similar fractal rupture mechanics in spreading cell membranes.

Self-organizing actin patterns shape membrane architecture but not cell mechanics

NASA Astrophysics Data System (ADS)

Fritzsche, M.; Li, D.; Colin-York, H.; Chang, V. T.; Moeendarbary, E.; Felce, J. H.; Sezgin, E.; Charras, G.; Betzig, E.; Eggeling, C.

2017-02-01

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

Self-organizing actin patterns shape membrane architecture but not cell mechanics

PubMed Central

Fritzsche, M.; Li, D.; Colin-York, H.; Chang, V. T.; Moeendarbary, E.; Felce, J. H.; Sezgin, E.; Charras, G.; Betzig, E.; Eggeling, C.

2017-01-01

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties. PMID:28194011

Response of MG63 osteoblast-like cells onto polycarbonate membrane surfaces with different micropore sizes.

PubMed

Lee, Sang Jin; Choi, Jin San; Park, Ki Suk; Khang, Gilson; Lee, Young Moo; Lee, Hai Bang

2004-08-01

Response of different types of cells on materials is important for the applications of tissue engineering and regenerative medicine. It is recognized that the behavior of the cell adhesion, proliferation, and differentiation on materials depends largely on surface characteristics such as wettability, chemistry, charge, rigidity, and roughness. In this study, we examined the behavior of MG63 osteoblast-like cells cultured on a polycarbonate (PC) membrane surfaces with different micropore sizes (0.2-8.0 microm in diameter). Cell adhesion and proliferation to the PC membrane surfaces were determined by cell counting and MTT assay. The effect of surface micropore on the MG63 cells was evaluated by cell morphology, protein content, and alkaline phosphatase (ALP) specific activity. It seems that the cell adhesion and proliferation were progressively inhibited as the PC membranes had micropores with increasing size, probably due to surface discontinuities produced by track-etched pores. Increasing micropore size of the PC membrane results in improved protein synthesis and ALP specific activity in isolated cells. There was a statistically significant difference (P<0.05) between different micropore sizes. The MG63 cells also maintained their phenotype under conditions that support a round cell shape. RT-PCR analysis further confirmed the osteogenic phenotype of the MG63 cells onto the PC membranes with different micropore sizes. In results, as micropore size is getting larger, cell number is reduced and cell differentiation and matrix production is increased. This study demonstrated that the surface topography plays an important role for phenotypic expression of the MG63 osteoblast-like cells.

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane

PubMed Central

Park, Se Won; Schonhoff, Christopher M.; Webster, Cynthia R. L.

2012-01-01

Cyclic AMP stimulates translocation of Na+/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation. PMID:22744337

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane.

PubMed

Park, Se Won; Schonhoff, Christopher M; Webster, Cynthia R L; Anwer, M Sawkat

2012-09-01

Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.

Live-cell confocal microscopy and quantitative 4D image analysis of anchor cell invasion through the basement membrane in C. elegans

PubMed Central

Kelley, Laura C.; Wang, Zheng; Hagedorn, Elliott J.; Wang, Lin; Shen, Wanqing; Lei, Shijun; Johnson, Sam A.; Sherwood, David R.

2018-01-01

Cell invasion through basement membrane (BM) barriers is crucial during development, leukocyte trafficking, and for the spread of cancer. Despite its importance in normal and diseased states, the mechanisms that direct invasion are poorly understood, in large part because of the inability to visualize dynamic cell-basement membrane interactions in vivo. This protocol describes multi-channel time-lapse confocal imaging of anchor cell invasion in live C. elegans. Methods presented include outline slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min), and quantitative analysis (variable timing). Images acquired enable direct measurement of invasive dynamics including invadopodia formation, cell membrane protrusions, and BM removal. This protocol can be combined with genetic analysis, molecular activity probes, and optogenetic approaches to uncover molecular mechanisms underlying cell invasion. These methods can also be readily adapted for real-time analysis of cell migration, basement membrane turnover, and cell membrane dynamics by any worm laboratory. PMID:28880279

Ellagitannin HeT obtained from strawberry leaves is oxidized by bacterial membranes and inhibits the respiratory chain.

PubMed

Martos, Gustavo G; Mamani, Alicia; Filippone, María P; Abate, Pedro O; Katz, Néstor E; Castagnaro, Atilio P; Díaz Ricci, Juan C

2018-02-01

Plant secondary metabolism produces a variety of tannins that have a wide range of biological activities, including activation of plant defenses and antimicrobial, anti-inflammatory and antitumoral effects. The ellagitannin HeT (1- O -galloyl-2,3;4,6-bis-hexahydroxydiphenoyl-β-d-glucopyranose) from strawberry leaves elicits a strong plant defense response, and exhibits antimicrobial activity associated to the inhibition of the oxygen consumption, but its mechanism of action is unknown. In this paper we investigate the influence of HeT on bacterial cell membrane integrity and its effect on respiration. A β-galactosidase unmasking experiment showed that HeT does not disrupt membrane integrity. Raman spectroscopy analysis revealed that HeT strongly interacts with the cell membrane. Spectrochemical analysis indicated that HeT is oxidized in contact with bacterial cell membranes, and functional studies showed that HeT inhibits oxygen consumption, NADH and MTT reduction. These results provide evidence that HeT inhibits the respiratory chain.

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

PubMed Central

Caiolfa, Valeria R.; Zamai, Moreno; Malengo, Gabriele; Andolfo, Annapaola; Madsen, Chris D.; Sutin, Jason; Digman, Michelle A.; Gratton, Enrico; Blasi, Francesco; Sidenius, Nicolai

2007-01-01

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents. PMID:18056417

Redesigned Spider Peptide with Improved Antimicrobial and Anticancer Properties.

PubMed

Troeira Henriques, Sónia; Lawrence, Nicole; Chaousis, Stephanie; Ravipati, Anjaneya S; Cheneval, Olivier; Benfield, Aurélie H; Elliott, Alysha G; Kavanagh, Angela Maria; Cooper, Matthew A; Chan, Lai Yue; Huang, Yen-Hua; Craik, David J

2017-09-15

Gomesin, a disulfide-rich antimicrobial peptide produced by the Brazilian spider Acanthoscurria gomesiana, has been shown to be potent against Gram-negative bacteria and to possess selective anticancer properties against melanoma cells. In a recent study, a backbone cyclized analogue of gomesin was shown to be as active but more stable than its native form. In the current study, we were interested in improving the antimicrobial properties of the cyclic gomesin, understanding its selectivity toward melanoma cells and elucidating its antimicrobial and anticancer mode of action. Rationally designed analogues of cyclic gomesin were examined for their antimicrobial potency, selectivity toward cancer cells, membrane-binding affinity, and ability to disrupt cell and model membranes. We improved the activity of cyclic gomesin by ∼10-fold against tested Gram-negative and Gram-positive bacteria without increasing toxicity to human red blood cells. In addition, we showed that gomesin and its analogues are more toxic toward melanoma and leukemia cells than toward red blood cells and act by selectively targeting and disrupting cancer cell membranes. Preference toward some cancer types is likely dependent on their different cell membrane properties. Our findings highlight the potential of peptides as antimicrobial and anticancer leads and the importance of selectively targeting cancer cell membranes for drug development.

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

Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to Simulated Food Processing Treatments, Determined Using Fluorescence-Activated Cell Sorting and Plate Counting▿

PubMed Central

Kennedy, Deirdre; Cronin, Ultan P.; Wilkinson, Martin G.

2011-01-01

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique. PMID:21602370

Membrane recycling at the infranuclear pole of the outer hair cell

NASA Astrophysics Data System (ADS)

Harasztosi, Csaba; Harasztosi, Emese; Gummer, Anthony W.

2015-12-01

Rapid endocytic activity of outer hair cells (OHCs) in the guinea-pig cochlea has been already studied using the fluorescent membrane marker FM1-43. It was demonstrated that vesicles were endocytosed at the apical pole of OHCs and transcytosed to the basolateral membrane and through a central strand towards the nucleus. The significance of endocytic activity in the infranuclear region is still not clear. Therefore, in this study endocytic activity at the synaptic pole of OHCs was investigated. Confocal laser scanning microscopy was used to visualize dye uptake of OHCs isolated from the guinea-pig cochlea. Signal intensity changes were quantified in the apical and basal poles relative to the signal at the membrane. Data showed no significant difference in fluorescent signal intensity changes between the opposite poles of the OHC. These results suggest that endocytic activities in both the basal and the apical poles contribute equally to the membrane recycling of OHCs.

Preserved function of the plasma membrane calcium pump of red blood cells from diabetic subjects with high levels of glycated haemoglobin.

PubMed

Bookchin, Robert M; Etzion, Zipora; Lew, Virgilio L; Tiffert, Teresa

2009-03-01

The activity of the plasma membrane Ca(2+)-pump decreases steeply throughout the 120 days lifespan of normal human red blood cells. Experiments with isolated membrane preparations showed that glycation of a lysine residue near the catalytic site of the pump ATPase had a powerful inhibitory effect. This prompted the question of whether glycation is the mechanism of age-related decline in pump activity in vivo. It is important to investigate this mechanism because the Ca(2+) pump is a major regulator of Ca(2+) homeostasis in all cells. Its impaired activity in diabetic patients, continuously exposed to high glycation rates, may thus contribute to varied tissue pathology in this disease. We measured Ca(2+)-pump activity as a function of red cell age in red cells from diabetics continuously exposed to high glucose concentrations, as documented by their high mean levels of glycated haemoglobin. The distribution of Ca(2+)-pump activities was indistinguishable from that in non-diabetics, and the pattern of activity decline with cell age in the diabetics' red cells was identical to that observed in red cells from non-diabetics. These results indicate that in intact cells the Ca(2+) pump is protected from glycation-induced inactivation.

Differential Activation of Acid Sphingomyelinase and Ceramide Release Determines Invasiveness of Neisseria meningitidis into Brain Endothelial Cells

PubMed Central

Simonis, Alexander; Hebling, Sabrina; Gulbins, Erich; Schneider-Schaulies, Sibylle; Schubert-Unkmeir, Alexandra

2014-01-01

The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains. PMID:24945304

75 FR 39667 - Availability for Non-Exclusive or Partially Exclusive Licensing of a U.S. Patent Application

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-12

..., which issued on December 15, 2009, entitled ``Use of Shigella Invaplex to Transport Functional Proteins... Functional Proteins and Transcriptionally Active Nucleic Acids Across Mammalian Cell Membranes In Vitro and... functional proteins and biologically active nucleic acids, across eukaryotic cell membranes. Brenda S. Bowen...

Anti-GM1 ganglioside antibodies modulate membrane-associated sphingomyelin metabolism by altering neutral sphingomyelinase activity.

PubMed

Ueda, Akihiro; Shima, Sayuri; Murate, Kenitiroh; Kikuchi, Kouichi; Nagao, Ryunosuke; Maeda, Toshiki; Muto, Eri; Niimi, Yoshiki; Mizutani, Yasuaki; Mutoh, Tatsuro

2018-06-01

Previous studies have shown that patients with Guillain-Barré syndrome express autoantibodies against ganglioside GM1 (GM1), although its pathogenic significance for the development of the disease remains to be elucidated. nSMase2 is the best characterized neutral sphingomyelinase (nSMase) found in neuronal cells. Activation of this enzyme leads to ceramide production, which is a known second messenger of the cell-death program in neuronal cells. We have explored the effects of anti-GM1 antibodies on sphingomyelin metabolism of PC12 cells stably transfected with human trk cDNA (PCtrk cells) by determining their effects on nSMase2 activity. The data we present here strongly suggest that anti-GM1 caused a significant change in sphingomyelin content of the membrane fraction in PCtrk cells. Both nSMase2 activity and the level of nSMase2 protein were significantly decreased by anti-GM1 treatment of PCtrk cells, while acidic SMase activities remained unchanged. Our results indicate, for the first time, that anti-GM1 may produce profound impacts on lipid metabolism in neuronal cell membranes. Copyright © 2018 Elsevier Inc. All rights reserved.

Hydroxyethyl cellulose hydrogel for wound dressing: Fabrication, characterization and in vitro evaluation.

PubMed

El Fawal, Gomaa F; Abu-Serie, Marwa M; Hassan, Mohamed A; Elnouby, Mohamed S

2018-05-01

In this study, new hydrogel membranes were developed based on hydroxyethyl cellulose (HEC) supplemented with tungsten oxide for further implementing in wound treatment. HEC hydrogel membranes were fabricated and crosslinked using citric acid (CA). Various tests were carried out including FTIR, XRD, porosity measurements, swelling, mechanical properties, gel fraction, and thermal gravimetric analysis to evaluate the efficiency of the prepared membranes as wound dressing material. In addition, wound healing activity of the examined membranes for human dermal fibroblast cell line was investigated employing in vitro scratching model. Furthermore, the potency of the prepared membranes to suppress wound complications was studied via determination of their anti-inflammatory and antibacterial activities exploiting MTT, ELISA, and disk agar diffusion methods. The results demonstrated that the HEC hydrogel membranes revealed an anti-inflammatory and antibacterial efficacy. Moreover, HEC improved the safety of tungsten oxide toward normal human cells (white blood cells and dermal fibroblast). Furthermore, HEC membranes loaded with WO 3 revealed the highest activities against Salmonella sp. pursued by P. aeruginosa in compared with the negative HEC hydrogel membrane. The current approach corroborated that HEC amended by tungsten oxide could be applied as a promising safe candidate for wound dressing material. Copyright © 2018 Elsevier B.V. All rights reserved.

The in vitro comparative study of the effect of BPA, BPS, BPF and BPAF on human erythrocyte membrane; perturbations in membrane fluidity, alterations in conformational state and damage to proteins, changes in ATP level and Na+/K+ ATPase and AChE activities.

PubMed

Maćczak, Aneta; Duchnowicz, Piotr; Sicińska, Paulina; Koter-Michalak, Maria; Bukowska, Bożena; Michałowicz, Jaromir

2017-12-01

Bisphenols are massively used in the industry, and thus the exposure of biota including humans to these substances has been noted. In this study we have assessed the effect of BPA and its selected analogs, i.e. BPS, BPF and BPAF on membrane of human red blood cells, which is the first barrier that must be overcome by xenobiotics penetrating the cell, and is commonly utilized as a model in the investigation of the effect of different xenobiotics on various cell types. Red blood cells were incubated with BPA and its analogs in the concentrations ranging from 0.1 to 250 μg/ml for 4 h and 24 h. We have noted that the compounds studied altered membrane fluidity at its hydrophobic region, increased internal viscosity and osmotic fragility of the erythrocytes and altered conformational state of membrane proteins. Moreover, bisphenols examined increased thiol groups level, caused oxidative damage to membrane proteins, decreased ATP level, depleted the activity of Na+/K + ATPase and changed the activity of AChE in human red blood cells. It has been shown that the strongest changes were noted in cells treated with BPAF, while BPS caused the weakest (or none) alterations in the parameters studied. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antibacterial activity and mode of action of ε-polylysine against Escherichia coli O157:H7.

PubMed

Zhang, Xiaowei; Shi, Ce; Liu, Zuojia; Pan, Fengguang; Meng, Rizeng; Bu, Xiujuan; Xing, Heqin; Deng, Yanhong; Guo, Na; Yu, Lu

2018-04-10

Gram-negative Escherichia coli O157:H7 were chosen as model bacteria to evaluate the antimicrobial mechanism of ε-polylysine (ε-PL). The antibacterial activity of ε-PL was detected by measuring the minimum inhibitory concentration values as well as the time-kill curve. The membrane integrity was determined by ultraviolet (UV) absorption, membrane potential (MP) assay and flow cytometry (FCM) experiments. The permeability of the inner membrane was detected by β-galactosidase activity assay. Furthermore, electron microscopy [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] was utilized to observe bacterial morphology. These results demonstrated that ε-PL showed its antibacterial activity by changing the integrity and permeability of cell membranes, leading to rapid cell death. The electron microscopy analysis (SEM and TEM) results indicated that the bacterial cell morphology, membrane integrity and permeability were spoiled when the E. coli O157:H7 cells were exposed to minimum inhibitory concentrations of ε-PL (16 µg ml -1 ). In addition, the bacterial membrane was damaged more severely when the concentration of ε-PL was increased. The present study investigated the antimicrobial mechanism of ε-PL by measuring the content of cytoplasmic β-galactosidase, proteins and DNA. In addition, SEM and TEM were carried out to assess the mechanism. These results show that ε-PL has the ability to decrease the content of large molecules, cellular soluble proteins and nucleic acids associated with increasing the content of cytoplasmic β-galactosidase in supernatant by causing damage to the cell membranes. Consequently, the use of ε-PL as a natural antimicrobial agent should eventually become an appealing method in the field of food preservation.

Effects of antibacterial mineral leachates on the cellular ultrastructure, morphology, and membrane integrity of Escherichia coli and methicillin-resistant Staphylococcus aureus

PubMed Central

2010-01-01

Background We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates. Methods We used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Baclight LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity. Results Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates. Conclusions These data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells. PMID:20846374

Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites

PubMed Central

1996-01-01

The protein ankyrin links integral membrane proteins to the spectrin- based membrane skeleton. Ankyrin is often concentrated within restricted membrane domains of polarized epithelia and neurons, but the mechanisms responsible for membrane targeting and its segregation within a continuous lipid bilayer remain unexplained. We provide evidence that neuroglian, a cell adhesion molecule related to L1 and neurofascin, can transmit positional information directly to ankyrin and thereby polarize its distribution in Drosophila S2 tissue culture cells. Ankyrin was not normally associated with the plasma membrane of these cells. Upon expression of an inducible neuroglian minigene, however, cells aggregated into large clusters and ankyrin became concentrated at sites of cell-cell contact. Spectrin was also recruited to sites of cell contact in response to neuroglian expression. The accumulation of ankyrin at cell contacts required the presence of the cytoplasmic domain of neuroglian since a glycosyl phosphatidylinositol- linked form of neuroglian failed to recruit ankyrin to sites of cell- cell contact. Double-labeling experiments revealed that, whereas ankyrin was strictly associated with sites of cell-cell contact, neuroglian was more broadly distributed over the cell surface. A direct interaction between neuroglian and ankyrin was demonstrated using yeast two-hybrid analysis. Thus, neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of cell contact and not with other regions of the plasma membrane. PMID:8636238

Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites.

PubMed

Dubreuil, R R; MacVicar, G; Dissanayake, S; Liu, C; Homer, D; Hortsch, M

1996-05-01

The protein ankyrin links integral membrane proteins to the spectrin-based membrane skeleton. Ankyrin is often concentrated within restricted membrane domains of polarized epithelia and neurons, but the mechanisms responsible for membrane targeting and its segregation within a continuous lipid bilayer remain unexplained. We provide evidence that neuroglian, a cell adhesion molecule related to L1 and neurofascin, can transmit positional information directly to ankyrin and thereby polarize its distribution in Drosophila S2 tissue culture cells. Ankyrin was not normally associated with the plasma membrane of these cells. Upon expression of an inducible neuroglian minigene, however, cells aggregated into large clusters and ankyrin became concentrated at sites of cell-cell contact. Spectrin was also recruited to sites of cell contact in response to neuroglian expression. The accumulation of ankyrin at cell contacts required the presence of the cytoplasmic domain of neuroglian since a glycosyl phosphatidylinositol-linked form of neuroglian failed to recruit ankyrin to sites of cell-cell contact. Double-labeling experiments revealed that, whereas ankyrin was strictly associated with sites of cell-cell contact, neuroglian was more broadly distributed over the cell surface. A direct interaction between neuroglian and ankyrin was demonstrated using yeast two-hybrid analysis. Thus, neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of cell contact and not with other regions of the plasma membrane.

Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor

NASA Technical Reports Server (NTRS)

Morre, D. James

2002-01-01

The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

Active processes make mixed lipid membranes either flat or crumpled

NASA Astrophysics Data System (ADS)

Banerjee, Tirthankar; Basu, Abhik

2018-01-01

Whether live cell membranes show miscibility phase transitions (MPTs), and if so, how they fluctuate near the transitions remain outstanding unresolved issues in physics and biology alike. Motivated by these questions we construct a generic hydrodynamic theory for lipid membranes that are active, due for instance, to the molecular motors in the surrounding cytoskeleton, or active protein components in the membrane itself. We use this to uncover a direct correspondence between membrane fluctuations and MPTs. Several testable predictions are made: (i) generic active stiffening with orientational long range order (flat membrane) or softening with crumpling of the membrane, controlled by the active tension and (ii) for mixed lipid membranes, capturing the nature of putative MPTs by measuring the membrane conformation fluctuations. Possibilities of both first and second order MPTs in mixed active membranes are argued for. Near second order MPTs, active stiffening (softening) manifests as a super-stiff (super-soft) membrane. Our predictions are testable in a variety of in vitro systems, e.g. live cytoskeletal extracts deposited on liposomes and lipid membranes containing active proteins embedded in a passive fluid.

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.

Effect of primycin on growth-arrested cultures and cell integrity of Staphylococcus aureus.

PubMed

Feiszt, Péter; Schneider, György; Emődy, Levente

2017-06-01

Bactericidal effect against non-dividing bacteria is a very advantageous, but rare characteristic among antimicrobial agents, mostly possessed by those affecting the cell membrane. These kinds of agents can kill bacterial cells without lysis. We assessed these characteristics on primycin, a topical anti-staphylococcal agent highly effective against prevalent multiresistant strains, as it also acts on the cell membrane. In time-kill studies, primycin preserved its bactericidal activity against growth-arrested Staphylococcus aureus cultures. The bactericidal action was slower against growth-arrested cultures compared to the exponentially growing ones to different extents depending on the manner of arrest. The bactericidal effect was less influenced by stringent response and by protein synthesis inhibition, proving that it does not depend on metabolic activity. In contrast, uncoupling of the membrane potential predominantly slowed, and low temperature almost stopped killing of bacteria. In consideration of published data, these facts suggest that the antibacterial action of primycin involves disrupting of the membrane potential, and is predominantly influenced by the membrane fluidity. Optical density measurements and transmission electron microscopy verified that primycin kills bacterial cells without lysis. These results reveal favorable characteristics of primycin and point to, and broaden the knowledge on its membrane-targeted effect.

Potassium Channels in Regulation of Vascular Smooth Muscle Contraction and Growth

PubMed Central

Jackson, William F.

2017-01-01

Potassium channels importantly contribute to the regulation of vascular smooth muscle (VSM) contraction and growth. They are the dominant ion conductance of the VSM cell membrane and importantly determine and regulate membrane potential. Membrane potential, in turn, regulates the open-state probability of voltage-gated Ca2+ channels (VGCC), Ca2+ influx through VGCC, intracellular Ca2+ and VSM contraction. Membrane potential also affects release of Ca2+ from internal stores and the Ca2+ sensitivity of the contractile machinery such that K+ channels participate in all aspects of regulation of VSM contraction. Potassium channels also regulate proliferation of VSM cells through membrane potential-dependent and membrane potential-independent mechanisms. Vascular smooth muscle cells express multiple isoforms of at least five classes of K+ channels contribute to the regulation of contraction and cell proliferation (growth). This review will examine the structure, expression and function of large-conductance, Ca2+-activated K+ (BKCa) channels, intermediate-conductance Ca2+-activated K+ (KCa3.1) channels, multiple isoforms of voltage-gated K+ (KV) channels, ATP-sensitive K+ (KATP) channels, and inward-rectifier K+ (KIR) channels in both contractile and proliferating VSM cells. PMID:28212804

Cell-autonomous defense, re-organization and trafficking of membranes in plant-microbe interactions.

PubMed

Dörmann, Peter; Kim, Hyeran; Ott, Thomas; Schulze-Lefert, Paul; Trujillo, Marco; Wewer, Vera; Hückelhoven, Ralph

2014-12-01

Plant cells dynamically change their architecture and molecular composition following encounters with beneficial or parasitic microbes, a process referred to as host cell reprogramming. Cell-autonomous defense reactions are typically polarized to the plant cell periphery underneath microbial contact sites, including de novo cell wall biosynthesis. Alternatively, host cell reprogramming converges in the biogenesis of membrane-enveloped compartments for accommodation of beneficial bacteria or invasive infection structures of filamentous microbes. Recent advances have revealed that, in response to microbial encounters, plasma membrane symmetry is broken, membrane tethering and SNARE complexes are recruited, lipid composition changes and plasma membrane-to-cytoskeleton signaling is activated, either for pre-invasive defense or for microbial entry. We provide a critical appraisal on recent studies with a focus on how plant cells re-structure membranes and the associated cytoskeleton in interactions with microbial pathogens, nitrogen-fixing rhizobia and mycorrhiza fungi. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Membrane peptidases in the pig choroid plexus and on other cell surfaces in contact with the cerebrospinal fluid.

PubMed Central

Bourne, A; Barnes, K; Taylor, B A; Turner, A J; Kenny, A J

1989-01-01

A comprehensive survey of 11 peptidases, all of which are markers for renal microvillar membranes, has been made in membrane fractions prepared from pig choroid plexus. Two fractionation schemes were explored, both depending on a MgCl2-precipitation step, the preferred one having advantages in speed and yield of the activities. The specific activities of the peptidases in the choroid-plexus membranes were, with the exception of carboxypeptidase M, lower than in renal microvillar membranes: those of aminopeptidase N, peptidyl dipeptidase A ('angiotensin-converting enzyme') and gamma-glutamyltransferase were 3-5-fold lower, those of aminopeptidase A and endopeptidase-24.11 were 12-15 fold lower, and those of dipeptidyl peptidase IV and aminopeptidase W were 50-70-fold lower. Carboxypeptidase M had a similar activity in both membranes. Alkaline phosphatase and (Na+ + K+)-activated ATPase were more active in the choroid-plexus membranes. No activity for microsomal dipeptidase, aminopeptidase P and carboxypeptidase P could be detected. Six of the peptidases and (Na+ + K+)-activated ATPase were also studied by immunoperoxidase histochemistry at light- and electron-microscopic levels. Endopeptidase-24.11 and (Na+ + K+)-activated ATPase were uniquely located on the brush border, and the other two peptidases appeared to be much more abundant on the endothelial lining of microvessels. Dipeptidyl peptidase IV and aminopeptidase W were also detected in microvasculature. Pial membranes associated with the brain and spinal cord also stained positively for endopeptidase-24.11, aminopeptidase N and peptidyl dipeptidase A. The immunohistochemical studies indicated the subcellular fractionation did not discriminate between membranes derived from epithelial cells (i.e. microvilli) and those from endothelial cells. The possible significance of these studies in relation to neuropeptide metabolism and the control of cerebrospinal fluid production is discussed. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2655579

Sterol-recognition ability and membrane-disrupting activity of Ornithogalum saponin OSW-1 and usual 3-O-glycosyl saponins.

PubMed

Malabed, Raymond; Hanashima, Shinya; Murata, Michio; Sakurai, Kaori

2017-12-01

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes

PubMed Central

Ohta, Naoki; Kato, Yasuhiko; Watanabe, Hajime; Mori, Hirotada; Matsuura, Tomoaki

2016-01-01

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach. PMID:27808179

Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.

PubMed

Biener, Eva; Charlier, Madia; Ramanujan, V Krishnan; Daniel, Nathalie; Eisenberg, Avital; Bjørbaek, Christian; Herman, Brian; Gertler, Arieh; Djiane, Jean

2005-12-01

Leptin, an adipocyte-secreted hormone, signals through activation of its membrane-embedded receptor (LEPR). To study the leptin-induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C-terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared. The constructs encoding mLEPRa (mouse LEPRa)-YFP and mLEPRa-CFP, mLEPRb-YFP and mLEPRb-CFP were tested for biological activity in transiently transfected CHO cells (Chinese-hamster ovary cells) and HEK-293T cells (human embryonic kidney 293 T cells) for activation of STAT3 (signal transduction and activators of transcription 3)-mediated LUC (luciferase) activity and binding of radiolabelled leptin. All four constructs were biologically active and were as potent as their untagged counterparts. The localization pattern of the fused protein appeared to be confined almost entirely to the cell membrane. The leptin-dependent interaction between various types of receptors in fixed cells were studied by measuring FRET, using fluorescence lifetime imaging microscopy and acceptor photobleaching methods. Both methods yielded similar results, indicating that (1) leptin receptors expressed in the cell membrane exist mostly as preformed LEPRa/LEPRa or LEPRb/LEPRb homo-oligomers but not as LEPRb/LEPRa hetero-oligomers; (2) the appearance of transient leptin-induced FRET in cells transfected with LEPRb/LEPRb reflects both a conformational change that leads to closer interaction in the cytosolic part and a higher FRET signal, as well as de novo homo-oligomerization; (3) in LEPRa/LEPRa, exposure to leptin does not lead to any increase in FRET signalling as the proximity of CFP and YFP fluorophores in space already gives maximal FRET efficiency of the preoligomerized receptors.

Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells.

PubMed

Manni, Marco M; Sot, Jesús; Goñi, Félix M

2015-03-01

Epsilon-toxin (ETX) is a powerful toxin produced by some strains of Clostridium perfringens (classified as types B and D) that is responsible for enterotoxemia in animals. ETX forms pores through the plasma membrane of eukaryotic cells, consisting of a β-barrel of 14 amphipathic β-strands. ETX shows a high specificity for certain cell lines, of which Madin-Darby canine kidney (MDCK) is the first sensitive cell line identified and the most studied one. The aim of this study was to establish the role of lipids in the toxicity caused by ETX and the correlation of its activity in model and biological membranes. In MDCK cells, using cell counting and confocal microscopy, we have observed that the toxin causes cell death mediated by toxin binding to plasma membrane. Moreover, ETX binds and permeabilizes the membranes of giant plasma membrane vesicles (GPMV). However, little effect is observed on protein-free vesicles. The data suggest the essential role of a protein receptor for the toxin in cell membranes. Copyright © 2014 Elsevier B.V. All rights reserved.

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Neurokinin 1 Receptor Mediates Membrane Blebbing and Sheer Stress-Induced Microparticle Formation in HEK293 Cells

PubMed Central

Chen, Panpan; Douglas, Steven D.; Meshki, John; Tuluc, Florin

2012-01-01

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing. PMID:23024816

Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle formation in HEK293 cells.

PubMed

Chen, Panpan; Douglas, Steven D; Meshki, John; Tuluc, Florin

2012-01-01

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2-10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.

Conditions of activation of yeast plasma membrane ATPase.

PubMed

Sychrová, H; Kotyk, A

1985-04-08

The in vivo activation of the H+-ATPase of baker's yeast plasma membrane found by Serrano in 1983 was demonstrated with D-glucose aerobically and anaerobically (as well as in a respiration-deficient mutant) and, after suitable induction, with maltose, trehalose, and galactose. The activated but not the control ATPase was sensitive to oligomycin. No activation was possible in a cell-free extract with added glucose. The ATPase was not activated in yeast protoplasts which may account for the absence of glucose-stimulated secondary active transports in these wall-less cells and provide support for a microscopic coupling between ATPase activity and these transports in yeast cells.

Membrane Tension Inhibits Rapid and Slow Endocytosis in Secretory Cells.

PubMed

Wu, Xin-Sheng; Elias, Sharon; Liu, Huisheng; Heureaux, Johanna; Wen, Peter J; Liu, Allen P; Kozlov, Michael M; Wu, Ling-Gang

2017-12-05

Endocytosis generates spherical or ellipsoid-like vesicles from the plasma membrane, which recycles vesicles that fuse with the plasma member during exocytosis in neurons and endocrine secretory cells. Although tension in the plasma membrane is generally considered to be an important factor in regulating endocytosis, whether membrane tension inhibits or facilitates endocytosis remains debated in the endocytosis field, and has been rarely studied for vesicular endocytosis in secretory cells. Here we report that increasing membrane tension by adjusting osmolarity inhibited both the rapid (a few seconds) and slow (tens of seconds) endocytosis in calyx-type nerve terminals containing conventional active zones and in neuroendocrine chromaffin cells. We address the mechanism of this phenomenon by computational modeling of the energy barrier that the system must overcome at the stage of membrane budding by an assembling protein coat. We show that this barrier grows with increasing tension, which may slow down or prevent membrane budding. These results suggest that in live secretory cells, membrane tension exerts inhibitory action on endocytosis. Published by Elsevier Inc.

Preparation and electrochemical characterization of gel polymer electrolyte based on electrospun polyacrylonitrile nonwoven membranes for lithium batteries

NASA Astrophysics Data System (ADS)

Raghavan, Prasanth; Manuel, James; Zhao, Xiaohui; Kim, Dul-Sun; Ahn, Jou-Hyeon; Nah, Changwoon

Electrospun membranes of polyacrylonitrile are prepared, and the electrospinning parameters are optimized to get fibrous membranes with uniform bead-free morphology. The polymer solution of 16 wt.% in N, N-dimethylformamide at an applied voltage of 20 kV results in the nanofibrous membrane with average fiber diameter of 350 nm and narrow fiber diameter distribution. Gel polymer electrolytes are prepared by activating the nonwoven membranes with different liquid electrolytes. The nanometer level fiber diameter and fully interconnected pore structure of the host polymer membranes facilitate easy penetration of the liquid electrolyte. The gel polymer electrolytes show high electrolyte uptake (>390%) and high ionic conductivity (>2 × 10 -3 S cm -1). The cell fabricated with the gel polymer electrolytes shows good interfacial stability and oxidation stability >4.7 V. Prototype coin cells with gel polymer electrolytes based on a membrane activated with 1 M LiPF 6 in ethylene carbonate/dimethyl carbonate or propylene carbonate are evaluated for discharge capacity and cycle property in Li/LiFePO 4 cells at room temperature. The cells show remarkably good cycle performance with high initial discharge properties and low capacity fade under continuous cycling.

Entorhinal stellate cells show preferred spike phase-locking to theta inputs that is enhanced by correlations in synaptic activity

PubMed Central

Fernandez, Fernando R.; Malerba, Paola; Bressloff, Paul C.; White, John A.

2013-01-01

In active networks, excitatory and inhibitory synaptic inputs generate membrane voltage fluctuations that drive spike activity in a probabilistic manner. Despite this, some cells in vivo show a strong propensity to precisely lock to the local field potential and maintain a specific spike-phase relationship relative to other cells. In recordings from rat medial entorhinal cortical stellate cells, we measured spike phase-locking in response to sinusoidal “test” inputs in the presence of different forms of background membrane voltage fluctuations, generated via dynamic clamp. We find that stellate cells show strong and robust spike phase-locking to theta (4–12 Hz) inputs. This response occurs under a wide variety of background membrane voltage fluctuation conditions that include a substantial increase in overall membrane conductance. Furthermore, the IH current present in stellate cells is critical to the enhanced spike phase-locking response at theta. Finally, we show that correlations between inhibitory and excitatory conductance fluctuations, which can arise through feed-back and feed-forward inhibition, can substantially enhance the spike phase-locking response. The enhancement in locking is a result of a selective reduction in the size of low frequency membrane voltage fluctuations due to cancelation of inhibitory and excitatory current fluctuations with correlations. Hence, our results demonstrate that stellate cells have a strong preference for spike phase-locking to theta band inputs and that the absolute magnitude of locking to theta can be modulated by the properties of background membrane voltage fluctuations. PMID:23554484

Recycling Endosomes of Polarized Epithelial Cells Actively Sort Apical and Basolateral Cargos into Separate Subdomains

PubMed Central

Thompson, Anthony; Nessler, Randy; Wisco, Dolora; Anderson, Eric; Winckler, Bettina

2007-01-01

The plasma membranes of epithelial cells plasma membranes contain distinct apical and basolateral domains that are critical for their polarized functions. However, both domains are continuously internalized, with proteins and lipids from each intermixing in supranuclear recycling endosomes (REs). To maintain polarity, REs must faithfully recycle membrane proteins back to the correct plasma membrane domains. We examined sorting within REs and found that apical and basolateral proteins were laterally segregated into subdomains of individual REs. Subdomains were absent in unpolarized cells and developed along with polarization. Subdomains were formed by an active sorting process within REs, which precedes the formation of AP-1B–dependent basolateral transport vesicles. Both the formation of subdomains and the fidelity of basolateral trafficking were dependent on PI3 kinase activity. This suggests that subdomain and transport vesicle formation occur as separate sorting steps and that both processes may contribute to sorting fidelity. PMID:17494872

Studies of the Mu-Opioid Receptor/G-protein Complex Affinity Co-Purified and Membrane Preparations from 7315c Cells

DTIC Science & Technology

1989-06-09

centrifugational sedimentation rates of the GTPys- activated and inactivated purified Gs (Birnbaumer et al., 1985). Recently, different antibodies raised against...and Membrane Preparations from 7315c Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e...were shown to be fused with the 1 membranes from turkey erythrocytes that have ~-adrenergic receptors but inactive adenylyl cyclase activity due to

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

Phorbol esters alter adenylate cyclase responses to vasoactive intestinal peptide and forskolin in the GH cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Summers, S.; Florio, T.; Cronin, M.

1986-05-01

Activation of protein kinase C with phorbol ester modifies cyclic AMP production in several anterior pituitary cell systems. In the GH cell line from a rat pituitary tumor, exposure to phorbol 12-myristate 13-acetate (PMA: 100 nM) for 30 minutes significantly reduces vasoactive intestinal peptide (VIP: 100 nM) stimulated adenylate cyclase (AC) activity in subsequent membrane preparations to 62 + 4% of control (n = 6 independent studies). In contrast, these same membrane preparations respond to forskolin (1 ..mu..M) with significantly more activity, 130 +/- 6% of controls (n = 6 independent studies). Finally, phorbol ester does not block an inhibitorymore » hormone input into the AC system; somatostatin (100 nM) reduction of VIP-stimulated AC activity is not significantly different in membrane preparations from PMA treated and control cells (n = 3 independent studies). These other findings lead the authors to propose that protein kinase C can modify several sites in the AC complex in anterior pituitary cells.« less

Evidence for the presence of a mammalian-like cholinesterase in Paramecium primaurelia (Protista, Ciliophora) developmental cycle.

PubMed

Delmonte Corrado, M U; Politi, H; Trielli, F; Angelini, C; Falugi, C

1999-01-01

By histochemical and immunohistochemical methods, the presence of cholinergic-like molecules has previously been demonstrated in Paramecium primaurelia, and their functional role in mating-cell pairing was suggested. In this work, both true acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were electrophoretically investigated, and the presence of molecules immunologically related to BuChE was checked by immunoblotting. The AChE activity, shown in the membrane protein fraction of mating-competent cells and in the cytoplasmic fraction of immature cells, is due to a 260-kDa molecular form, similar to the membrane-bound tetrameric form present in human erythrocytes. This AChE activity does not appear in either the cytoplasmic fraction of mating-competent cells or in the membrane protein fraction of immature cells. No evidence was found for the presence or the activity of BuChE-like molecules. The role of AChE in P. primaurelia developmental cycle is discussed.

Voltage-Gated Potassium Channels Kv1.3--Potentially New Molecular Target in Cancer Diagnostics and Therapy.

PubMed

Teisseyre, Andrzej; Gąsiorowska, Justyna; Michalak, Krystyna

2015-01-01

Voltage-gated potassium channels, Kv1.3, which were discovered in 1984, are integral membrane proteins which are activated ("open") upon change of the cell membrane potential, enabling a passive flux of potassium ions across the cell membrane. The channels are expressed in many different tissues, both normal and cancer. Since 2005 it has been known that the channels are expressed not only in the plasma membrane, but also in the inner mitochondrial membrane. The activity of Kv1.3 channels plays an important role, among others, in setting the cell resting membrane potential, cell proliferation, apoptosis and volume regulation. For some years, these channels have been considered a potentially new molecular target in both the diagnostics and therapy of some cancer diseases. This review article focuses on: 1) changes of expression of the channels in cancer disorders with special regard to correlations between the channels' expression and stage of the disease, 2) influence of inhibitors of Kv1.3 channels on proliferation and apoptosis of cancer cells, 3) possible future applications of Kv1.3 channels' inhibitors in therapy of some cancer diseases. In the last section, the results of studies performed in our Laboratory of Bioelectricity on the influence of selected biologically active plant-derived compounds from the groups of flavonoids and stilbenes and their natural and synthetic derivatives on the activity of Kv1.3 channels in normal and cancer cells are reviewed. A possible application of some compounds from these groups to support therapy of cancer diseases, such as breast, colon and lymph node cancer, and melanoma or chronic lymphocytic leukemia (B-CLL), is announced.

The Effects of Altered Membrane Cholesterol Levels on Sodium Pump Activity in Subclinical Hypothyroidism.

PubMed

Roy, Suparna; Dasgupta, Anindya

2017-03-01

Metabolic dysfunctions characteristic of overt hypothyroidism (OH) start at the early stage of subclinical hypothyroidism (SCH). Na⁺/K⁺-ATPase (the sodium pump) is a transmembrane enzyme that plays a vital role in cellular activities in combination with membrane lipids. We evaluated the effects of early changes in thyroid hormone and membrane cholesterol on sodium pump activity in SCH and OH patients. In 32 SCH patients, 35 OH patients, and 34 euthyroid patients, sodium pump activity and cholesterol levels in red blood cell membranes were measured. Serum thyroxine (T₄) and thyroid stimulating hormone (TSH) levels were measured using enzyme-linked immunosorbent assays. Differences in their mean values were analysed using post hoc analysis of variance. We assessed the dependence of the sodium pump on other metabolites by multiple regression analysis. Sodium pump activity and membrane cholesterol were lower in both hypothyroid groups than in control group, OH group exhibiting lower values than SCH group. In SCH group, sodium pump activity showed a significant direct dependence on membrane cholesterol with an inverse relationship with serum TSH levels. In OH group, sodium pump activity depended directly on membrane cholesterol and serum T₄ levels. No dependence on serum cholesterol was observed in either case. Despite the presence of elevated serum cholesterol in hypothyroidism, membrane cholesterol contributed significantly to maintain sodium pump activity in the cells. A critical reduction in membrane cholesterol levels heralds compromised enzyme activity, even in the early stage of hypothyroidism, and this can be predicted by elevated TSH levels alone, without any evident clinical manifestations. Copyright © 2017 Korean Endocrine Society

Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency.

PubMed

Yan, Feng; Zhu, Yiyong; Müller, Caroline; Zörb, Christian; Schubert, Sven

2002-05-01

White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.

THE LOCALIZATION OF ENZYME ACTIVITIES IN THE RAT BRAIN

PubMed Central

Becker, Norwin H.; Goldfischer, Sidney; Shin, Woo-Yung; Novikoff, Alex B.

1960-01-01

Studies with rat brain illustrate the usefulness of formol-calcium-fixed tissue for studying both enzymatic "chemoarchitectonics" and intracellular organelles. Unembedded frozen sections and polyvinyl alcohol-embedded sections may be used to demonstrate the activities of DPNH-tetrazolium reductase localized in mitochondria and ergastoplasm, TPNH-tetrazolium reductase localized in mitochondria, ATPase (and/or apyrase or ADPase) in cell membranes, and acid phosphatase in lysosomes.1 Among the observations recorded are: (1) the presence of lysosomes in all cells of the brain; (2) the presence of numerous large lysosomes near the nuclei of capillary endothelial cells; (3) a polarized arrangement of large lysosomes in epithelial cells of the ependyma and choroid plexus; (4) the presence of ATPase activity in the cell membranes of some neurons; (5) the presence of either an apyrase or combination of ATPase and ADPase in the cell membranes of neuroglia and capillaries; (6) the presence of both DPNH- and TPNH-tetrazolium reductase activities in neuroglia; (7) the presence of DPNH- and TPNH-tetrazolium reductase activities in mitochondria and of DPNH-tetrazolium reductase activity in Nissl substance. The possible functional significance of these localizations is briefly discussed, as is their relation to "quantitative histochemistry" data available in the literature. PMID:13688468

The localization of enzyme activities in the rat brain.

PubMed

BECKER, N H; GOLDFISCHER, S; SHIN, W Y; NOVIKOFF, A B

1960-12-01

Studies with rat brain illustrate the usefulness of formol-calcium-fixed tissue for studying both enzymatic "chemoarchitectonics" and intracellular organelles. Unembedded frozen sections and polyvinyl alcohol-embedded sections may be used to demonstrate the activities of DPNH-tetrazolium reductase localized in mitochondria and ergastoplasm, TPNH-tetrazolium reductase localized in mitochondria, ATPase (and/or apyrase or ADPase) in cell membranes, and acid phosphatase in lysosomes.(1) Among the observations recorded are: (1) the presence of lysosomes in all cells of the brain; (2) the presence of numerous large lysosomes near the nuclei of capillary endothelial cells; (3) a polarized arrangement of large lysosomes in epithelial cells of the ependyma and choroid plexus; (4) the presence of ATPase activity in the cell membranes of some neurons; (5) the presence of either an apyrase or combination of ATPase and ADPase in the cell membranes of neuroglia and capillaries; (6) the presence of both DPNH- and TPNH-tetrazolium reductase activities in neuroglia; (7) the presence of DPNH- and TPNH-tetrazolium reductase activities in mitochondria and of DPNH-tetrazolium reductase activity in Nissl substance. The possible functional significance of these localizations is briefly discussed, as is their relation to "quantitative histochemistry" data available in the literature.

Phosphatidylserine lipids and membrane order precisely regulate the activity of Polybia-MP1 peptide.

PubMed

Alvares, Dayane S; Ruggiero Neto, João; Ambroggio, Ernesto E

2017-06-01

Polybia-MP1 (IDWKKLLDAAKQIL-NH 2 ) is a lytic peptide from the Brazilian wasp venom with known anti-cancer properties. Previous evidence indicates that phosphatidylserine (PS) lipids are relevant for the lytic activity of MP1. In agreement with this requirement, phosphatidylserine lipids are translocated to the outer leaflet of cells, and are available for MP1 binding, depending on the presence of liquid-ordered domains. Here, we investigated the effect of PS on MP1 activity when this lipid is reconstituted in membranes of giant or large liposomes with different lipid-phase states. By monitoring the membrane and soluble luminal content of giant unilamellar vesicles (GUVs), using fluorescence confocal microscopy, we were able to determine that MP1 has a pore-forming activity at the membrane level. Liquid-ordered domains, which were phase-separated within the membrane of GUVs, influenced the pore-forming activity of MP1. Experiments evaluating the membrane-binding and lytic activity of MP1 on large unilamellar vesicles (LUVs), with the same lipid composition as GUVs, demonstrated that there was synergy between liquid-ordered domains and PS, which enhanced both activities. Based on our findings, we propose that the physicochemical properties of cancer cell membranes, which possess a much higher concentration of PS than normal cells, renders them susceptible to MP1 binding and lytic pore formation. These results can be correlated with MP1's potent and selective anti-cancer activity and pave the way for future research to develop cancer therapies that harness and exploit the properties of MP1. Copyright © 2017 Elsevier B.V. All rights reserved.

Roles of Specific Membrane Lipid Domains in EGF Receptor Activation and Cell Adhesion Molecule Stabilization in a Developing Olfactory System

PubMed Central

Gibson, Nicholas J.; Tolbert, Leslie P.; Oland, Lynne A.

2009-01-01

Background Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. Methodology/Principal Findings We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-β-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. Conclusions/Significance We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons. PMID:19787046

Manifestation of cryptic fibroblast tissue factor occurs at detergent concentrations which dissolve the plasma membrane.

PubMed

Carson, S D

1996-04-01

Cultured fibroblasts treated with increasing concentrations of detergents expressed only encrypted levels of tissue factor activity (measured by fX activation in the presence of fVIIa), characteristic of undamaged cells, until each detergent reached a critical concentration at which the cryptic tissue factor activity was manifested. Beyond the narrow ranges of concentrations over which the detergents stimulated tissue factor activity, the detergents were inhibitory. Studies with Triton X-100 and octyl glucoside revealed that manifestation of tissue factor activity coincided with breakdown of the plasma membrane. The magnitude of the increased tissue factor activity differed among detergents, with octyl glucoside giving the largest response. The tissue factor that was active after Triton X-100 treatment remained mostly associated with the insoluble cell residue, whereas the concentration of octyl glucoside which stimulated activity released tissue factor activity into the supernatant. Radiolabeled antibody against human tissue factor was used to show that a small percentage of the total accessible tissue factor remained in the insoluble fraction after treatment with either non-ionic detergent. Chromatographic analysis of lipids extracted from cells treated with detergents and dansyl chloride showed dansyl-reactivity of phosphatidylserine on intact cells, and solubilization of membrane lipids at sublytic concentrations of detergents. These findings reveal that there is a critical level of detergent-induced membrane damage at which tissue factor activity is maximally expressed, in essentially an all-or-none manner. The results are consistent with a major role for phospholipid asymmetry in regulation of tissue factor specific activity, but require either maintenance of asymmetry during sublytic detergent perturbation of the plasma membrane or additional control mechanisms.

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Membrane Capacitive Memory Alters Spiking in Neurons Described by the Fractional-Order Hodgkin-Huxley Model

PubMed Central

Weinberg, Seth H.

2015-01-01

Excitable cells and cell membranes are often modeled by the simple yet elegant parallel resistor-capacitor circuit. However, studies have shown that the passive properties of membranes may be more appropriately modeled with a non-ideal capacitor, in which the current-voltage relationship is given by a fractional-order derivative. Fractional-order membrane potential dynamics introduce capacitive memory effects, i.e., dynamics are influenced by a weighted sum of the membrane potential prior history. However, it is not clear to what extent fractional-order dynamics may alter the properties of active excitable cells. In this study, we investigate the spiking properties of the neuronal membrane patch, nerve axon, and neural networks described by the fractional-order Hodgkin-Huxley neuron model. We find that in the membrane patch model, as fractional-order decreases, i.e., a greater influence of membrane potential memory, peak sodium and potassium currents are altered, and spike frequency and amplitude are generally reduced. In the nerve axon, the velocity of spike propagation increases as fractional-order decreases, while in a neural network, electrical activity is more likely to cease for smaller fractional-order. Importantly, we demonstrate that the modulation of the peak ionic currents that occurs for reduced fractional-order alone fails to reproduce many of the key alterations in spiking properties, suggesting that membrane capacitive memory and fractional-order membrane potential dynamics are important and necessary to reproduce neuronal electrical activity. PMID:25970534

Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15-34), antimicrobial peptides from rattlesnake venom.

PubMed

Pérez-Peinado, Clara; Dias, Susana Almeida; Domingues, Marco M; Benfield, Aurélie H; Freire, João Miguel; Rádis-Baptista, Gandhi; Gaspar, Diana; Castanho, Miguel A R B; Craik, David J; Henriques, Sónia Troeira; Veiga, Ana Salomé; Andreu, David

2018-02-02

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

A high-performance polydimethylsiloxane electrospun membrane for cell culture in lab-on-a-chip.

PubMed

Moghadas, Hajar; Saidi, Mohammad Said; Kashaninejad, Navid; Nguyen, Nam-Trung

2018-03-01

Thin porous membranes are important components in a microfluidic device, serving as separators, filters, and scaffolds for cell culture. However, the fabrication and the integration of these membranes possess many challenges, which restrict their widespread applications. This paper reports a facile technique to fabricate robust membrane-embedded microfluidic devices. We integrated an electrospun membrane into a polydimethylsiloxane (PDMS) device using the simple plasma-activated bonding technique. To increase the flexibility of the membrane and to address the leakage problem, the electrospun membrane was fabricated with the highest weight ratio of PDMS to polymethylmethacrylate (i.e., 6:1 w/w). The membrane-integrated microfluidic device could withstand a flow rate of up to 50  μ l/min. As a proof of concept, we demonstrated that such a compartmentalized microfluidic platform could be successfully used for cell culture with the capability of providing a more realistic in vivo -like condition. Human lung cancer epithelial cells (A549) were seeded on the membrane from the top microchannel, while the continuous flow of the culture medium through the bottom microchannel provided a shear-free cell culture condition. The tortuous micro-/nanofibers of the membrane immobilized the cells within the hydrophobic micropores and with no need of extracellular matrix for cell adhesion and cell growth. The hydrophobic surface conditions of the membrane were suitable for anchorage-independent cell types. To further extend the application of the device, we qualitatively showed that rinsing the membrane with ethanol prior to cell seeding could temporarily render the membrane hydrophilic and the platform could also be used for anchorage-dependent cells. Due to the three-dimensional (3D) topography of the membranes, three different configurations were observed, including individual single cells, monolayer cells, and 3D cell clusters. This cost-effective and robust compartmentalized microfluidic device may open up new avenues in translational medicine and pharmacodynamics research.

Peroxiredoxin 6 in the repair of peroxidized cell membranes and cell signaling.

PubMed

Fisher, Aron B

2017-03-01

Peroxiredoxin 6 represents a widely distributed group of peroxiredoxins that contain a single conserved cysteine in the protein monomer (1-cys Prdx). The cys when oxidized to the sulfenic form is reduced with glutathione (GSH) catalyzed by the π isoform of GSH-S-transferase. Three enzymatic activities of the protein have been described:1) peroxidase with H 2 O 2 , short chain hydroperoxides, and phospholipid hydroperoxides as substrates; 2) phospholipase A 2 (PLA 2 ); and 3) lysophosphatidylcholine acyl transferase (LPCAT). These activities have important physiological roles in antioxidant defense, turnover of cellular phospholipids, and the generation of superoxide anion via initiation of the signaling cascade for activation of NADPH oxidase (type 2). The ability of Prdx6 to reduce peroxidized cell membrane phospholipids (peroxidase activity) and also to replace the oxidized sn-2 fatty acyl group through hydrolysis/reacylation (PLA 2 and LPCAT activities) provides a complete system for the repair of peroxidized cell membranes. Copyright © 2016 Elsevier Inc. All rights reserved.

Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes

NASA Astrophysics Data System (ADS)

Weinberger, Andreas; Walter, Vivien; MacEwan, Sarah R.; Schmatko, Tatiana; Muller, Pierre; Schroder, André P.; Chilkoti, Ashutosh; Marques, Carlos M.

2017-03-01

Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed on model giant unilamellar vesicles under a confocal laser scanning microscope show that a family of thermally responsive elastin-like polypeptides that exhibit temperature-triggered micellization can promote temperature triggered attachment of the micelles to membranes, thus rescuing by self-assembly the cargo-induced loss of the CPP affinity to bio-membranes.

Cholesterol depletion in cell membranes of human airway epithelial cells suppresses MUC5AC gene expression.

PubMed

Song, Kee Jae; Kim, Na Hyun; Lee, Gi Bong; Kim, Ji Hoon; Kwon, Jin Ho; Kim, Kyung-Su

2013-05-01

If cholesterol in the cell membrane is depleted by treating cells with methyl-β-cyclodextrin (MβCD), the activities of transmembrane receptors are altered in a cell-specific and/or receptor-specific manner. The proinflammatory cytokines, IL-1β is potent inducers of MUC5AC mRNA and protein synthesis in human airway epithelial cells. Cells activated by IL-1β showed increased phosphorylation of extracellular signal regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Thus, we investigated the effects of cholesterol depletion on the expression of MUC5AC in human airway epithelial cells and whether these alterations to MUC5AC expression were related to MAPK activity. After NCI-H292 cells were pretreated with 1% MβCD before adding IL-1β for 24 hours, MUC5AC mRNA expression was determined by reverse transcription- polymerase chain reaction (RT-PCR) and real time-PCR. Cholesterol depletion by MβCD was measured by modified microenzymatic fluorescence assay and filipin staining. The phosphorylation of IL-1 receptor, ERK and p38 MAPK, was analyzed by western blot. Cholesterol in the cell membrane was significantly depleted by treatment with MβCD on cells. IL-1β-induced MUC5AC mRNA expression was decreased by MβCD and this decrease occurred IL-1-receptor- specifically. Moreover, we have shown that MβCD suppressed the activation of ERK1/2 and p38 MAPK in cells activated with IL-1β. This result suggests that MβCD-mediated suppression of IL-1β-induced MUC5AC mRNA operated via the ERK- and p38 MAPK-dependent pathway. Cholesterol depletion in NCI-H292 cell membrane may be considered an anti-hypersecretory method since it effectively inhibits mucus secretion of respiratory epithelial cells.

Antimicrobial Activity of Ferulic Acid Against Cronobacter sakazakii and Possible Mechanism of Action.

PubMed

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

2016-04-01

Cronobacter sakazakii is an opportunistic pathogen transmitted by food that affects mainly newborns, infants, and immune-compromised adults. In this study, the antibacterial activity of ferulic acid was tested against C. sakazakii strains. Minimum inhibitory concentration of ferulic acid against C. sakazakii strains was determined using the agar dilution method. Changes in intracellular pH, membrane potential and intracellular ATP concentration were measured to elucidate the possible antibacterial mechanism. Moreover, SYTO 9 nucleic acid staining was used to assess the effect of ferulic acid on bacterial membrane integrity. Cell morphology changes were observed under a field emission scanning electron microscope. The minimum inhibitory concentrations of ferulic acid against C. sakazakii strains ranged from 2.5 to 5.0 mg/mL. Addition of ferulic acid exerted an immediate and sustained inhibition of C. sakazakii proliferation. Ferulic acid affected the membrane integrity of C. sakazakii, as evidenced by intracellular ATP concentration decrease. Moreover, reduction of intracellular pH and cell membrane hyperpolarization were detected in C. sakazakii after exposure to ferulic acid. Reduction of green fluorescence indicated the injury of cell membrane. Electronic microscopy confirmed that cell membrane of C. sakazakii was damaged by ferulic acid. Our results demonstrate that ferulic acid has moderate antimicrobial activity against C. sakazakii. It exerts its antimicrobial action partly through causing cell membrane dysfunction and changes in cellular morphology. Considering its antimicrobial properties, together with its well-known nutritional functions, ferulic acid has potential to be developed as a supplement in infant formula or other foods to control C. sakazakii.

Changes in membrane lipids drive increased endocytosis following Fas ligation.

PubMed

Degli Esposti, Mauro; Matarrese, Paola; Tinari, Antonella; Longo, Agostina; Recalchi, Serena; Khosravi-Far, Roya; Malorni, Walter; Misasi, Roberta; Garofalo, Tina; Sorice, Maurizio

2017-05-01

Once activated, some surface receptors promote membrane movements that open new portals of endocytosis, in part to facilitate the internalization of their activated complexes. The prototypic death receptor Fas (CD95/Apo1) promotes a wave of enhanced endocytosis that induces a transient intermixing of endosomes with mitochondria in cells that require mitochondria to amplify death signaling. This initiates a global alteration in membrane traffic that originates from changes in key membrane lipids occurring in the endoplasmic reticulum (ER). We have focused the current study on specific lipid changes occurring early after Fas ligation. We analyzed the interaction between endosomes and mitochondria in Jurkat T cells by nanospray-Time-of-flight (ToF) Mass Spectrometry. Immediately after Fas ligation, we found a transient wave of lipid changes that drives a subpopulation of early endosomes to merge with mitochondria. The earliest event appears to be a decrease of phosphatidylcholine (PC), linked to a metabolic switch enhancing phosphatidylinositol (PI) and phosphoinositides, which are crucial for the formation of vacuolar membranes and endocytosis. Lipid changes occur independently of caspase activation and appear to be exacerbated by caspase inhibition. Conversely, inhibition or compensation of PC deficiency attenuates endocytosis, endosome-mitochondria mixing and the induction of cell death. Deficiency of receptor interacting protein, RIP, also limits the specific changes in membrane lipids that are induced by Fas activation, with parallel reduction of endocytosis. Thus, Fas activation rapidly changes the interconversion of PC and PI, which then drives enhanced endocytosis, thus likely propagating death signaling from the cell surface to mitochondria and other organelles.

Influence of cell shape, inhomogeneities and diffusion barriers in cell polarization models

NASA Astrophysics Data System (ADS)

Giese, Wolfgang; Eigel, Martin; Westerheide, Sebastian; Engwer, Christian; Klipp, Edda

2015-12-01

In silico experiments bear the potential for further understanding of biological transport processes by allowing a systematic modification of any spatial property and providing immediate simulation results. Cell polarization and spatial reorganization of membrane proteins are fundamental for cell division, chemotaxis and morphogenesis. We chose the yeast Saccharomyces cerevisiae as an exemplary model system which entails the shuttling of small Rho GTPases such as Cdc42 and Rho, between an active membrane-bound form and an inactive cytosolic form. We used partial differential equations to describe the membrane-cytosol shuttling of proteins. In this study, a consistent extension of a class of 1D reaction-diffusion systems into higher space dimensions is suggested. The membrane is modeled as a thin layer to allow for lateral diffusion and the cytosol is modeled as an enclosed volume. Two well-known polarization mechanisms were considered. One shows the classical Turing-instability patterns, the other exhibits wave-pinning dynamics. For both models, we investigated how cell shape and diffusion barriers like septin structures or bud scars influence the formation of signaling molecule clusters and subsequent polarization. An extensive set of in silico experiments with different modeling hypotheses illustrated the dependence of cell polarization models on local membrane curvature, cell size and inhomogeneities on the membrane and in the cytosol. In particular, the results of our computer simulations suggested that for both mechanisms, local diffusion barriers on the membrane facilitate Rho GTPase aggregation, while diffusion barriers in the cytosol and cell protrusions limit spontaneous molecule aggregations of active Rho GTPase locally.

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

PubMed

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

2015-09-01

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

Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane.

PubMed

Filardo, E; Quinn, J; Pang, Y; Graeber, C; Shaw, S; Dong, J; Thomas, P

2007-07-01

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17beta-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17beta-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

Hydrodynamic collective effects of active protein machines in solution and lipid bilayers

PubMed Central

Mikhailov, Alexander S.; Kapral, Raymond

2015-01-01

The cytoplasm and biomembranes in biological cells contain large numbers of proteins that cyclically change their shapes. They are molecular machines that can function as molecular motors or carry out various other tasks in the cell. Many enzymes also undergo conformational changes within their turnover cycles. We analyze the advection effects that nonthermal fluctuating hydrodynamic flows induced by active proteins have on other passive molecules in solution or membranes. We show that the diffusion constants of passive particles are enhanced substantially. Furthermore, when gradients of active proteins are present, a chemotaxis-like drift of passive particles takes place. In lipid bilayers, the effects are strongly nonlocal, so that active inclusions in the entire membrane contribute to local diffusion enhancement and the drift. All active proteins in a biological cell or in a membrane contribute to such effects and all passive particles, and the proteins themselves, will be subject to them. PMID:26124140

Selective Antimicrobial Activities and Action Mechanism of Micelles Self-Assembled by Cationic Oligomeric Surfactants.

PubMed

Zhou, Chengcheng; Wang, Fengyan; Chen, Hui; Li, Meng; Qiao, Fulin; Liu, Zhang; Hou, Yanbo; Wu, Chunxian; Fan, Yaxun; Liu, Libing; Wang, Shu; Wang, Yilin

2016-02-17

This work reports that cationic micelles formed by cationic trimeric, tetrameric, and hexameric surfactants bearing amide moieties in spacers can efficiently kill Gram-negative E. coli with a very low minimum inhibitory concentration (1.70-0.93 μM), and do not cause obvious toxicity to mammalian cells at the concentrations used. With the increase of the oligomerization degree, the antibacterial activity of the oligomeric surfactants increases, i.e., hexameric surfactant > tetrameric surfactant > trimeric surfactant. Isothermal titration microcalorimetry, scanning electron microscopy, and zeta potential results reveal that the cationic micelles interact with the cell membrane of E. coli through two processes. First, the integrity of outer membrane of E. coli is disrupted by the electrostatic interaction of the cationic ammonium groups of the surfactants with anionic groups of E. coli, resulting in loss of the barrier function of the outer membrane. The inner membrane then is disintegrated by the hydrophobic interaction of the surfactant hydrocarbon chains with the hydrophobic domains of the inner membrane, leading to the cytoplast leakage. The formation of micelles of these cationic oligomeric surfactants at very low concentration enables more efficient interaction with bacterial cell membrane, which endows the oligomeric surfactants with high antibacterial activity.

Poliovirus Proteins Induce Membrane Association of GTPase ADP-Ribosylation Factor

PubMed Central

Belov, George A.; Fogg, Mark H.; Ehrenfeld, Ellie

2005-01-01

Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases. During normal membrane trafficking in uninfected cells, ARFs are involved in vesicle formation from different intracellular sites through interaction with numerous regulatory and coat proteins as well as in regulation of phospholipase D activity and cytoskeleton modifications. We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA. The accumulation of ARFs on membranes correlates with active replication of poliovirus RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of poliovirus 3A or 3CD proteins, and we describe mutations that abolished this activity. In infected HeLa cells, an ARF1-enhanced green fluorescent protein fusion redistributes from Golgi stacks to the perinuclear region, where poliovirus RNA replication occurs. Taken together, the data suggest an involvement of ARF in poliovirus RNA replication. PMID:15890959

Shape Changes and Interaction Mechanism of Escherichia coli Cells Treated with Sericin and Use of a Sericin-Based Hydrogel for Wound Healing

PubMed Central

Xue, Rui; Liu, Yalong; Liang, Congcong; Qin, Huazhen; Liu, Pengfei; Wang, Ke; Zhang, Xiaoyong; Chen, Li

2016-01-01

ABSTRACT To verify the interaction mechanism between sericin and Escherichia coli, especially the morphological and structural changes in the bacterial cells, the antimicrobial activity of sericin against E. coli as a model for Gram-negative bacteria was investigated. The antibacterial activity of sericin on E. coli and the interaction mechanism were investigated in this study by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with sericin. The changes in morphology and cellular compositions of bacterial cells treated with sericin were observed by an inverted fluorescence microscope, scanning electron microscopy, and transmission electron microscopy. Changes in electrical conductivity, total sugar concentration of the broth for the bacteria, and protein expression of the bacteria were determined to investigate the permeability of the cell membrane. A sericin-based hydrogel was prepared for an in vivo study of wound dressing. The results showed that the antibacterial activity of the hydrogel increased with the increase in the concentration of sericin from 10 g/liter to 40 g/liter. The introduction of sericin induces membrane blebbing of E. coli cells caused by antibiotic action on the cell membrane. The cytoplasm shrinkage phenomenon was accompanied by blurring of the membrane wall boundaries. When E. coli cells were treated with sericin, release of intracellular components quickly increased. The electrical conductivity assay indicated that the charged ions are reduced after exposure to sericin so that the integrity of the cell membrane is weakened and metabolism is blocked. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that sericin hinders the expression of bacterial protein. Sericin may damage the integrity of the bacterial cell membrane, thereby eventually inhibiting the growth and reproduction of E. coli. Compared to sterile gauze, the sericin-based hydrogel promoted fibroblast cell proliferation and accelerated the formation of granulation tissues and neovessels. IMPORTANCE The specific relationship and interaction mechanism between sericin and E. coli cells were investigated and elucidated. The results show that after 12 h of treatment, sericin molecules induce membrane blebbing of E. coli cells, and the bacteria show decreases in liquidity and permeability of biological membrane, resulting in alterations in the conductivity of the culture medium and the integrity of the outer membrane. The subsequent in vivo results demonstrate that the sericin-poly(N-isopropylacrylamide-N,N′-methylene-bis-acrylamide [NIPAm-MBA]) hydrogel accelerated wound healing compared to that with sterile gauze, which is a beneficial result for future applications in clinical medicine and the textile, food, and coating industries. PMID:27235427

A dual mechanism involved in membrane and nucleic acid disruption of AvBD103b, a new avian defensin from the king penguin, against Salmonella enteritidis CVCC3377.

PubMed

Teng, Da; Wang, Xiumin; Xi, Di; Mao, Ruoyu; Zhang, Yong; Guan, Qingfeng; Zhang, Jun; Wang, Jianhua

2014-10-01

The food-borne bacterial gastrointestinal infection is a serious public health threat. Defensins are evolutionarily conserved innate immune components with broad-spectrum antibacterial activity that do not easily induce resistance. AvBD103b, an avian defensin with potent activity against Salmonella enteritidis, was isolated from the stomach contents of the king penguin (Aptenodytes patagonicus). To elucidate further the antibacterial mechanism of AvBD103b, its effect on the S. enteritidis CVCC3377 cell membrane and intracellular DNA was researched. The cell surface hydrophobicity and a N-phenyl-1-naphthylamine uptake assay demonstrated that AvBD103b treatment increased the cell surface hydrophobicity and outer membrane permeability. Atomic absorption spectrometry, ultraviolet spectrophotometry, flow cytometry, and transmission electron microscopy (TEM) indicated that AvBD103b treatment can lead to the release of the cellular contents and cell death through damage of the membrane. DNA gel retardation and circular dichroism analysis demonstrated that AvBD103b interacted with DNA and intercalated into the DNA base pairs. A cell cycle assay demonstrated that AvBD103b affected cellular functions, such as DNA synthesis. Our results confirmed that AvBD103b exerts its antibacterial activity by damaging the cell membrane and interfering with intracellular DNA, ultimately causing cell death, and suggested that AvBD103b may be a promising candidate as an alternative to antibiotics against S. enteritidis.

Repair of Nerve Cell Membrance Damage by Calcium-Dependent, Membrane-Binding Proteins

DTIC Science & Technology

2013-09-01

In acute spinal cord injury the plasma membranes of spinal neurons are torn allowing high concentrations of calcium to enter the cytoplasm, activating...repairing the cell membrane as soon as the increase in intracellular calcium is sensed by calcium -binding proteins. If these repair mechanisms can be...testing the hypothesis that the action of copine, a human calcium -dependent-membrane-binding protein, in model systems can promote a stable repair of

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

PubMed

Christensen, Inga Baasch; Mogensen, Esben Nees; Damkier, Helle Hasager; Praetorius, Jeppe

2018-05-01

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na + -K + -ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP 2 ) staining was most prominent in the luminal membrane domain along with the PIP 3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.

Dynamic nanoplatforms in biosensor and membrane constitutional systems.

PubMed

Mahon, Eugene; Aastrup, Teodor; Barboiu, Mihail

2012-01-01

Molecular recognition in biological systems occurs mainly at interfacial environments such as membrane surfaces, enzyme active sites, or the interior of the DNA double helix. At the cell membrane surface, carbohydrate-protein recognition principles apply to a range of specific non-covalent interactions including immune response, cell proliferation, adhesion and death, cell-cell interaction and communication. Protein-protein recognition meanwhile accounts for signalling processes and ion channel structure. In this chapter we aim to describe such constitutional dynamic interfaces for biosensing and membrane transport applications. Constitutionally adaptive interfaces may mimic the recognition capabilities intrinsic to natural recognition processes. We present some recent examples of 2D and 3D constructed sensors and membranes of this type and describe their sensing and transport capabilities.

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

NASA Technical Reports Server (NTRS)

Kristjansson, H.; Hochstein, L. I.

1986-01-01

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

Activation Mobilizes the Cholesterol in the Late Endosomes-Lysosomes of Niemann Pick Type C Cells

PubMed Central

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

2012-01-01

A variety of intercalating amphipaths increase the chemical activity of plasma membrane cholesterol. To test whether intracellular cholesterol can be similarly activated, we examined NPC1 and NPC2 fibroblasts, since they accumulate large amounts of cholesterol in their late endosomes and lysosomes (LE/L). We gauged the mobility of intracellular sterol from its appearance at the surface of the intact cells, as determined by its susceptibility to cholesterol oxidase and its isotope exchange with extracellular 2-(hydroxypropyl)-β-cyclodextrin-cholesterol. The entire cytoplasmic cholesterol pool in these cells was mobile, exchanging with the plasma membrane with an apparent half-time of ∼3–4 hours, ∼4–5 times slower than that for wild type human fibroblasts (half-time ∼0.75 hours). The mobility of the intracellular cholesterol was increased by the membrane-intercalating amphipaths chlorpromazine and 1-octanol. Chlorpromazine also promoted the net transfer of LE/L cholesterol to serum and cyclodextrin. Surprisingly, the mobility of LE/L cholesterol was greatly stimulated by treating intact NPC cells with glutaraldehyde or formaldehyde. Similar effects were seen with wild type fibroblasts in which the LE/L cholesterol pool had been expanded using U18666A. We also showed that the cholesterol in the intracellular membranes of fixed wild-type fibroblasts was mobile; it was rapidly oxidized by cholesterol oxidase and was rapidly replenished by exogenous sterol. We conclude that a) the cholesterol in NPC cells can exit the LE/L (and the extensive membranous inclusions therein) over a few hours; b) this mobility is stimulated by the activation of the cholesterol with intercalating amphipaths; c) intracellular cholesterol is even more mobile in fixed cells; and d) amphipaths that activate cholesterol might be useful in treating NPC disease. PMID:22276143

Increased Degree of Unsaturation in the Lipid of Antifungal Cationic Amphiphiles Facilitates Selective Fungal Cell Disruption.

PubMed

Steinbuch, Kfir B; Benhamou, Raphael I; Levin, Lotan; Stein, Reuven; Fridman, Micha

2018-05-11

Antimicrobial cationic amphiphiles derived from aminoglycosides act through cell membrane permeabilization but have limited selectivity for microbial cell membranes. Herein, we report that an increased degree of unsaturation in the fatty acid segment of antifungal cationic amphiphiles derived from the aminoglycoside tobramycin significantly reduced toxicity to mammalian cells. A collection of tobramycin-derived cationic amphiphiles substituted with C 18 lipid chains varying in degree of unsaturation and double bond configuration were synthesized. All had potent activity against a panel of important fungal pathogens including strains with resistance to a variety of antifungal drugs. The tobramycin-derived cationic amphiphile substituted with linolenic acid with three cis double bonds (compound 6) was up to an order of magnitude less toxic to mammalian cells than cationic amphiphiles composed of lipids with a lower degree of unsaturation and than the fungal membrane disrupting drug amphotericin B. Compound 6 was 12-fold more selective (red blood cell hemolysis relative to antifungal activity) than compound 1, the derivative with a fully saturated lipid chain. Notably, compound 6 disrupted the membranes of fungal cells without affecting the viability of cocultured mammalian cells. This study demonstrates that the degree of unsaturation and the configuration of the double bond in lipids of cationic amphiphiles are important parameters that, if optimized, result in compounds with broad spectrum and potent antifungal activity as well as reduced toxicity toward mammalian cells.

Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity.

PubMed

López-Pérez, Luis; Martínez-Ballesta, María Del Carmen; Maurel, Christophe; Carvajal, Micaela

2009-03-01

Salinity stress is known to modify the plasma membrane lipid and protein composition of plant cells. In this work, we determined the effects of salt stress on the lipid composition of broccoli root plasma membrane vesicles and investigated how these changes could affect water transport via aquaporins. Brassica oleracea L. var. Italica plants treated with different levels of NaCl (0, 40 or 80mM) showed significant differences in sterol and fatty acid levels. Salinity increased linoleic (18:2) and linolenic (18:3) acids and stigmasterol, but decreased palmitoleic (16:1) and oleic (18:1) acids and sitosterol. Also, the unsaturation index increased with salinity. Salinity increased the expression of aquaporins of the PIP1 and PIP2 subfamilies and the activity of the plasma membrane H(+)-ATPase. However, there was no effect of NaCl on water permeability (P(f)) values of root plasma membrane vesicles, as determined by stopped-flow light scattering. The counteracting changes in lipid composition and aquaporin expression observed in NaCl-treated plants could allow to maintain the membrane permeability to water and a higher H(+)-ATPase activity, thereby helping to reduce partially the Na(+) concentration in the cytoplasm of the cell while maintaining water uptake via cell-to-cell pathways. We propose that the modification of lipid composition could affect membrane stability and the abundance or activity of plasma membrane proteins such as aquaporins or H(+)-ATPase. This would provide a mechanism for controlling water permeability and for acclimation to salinity stress.

Antifungal effect and pore-forming action of lactoferricin B like peptide derived from centipede Scolopendra subspinipes mutilans.

PubMed

Choi, Hyemin; Hwang, Jae-Sam; Lee, Dong Gun

2013-11-01

The centipede Scolopendra subspinipes mutilans has been a medically important arthropod species by using it as a traditional medicine for the treatment of various diseases. In this study, we derived a novel lactoferricin B like peptide (LBLP) from the whole bodies of adult centipedes, S. s. mutilans, and investigated the antifungal effect of LBLP. LBLP exerted an antifungal and fungicidal activity without hemolysis. To investigate the antifungal mechanism of LBLP, a membrane study with propidium iodide was first conducted against Candida albicans. The result showed that LBLP caused fungal membrane permeabilization. The assays of the three dimensional flow cytometric contour plot and membrane potential further showed cell shrinkage and membrane depolarization by the membrane damage. Finally, we confirmed the membrane-active mechanism of LBLP by synthesizing model membranes, calcein and FITC-dextran loaded large unilamellar vesicles. These results showed that the antifungal effect of LBLP on membrane was due to the formation of pores with radii between 0.74nm and 1.4nm. In conclusion, this study suggests that LBLP exerts a potent antifungal activity by pore formation in the membrane, eventually leading to fungal cell death. © 2013.

Activation of AMPK in human fetal membranes alleviates infection-induced expression of pro-inflammatory and pro-labour mediators.

PubMed

Lim, R; Barker, G; Lappas, M

2015-04-01

In non-gestational tissues, the activation of adenosine monophosphate (AMP)-activated kinase (AMPK) is associated with potent anti-inflammatory actions. Infection and/or inflammation, by stimulating pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9, play a central role in the rupture of fetal membranes. However, no studies have examined the role of AMPK in human labour. Fetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and after preterm pre-labour rupture of membranes (PPROM). AMPK activity was assessed by Western blotting of phosphorylated AMPK expression. To determine the effect of AMPK activators on pro-inflammatory cytokines, fetal membranes were pre-treated with AMPK activators then stimulated with bacterial products LPS and flagellin or viral dsDNA analogue poly(I:C). Primary amnion cells were used to determine the effect of AMPK activators on IL-1β-stimulated MMP-9 expression. AMPK activity was decreased with term labour. There was no effect of preterm labour. AMPK activity was also decreased in preterm fetal membranes, in the absence of labour, with PROM compared to intact membranes. AMPK activators AICAR, phenformin and A769662 significantly decreased IL-6 and IL-8 stimulated by LPS, flagellin and poly(I:C). Primary amnion cells treated with AMPK activators significantly decreased IL-1β-induced MMP-9 expression. The decrease in AMPK activity in fetal membranes after spontaneous term labour and PPROM indicates an anti-inflammatory role for AMPK in human labour and delivery. The use of AMPK activators as possible therapeutics for threatened preterm labour would be an exciting future avenue of research. Copyright © 2015 Elsevier Ltd. All rights reserved.

A role for the thermal environment in defining co-stimulation requirements for CD4+ T cell activation

PubMed Central

Zynda, Evan R; Grimm, Melissa J; Yuan, Min; Zhong, Lingwen; Mace, Thomas A; Capitano, Maegan; Ostberg, Julie R; Lee, Kelvin P; Pralle, Arnd; Repasky, Elizabeth A

2015-01-01

Maintenance of normal core body temperature is vigorously defended by long conserved, neurovascular homeostatic mechanisms that assist in heat dissipation during prolonged, heat generating exercise or exposure to warm environments. Moreover, during febrile episodes, body temperature can be significantly elevated for at least several hours at a time. Thus, as blood cells circulate throughout the body, physiologically relevant variations in surrounding tissue temperature can occur; moreover, shifts in core temperature occur during daily circadian cycles. This study has addressed the fundamental question of whether the threshold of stimulation needed to activate lymphocytes is influenced by temperature increases associated with physiologically relevant increases in temperature. We report that the need for co-stimulation of CD4+ T cells via CD28 ligation for the production of IL-2 is significantly reduced when cells are exposed to fever-range temperature. Moreover, even in the presence of sufficient CD28 ligation, provision of extra heat further increases IL-2 production. Additional in vivo and in vitro data (using both thermal and chemical modulation of membrane fluidity) support the hypothesis that the mechanism by which temperature modulates co-stimulation is linked to increases in membrane fluidity and membrane macromolecular clustering in the plasma membrane. Thermally-regulated changes in plasma membrane organization in response to physiological increases in temperature may assist in the geographical control of lymphocyte activation, i.e., stimulating activation in lymph nodes rather than in cooler surface regions, and further, may temporarily and reversibly enable CD4+ T cells to become more quickly and easily activated during times of infection during fever. PMID:26131730

Controlled permeation of cell membrane by single bubble acoustic cavitation

PubMed Central

Zhou, Y.; Yang, K.; Cui, J.; Ye, J. Y.; Deng, C. X.

2011-01-01

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustic, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 µs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 µm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 µs), and increased to 0.171 ± 0.030 µm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 µm (n=112) for a pulse duration of 40 µs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level. PMID:21945682

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.

Suppression of KV7/KCNQ potassium channel enhances neuronal differentiation of PC12 cells.

PubMed

Zhou, Najing; Huang, Sha; Li, Li; Huang, Dongyang; Yan, Yunli; Du, Xiaona; Zhang, Hailin

2016-10-01

Membrane potential shift driven by electrical activity is critical in determining the cell fate of proliferation or differentiation. As such, the ion channels that underlie the membrane electrical activity play an important role in cell proliferation/differentiation. KV7/KCNQ potassium channels are critical in determining the resting membrane potentials in many neuronal cells. However, the role of these channels in cell differentiation is not well studied. In the present study, we used PC12 cells as well as primary cultured rat cortical neurons to study the role and mechanism of KV7/KCNQ in neuronal differentiation. NGF induced PC12 cell differentiation into neuron-like cells with growth of neurites showing typical growth cone-like extensions. The Kv7/KCNQ blocker XE991 promoted NGF-induced neurite outgrowth, whereas Kv7/KCNQ opener retigabine (RTG) inhibited outgrowth. M-type Kv7 channels are likely involved in regulating neurite growth because overexpression of KCNQ2/Q3 inhibited neurite growth whereas suppression of KCNQ2/Q3 with shRNA promoted neurite growth. Membrane depolarization possibly underpins enhanced neurite growth induced by the suppression of Kv7/KCNQ. Additionally, high extracellular K(+) likely induced membrane depolarization and also promoted neurite growth. Finally, T-type Ca(2+) channels may be involved in membrane-depolarization-induced neurite growth. This study provides a new perspective for understanding neuronal differentiation as well as KV7/KCNQ channel function. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

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

Anti-methicillin-resistance Staphylococcus aureus (MRSA) compounds from Bauhinia kockiana Korth. And their mechanism of antibacterial activity.

PubMed

Chew, Yik Ling; Mahadi, Adlina Maisarah; Wong, Kak Ming; Goh, Joo Kheng

2018-02-20

Bauhinia kockiana originates from Peninsular Malaysia and it is grown as a garden ornamental plant. Our previous study reported that this plant exhibited fairly strong antioxidant and antimicrobial activities. This paper focused on the assessment of the antibacterial activity of B. kockiana towards methicillin-resistance Staphylococcus aureus (MRSA), to purify and to identify the antibacterial compounds, and to determine the mechanism of antibacterial activity. Antibacterial activity of B. kockiana flower was evaluated qualitatively and quantitatively using disc diffusion assay and microbroth dilution method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of extracts were examined. Phytochemical analysis was performed to determine the classes of phytochemicals in the extracts. Bioactivity guided isolation was employed to purify the antibacterial agents and identified via various spectroscopy methods. Scanning electron microscopy (SEM) technique was used to evaluate the antibacterial mechanism of extract and compounds isolated. B. kockiana flower was found to exhibit fairly strong antibacterial activity towards both strains of MRSA bacteria used, MIC varies from 62.5-250 μg/mL. Tannins and flavonoids have been detected in the phytochemical analysis. Gallic acid and its ester derivatives purified from ethyl acetate extract could inhibit MRSA at 250-500 μg/mL. SEM revealed that the cells have undergone plasmolysis upon treatment with the extract and compounds. Tannins and polyphenols are the antibacterial components towards MRSA in B. kockiana. Massive leakage of the cell content observed in treated cells showed that the phytochemicals have changed the properties of the cell membranes. Amphiphilic nature of the compounds exhibited the antibacterial activity towards MRSA via three stages: (1) cell membrane attachment; (2) cell membrane fluidity modification; and (3) cell membrane structure disruption.

Using 1H2O MR to measure and map sodium pump activity in vivo

NASA Astrophysics Data System (ADS)

Springer, Charles S.

2018-06-01

The cell plasma membrane Na+,K+-ATPase [NKA] is one of biology's most [if not the most] significant enzymes. By actively transporting Na+ out [and K+ in], it maintains the vital trans-membrane ion concentration gradients and the membrane potential. The forward NKA reaction is shown in the Graphical Abstract [which is elaborated in the text]. Crucially, NKA does not operate in isolation. There are other transporters that conduct K+ back out of [II, Graphical Abstract] and Na+ back into [III, Graphical Abstract] the cell. Thus, NKA must function continually. Principal routes for ATP replenishment include mitochondrial oxidative phosphorylation, glycolysis, and creatine kinase [CrK] activity. However, it has never been possible to measure, let alone map, this integrated, cellular homeostatic NKA activity in vivo. Active trans-membrane water cycling [AWC] promises a way to do this with 1H2O MR. In the Graphical Abstract, the AWC system is characterized by active contributions to the unidirectional rate constants for steady-state water efflux and influx, respectively, kio(a) and koi(a). The discovery, validation, and initial exploration of active water cycling are reviewed here. Promising applications in cancer, cardiological, and neurological MRI are covered. This initial work employed paramagnetic Gd(III) chelate contrast agents [CAs]. However, the significant problems associated with in vivo CA use are also reviewed. A new analysis of water diffusion-weighted MRI [DWI] is presented. Preliminary results suggest a non-invasive way to measure the cell number density [ρ (cells/μL)], the mean cell volume [V (pL)], and the cellular NKA metabolic rate [cMRNKA (fmol(ATP)/s/cell)] with high spatial resolution. These crucial cell biology properties have not before been accessible in vivo. Furthermore, initial findings indicate their absolute values can be determined.

Membrane of Candida albicans as a target of berberine.

PubMed

Zorić, Nataša; Kosalec, Ivan; Tomić, Siniša; Bobnjarić, Ivan; Jug, Mario; Vlainić, Toni; Vlainić, Josipa

2017-05-17

We investigated the mechanisms of anti-Candida action of isoquinoline alkaloid berberine, active constituent of medically important plants of Barberry species. The effects on membrane, morphological transition, synthesis of ergosterol and the consequent changes in membrane permeability have been studied. Polarization and lipid peroxidation level of the membrane following berberine treatment have been addressed. Minimal inhibitory concentration (MIC) of berberine against C. albicans was 17.75 μg/mL. Cytotoxic effect of berberine was concentration dependent, and in sub-MIC concentrations inhibit morphological transition of C. albicans cells to its filamentous form. Results showed that berberine affects synthesis of membrane ergosterol dose-dependently and induces increased membrane permeability causing loss of intracellular material to the outer space (DNA/protein leakage). Berberine also caused membrane depolarization and lipid peroxidation of membrane constituents indicating its direct effect on the membrane. Moreover, ROS levels were also increased following berberine treatment indicating further the possibility of membrane damage. Based on the obtained results it seems that berberine achieves its anti-Candida activity by affecting the cell membrane.

On the role of adenylate cyclase, tyrosine kinase, and tyrosine phosphatase in the response of nerve and glial cells to photodynamic impact

NASA Astrophysics Data System (ADS)

Kolosov, Mikhail S.; Bragin, D. E.; Dergacheva, Olga Y.; Vanzha, O.; Oparina, L.; Uzdensky, Anatoly B.

2004-08-01

The role of different intercellular signaling pathways involving adenylate cyclase (AC), receptor tyrosine kinase (RTK), tyrosine and serine/threonine protein phosphatases (PTP or PP, respectively) in the response of crayfish mechanoreceptor neuron (MRN) and surrounding glial cells to photodynamic effect of aluminum phthalocyanine Photosens have been studied. AC inhibition by MDL-12330A decreased neuron lifetime, whereas AC activation by forskolin increase it. Thus, increase in cAMP produced by activated AC protects SRN against photodynamic inactivation. Similarly, RTK inhibition by genistein decreased neuron lifetime, while inhibition of PTP or PP that remove phosphate groups from proteins, prolonged neuronal activity. AC inhibition reduced photoinduced damage of the plasma membrane, and, therefore, necrosis in neuronal and glial cells. RTK inhibition protected only neurons against PDT-induced membrane permeabilization while glial cells became lesser permeable under ortovanadate-mediated PTP inhibition. AC activation also prevented PDT-induced apoptosis in glial cells. PP inhibition enhanced apoptotic processes in photosensitized glial cells. Therefore, both intercellular signaling pathways involving AC and TRK are involved in the maintenance of neuronal activity, integrity of the neuronal and glial plasma membranes and in apoptotic processes in glia under photosensitization.

The Chemical Potential of Plasma Membrane Cholesterol: Implications for Cell Biology.

PubMed

Ayuyan, Artem G; Cohen, Fredric S

2018-02-27

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

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation

PubMed Central

Naegeli, Kaleb M.; Chi, Qiuyi; Ziel, Joshua W.; Hagedorn, Elliott J.; Park, Jieun E.; Jayadev, Ranjay; Sherwood, David R.

2016-01-01

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue. PMID:26765257

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

PubMed

Lohmer, Lauren L; Clay, Matthew R; Naegeli, Kaleb M; Chi, Qiuyi; Ziel, Joshua W; Hagedorn, Elliott J; Park, Jieun E; Jayadev, Ranjay; Sherwood, David R

2016-01-01

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus.

PubMed

Meier, Elizabeth L; Razavi, Shiva; Inoue, Takanari; Goley, Erin D

2016-07-01

In most bacteria, the tubulin-like GTPase FtsZ forms an annulus at midcell (the Z-ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z-ring assembly and early FtsZ-directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C-terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane-anchored FtsZ in the regulation of cell wall hydrolysis. © 2016 John Wiley & Sons Ltd.

Decreased membrane potassium permeability and transport in human chronic leukemic and tonsillar lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Segel, G.B.; Lichtman, M.A.

Human blood T-lymphocytes increase their potassium (K/sup +/) permeability and active K/sup +/ transport following lectin or antigen stimulation. We have studied the permeability and active transport of K/sup +/ by lymphocytes in chronic lymphocytic leukemia (CLL) to determine if their membrane K/sup +/ transport was similar to resting or lectin-stimulated normal blood lymphocytes. K/sup +/ transport was assessed both by the rate of isotopic /sup 42/K/sup +/ uptake and by the rate of change in cell K/sup +/ concentration after inhibition of the K/sup +/ transport system with ouabain. CLL lymphocytes had a marked decrease in membrane K/sup +/more » permeability and active transport of K/sup +/ when compared to blood T lymphocytes. K/sup +/ transport in five subjects with CLL (10 mmol . 1 cell water/sup -1/ . h/sup -1/) was half that in normal blood T-lymphocytes (20 mmol . 1 cell water/sup -1/ h/sup -1/). Phytohemagglutinin (PHA) treatment of CLL lymphocytes did not increase significantly their active K/sup +/ transport, whereas K/sup +/ transport by normal T-lymphocytes increased by 100%. Since there were 73% T-lymphocytes in normal blood and 14% in CLL blood, the difference in membrane K/sup +/ turnover could be related either to neoplasia or to the proposed B-lymphocyte origin of CLL. We studied human tonsillar lymphocytes which contained a mean of 34% T-cells. In five studies of tonsils, K/sup +/ transport was 14 mmol . 1 cell water/sup -1/ . h/sup -1/ and treatment with PHA increased K/sup +/ transport only 30%. The intermediate values for basal K/sup +/ transport and K/sup +/ transport in response to PHA in tonsillar lymphocytes were consistent with the proportion of T-lymphocytes present. These data sugges t that B-lymphocytes have reduced membrane permeability and active transport of K/sup +/. Thus the marked decrease in CLL lymphocyte membrane K/sup +/ permeability and transport may be a reflection of its presumed B-cell origin, rather than a membrane alteration related to malignant transformation.« less

Influence of cell integrity on textural properties of raw, high pressure, and thermally processed onions.

PubMed

Gonzalez, M E; Jernstedt, J A; Slaughter, D C; Barrett, D M

2010-09-01

The integrity of onion cells and its impact on tissue texture after high pressure and thermal processing was studied. The contribution of cell membranes and the pectic component of cell walls on the texture properties of onion tissue were analyzed. Neutral red (NR) staining of onion parenchyma cell vacuoles was used for the evaluation of cell membrane integrity and microscopic image analysis was used for its quantification. The content of methanol in tissue as a result of pectin methylesterase activity was used to evaluate the pectin component of the middle lamella and cell walls and the hardening effect on the tissue after processing. High pressure treatments consisted of 5-min holding times at 50, 100, 200, 300, or 600 MPa. Thermal treatments consisted of 30-min water bath exposure to 40, 50, 60, 70, or 90 °C. In the high pressure treatments, loss of membrane integrity commenced at 200 MPa and total loss of membrane integrity occurred at 300 MPa and above. In the thermal treatments, membrane integrity was lost between 50 and 60 °C. The texture of onions was influenced by the state of the membranes and texture profiles were abruptly modified once membrane integrity was lost. Hardening of the tissue corresponded with pressure and temperature PME activation and occurred after membrane integrity loss. The texture of vegetables is an important quality attribute that affects consumer preference. Loss of textural integrity also indicates that other biochemical reactions that affect color, flavor, and nutrient content may occur more rapidly. In this study, we analyzed changes in the texture of onions after preservation with heat and high pressure.

Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

NASA Astrophysics Data System (ADS)

Carlsson, Anders

Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

The antimicrobial peptide, lactoferricin B, is cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in vivo.

PubMed

Eliassen, Liv Tone; Berge, Gerd; Leknessund, Arild; Wikman, Mari; Lindin, Inger; Løkke, Cecilie; Ponthan, Frida; Johnsen, John Inge; Sveinbjørnsson, Baldur; Kogner, Per; Flaegstad, Trond; Rekdal, Øystein

2006-08-01

Antimicrobial peptides have been shown to exert cytotoxic activity towards cancer cells through their ability to interact with negatively charged cell membranes. In this study the cytotoxic effect of the antimicrobial peptide, LfcinB was tested in a panel of human neuroblastoma cell lines. LfcinB displayed a selective cytotoxic activity against both MYCN-amplified and non-MYCN-amplified cell lines. Non-transformed fibroblasts were not substantially affected by LfcinB. Treatment of neuroblastoma cells with LfcinB induced rapid destabilization of the cytoplasmic membrane and formation of membrane blebs. Depolarization of the mitochondria membranes and irreversible changes in the mitochondria morphology was also evident. Immuno- and fluorescence-labeled LfcinB revealed that the peptide co-localized with mitochondria. Furthermore, treatment of neuroblastoma cells with LfcinB induced cleavage of caspase-6, -7 and -9 followed by cell death. However, neither addition of the pan-caspase inhibitor, zVAD-fmk, or specific caspase inhibitors could reverse the cytotoxic effect induced by LfcinB. Treatment of established SH-SY-5Y neuroblastoma xenografts with repeated injections of LfcinB resulted in significant tumor growth inhibition. These results revealed a selective destabilizing effect of LfcinB on two important targets in the neuroblastoma cells, the cytoplasmic- and the mitochondria membrane. Copyright (c) 2006 Wiley-Liss, Inc.

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.

Combined fluorimetric caspase 3/7 assay and bradford protein determination for assessment of polycation-mediated cytotoxicity.

PubMed

Larsen, Anna K; Hall, Arnaldur; Lundsgart, Henrik; Moghimi, S Moein

2013-01-01

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

Evaluation of the antioxidants activities of four Slovene medicinal plant species by traditional and novel biosensory assays.

PubMed

Kintzios, Spiridon; Papageorgiou, Katerina; Yiakoumettis, Iakovos; Baricevic, Dea; Kusar, Anita

2010-11-02

We investigated the antioxidant activity of methanolic and water extracts of Slovene accessions of four medicinal plant species (Salvia officinalis, Achillea millefolium, Origanum vulgare subsp. vulgare and Gentiana lutea). Their free radical-scavenging activity against the DPPH. free radical was studied with a spectrophotometric assay, while their biological activity with the help of a laboratory-made biosensor based on immobilized fibroblast cells (assay duration: 3 min). The observed antioxidant activity of the extracts from the four investigated medicinal plant species was dependent on both the solvent used for extraction and the assay method (conventional or biosensor-based). Independently from the assay method and the solvent used for extraction, the lowest scavenging activity was observed in root extracts of G. lutea. Treatment of the immobilized cells with the plant extracts resulted in an increase of the cell membrane potential (membrane hyperpolarization), possibly due to the reduction of membrane damage due to oxidation. The novel cell biosensor could be utilized as a rapid, high throughput tool for screening the antioxidant properties of plant-derived compounds. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Potassium accumulation by the glial membrane pump as revealed by membrane potential recording from isolated rabbit retinal Müller cells.

PubMed

Reichenbach, A; Nilius, B; Eberhardt, W

1986-01-30

Müller (glial) cells were isolated from rabbit retinae by papaine and mechanical dissociation. In a special perfusion chamber, the cells were penetrated with a recording electrode. When high-K+ solutions were applied into the environment of the cells by means of a second micropipette, the cell membrane depolarized strongly. During prolonged application of high-K+ solutions, however, there occurred a marked repolarization, and after cessation of high-K+ application, a strong hyperpolarization was observed. Both effects disappeared under the influence of ouabain, suggesting the accumulation of intracellular K+ by an active membrane pump. The data were used for calculation of the membrane's Na+:K+ permeability ratio, the intracellular K+ concentration, the pump rate and the mean pump site density. The calculated values are in good agreement with published data from mammalian astrocytes and are compared with those from amphibian Müller cells.

Transport across the cell-membrane dictates nanoparticle fate and toxicity: a new paradigm in nanotoxicology

NASA Astrophysics Data System (ADS)

Guarnieri, Daniela; Sabella, Stefania; Muscetti, Ornella; Belli, Valentina; Malvindi, Maria Ada; Fusco, Sabato; de Luca, Elisa; Pompa, Pier Paolo; Netti, Paolo A.

2014-08-01

The toxicity of metallic nanoparticles (MNPs) has been fully ascertained, but the mechanisms underlying their cytotoxicity remain still largely unclear. Here we demonstrate that the cytotoxicity of MNPs is strictly reliant on the pathway of cellular internalization. In particular, if otherwise toxic gold, silver, and iron oxide NPs are forced through the cell membrane bypassing any form of active mechanism (e.g., endocytosis), no significant cytotoxic effect is registered. Pneumatically driven NPs across the cell membrane show a different distribution within the cytosol compared to NPs entering the cell by active endocytosis. Specifically, they exhibit free random Brownian motions within the cytosol and do not accumulate in lysosomes. Results suggest that intracellular accumulation of metallic nanoparticles into endo-lysosomal compartments is the leading cause of nanotoxicity, due to consequent nanoparticle degradation and in situ release of metal ions.The toxicity of metallic nanoparticles (MNPs) has been fully ascertained, but the mechanisms underlying their cytotoxicity remain still largely unclear. Here we demonstrate that the cytotoxicity of MNPs is strictly reliant on the pathway of cellular internalization. In particular, if otherwise toxic gold, silver, and iron oxide NPs are forced through the cell membrane bypassing any form of active mechanism (e.g., endocytosis), no significant cytotoxic effect is registered. Pneumatically driven NPs across the cell membrane show a different distribution within the cytosol compared to NPs entering the cell by active endocytosis. Specifically, they exhibit free random Brownian motions within the cytosol and do not accumulate in lysosomes. Results suggest that intracellular accumulation of metallic nanoparticles into endo-lysosomal compartments is the leading cause of nanotoxicity, due to consequent nanoparticle degradation and in situ release of metal ions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr02008a

Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells.

PubMed

Migocka, Magdalena; Papierniak, Anna; Kosatka, Ewelina; Klobus, Grazyna

2011-10-01

The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

Simultaneous screening of four epidermal growth factor receptor antagonists from Curcuma longa via cell membrane chromatography online coupled with HPLC-MS.

PubMed

Sun, Meng; Ma, Wei-na; Guo, Ying; Hu, Zhi-gang; He, Lang-chong

2013-07-01

The epidermal growth factor receptors (EGFRs) are significant targets for screening active compounds. In this work, an analytical method was established for rapid screening, separation, and identification of EGFRs antagonists from Curcuma longa. Human embryonic kidney 293 cells with a steadily high expression of EGFRs were used to prepare the cell membrane stationary phase in a cell membrane chromatography model for screening active compounds. Separation and identification of the retention chromatographic peaks was achieved by HPLC-MS. The active sites, docking extents and inhibitory effects of the active compounds were also demonstrated. The screening result found that ar-turmerone, curcumin, demethoxycurcumin, and bisdemethoxycurcumin from Curcuma longa could be active components in a similar manner to gefitinib. Biological trials showed that all of four compounds can inhibit EGFRs protein secretion and cell growth in a dose-dependent manner, and downregulate the phosphorylation of EGFRs. This analytical method demonstrated fast and effective characteristics for screening, separation and identification of the active compounds from a complex system and should be useful for drug discovery with natural medicinal herbs. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation of sulfonated polysulfone membranes as electrolyte in a passive-mode direct methanol fuel cell mini-stack

NASA Astrophysics Data System (ADS)

Lufrano, F.; Baglio, V.; Staiti, P.; Stassi, A.; Aricò, A. S.; Antonucci, V.

This paper reports on the development of polymer electrolyte membranes (PEMs) based on sulfonated polysulfone for application in a DMFC mini-stack operating at room temperature in passive mode. The sulfonated polysulfone (SPSf) with two degrees of sulfonation (57 and 66%) was synthesized by a well-known sulfonation process. SPSf membranes with different thicknesses were prepared and investigated. These membranes were characterized in terms of methanol/water uptake, proton conductivity, and fuel cell performance in a DMFC single cell and mini-stack operating at room temperature. The study addressed (a) control of the synthesis of sulfonated polysulfone, (b) optimization of the assembling procedure, (c) a short lifetime investigation and (d) a comparison of DMFC performance in active-mode operation vs. passive-mode operation. The best passive DMFC performance was 220 mW (average cell power density of about 19 mW cm -2), obtained with a thin SPSf membrane (70 μm) at room temperature, whereas the performance of the same membrane-based DMFC in active mode was 38 mW cm -2. The conductivity of this membrane, SPSf (IEC = 1.34 mequiv. g -1) was 2.8 × 10 -2 S cm -1. A preliminary short-term test (200 min) showed good stability during chrono-amperometry measurements.

Membrane Repair: Mechanisms and Pathophysiology

PubMed Central

Cooper, Sandra T.; McNeil, Paul L.

2015-01-01

Eukaryotic cells have been confronted throughout their evolution with potentially lethal plasma membrane injuries, including those caused by osmotic stress, by infection from bacterial toxins and parasites, and by mechanical and ischemic stress. The wounded cell can survive if a rapid repair response is mounted that restores boundary integrity. Calcium has been identified as the key trigger to activate an effective membrane repair response that utilizes exocytosis and endocytosis to repair a membrane tear, or remove a membrane pore. We here review what is known about the cellular and molecular mechanisms of membrane repair, with particular emphasis on the relevance of repair as it relates to disease pathologies. Collective evidence reveals membrane repair employs primitive yet robust molecular machinery, such as vesicle fusion and contractile rings, processes evolutionarily honed for simplicity and success. Yet to be fully understood is whether core membrane repair machinery exists in all cells, or whether evolutionary adaptation has resulted in multiple compensatory repair pathways that specialize in different tissues and cells within our body. PMID:26336031

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

PubMed Central

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

2016-01-01

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70–80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids. PMID:27872310

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

PubMed

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

2016-12-06

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

Differential effects of eicosapentaenoic acid and docosahexaenoic acid on human skin fibroblasts.

PubMed

Brown, E R; Subbaiah, P V

1994-12-01

To better understand the mode of action of omega 3 fatty acids in cell membranes, human foreskin fibroblasts were grown in serum-free medium supplemented with 50 microM oleic acid linoleic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and the effects on membrane composition, fluorescence polarization and enzyme activities were followed. The cells were enriched with EPA and DHA up to 7 and 13% of total lipids, respectively, of which > 95% was associated with phospholipids. In addition, the concentration of 22:5n-3 increased with both EPA and DHA to 7.5, and 2.1% of the total fatty acids, respectively. When compared to controls (oleic acid), cells treated with DHA showed a decrease in cholesterol, phospholipids, arachidonic acid (AA) and free cholesterol/phospholipid ratio (P < 0.05). In the presence of EPA, only decreases in AA and cholesterol were significant (P < 0.05). Membrane fluidity, assessed by fluorescence anisotropy, was increased 16% in cells enriched with DHA (P < 0.05), but showed no change with EPA or linoleic acid. There was an increase in membrane-associated 5'-nucleotidase (+27%) and adenylate cyclase (+19%) activities (P < 0.05), in DHA-enriched, but not in EPA-enriched cells, when compared with oleate controls. The studies show that incorporation of DHA, but not EPA, into cell membranes of fibroblasts alters membrane biophysical characteristics and function. We suggest that these two major n-3 fatty acids of fish oils have differential effects on cell membranes, and this may be related to the known differences in their physiological effects.

[Protective effect of hydrogen against hyperoxia-induced type II alveolar epithelial cell injury].

PubMed

Yao, Lan; Xu, Feng; Luo, Chong; Yu, Pan; Dong, Xinxin; Sun, Xuejun; Liu, Chengjun

2013-02-01

To investigate the protective effect of hydrogen against hyperoxia-induced oxidative stress injury in premature rat type II alveolar epithelial cells (AECs). The type II AECs isolated from premature rats were randomly divided into air (21% oxygen) control group, hyperoxia (95% oxygen) control group, air + hydrogen group, and hyperoxia+ hydrogen group. The cells with hydrogen treatment were cultured in the presence of rich hydrogen. After the corresponding exposure for 24 h, the cell morphology was observed microscopically. MTT assay was used to evaluated the cell proliferation ability, and JC-1 fluorescence probe was used to detect the mitochondrial membrane potential (δφ) changes of the type II AECs. The concentration of maleic dialdehyde (MDA) and superoxide dismutase (SOD) activity in the cell supernatant were detected using colorimetric method. No significant differences were found in cell growth or measurements between air control and air + hydrogen groups. Compared with air control group, the cells exposed to hyperoxia showed significantly suppressed proliferation, reduced mitochondrial membrane potential, increased MDA content, and decreased SOD activity. Intervention with hydrogen resulted in significantly increased cell proliferation and SOD activity and lowered MDA content, and restored the mitochondrial membrane potential in the cells with hyperoxia exposure (P<0.05). Hydrogen can significantly reduce hyperoxia-induced oxidative stress injury in premature rat type II AECs, improve the cellular antioxidant capacity, stabilize the mitochondrial membrane potential, and reduce the inhibitory effect of hyperoxia on cell proliferation.

Membrane stability and mitochondrial activity of human-ejaculated spermatozoa during in vitro experimental infection with Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus.

PubMed

Fraczek, M; Piasecka, M; Gaczarzewicz, D; Szumala-Kakol, A; Kazienko, A; Lenart, S; Laszczynska, M; Kurpisz, M

2012-10-01

The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim-up-separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC-1 test as well as the NADH-dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility. © 2012 Blackwell Verlag GmbH.

Plasma membrane disruption: repair, prevention, adaptation

NASA Technical Reports Server (NTRS)

McNeil, Paul L.; Steinhardt, Richard A.

2003-01-01

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

Novel Outer Membrane Protein Involved in Cellulose and Cellooligosaccharide Degradation by Cytophaga hutchinsonii

PubMed Central

Ji, Xiaofei; Wang, Ying; Zhang, Cong; Bai, Xinfeng; Zhang, Weican

2014-01-01

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and β-glucosidase activity was distributed on the cell surface of C. hutchinsonii. Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the β-glucosidase activity, it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the β-glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface β-glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii. PMID:24837387

Crude venom from nematocysts of Pelagia noctiluca (Cnidaria: Scyphozoa) elicits a sodium conductance in the plasma membrane of mammalian cells

NASA Astrophysics Data System (ADS)

Morabito, Rossana; Costa, Roberta; Rizzo, Valentina; Remigante, Alessia; Nofziger, Charity; La Spada, Giuseppa; Marino, Angela; Paulmichl, Markus; Dossena, Silvia

2017-01-01

Cnidarians may negatively impact human activities and public health but concomitantly their venom represents a rich source of bioactive substances. Pelagia noctiluca is the most venomous and abundant jellyfish of the Mediterranean Sea and possesses a venom with hemolytic and cytolytic activity for which the mechanism is largely unknown. Here we show that exposure of mammalian cells to crude venom from the nematocysts of P. noctiluca profoundly alters the ion conductance of the plasma membrane, therefore affecting homeostatic functions such as the regulation and maintenance of cellular volume. Venom-treated cells exhibited a large, inwardly rectifying current mainly due to permeation of Na+ and Cl-, sensitive to amiloride and completely abrogated following harsh thermal treatment of crude venom extract. Curiously, the plasma membrane conductance of Ca2+ and K+ was not affected. Current-inducing activity was also observed following delivery of venom to the cytosolic side of the plasma membrane, consistent with a pore-forming mechanism. Venom-induced NaCl influx followed by water and consequent cell swelling most likely underlie the hemolytic and cytolytic activity of P. noctiluca venom. The present study underscores unique properties of P. noctiluca venom and provides essential information for a possible use of its active compounds and treatment of envenomation.

Changes in E-cadherin rigidity sensing regulate cell adhesion.

PubMed

Collins, Caitlin; Denisin, Aleksandra K; Pruitt, Beth L; Nelson, W James

2017-07-18

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.

The direct anti-MRSA effect of emodin via damaging cell membrane.

PubMed

Liu, Ming; Peng, Wei; Qin, Rongxin; Yan, Zifei; Cen, Yanyan; Zheng, Xinchuan; Pan, Xichun; Jiang, Weiwei; Li, Bin; Li, Xiaoli; Zhou, Hong

2015-09-01

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important bacterium for nosocomial infection. Only a few antibiotics can be effective against MRSA. Therefore, searching for new drugs against MRSA is important. Herein, anti-MRSA activities of emodin and its mechanisms were investigated. Firstly, in vitro antimicrobial activity was investigated by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-growth curve, and multipassage resistance testing was performed. Secondly, protection of emodin on mice survival and blood bacterial load in mice challenged with lethal or sublethal dose of MRSA were investigated. Subsequently, the influences of emodin on the bacterial morphology, messenger RNA (mRNA) expressions related to cell wall synthesis and lysis, β-lactamase activity, drug accumulation, membrane fluidity, and integrity were performed to investigate its mechanisms. Lastly, in vitro cytotoxicity assay were performed using the 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) method. The results showed MICs and MBCs of emodin against MRSA252 and 36 clinical MRSA strains were among 2-8 and 4-32 μg/mL, respectively. There was no MIC increase for emodin during 20 passages. In vivo, emodin dose-dependently protected mice challenged with lethal dose of MRSA and decreased bacterial load in mice challenged with sublethal dose of MRSA. Morphology observation showed emodin might disrupt cell wall and membrane of MRSA. Although emodin had no influence on genes related to cell wall synthesis and lysis as well as β-lactamase activity and drug accumulation, emodin reduced membrane fluidity and disrupted membrane integrity. Based on the fact that emodin had no significant cytotoxicity against mammalian cells, it could be further investigated as a membrane-damage bactericide against MRSA in the future.

Release characteristics and bioactivity of gelatin-tricalcium phosphate membranes covalently immobilized with nerve growth factors.

PubMed

Chen, Pei-Ru; Chen, Ming-Hong; Lin, Feng-Huei; Su, Wen-Yu

2005-11-01

The gelatin-tricalcium phosphate membranes were cross-linking with low concentration glutaraldehyde solution (GTG). This material has good mechanical property, biocompatibility, and is feasible for surgical manipulation. For axonal regeneration, nerve growth factors (NGF) were immobilized onto the composite (GTG) with carbodiimide. The purpose of this study was to evaluate the release characteristics and bioactivity of NGF after covalent immobilization onto the GTG membranes (GEN). NGF immobilized onto and released from the composite was quantified using ELISA method. PC 12 cells were cultured on the GTG and GEN composites. Cell survival, cytotoxicity, and cellular activity were evaluated by total protein content, LDH activity, and MTT assay respectively. Neurite outgrowth assay was used to evaluate the biological activity of NGF released from GEN composite. From ELISA measurement, the releasing curve for NGF showing two distinctive parts with different slopes indicated that NGF were released from the composite in diffusion-controlled mechanism and degradation-controlled mechanism respectively. While culturing with PC 12 cells, LDH leakage results implied that whether GTG composite cross-linked with NGF or not showed little cytotoxicity. The total protein content and cellular activity of PC 12 cells were lower on GTG and GEN membranes than control group. However, 56%+/-3.98 of PC 12 cells showed significant neurite outgrowth on GEN membranes which was statistically higher than GTG without NGF immobilization. In addition, sustained release of bioactive NGF for two months had been demonstrated by neurite outgrowth assay. From these experiments, it can be concluded that the technique used in the present study is capable of immobilizing NGF onto GTG membranes covalently and remaining the bioactivity of NGF. Therefore, GEN composite can be materials for sustained release of bioactive NGF and a candidate for future therapeutic application in nerve repair.

The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum

PubMed Central

Green, Anita A.; Newell, Peter C.

1974-01-01

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N2 and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [125I]iodide. 5′-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH–cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH–cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants. ImagesPLATE 1 PMID:4156170

Testosterone decreases urinary bladder smooth muscle excitability via novel signaling mechanism involving direct activation of the BK channels

PubMed Central

Hristov, Kiril L.; Parajuli, Shankar P.; Provence, Aaron

2016-01-01

In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large-conductance voltage- and Ca2+-activated K+ (BK) channels with paxilline (1 μM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of −20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a nongenomic mechanism. Live-cell Ca2+ imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca2+ concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent from genomic testosterone receptors, and thus regulates DSM excitability. PMID:27605581

Activation of phospholipase activity during Semliki Forest virus infection.

PubMed

Pérez, L; Irurzun, A; Carrasco, L

1993-05-01

Infection of animal cells by a number of cytolytic viruses leads to increased membrane permeability. Thus, Semliki Forest virus (SFV) infection of susceptible cells modifies the permeability of the membrane for a number of cations and metabolites (Muñoz et al. (1985), Virology 146, 203-212). The molecular basis of this modification of the cell membrane has not been investigated in detail. We report that during the infection of HeLa cells with SFV, or BHK cells with vesicular stomatitis virus, there is a significant increase in the release of choline and arachidonic acid into the culture medium, suggesting that both phospholipases (PLases) C and A2 become activated during infection. Both choline and phosphorylcholine are released into the medium as expected when PLase C is activated. Cells prelabeled with arachidonic acid release a significant amount of radioactivity from the third hour postinfection. Most of this radioactivity is present in the medium of SFV-infected cells in the form of free fatty acid, suggesting that phospholipid hydrolysis has occurred; no intact phospholipids are detected in the culture medium. Finally, the action of several inhibitors of PLases, such as zinc and cadmium ions, chloroquine, chlorpromazine, amantadine, and dansylcadaverine were assayed. Our findings indicate that the release of choline or arachidonic acid is potently blocked by some of these lipase inhibitors. Following infection by SFV HeLa cells become susceptible to the inhibition of protein synthesis by hygromycin B due to increased uptake of this antibiotic. Entry of hygromycin B was prevented by zinc ions or chloroquine, suggesting that the increase in membrane permeability in SFV-infected cells may be mediated in part by lipase activation.

Live cell plasma membranes do not exhibit a miscibility phase transition over a wide range of temperatures.

PubMed

Lee, Il-Hyung; Saha, Suvrajit; Polley, Anirban; Huang, Hector; Mayor, Satyajit; Rao, Madan; Groves, Jay T

2015-03-26

Lipid/cholesterol mixtures derived from cell membranes as well as their synthetic reconstitutions exhibit well-defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid/cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid-phase separation is not generally observed in cell membranes, and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown. A fundamental property of phase transitions is that the variation of tagged particle diffusion with temperature exhibits an abrupt change as the system passes through the transition, even when the two phases are distributed in a nanometer-scale emulsion. We support this using a variety of Monte Carlo and atomistic simulations on model lipid membrane systems. However, temperature-dependent fluorescence correlation spectroscopy of labeled lipids and membrane-anchored proteins in live cell membranes shows a consistently smooth increase in the diffusion coefficient as a function of temperature. We find no evidence of a discrete miscibility phase transition throughout a wide range of temperatures: 14-37 °C. This contrasts the behavior of giant plasma membrane vesicles (GPMVs) blebbed from the same cells, which do exhibit phase transitions and macroscopic phase separation. Fluorescence lifetime analysis of a DiI probe in both cases reveals a significant environmental difference between the live cell and the GPMV. Taken together, these data suggest the live cell membrane may avoid the miscibility phase transition inherent to its lipid constituents by actively regulating physical parameters, such as tension, in the membrane.

A split ubiquitin system to reveal topology and released peptides of membrane proteins.

PubMed

Li, Qiu-Ping; Wang, Shuai; Gou, Jin-Ying

2017-09-02

Membrane proteins define biological functions of membranes in cells. Extracellular peptides of transmembrane proteins receive signals from pathogens or environments, and are the major targets of drug developments. Despite of their essential roles, membrane proteins remain elusive in topological studies due to technique difficulties in their expressions and purifications. First, the target gene is cloned into a destination vector to fuse with C terminal ubiquitin at the N or C terminus. Then, Cub vector with target gene and Nub WT or Nub G vectors are transformed into AP4 or AP5 yeast cells, respectively. After mating, the diploid cells are dipped onto selection medium to check the growth. Topology of the target protein is determined according to Table 1. We present a split ubiquitin topology (SUT) analysis system to study the topology and truncation peptide of membrane proteins in a simple yeast experiment. In the SUT system, transcription activator (TA) fused with a nucleo-cytoplasmic protein shows strong auto-activation with both positive and negative control vectors. TA fused with the cytoplasmic end of membrane proteins activates reporter genes only with positive control vector with a wild type N terminal ubiquitin (Nub WT ). However, TA fused with the extracellular termini of membrane proteins can't activate reporter genes even with Nub WT . Interestingly,TA fused with the released peptide of a membrane protein shows autoactivation in the SUT system. The SUT system is a simple and fast experimental procedure complementary to computational predictions and large scale proteomic techniques. The preliminary data from SUT are valuable for pathogen recognitions and new drug developments.

Interleukin-4 activates large-conductance, calciumactivated potassium (BKCa) channels in human airway smooth muscle cells

PubMed Central

Martin, Gilles; O’Connell, Robert J.; Pietrzykowski, Andrzej Z.; Treistman, Steven N.; Ethier, Michael F.; Madison, J. Mark

2014-01-01

Large-conductance, calcium-activated potassium (BKCa) channels are regulated by voltage and near-membrane calcium concentrations and are determinants of membrane potential and excitability in airway smooth muscle cells. Since the T helper–2 (Th2) cytokine, interleukin (IL)-4, is an important mediator of airway inflammation, we investigated whether IL-4 rapidly regulated BKCa activity in normal airway smooth muscle cells. On-cell voltage clamp recordings were made on subconfluent, cultured human bronchial smooth muscle cells (HBSMC). Interleukin-4 (50 ng ml−1), IL-13 (50 ng ml−1) or histamine (10 μm) was added to the bath during the recordings. Immunofluorescence studies with selective antibodies against the α and β1 subunits of BKCa were also performed. Both approaches demonstrated that HBSMC membranes contained large-conductance channels (>200 pS) with both calcium and voltage sensitivity, all of which is characteristic of the BKCa channel. Histamine caused a rapid increase in channel activity, as expected. A new finding was that perfusion with IL-4 stimulated rapid, large increases in BKCa channel activity (77.2 ± 63.3-fold increase, P < 0.05, n = 18). This large potentiation depended on the presence of external calcium. In contrast, IL-13 (50 ng ml−1) had little effect on BKCa channel activity, but inhibited the effect of IL-4. Thus, HBSMC contain functional BKCa channels whose activity is rapidly potentiated by the cytokine, IL-4, but not by IL-13.These findings are consistent with a model in which IL-4 rapidly increases near-membrane calcium concentrations to regulate BKCa activity. PMID:18403443

Antimicrobial peptides and induced membrane curvature: geometry, coordination chemistry, and molecular engineering

PubMed Central

Schmidt, Nathan W.; Wong, Gerard C. L.

2013-01-01

Short cationic, amphipathic antimicrobial peptides are multi-functional molecules that have roles in host defense as direct microbicides and modulators of the immune response. While a general mechanism of microbicidal activity involves the selective disruption and permeabilization of cell membranes, the relationships between peptide sequence and membrane activity are still under investigation. Here, we review the diverse functions that AMPs collectively have in host defense, and show that these functions can be multiplexed with a membrane mechanism of activity derived from the generation of negative Gaussian membrane curvature. As AMPs preferentially generate this curvature in model bacterial cell membranes, the selective generation of negative Gaussian curvature provides AMPs with a broad mechanism to target microbial membranes. The amino acid constraints placed on AMPs by the geometric requirement to induce negative Gaussian curvature are consistent with known AMP sequences. This ‘saddle-splay curvature selection rule’ is not strongly restrictive so AMPs have significant compositional freedom to multiplex membrane activity with other useful functions. The observation that certain proteins involved in cellular processes which require negative Gaussian curvature contain domains with similar motifs as AMPs, suggests this rule may be applicable to other curvature-generating proteins. Since our saddle-splay curvature design rule is based upon both a mechanism of activity and the existing motifs of natural AMPs, we believe it will assist the development of synthetic antimicrobials. PMID:24778573

Distinct subcellular patterns of neprilysin protein and activity in the brains of Alzheimer’s disease patients, transgenic mice and cultured human neuronal cells

PubMed Central

Zhou, Li; Wei, Chunsheng; Huang, Wei; Bennett, David A; Dickson, Dennis W; Wang, Rui; Wang, Dengshun

2013-01-01

We investigated the subcellular distribution of NEP protein and activity in brains of human individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD dementia, as well as double transgenic mice and human neuronal cell line treated with Aβ and 4-hydroxy-2-nonenal (HNE). Total cortical neuronal-related NEP was significantly increased in MCI compared to NCI brains. NeuN was decreased in both MCI and AD, consistent with neuronal loss occurring in MCI and AD. Negative relationship between NEP protein and NeuN in MCI brains, and positive correlation between NEP and pan-cadherin in NCI and MCI brains, suggesting the increased NEP expression in NCI and MCI might be due to membrane associated NEP in non-neuronal cells. In subcellular extracts, NEP protein decreased in cytoplasmic fractions in MCI and AD, but increased in membrane fractions, with a significant increase in the membrane/cytoplasmic ratio of NEP protein in AD brains. By contrast, NEP activity was decreased in AD. Similar results were observed in AD-mimic transgenic mice. Studies of SH-SY5Y neuroblastoma showed an up-regulation of NEP protein in the cytoplasmic compartment induced by HNE and Aβ; however, NEP activity decreased in cytoplasmic fractions. Activity of NEP in membrane fractions increased at 48 hours and then significantly decreased after treatment with HNE and Aβ. The cytoplasmic/membrane ratio of NEP protein increased at 24 hours and then decreased in both HNE and Aβ treated cells. Both HNE and Aβ up-regulate NEP expression, but NEP enzyme activity did not show the same increase, possibly indicating immature cytoplasmic NEP is less active than membrane associated NEP. These observations indicate that modulation of NEP protein levels and its subcellular location influence the net proteolytic activity and this complex association might participate in deficiency of Aβ degradation that is associated with amyloid deposition in AD. PMID:24093058

Plasma membrane wounding and repair in pulmonary diseases.

PubMed

Cong, Xiaofei; Hubmayr, Rolf D; Li, Changgong; Zhao, Xiaoli

2017-03-01

Various pathophysiological conditions such as surfactant dysfunction, mechanical ventilation, inflammation, pathogen products, environmental exposures, and gastric acid aspiration stress lung cells, and the compromise of plasma membranes occurs as a result. The mechanisms necessary for cells to repair plasma membrane defects have been extensively investigated in the last two decades, and some of these key repair mechanisms are also shown to occur following lung cell injury. Because it was theorized that lung wounding and repair are involved in the pathogenesis of acute respiratory distress syndrome (ARDS) and idiopathic pulmonary fibrosis (IPF), in this review, we summarized the experimental evidence of lung cell injury in these two devastating syndromes and discuss relevant genetic, physical, and biological injury mechanisms, as well as mechanisms used by lung cells for cell survival and membrane repair. Finally, we discuss relevant signaling pathways that may be activated by chronic or repeated lung cell injury as an extension of our cell injury and repair focus in this review. We hope that a holistic view of injurious stimuli relevant for ARDS and IPF could lead to updated experimental models. In addition, parallel discussion of membrane repair mechanisms in lung cells and injury-activated signaling pathways would encourage research to bridge gaps in current knowledge. Indeed, deep understanding of lung cell wounding and repair, and discovery of relevant repair moieties for lung cells, should inspire the development of new therapies that are likely preventive and broadly effective for targeting injurious pulmonary diseases. Copyright © 2017 the American Physiological Society.

NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes*

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-05-13

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells.

PubMed

Ehre, Camille; Rossi, Andrea H; Abdullah, Lubna H; De Pestel, Kathleen; Hill, Sandra; Olsen, John C; Davis, C William

2005-01-01

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G(q)-coupled P2Y(2) receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of beta- and gamma-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of beta- or gamma-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca(2+)-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

The antibacterial activity and action mechanism of emodin from Polygonum cuspidatum against Haemophilus parasuis in vitro.

PubMed

Li, Li; Song, Xu; Yin, Zhongqiong; Jia, Renyong; Li, Zhengwen; Zhou, Xun; Zou, Yuanfeng; Li, Lixia; Yin, Lizi; Yue, Guizhou; Ye, Gang; Lv, Cheng; Shi, Wenjing; Fu, Yuping

2016-01-01

Haemophilus parasuis is the causative agent of Glässer's disease, which leads to serious economic loss to the swine industry. Although antibiotics are widely used to control infections, outbreaks of this disease repeatedly happen. In this study, emodin from Polygonum cuspidatum showed potent inhibitory effect against H. parasuis. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of emodin were 32 and 64μg/mL, respectively. The antibacterial kinetic curves indicated the antibacterial activity of emodin was in a concentration-dependent manner. Cell membrane permeability and flow cytometry assays proved that emodin could destroy cell membrane integrity and increase membrane permeability, and fluorescence spectra assay indicated emodin has influenced conformation of membrane protein. Under transmission electron microscopy, serious lesions of H. parasuis exposed to emodin (64μg/mL) were found, including irregular cell shape, plasmolysis, ruptured cell wall and membrane and cytoplasmic vacuolation. These results suggested that emodin could be used as candidate for treating Glässer's disease. Copyright © 2016 Elsevier GmbH. All rights reserved.

Nanoscale Photosynthesis and the Photophysics of Neural Cells

NASA Astrophysics Data System (ADS)

Greenbaum, Elias; Kuritz, Tanya; Owens, Elizabeth; Lee, Ida; Humayun, Mark

2004-03-01

We extracted and purified integral membrane Photosystem I (PSI) reaction centers from spinach leaves and measured their open and closed circuit photovoltages. The open circuit value is at least 1 V whereas the closed circuit value is at least 0.6 V. A quantitative analysis of the physical properties of PSI reaction centers and voltage-gated ion channels indicates that PSI should be able to trigger the opening of the channels. The cell membrane can be depolarized or hyperpolarized depending on the orientation of the PSI reaction center in the membrane. PSI-proteoliposomes were used as the delivery vehicle. We inserted PSI reaction centers into liposome membranes and, using P700 absorption spectroscopy, demonstrated that the reaction centers retain their functional activity in the liposomes. We have also obtained microscopic evidence that the liposomes are capable of fusing with the membranes of retinoblastoma cells. We report the creation of photoreceptor activity in retinoblastoma cells by PSI reaction centers as indicated by light-induced movement of calcium ions. These results may have application in the field of artificial sight in treating age-related macular degeneration and retinitis pigmentosa.

PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae.

PubMed

Zucchini, Laure; Mercy, Chryslène; Garcia, Pierre Simon; Cluzel, Caroline; Gueguen-Chaignon, Virginie; Galisson, Frédéric; Freton, Céline; Guiral, Sébastien; Brochier-Armanet, Céline; Gouet, Patrice; Grangeasse, Christophe

2018-02-01

Eukaryotic-like serine/threonine kinases (eSTKs) with extracellular PASTA repeats are key membrane regulators of bacterial cell division. How PASTA repeats govern eSTK activation and function remains elusive. Using evolution- and structural-guided approaches combined with cell imaging, we disentangle the role of each PASTA repeat of the eSTK StkP from Streptococcus pneumoniae. While the three membrane-proximal PASTA repeats behave as interchangeable modules required for the activation of StkP independently of cell wall binding, they also control the septal cell wall thickness. In contrast, the fourth and membrane-distal PASTA repeat directs StkP localization at the division septum and encompasses a specific motif that is critical for final cell separation through interaction with the cell wall hydrolase LytB. We propose a model in which the extracellular four-PASTA domain of StkP plays a dual function in interconnecting the phosphorylation of StkP endogenous targets along with septal cell wall remodelling to allow cell division of the pneumococcus.

Unique battery with an active membrane separator having uniform physico-chemically functionalized ion channels and a method making the same

DOEpatents

Gerald, II, Rex E.; Ruscic, Katarina J [Chicago, IL; Sears, Devin N [Spruce Grove, CA; Smith, Luis J [Natick, MA; Klingler, Robert J [Glenview, IL; Rathke, Jerome W [Homer Glen, IL

2012-02-21

The invention relates to a unique battery having an active, porous membrane and method of making the same. More specifically the invention relates to a sealed battery system having a porous, metal oxide membrane with uniform, physicochemically functionalized ion channels capable of adjustable ionic interaction. The physicochemically-active porous membrane 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. 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.

Studying Mechanosensitivity of Two-Pore Domain K+ Channels in Cellular and Reconstituted Proteoliposome Membranes.

PubMed

Del Mármol, Josefina; Rietmeijer, Robert A; Brohawn, Stephen G

2018-01-01

Mechanical force sensation is fundamental to a wide breadth of biology from the classic senses of touch, pain, hearing, and balance to less conspicuous sensations of proprioception, blood pressure, and osmolarity and basic aspects of cell growth, differentiation, and development. These diverse and essential systems use force-gated (or mechanosensitive) ion channels that convert mechanical stimuli into cellular electrical signals. TRAAK, TREK1, and TREK2 are K + -selective ion channels of the two-pore domain K + (K2P) family that are mechanosensitive: they are gated open by increasing membrane tension. TRAAK and TREK channels are thought to play roles in somatosensory and other mechanosensory processes in neuronal and non-neuronal tissues. Here, we present protocols for three assays to study mechanical activation of these channels in cell membranes: (1) cell swelling, (2) cell poking, and (3) patched membrane stretching. Patched membrane stretching is also applicable to the study of mechanosensitive K2P channel activity in a cell-free system and a procedure for proteoliposome reconstitution and patching is also presented. These approaches are also readily applicable to the study of other mechanosensitive ion channels.

Performance enhancement of polymer electrolyte membrane fuel cells by dual-layered membrane electrode assembly structures with carbon nanotubes.

PubMed

Jung, Dong-Won; Kim, Jun-Ho; Kim, Se-Hoon; Kim, Jun-Bom; Oh, Eun-Suok

2013-05-01

The effect of dual-layered membrane electrode assemblies (d-MEAs) on the performance of a polymer electrolyte membrane fuel cell (PEMFC) was investigated using the following characterization techniques: single cell performance test, electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). It has been shown that the PEMFC with d-MEAs has better cell performance than that with typical mono-layered MEAs (m-MEAs). In particular, the d-MEA whose inner layer is composed of multi-walled carbon nanotubes (MWCNTs) showed the best fuel cell performance. This is due to the fact that the d-MEAs with MWCNTs have the highest electrochemical surface area and the lowest activation polarization, as observed from the CV and EIS test.

Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121[W

PubMed Central

Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S.; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R.; Chaumont, François

2012-01-01

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K+ channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K+ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis. PMID:22942383

Selective regulation of maize plasma membrane aquaporin trafficking and activity by the SNARE SYP121.

PubMed

Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R; Chaumont, François

2012-08-01

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K(+) channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K(+) channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.

Calcium and Magnesium Ions Are Membrane-Active against Stationary-Phase Staphylococcus aureus with High Specificity

NASA Astrophysics Data System (ADS)

Xie, Yuntao; Yang, Lihua

2016-02-01

Staphylococcus aureus (S. aureus) is notorious for its ability to acquire antibiotic-resistance, and antibiotic-resistant S. aureus has become a wide-spread cause of high mortality rate. Novel antimicrobials capable of eradicating S. aureus cells including antibiotic-resistant ones are thus highly desired. Membrane-active bactericides and species-specific antimicrobials are two promising sources of novel anti-infective agents for fighting against bacterial antibiotic-resistance. We herein show that Ca2+ and Mg2+, two alkaline-earth-metal ions physiologically essential for diverse living organisms, both disrupt model S. aureus membranes and kill stationary-phase S. aureus cells, indicative of membrane-activity. In contrast to S. aureus, Escherichia coli and Bacillus subtilis exhibit unaffected survival after similar treatment with these two cations, indicative of species-specific activity against S. aureus. Moreover, neither Ca2+ nor Mg2+ lyses mouse red blood cells, indicative of hemo-compatibility. This works suggests that Ca2+ and Mg2+ may have implications in targeted eradication of S. aureus pathogen including the antibiotic-resistant ones.

Effect of a membrane-targeted sphingosine kinase 1 on cell proliferation and survival

PubMed Central

2005-01-01

Numerous extracellular stimuli activate SK1 (sphingosine kinase type 1) to catalyse the production of sphingosine 1-phosphate, a bioactive lipid that functions as both an extracellular ligand for a family of G-protein-linked receptors and as a putative intracellular messenger. Phorbol esters, calcium or immunoglobulin receptors stimulate SK1 by promoting its translocation to the plasma membrane, which brings it into proximity both to its substrate (i.e. sphingosine) and to activating acidic phospholipids (e.g. phosphatidylserine). To evaluate the consequence of SK translocation, we generated an SK1-derivative tagged with a myristoylation sequence (Myr-SK1) on its N-terminus and overexpressed the construct in 3T3-L1 fibroblasts using recombinant retrovirus. Myr-SK1 overexpression increased SK activity by more than 50-fold in crude membranes, while only stimulating cytoplasmic SK activity by 4-fold. In contrast, the overexpression of WT-SK1 (wild-type SK1), as well as that of a construct containing a false myristoylation sequence (A2-Myr-SK1), markedly increased SK activity in both membrane and cytoplasmic compartments. Immunofluorescence confirmed that Myr-SK1 preferentially localized at the plasma membrane, whereas WT-SK1 and A2-Myr-SK1 partitioned in cytoplasmic/perinuclear cellular regions. Surprisingly, Myr-SK1 overexpression significantly decreased the rates of cell proliferation by delaying exit from G0/G1 phase. Moreover, expression of Myr-SK1 but not WT-SK1 or A2-Myr-SK1 protected cells from apoptosis induced by serum withdrawal. Collectively, these findings reveal that altering the subcellular location of SK1 has marked effects on cell function, with plasma membrane-associated SK having a potent inhibitory effect on the G1–S phase transition. PMID:15693752

Hemin-induced suicidal erythrocyte death.

PubMed

Gatidis, Sergios; Föller, Michael; Lang, Florian

2009-08-01

Several diseases, such as malaria, sickle cell disease, and ischemia/reperfusion may cause excessive formation of hemin, which may in turn trigger hemolysis. A variety of drugs and diseases leading to hemolysis triggers suicidal erythrocyte death or eryptosis, i.e., cell membrane scrambling and cell shrinkage. Eryptosis is elicited by increased cytosolic Ca(2+) activity and by ceramide. The present study explored whether hemin stimulates eryptosis. Cell membrane scrambling was estimated from annexin V-binding to phosphatidylserine exposed at the cell surface, cell shrinkage from forward scatter in fluorescence-activated cell sorter analysis, cytosolic Ca(2+) activity from Fluo3 fluorescence and ceramide formation from fluorescence-labeled antibody binding. Exposure to hemin (1-10 microM) within 48 h significantly increased annexin V-binding, decreased forward scatter, increased cytosolic Ca(2+) activity, and stimulated ceramide formation. In conclusion, hemin stimulates suicidal cell death, which may in turn contribute to the clearance of circulating erythrocytes and thus to anemia.

Functional analysis of corin protein domains required for PCSK6-mediated activation.

PubMed

Chen, Shenghan; Wang, Hao; Li, Heng; Zhang, Yue; Wu, Qingyu

2018-01-01

Atrial natriuretic peptide (ANP) is a cardiac hormone essential for normal blood pressure and cardiac function. Corin is a transmembrane serine protease that activates ANP. Recently, we identified proprotein convertase subtilisin/kexin-6 (PCSK6), also called PACE4, as the long-sought corin activator. Both corin and PCSK6 are expressed in cardiomyocytes, but corin activation occurs only on the cell surface. It remains unknown if cell membrane association is needed for PCSK6 to activate corin. Here we expressed corin deletion mutants in HEK293 cells to analyze the domain structures required for PCSK6-mediated activation. Our results show that soluble corin lacking the transmembrane domain was activated by PCSK6 in the conditioned medium but not intracellularly. Recombinant PCSK6 also activated the soluble corin under cell-free conditions. Moreover, PCSK6-mediated corin activation was not enhanced by cell membrane fractions. These results indicate that cell membrane association is unnecessary for PCSK6 to activate corin. Experiments with monensin that blocks PCSK6 secretion and immunostaining indicated that the soluble corin and PCSK6 were secreted via different intracellular pathways, which may explain the lack of corin activation inside the cell. We also found that the protein domains in the corin pro-peptide region were dispensable for PCSK6-mediated activation and that addition of heparan sulfate and chondroitin sulfate or treatment with heparinase or chondroitinase did not alter corin activation by PCSK6 in HEK293 cells. Together, our results provide important insights into the molecular and cellular mechanisms underlying PCSK6-mediated corin activation that is critical for cardiovascular homeostasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

PubMed Central

1984-01-01

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron- glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells. PMID:6725397

A Comparison of Water Diffusion in Polymer Based Fuel Cell and Reverse Osmosis Membrane Materials

NASA Astrophysics Data System (ADS)

Soles, Christopher; Frieberg, Bradley; Tarver, Jacob; Tyagi, Madhusudan; Jeong, Cheol; Chan, Edwin; Stafford, Christopher

Hydrated polymer membranes are critical in both fuel cells and water filtration and desalination. In both of these applications the membrane function (selectively transporting or separating ions) is coupled with the transport of water through the membrane. There is a significant need to understand the nature by which the water and ions distribute and move through these membranes. This presentation compares the transport mechanisms in in an ion containing block copolymer alkaline fuel cell membrane with that of a polyamide membrane that is used as the active layer in a reverse osmosis water desalination membrane. Small angle neutron scattering measurements are used to locally probe how water swells the different materials and quantitatively describe the distribution of water within the membrane microstructures. Quasielastic neutron scattering measurements are then used to separate the polymer dynamics of the host membranes from the dynamics of the water inside the membranes. This reveals that water moves at least an order of magnitude slower through the ion containing fuel cell membrane materials, consistent with a solution-diffusion model, while the water in the polyamide membranes moves faster, consistent with a pore-flow diffusion mechanism. These insights will be discussed in terms of a coupling of the water and polymer dynamics and design cues for high performance membrane materials.

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose.

PubMed

Szamel, M; Goppelt, M; Resch, K

1985-12-19

Purified plasma membranes of mouse EL4 lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including gamma-glutamyl transpeptidase, 5'-nucleotidase and Mg2+-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL4 lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.

Study of Raft Domains in Model Membrane of DPPC/PE/Cholesterol

NASA Astrophysics Data System (ADS)

Lor, Chai; Hirst, Linda

2010-10-01

Raft domains in bilayer membrane are thought to play an important role in many cell functions such as cell signaling or trans-membrane protein activation. Here we use a model membrane consisting of DPPC/PE/cholesterol to examine the structure of membrane rafts and phase interactions. In particular we are interested in lipids containing the highly polyunsaturated fatty acid DHA. We use both atomic force microscopy (AFM) and fluorescence microscopy to obtain information on the structural properties of raft regions and track cholesterol. As expected, we find phase separation of raft regions between saturated and unsaturated lipids. Moreover, we find that the roughness of the domains change with varying cholesterol concentration possibly due to overpacking. This model study provides further understanding of the role of cholesterol in bilayer membrane leading towards a better knowledge of cell membranes.

Segregation of two spectrin isoforms: polarized membrane-binding sites direct polarized membrane skeleton assembly.

PubMed

Dubreuil, R R; Maddux, P B; Grushko, T A; MacVicar, G R

1997-10-01

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and beta spectrin are recruited to sites of cell-cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (alpha beta H), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and alpha beta spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, alpha beta spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, alpha beta H spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell-cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.

Endothelial cell membrane vesicles in the study of organ preference of metastasis.

PubMed

Johnson, R C; Augustin-Voss, H G; Zhu, D Z; Pauli, B U

1991-01-01

Many malignancies exhibit distinct patterns of metastasis that appear to be mediated by receptor/ligand-like interactions between tumor cells and organ-specific vascular endothelium. In order to study endothelial cell surface molecules involved in the binding of metastatic cells, we developed a perfusion method to isolate outside-out membrane vesicles from the lumenal surface of rat lung microvascular endothelium. Lungs were perfused in situ for 4 h at 37 degrees C with a solution of 100 mM formaldehyde, 2 mM dithiothreitol in phosphate-buffered saline to induce endothelial cell vesiculation. Radioiodinated rat lung endothelial cell membrane vesicles bound lung-metastatic tumor cells (B16F10, R323OAC-MET) in significantly higher numbers than their low or nonmetastatic counterparts (B16F0, R323OAC-LR). In contrast, leg endothelial membrane vesicle showed no binding preference for either cell line. Neuraminidase treatment of vesicles abolished specificity of adhesion of lung-derived vesicles to lung metastatic tumor cells. These results demonstrate that in situ perfusion is an appropriate technique to obtain pure endothelial cell membrane vesicles containing functionally active adhesion molecules. The preferential binding of lung-derived endothelial cell membrane vesicles by lung metastatic tumor cells is evidence of the importance of endothelial cell adhesion molecules in the formation of metastases.

YAP and TAZ in epithelial stem cells: A sensor for cell polarity, mechanical forces and tissue damage.

PubMed

Elbediwy, Ahmed; Vincent-Mistiaen, Zoé I; Thompson, Barry J

2016-07-01

The YAP/TAZ family of transcriptional co-activators drives cell proliferation in epithelial tissues and cancers. Yet, how YAP and TAZ are physiologically regulated remains unclear. Here we review recent reports that YAP and TAZ act primarily as sensors of epithelial cell polarity, being inhibited when cells differentiate an apical membrane domain, and being activated when cells contact the extracellular matrix via their basal membrane domain. Apical signalling occurs via the canonical Crumbs/CRB-Hippo/MST-Warts/LATS kinase cascade to phosphorylate and inhibit YAP/TAZ. Basal signalling occurs via Integrins and Src family kinases to phosphorylate and activate YAP/TAZ. Thus, YAP/TAZ is localised to the nucleus in basal stem/progenitor cells and cytoplasm in differentiated squamous cells or columnar cells. In addition, other signals such as mechanical forces, tissue damage and possibly receptor tyrosine kinases (RTKs) can influence MST-LATS or Src family kinase activity to modulate YAP/TAZ activity. © 2016 The Authors BioEssays Published by WILEY Periodicals, Inc.

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

PubMed

Luo, Yu; Russinova, Eugenia

2017-01-01

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

Immunological Functions of the Membrane Proximal Region of MHC Class II Molecules

PubMed Central

Harton, Jonathan; Jin, Lei; Hahn, Amy; Drake, Jim

2016-01-01

Major histocompatibility complex (MHC) class II molecules present exogenously derived antigen peptides to CD4 T cells, driving activation of naïve T cells and supporting CD4-driven immune functions. However, MHC class II molecules are not inert protein pedestals that simply bind and present peptides. These molecules also serve as multi-functional signaling molecules delivering activation, differentiation, or death signals (or a combination of these) to B cells, macrophages, as well as MHC class II-expressing T cells and tumor cells. Although multiple proteins are known to associate with MHC class II, interaction with STING (stimulator of interferon genes) and CD79 is essential for signaling. In addition, alternative transmembrane domain pairing between class II α and β chains influences association with membrane lipid sub-domains, impacting both signaling and antigen presentation. In contrast to the membrane-distal region of the class II molecule responsible for peptide binding and T-cell receptor engagement, the membrane-proximal region (composed of the connecting peptide, transmembrane domain, and cytoplasmic tail) mediates these “non-traditional” class II functions. Here, we review the literature on the function of the membrane-proximal region of the MHC class II molecule and discuss the impact of this aspect of class II immunobiology on immune regulation and human disease. PMID:27006762

Delivery of molecules into cells using carbon nanoparticles activated by femtosecond laser pulses

PubMed Central

Chakravarty, Prerona; Qian, Wei; El-Sayed, Mostafa A.; Prausnitz, Mark R.

2010-01-01

A major barrier to drug and gene delivery is crossing the cell's plasma membrane. Physical forces applied to cells via electroporation1, ultrasound2 and laser-irradiation3–6 generate nanoscale holes in the plasma membrane for direct delivery of drugs into the cytoplasm. Inspired by previous work showing that laser excitation of carbon nanoparticles can drive the carbon-steam reaction to generate highly controlled shock waves7–10, here we show carbon black (CB) nanoparticles activated by femtosecond laser pulses can facilitate the delivery of small molecules, proteins and DNA into two types of cells. Our initial results suggest that interaction between the laser energy and CB nanoparticles may generate photoacoustic forces by chemical reaction to create transient holes in the membrane for delivery. PMID:20639882

Interfacial Water-Transport Effects in Proton-Exchange Membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kienitz, Brian; Yamada, Haruhiko; Nonoyama, Nobuaki

2009-11-19

It is well known that the proton-exchange membrane is perhaps the most critical component of a polymer-electrolyte fuel cell. Typical membranes, such as Nafion(R), require hydration to conduct efficiently and are instrumental in cell water management. Recently, evidence has been shown that these membranes might have different interfacial morphology and transport properties than in the bulk. In this paper, experimental data combined with theoretical simulations will be presented that explore the existence and impact of interfacial resistance on water transport for Nafion(R) 21x membranes. A mass-transfer coefficient for the interfacial resistance is calculated from experimental data using different permeation cells.more » This coefficient is shown to depend exponentially on relative humidity or water activity. The interfacial resistance does not seem to exist for liquid/membrane or membrane/membrane interfaces. The effect of the interfacial resistance is to flatten the water-content profiles within the membrane during operation. Under typical operating conditions, the resistance is on par with the water-transport resistance of the bulk membrane. Thus, the interfacial resistance can be dominant especially in thin, dry membranes and can affect overall fuel-cell performance.« less

Inflammatory Caspases: Activation and Cleavage of Gasdermin-D In Vitro and During Pyroptosis.

PubMed

Zhao, Yue; Shi, Jianjin; Shao, Feng

2018-01-01

Gasdermin-D (also known as GSDMD), the newly identified executioner of pyroptotic cell death, is cleaved by activated caspase-1 downstream of canonical inflammasome activation or caspase-4, 5, and 11 upon their ligation and activation by cytosolic LPS. Upon a single cleavage between the two domains in Gasdermin-D, the N-terminal domain binds to membrane lipids and lyses cells by forming pores of an inner diameter of 10-14 nm within the membrane. The inter-domain cleavage of Gasdermin-D is a reliable marker for the activation of inflammatory caspases and cell pyroptosis. Here, we describe the methods for examining Gasdermin-D cleavage by activated inflammatory caspases in vitro and upon inflammasome activation in vivo.

Erythrocyte ion channels in regulation of apoptosis.

PubMed

Lang, Florian; Birka, Christina; Myssina, Svetlana; Lang, Karl S; Lang, Philipp A; Tanneur, Valerie; Duranton, Christophe; Wieder, Thomas; Huber, Stephan M

2004-01-01

Erythrocytes lack mitochondria and nuclei, key organelles in the regulation of apoptosis. Until recently, erythrocytes were thus not considered subject to this type of cell death. However, exposure of erythrocytes to the Ca2+ ionophore ionomycin was shown to induce cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the cell surface, all typical features of apoptosis. Further studies revealed the participation of ion channels in the regulation of erythrocyte "apoptosis." Osmotic shock, oxidative stress and energy depletion all activate a Ca2(+)-permeable non-selective cation channel in the erythrocyte cell membrane. The subsequent increase of Ca2+ concentration stimulates a scramblase leading to breakdown of cell membrane phosphatidylserine asymmetry and activates Ca2+ sensitive K+ (Gardos) channels leading to KCl loss and (further) cell shrinkage. Phosphatidylserine exposure and cell shrinkage are blunted in the nominal absence of extracellular Ca2+, in the presence of the cation channel inhibitors amiloride or ethylisopropylamiloride, at increased extracellular K+ or in the presence of the Gardos channel inhibitors clotrimazole or charybdotoxin. Thus, increase of cytosolic Ca2+ and cellular loss of K+ participate in the triggering of erythrocyte scramblase. Nevertheless, phosphatidylserine exposure is not completely abrogated in the nominal absence of Ca2+, pointing to additional Ca2(+)-independent pathways. One of those is activation of sphingomyelinase with subsequent formation of ceramide which in turn leads to stimulation of erythrocyte scramblase. The exposure of phosphatidylserine at the extracellular face of the cell membrane stimulates phagocytes to engulf the apoptotic erythrocytes. Thus, sustained activation of the cation channels eventually leads to clearance of affected erythrocytes from peripheral blood. Erythropoietin inhibits the non-selective cation channel and thus interferes with erythrocyte "apoptosis." Susceptibility to scramblase activation is enhanced in thalassemia, sickle cell disease and glucose-6-phosphate dehydrogenase deficiency. Infection with Plasmodium falciparum leads to activation of the cation channel eventually triggering erythrocyte "apoptosis."

A calcium-permeable cGMP-activated cation conductance in hippocampal neurons

NASA Technical Reports Server (NTRS)

Leinders-Zufall, T.; Rosenboom, H.; Barnstable, C. J.; Shepherd, G. M.; Zufall, F.

1995-01-01

Whole-cell patch clamp recordings detected a previously unidentified cGMP-activated membrane conductance in cultured rat hippocampal neurons. This conductance is nonselectively permeable for cations and is completely but reversibly blocked by external Cd2+. The Ca2+ permeability of the hippocampal cGMP-activated conductance was examined in detail, indicating that the underlying ion channels display a high relative permeability for Ca2+. The results indicate that hippocampal neurons contain a cGMP-activated membrane conductance that has some properties similar to the cyclic nucleotide-gated channels previously shown in sensory receptor cells and retinal neurons. In hippocampal neurons this conductance similarly could mediate membrane depolarization and Ca2+ fluxes in response to intracellular cGMP elevation.

Spatial modeling of the membrane-cytosolic interface in protein kinase signal transduction

PubMed Central

Schröder, Andreas

2018-01-01

The spatial architecture of signaling pathways and the interaction with cell size and morphology are complex, but little understood. With the advances of single cell imaging and single cell biology, it becomes crucial to understand intracellular processes in time and space. Activation of cell surface receptors often triggers a signaling cascade including the activation of membrane-attached and cytosolic signaling components, which eventually transmit the signal to the cell nucleus. Signaling proteins can form steep gradients in the cytosol, which cause strong cell size dependence. We show that the kinetics at the membrane-cytosolic interface and the ratio of cell membrane area to the enclosed cytosolic volume change the behavior of signaling cascades significantly. We suggest an estimate of average concentration for arbitrary cell shapes depending on the cell volume and cell surface area. The normalized variance, known from image analysis, is suggested as an alternative measure to quantify the deviation from the average concentration. A mathematical analysis of signal transduction in time and space is presented, providing analytical solutions for different spatial arrangements of linear signaling cascades. Quantification of signaling time scales reveals that signal propagation is faster at the membrane than at the nucleus, while this time difference decreases with the number of signaling components in the cytosol. Our investigations are complemented by numerical simulations of non-linear cascades with feedback and asymmetric cell shapes. We conclude that intracellular signal propagation is highly dependent on cell geometry and, thereby, conveys information on cell size and shape to the nucleus. PMID:29630597

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

PubMed

Thuleau, P; Ward, J M; Ranjeva, R; Schroeder, J I

1994-07-01

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.

Effect of Thrombin on Human Amnion Mesenchymal Cells, Mouse Fetal Membranes, and Preterm Birth*

PubMed Central

Mogami, Haruta; Keller, Patrick W.; Shi, Haolin; Word, R. Ann

2014-01-01

Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE2 in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE2 content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion. PMID:24652285

Enzymatic Oxidation of Cholesterol: Properties and Functional Effects of Cholestenone in Cell Membranes

PubMed Central

Neuvonen, Maarit; Manna, Moutusi; Mokkila, Sini; Javanainen, Matti; Rog, Tomasz; Liu, Zheng; Bittman, Robert; Vattulainen, Ilpo; Ikonen, Elina

2014-01-01

Bacterial cholesterol oxidase is commonly used as an experimental tool to reduce cellular cholesterol content. That the treatment also generates the poorly degradable metabolite 4-cholesten-3-one (cholestenone) has received less attention. Here, we investigated the membrane partitioning of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human fibroblasts, cholestenone was released from membranes to physiological extracellular acceptors more avidly than cholesterol, but without acceptors it remained in cells over a day. To address the functional effects of cholestenone, we studied fibroblast migration during wound healing. When cells were either cholesterol oxidase treated or part of cellular cholesterol was exchanged for cholestenone with cyclodextrin, cell migration during 22 h was markedly inhibited. Instead, when a similar fraction of cholesterol was removed using cyclodextrin, cells replenished their cholesterol content in 3 h and migrated similarly to control cells. Thus, cholesterol oxidation produces long-term functional effects in cells and these are in part due to the generated membrane active cholestenone. PMID:25157633

Effect of uv light on formation and synthetic capacity of DNA-membrane complexes from T7-infected cells of E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wintermantel, G.

1974-02-01

The action of uv light on the attachment of T7-DNA to the bacterial cell membrane, and the RNA- and DNA-synthesizing activity of the DNA-membrane complex, were investigated. In E. coli H560 cells infected by uv-irradiated T7 phages, the amount of membrane-associated parental /sup 32/P-labeled T7-DNA was determined. To measure the RNA- and DNA-synthesizing activity, the isolated DNA- membrane complexes were incubated with an equimolar mixture of the corresponding four nucleoside triphosphates. The uv-sensitivities of the functions tested were calculated from the slopes of the dose-effect curves. In relation to the uv- sensitivity for plaque-forming ability of the T7bacteriophages they amountmore » to 0.07 ( plus or minus 0.01); 0.15 ( plus or minus 0.01); and 0.56 ( plus or minus 0.04), respectively. (auth)« less

Tuning B cell responses to antigens by cell polarity and membrane trafficking.

PubMed

Del Valle Batalla, Felipe; Lennon-Dumenil, Ana-María; Yuseff, María-Isabel

2018-06-20

The capacity of B lymphocytes to produce specific antibodies, particularly broadly neutralizing antibodies that provide immunity to viral pathogens has positioned them as valuable therapeutic targets for immunomodulation. To become competent as antibody secreting cells, B cells undergo a series of activation steps, which are triggered by the recognition of antigens frequently displayed on the surface of other presenting cells. Such antigens elicit the formation of an immune synapse (IS), where local cytoskeleton rearrangements coupled to mechanical forces and membrane trafficking orchestrate the extraction and processing of antigens in B cells. In this review, we discuss the molecular mechanisms that regulate polarized membrane trafficking and mechanical properties of the immune synapse, as well as the potential extracellular cues from the environment, which may impact the ability of B cells to sense and acquire antigens at the immune synapse. An integrated view of the diverse cellular mechanisms that shape the immune synapse will provide a better understanding on how B cells are efficiently activated. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differential protection by cell wall components of Lactobacillus amylovorus DSM 16698Tagainst alterations of membrane barrier and NF-kB activation induced by enterotoxigenic F4+ Escherichia coli on intestinal cells.

PubMed

Roselli, Marianna; Finamore, Alberto; Hynönen, Ulla; Palva, Airi; Mengheri, Elena

2016-09-29

The role of Lactobacillus cell wall components in the protection against pathogen infection in the gut is still largely unexplored. We have previously shown that L. amylovorus DSM 16698 T is able to reduce the enterotoxigenic F4 + Escherichia coli (ETEC) adhesion and prevent the pathogen-induced membrane barrier disruption through the regulation of IL-10 and IL-8 expression in intestinal cells. We have also demonstrated that L. amylovorus DSM 16698 T protects host cells through the inhibition of NF-kB signaling. In the present study, we investigated the role of L. amylovorus DSM 16698 T cell wall components in the protection against F4 + ETEC infection using the intestinal Caco-2 cell line. Purified cell wall fragments (CWF) from L. amylovorus DSM 16698 T were used either as such (uncoated, U-CWF) or coated with S-layer proteins (S-CWF). Differentiated Caco-2/TC7 cells on Transwell filters were infected with F4 + ETEC, treated with S-CWF or U-CWF, co-treated with S-CWF or U-CWF and F4 + ETEC for 2.5 h, or pre-treated with S-CWF or U-CWF for 1 h before F4 + ETEC addition. Tight junction (TJ) and adherens junction (AJ) proteins were analyzed by immunofluorescence and Western blot. Membrane permeability was determined by phenol red passage. Phosphorylated p65-NF-kB was measured by Western blot. We showed that both the pre-treatment with S-CWF and the co- treatment of S-CWF with the pathogen protected the cells from F4 + ETEC induced TJ and AJ injury, increased membrane permeability and activation of NF-kB expression. Moreover, the U-CWF pre-treatment, but not the co-treatment with F4 + ETEC, inhibited membrane damage and prevented NF-kB activation. The results indicate that the various components of L. amylovorus DSM 16698 T cell wall may counteract the damage caused by F4 + ETEC through different mechanisms. S-layer proteins are essential for maintaining membrane barrier function and for mounting an anti-inflammatory response against F4 + ETEC infection. U-CWF are not able to defend the cells when they are infected with F4 + ETEC but may activate protective mechanisms before pathogen infection.

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

PubMed Central

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

PubMed

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Changes in E-cadherin rigidity sensing regulate cell adhesion

PubMed Central

Collins, Caitlin; Pruitt, Beth L.; Nelson, W. James

2017-01-01

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin–dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell–cell adhesion assay and live cell imaging of cell–cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell–cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell–cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell–cell adhesion. PMID:28674019

Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers*

PubMed Central

Antonenko, Yuri N.; Avetisyan, Armine V.; Cherepanov, Dmitry A.; Knorre, Dmitry A.; Korshunova, Galina A.; Markova, Olga V.; Ojovan, Silvia M.; Perevoshchikova, Irina V.; Pustovidko, Antonina V.; Rokitskaya, Tatyana I.; Severina, Inna I.; Simonyan, Ruben A.; Smirnova, Ekaterina A.; Sobko, Alexander A.; Sumbatyan, Natalia V.; Severin, Fedor F.; Skulachev, Vladimir P.

2011-01-01

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C12R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H+ ions was generated in the presence of C12R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C12R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C12R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C12R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease. PMID:21454507

Cell signalling and phospholipid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boss, W.F.

1989-01-01

Our research for the past two years has involved the study of phosphoinositides and their potential role in regulating plant growth and development. Our initial goal was to document the sequence of events involved in inositol phospholipid metabolism in response to external stimuli. Our working hypothesis was that phosphatidylinositol bisphosphate (PIP/sub 2/) was in the plasma membrane of plants cells and would be hydrolyzed by phospholipase C to yield the second messengers inositol triphosphate (IP/sub 3/) and diacyglycerol (DAG) and that IP/sub 3/ would mobilize intracellular calcium as has been shown for animal cells. Our results with both carrot suspensionmore » culture cells and sunflower hypocotyl indicate that this paradigm is not the primary mechanism of signal transduction in these systems. We have observed very rapid, within 5 sec, stimulation of phosphatidylinositol monophosphate (PIP) kinase which resulted in an increase in PIP/sub 2/. However, there was no evidence for activation of phospholipase C. In addition, we have shown that PIP and PIP/sub 2/ can activate the plasma membrane ATPase. The results of these studies are described briefly in the paragraphs below. Inositol phospholipids are localized in distinct membrane fractions. If PIP and PIP/sub 2/ play a role in the transduction of external signals, they should be present in the plasma membrane. We used the fusogenic carrot suspension culture cells as a model system to study the distribution of inositol phospholipids in various membrane fractions and organelles. Cells were labeled 12 to 18 h with myo(2-/sup 3/H) inositol and the membranes were isolated by aqueous two-phase partitioning. The plasma membrane was enriched in PIP and PIP/sub 2/ compared to the intracellular membranes.« less

Mechanisms and significance of eryptosis.

PubMed

Lang, Florian; Lang, Karl S; Lang, Philipp A; Huber, Stephan M; Wieder, Thomas

2006-01-01

Suicidal death of erythrocytes (eryptosis) is characterized by cell shrinkage, membrane blebbing, activation of proteases, and phosphatidylserine exposure at the outer membrane leaflet. Exposed phosphatidylserine is recognized by macrophages that engulf and degrade the affected cells. Eryptosis is triggered by erythrocyte injury after several stressors, including oxidative stress. Besides caspase activation after oxidative stress, two signaling pathways converge to trigger eryptosis: (a) formation of prostaglandin E(2) leads to activation of Ca(2+)-permeable cation channels, and (b) the phospholipase A(2)-mediated release of platelet-activating factor activates a sphingomyelinase, leading to formation of ceramide. Increased cytosolic Ca(2+) activity and enhanced ceramide levels lead to membrane scrambling with subsequent phosphatidylserine exposure. Moreover, Ca(2+) activates Ca(2+)-sensitive K(2+) channels, leading to cellular KCl loss and cell shrinkage. In addition, Ca(2+) stimulates the protease calpain, resulting in degradation of the cytoskeleton. Eryptosis is inhibited by erythropoietin, which thus extends the life span of circulating erythrocytes. Eryptosis may be a mechanism of defective erythrocytes to escape hemolysis. Conversely, excessive eryptosis favors the development of anemia. Conditions with excessive eryptosis include iron deficiency, lead or mercury intoxication, sickle cell anemia, thalassemia, glucose 6- phosphate dehydrogenase deficiency, malaria, and infection with hemolysin-forming pathogens.

Decolorization of Malachite Green and Crystal Violet by Waterborne Pathogenic Mycobacteria

PubMed Central

Jones, Jefferson J.; Falkinham III, Joseph O.

2003-01-01

Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium marinum, and Mycobacterium chelonae tolerate high concentrations of the dyes malachite green and crystal violet. Cells of strains of those species decolorized (reduced) both malachite green and crystal violet. Because decolorized malachite green lacked antimicrobial activity, the resistance of these mycobacteria could be due, in part, to their ability to decolorize the dyes. Small amounts of malachite green and its reduced, decolorized product were detected in the lipid fraction of M. avium strain A5 cells grown in the presence of malachite green, suggesting that a minor component of resistance could be due to sequestering the dyes in the extensive mycobacterial cell surface lipid. The membrane fraction of M. avium strain A5 had at least a fivefold-higher specific decolorization rate than did the crude extract, suggesting that the decolorization activity is membrane associated. The malachite green-decolorizing activity of the membrane fraction of M. avium strain A5 was abolished by either boiling or proteinase exposure, suggesting that the decolorizing activity was due to a protein. Decolorization activity of membrane fractions was stimulated by ferrous ion and inhibited by dinitrophenol and metyrapone. PMID:12821489

The role of PIP2 and the IP3/DAG pathway in intracellular calcium release and cell survival during nanosecond electric pulse exposures

NASA Astrophysics Data System (ADS)

Steelman, Zachary A.; Tolstykh, Gleb P.; Estlack, Larry E.; Roth, Caleb C.; Ibey, Bennett L.

2015-03-01

Phosphatidylinositol4,5-biphosphate (PIP2) is a membrane phospholipid of particular importance in cell-signaling pathways. Hydrolysis of PIP2 releases inositol-1,4,5-triphosphate (IP3) from the membrane, activating IP3 receptors on the smooth endoplasmic reticulum (ER) and facilitating a release of intracellular calcium stores and activation of protein kinase C (PKC). Recent studies suggest that nanosecond pulsed electric fields (nsPEF) cause depletion of PIP2 in the cellular membrane, activating the IP3 signaling pathway. However, the exact mechanism(s) causing this observed depletion of PIP2 are unknown. Complicating the matter, nsPEF create nanopores in the plasma membrane, allowing calcium to enter the cell and thus causing an increase in intracellular calcium. While elevated intracellular calcium can cause activation of phospholipase C (PLC) (a known catalyst of PIP2 hydrolysis), PIP2 depletion has been shown to occur in the absence of both extracellular and intracellular calcium. These observations have led to the hypothesis that the high electric field itself may be playing a direct role in the hydrolysis of PIP2 from the plasma membrane. To support this hypothesis, we used edelfosine to block PLC and prevent activation of the IP3/DAG pathway in Chinese Hamster Ovarian (CHO) cells prior to applying nsPEF. Fluorescence microscopy was used to monitor intracellular calcium bursts during nsPEF, while MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survivability assays were utilized to determine whether edelfosine improved cell survival during nsPEF exposure. This work is critical to refine the role of PIP2 in the cellular response to nsPEF, and also to determine the fundamental biological effects of high electric field exposures.

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

PubMed

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

2015-10-20

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

Withaferin-A induces apoptosis in osteosarcoma U2OS cell line via generation of ROS and disruption of mitochondrial membrane potential.

PubMed

Li, A-X; Sun, M; Li, X

2017-03-01

Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania somnifera. It has marked pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti-cancer therapeutic development is to identify apoptosis-inducing drug-like molecules. Osteosarcoma is a rare type of bone cancer affecting humans. The objective of the present study was therefore to evaluate the antitumor potential of WF-A against several osteosarcoma cell lines. MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC50. DAPI staining was used to confirm the apoptosis-inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis. The results of the present study revealed that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC50 ranging from 0.32 to 7.6 µM. The lowest IC50 (0.32 µM) was observed against U2OS cell line and, therefore, it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced a ROS-mediated reduction in mitochondrial membrane potential in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells. We suggest that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential.

Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

PubMed

Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

2015-01-01

Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.

GDC-0941 enhances the lysosomal compartment via TFEB and primes glioblastoma cells to lysosomal membrane permeabilization and cell death.

PubMed

Enzenmüller, Stefanie; Gonzalez, Patrick; Karpel-Massler, Georg; Debatin, Klaus-Michael; Fulda, Simone

2013-02-01

Since phosphatidylinositol-3-kinase (PI3K) inhibitors are primarily cytostatic against glioblastoma, we searched for new drug combinations. Here, we discover that the PI3K inhibitor GDC-0941 acts in concert with the natural compound B10, a glycosylated derivative of betulinic acid, to induce cell death in glioblastoma cells. Importantly, parallel experiments in primary glioblastoma cultures similarly show that GDC-0941 and B10 cooperate to trigger cell death, underscoring the clinical relevance of this finding. Molecular studies revealed that treatment with GDC-0941 stimulates the expression and nuclear translocation of Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, the lysosomal membrane marker LAMP-1 and the mature form of cathepsin B. Also, GDC-0941 triggers a time-dependent increase of the lysosomal compartment in a TFEB-dependent manner, since knockdown of TFEB significantly reduces this GDC-0941-stimulated lysosomal enhancement. Importantly, GDC-0941 cooperates with B10 to trigger lysosomal membrane permeabilization, leading to increased activation of Bax, loss of mitochondrial membrane potential (MMP), caspase-3 activation and cell death. Addition of the cathepsin B inhibitor CA-074me reduces Bax activation, loss of MMP, caspase-3 activation and cell death upon treatment with GDC-0941/B10. By comparison, knockdown of caspase-3 or the broad-range caspase inhibitor zVAD.fmk inhibits GDC-0941/B10-induced DNA fragmentation, but does not prevent cell death, thus pointing to both caspase-dependent and -independent pathways. By identifying the combination of GDC-0941 and B10 as a new, potent strategy to trigger cell death in glioblastoma cells, our findings have important implications for the development of novel treatment approaches for glioblastoma. Copyright © 2012. Published by Elsevier Ireland Ltd.

Cross Talk Between Ceramide and Redox Signaling: Implications for Endothelial Dysfunction and Renal Disease

PubMed Central

Li, Pin-Lan; Zhang, Yang

2013-01-01

Recent studies have demonstrated that cross talk between ceramide and redox signaling modulates various cell activities and functions and contributes to the development of cardiovascular diseases and renal dysfunctions. Ceramide triggers the generation of reactive oxygen species (ROS) and increases oxidative stress in many mammalian cells and animal models. On the other hand, inhibition of ROS-generating enzymes or treatment of antioxidants impairs sphingomyelinase activation and ceramide production. As a mechanism, ceramide-enriched signaling platforms, special cell membrane rafts (MR) (formerly lipid rafts), provide an important microenvironment to mediate the cross talk of ceramide and redox signaling to exert a corresponding regulatory role on cell and organ functions. In this regard, activation of acid sphingomyelinase and generation of ceramide mediate the formation of ceramide-enriched membrane platforms, where trans-membrane signals are transmitted or amplified through recruitment, clustering, assembling, or integration of various signaling molecules. A typical such signaling platform is MR redox signaling platform that is centered on ceramide production and aggregation leading to recruitment and assembling of NADPH oxidase to form an active complex in the cell plasma membrane. This redox signaling platform not only conducts redox signaling or regulation but also facilitates a feedforward amplification of both ceramide and redox signaling. In addition to this membrane MR redox signaling platform, the cross talk between ceramide and redox signaling may occur in other cell compartments. This book chapter focuses on the molecular mechanisms, spatial–temporal regulations, and implications of this cross talk between ceramide and redox signaling, which may provide novel insights into the understanding of both ceramide and redox signaling pathways. PMID:23563657

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

PubMed

Morré, D M; Morre, D J

2000-06-23

Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum

Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells

NASA Technical Reports Server (NTRS)

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

2000-01-01

Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum

Shape Changes and Interaction Mechanism of Escherichia coli Cells Treated with Sericin and Use of a Sericin-Based Hydrogel for Wound Healing.

PubMed

Xue, Rui; Liu, Yalong; Zhang, Qingsong; Liang, Congcong; Qin, Huazhen; Liu, Pengfei; Wang, Ke; Zhang, Xiaoyong; Chen, Li; Wei, Yen

2016-08-01

To verify the interaction mechanism between sericin and Escherichia coli, especially the morphological and structural changes in the bacterial cells, the antimicrobial activity of sericin against E. coli as a model for Gram-negative bacteria was investigated. The antibacterial activity of sericin on E. coli and the interaction mechanism were investigated in this study by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with sericin. The changes in morphology and cellular compositions of bacterial cells treated with sericin were observed by an inverted fluorescence microscope, scanning electron microscopy, and transmission electron microscopy. Changes in electrical conductivity, total sugar concentration of the broth for the bacteria, and protein expression of the bacteria were determined to investigate the permeability of the cell membrane. A sericin-based hydrogel was prepared for an in vivo study of wound dressing. The results showed that the antibacterial activity of the hydrogel increased with the increase in the concentration of sericin from 10 g/liter to 40 g/liter. The introduction of sericin induces membrane blebbing of E. coli cells caused by antibiotic action on the cell membrane. The cytoplasm shrinkage phenomenon was accompanied by blurring of the membrane wall boundaries. When E. coli cells were treated with sericin, release of intracellular components quickly increased. The electrical conductivity assay indicated that the charged ions are reduced after exposure to sericin so that the integrity of the cell membrane is weakened and metabolism is blocked. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that sericin hinders the expression of bacterial protein. Sericin may damage the integrity of the bacterial cell membrane, thereby eventually inhibiting the growth and reproduction of E. coli Compared to sterile gauze, the sericin-based hydrogel promoted fibroblast cell proliferation and accelerated the formation of granulation tissues and neovessels. The specific relationship and interaction mechanism between sericin and E. coli cells were investigated and elucidated. The results show that after 12 h of treatment, sericin molecules induce membrane blebbing of E. coli cells, and the bacteria show decreases in liquidity and permeability of biological membrane, resulting in alterations in the conductivity of the culture medium and the integrity of the outer membrane. The subsequent in vivo results demonstrate that the sericin-poly(N-isopropylacrylamide-N,N'-methylene-bis-acrylamide [NIPAm-MBA]) hydrogel accelerated wound healing compared to that with sterile gauze, which is a beneficial result for future applications in clinical medicine and the textile, food, and coating industries. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Effective tension and fluctuations in active membranes.

PubMed

Loubet, Bastien; Seifert, Udo; Lomholt, Michael Andersen

2012-03-01

We calculate the fluctuation spectrum of the shape of a lipid vesicle or cell exposed to a nonthermal source of noise. In particular, we take constraints on the membrane area and the volume of fluid that it encapsulates into account when obtaining expressions for the dependency of the membrane tension on the noise. We then investigate three possible origins of the nonthermal noise taken from the literature: A direct force, which models an external medium pushing on the membrane, a curvature force, which models a fluctuating spontaneous curvature, and a permeation force coming from an active transport of fluid through the membrane. For the direct force and curvature force cases, we compare our results to existing experiments on active membranes.

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.

Impaired Bioenergetics in Mutant Mitochondrial DNA Determines Cell Fate During Seizure-Like Activity.

PubMed

Kovac, Stjepana; Preza, Elisavet; Houlden, Henry; Walker, Matthew C; Abramov, Andrey Y

2018-04-27

Mutations in genes affecting mitochondrial proteins are increasingly recognised in patients with epilepsy, but the factors determining cell fate during seizure activity in these mutations remain unknown. Fluorescent dye imaging techniques were applied to fibroblast cell lines from patients suffering from common mitochondrial mutations and to age-matched controls. Using live cell imaging techniques in fibroblasts, we show that fibroblasts with mutations in the mitochondrial genome had reduced mitochondrial membrane potential and NADH pools and higher redox indices, indicative of respiratory chain dysfunction. Increasing concentrations of ferutinin, a Ca 2+ ionophore, led to oscillatory Ca 2+ signals in fibroblasts resembling dynamic Ca 2+ changes that occur during seizure-like activity. Co-monitoring of mitochondrial membrane potential (ΔΨ m ) changes induced by ferutinin showed accelerated membrane depolarisation and cell collapse in fibroblasts with mutations in the mitochondrial genome when compared to controls. Ca 2+ flash photolysis using caged Ca 2+ confirmed impaired Ca 2+ handling in fibroblasts with mitochondrial mutations. Findings indicate that intracellular Ca 2+ levels cannot be compensated during periods of hyperexcitability, leading to Ca 2+ overload and subsequent cell death in mitochondrial diseases.

Investigation of Oxygen Reduction Activity of Catalysts Derived from Co and Co/Zn Methyl-Imidazolate Frameworks in Proton Exchange Membrane Fuel Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chong, Lina; Goenaga, Gabriel A.; Williams, Kia

We demonstrated that the oxygen reduction reaction (ORR) activity over the catalysts derived from pyrolyzed cobalt zeolitic imidazolate frameworks depends strongly on the imidazole ligand structure and cobalt content. The activity and durability of these catalysts were tested in the proton exchange membrane fuel cell for the first time. The membrane electrode assembly containing a catalyst derived from Co/Zn bimetallic ZIF at cathode achieved an open circuit voltage of 0.93 V, a current density of 28 mA cm-2 at 0.8 ViR-free and a peak power density of 374 mW cm-2.

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways.

PubMed

Qu, Yan; Dubyak, George R

2009-06-01

Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived microvesicles or microparticles; (4) PM blebbing; (5) cell-cell fusion resulting in formation of multinucleate cells; (6) phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes from multivesicular bodies.

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

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

2014-12-10

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.

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

NASA Astrophysics Data System (ADS)

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

2014-12-01

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.

Spring-Loaded Model Revisited: Paramyxovirus Fusion Requires Engagement of a Receptor Binding Protein beyond Initial Triggering of the Fusion Protein▿

PubMed Central

Porotto, Matteo; DeVito, Ilaria; Palmer, Samantha G.; Jurgens, Eric M.; Yee, Jia L.; Yokoyama, Christine C.; Pessi, Antonello; Moscona, Anne

2011-01-01

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry. PMID:21976650

Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein.

PubMed

Porotto, Matteo; Devito, Ilaria; Palmer, Samantha G; Jurgens, Eric M; Yee, Jia L; Yokoyama, Christine C; Pessi, Antonello; Moscona, Anne

2011-12-01

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.

Side Chain Degradable Cationic-Amphiphilic Polymers with Tunable Hydrophobicity Show in Vivo Activity.

PubMed

Uppu, Divakara S S M; Samaddar, Sandip; Hoque, Jiaul; Konai, Mohini M; Krishnamoorthy, Paramanandham; Shome, Bibek R; Haldar, Jayanta

2016-09-12

Cationic-amphiphilic antibacterial polymers with optimal amphiphilicity generally target the bacterial membranes instead of mammalian membranes. To date, this balance has been achieved by varying the cationic charge or side chain hydrophobicity in a variety of cationic-amphiphilic polymers. Optimal hydrophobicity of cationic-amphiphilic polymers has been considered as the governing factor for potent antibacterial activity yet minimal mammalian cell toxicity. However, the concomitant role of hydrogen bonding and hydrophobicity with constant cationic charge in the interactions of antibacterial polymers with bacterial membranes is not understood. Also, degradable polymers that result in nontoxic degradation byproducts offer promise as safe antibacterial agents. Here we show that amide- and ester (degradable)-bearing cationic-amphiphilic polymers with tunable side chain hydrophobicity can modulate antibacterial activity and cytotoxicity. Our results suggest that an amide polymer can be a potent antibacterial agent with lower hydrophobicity whereas the corresponding ester polymer needs a relatively higher hydrophobicity to be as effective as its amide counterpart. Our studies reveal that at higher hydrophobicities both amide and ester polymers have similar profiles of membrane-active antibacterial activity and mammalian cell toxicity. On the contrary, at lower hydrophobicities, amide and ester polymers are less cytotoxic, but the former have potent antibacterial and membrane activity compared to the latter. Incorporation of amide and ester moieties made these polymers side chain degradable, with amide polymers being more stable than the ester polymers. Further, the polymers are less toxic, and their degradation byproducts are nontoxic to mice. More importantly, the optimized amide polymer reduces the bacterial burden of burn wound infections in mice models. Our design introduces a new strategy of interplay between the hydrophobic and hydrogen bonding interactions keeping constant cationic charge density for developing potent membrane-active antibacterial polymers with minimal toxicity to mammalian cells.

Decreased sialidase activity in alveolar macrophages of guinea pigs exposed to coal mine dust.

PubMed Central

Terzidis-Trabelsi, H; Lefèvre, J P; Bignon, J; Lambré, C R

1992-01-01

The origin of immune dysfunctions that are observed in pneumoconiotic miners still remains unknown. There is evidence that the carbohydrate moiety of membrane glycoconjugates is of primary importance in many functions of immunocompetent cells. The glycosylation, and especially the sialylation level of membrane components of various lymphocyte and macrophage subsets, vary depending on the state of cellular differentiation and activation. Sialidases, which may regulate the amount of sialic acids exposed on the cell membrane, can thus be considered as immunoregulatory enzymes. In this report, the sialidase activity has been measured in alveolar macrophages (AM) and in cell-free bronchoalveolar lavage fluid (BALF) from guinea pigs exposed for 4 months to coal mine dust at a concentration of 300 mg/m3. The samples were collected by bronchoalveolar lavage 2 months after cessation of exposure. The sialidase activity in the cell-free fluid and in the purified alveolar macrophages showed a 10-fold decrease (p less than 0.001). Kinetic parameters of the enzyme such as Km and optimum pH did not change. This changed activity was specific for sialidase, as two other lysosomal glycosidases, beta-galactosidase and N-acetylglucosaminidase, showed unchanged activities. These results suggest the possibility that, by inducing a decreased sialidase activity, exposure to coal mine dust may lead to a modified expression of AM membrane-associated sialic acids giving rise to altered immune functions (i. e., phagocytosis, antigen processing, response to cytokines, etc.). PMID:1396442

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

PubMed

Huang, Chao; Zhang, Zhe; Chen, Lihan; Lee, Hank W; Ayrapetov, Marina K; Zhao, Ting C; Hao, Yimei; Gao, Jinsong; Yang, Chunzhang; Mehta, Gautam U; Zhuang, Zhengping; Zhang, Xiaoren; Hu, Guohong; Chin, Y Eugene

2018-06-01

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G 2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR . ©2018 American Association for Cancer Research.

Magnetic resonance imaging of water content across the Nafion membrane in an operational PEM fuel cell.

PubMed

Zhang, Ziheng; Martin, Jonathan; Wu, Jinfeng; Wang, Haijiang; Promislow, Keith; Balcom, Bruce J

2008-08-01

Water management is critical to optimize the operation of polymer electrolyte membrane fuel cells. At present, numerical models are employed to guide water management in such fuel cells. Accurate measurements of water content variation in polymer electrolyte membrane fuel cells are required to validate these models and to optimize fuel cell behavior. We report a direct water content measurement across the Nafion membrane in an operational polymer electrolyte membrane fuel cell, employing double half k-space spin echo single point imaging techniques. The MRI measurements with T2 mapping were undertaken with a parallel plate resonator to avoid the effects of RF screening. The parallel plate resonator employs the electrodes inherent to the fuel cell to create a resonant circuit at RF frequencies for MR excitation and detection, while still operating as a conventional fuel cell at DC. Three stages of fuel cell operation were investigated: activation, operation and dehydration. Each profile was acquired in 6 min, with 6 microm nominal resolution and a SNR of better than 15.

Segregation of Two Spectrin Isoforms: Polarized Membrane-binding Sites Direct Polarized Membrane Skeleton Assembly

PubMed Central

Dubreuil, Ronald R.; Maddux, Pratumtip Boontrakulpoontawee; Grushko, Tanya A.; Macvicar, Gary R.

1997-01-01

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and β spectrin are recruited to sites of cell–cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (αβH), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and αβ spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, αβ spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, αβH spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell–cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells. PMID:9348534

Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles.

PubMed

Martin, Katherine R; Kantari-Mimoun, Chahrazade; Yin, Min; Pederzoli-Ribeil, Magali; Angelot-Delettre, Fanny; Ceroi, Adam; Grauffel, Cédric; Benhamou, Marc; Reuter, Nathalie; Saas, Philippe; Frachet, Philippe; Boulanger, Chantal M; Witko-Sarsat, Véronique

2016-05-13

Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles*

PubMed Central

Martin, Katherine R.; Kantari-Mimoun, Chahrazade; Yin, Min; Pederzoli-Ribeil, Magali; Angelot-Delettre, Fanny; Ceroi, Adam; Grauffel, Cédric; Benhamou, Marc; Reuter, Nathalie; Saas, Philippe; Frachet, Philippe; Boulanger, Chantal M.; Witko-Sarsat, Véronique

2016-01-01

Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease. PMID:26961880

An Unconventional Diacylglycerol Kinase That Regulates Phospholipid Synthesis and Nuclear Membrane Growth*♦

PubMed Central

Han, Gil-Soo; O'Hara, Laura; Carman, George M.; Siniossoglou, Symeon

2008-01-01

Changes in nuclear size and shape during the cell cycle or during development require coordinated nuclear membrane remodeling, but the underlying molecular events are largely unknown. We have shown previously that the activity of the conserved phosphatidate phosphatase Pah1p/Smp2p regulates nuclear structure in yeast by controlling phospholipid synthesis and membrane biogenesis at the nuclear envelope. Two screens for novel regulators of phosphatidate led to the identification of DGK1. We show that Dgk1p is a unique diacylglycerol kinase that uses CTP, instead of ATP, to generate phosphatidate. DGK1 counteracts the activity of PAH1 at the nuclear envelope by controlling phosphatidate levels. Overexpression of DGK1 causes the appearance of phosphatidate-enriched membranes around the nucleus and leads to its expansion, without proliferating the cortical endoplasmic reticulum membrane. Mutations that decrease phosphatidate levels decrease nuclear membrane growth in pah1Δ cells. We propose that phosphatidate metabolism is a critical factor determining nuclear structure by regulating nuclear membrane biogenesis. PMID:18458075

Detecting Extracellular Carbonic Anhydrase Activity Using Membrane Inlet Mass Spectrometry

PubMed Central

Delacruz, Joannalyn; Mikulski, Rose; Tu, Chingkuang; Li, Ying; Wang, Hai; Shiverick, Kathleen T.; Frost, Susan C.; Horenstein, Nicole A.; Silverman, David N.

2010-01-01

Current research into the function of carbonic anhydrases in cell physiology emphasizes the role of membrane-bound carbonic anhydrases, such as carbonic anhydrase IX that has been identified in malignant tumors and is associated with extracellular acidification as a response to hypoxia. We present here a mass spectrometric method to determine the extent to which total carbonic anhydrase activity is due to extracellular carbonic anhydrase in whole cell preparations. The method is based on the biphasic rate of depletion of 18O from CO2 measured by membrane inlet mass spectrometry. The slopes of the biphasic depletion are a sensitive measure of the presence of carbonic anhydrase outside and inside of the cells. This property is demonstrated here using suspensions of human red cells in which external carbonic anhydrase was added to the suspending solution. It is also applied to breast and prostate cancer cells which both express exofacial carbonic anhydrase IX. Inhibition of external carbonic anhydrase is achieved by use of a membrane impermeant inhibitor that was synthesized for this purpose, p-aminomethylbenzenesulfonamide attached to a polyethyleneglycol polymer. PMID:20417171

Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

NASA Technical Reports Server (NTRS)

Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

2000-01-01

DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

The membrane may be an important factor in browning of fresh-cut pear.

PubMed

Li, Zhenghong; Zhang, Yuxing; Ge, Huibo

2017-09-01

Surface browning is an important cause of deterioration of fresh-cut fruit during postharvest handling. In this paper, four pear cultivars with different extents of natural browning were selected to analyse the factors involved in browning. The main results are as follows: the lipoxygenase (LOX) activity of 'Mantianhong' and 'Yali' pears was higher accompanied by a stronger degree of browning, while the LOX activity in 'Xueqing' and 'Xinli 7' pears was lower, with less browning. A higher unsaturated fatty acid ratio of pear resulted in reduced browning. The cell membranes disappeared 30min after being cut in 'Mantianhong' pear, which browns easily; however, the cell membranes were still intact 30min after being cut in 'Xueqing' pear, which does not brown easily. Therefore, it can be assumed that the stability of the cell membrane plays an important role in inhibiting browning of fresh-cut pears. Copyright © 2017. Published by Elsevier Ltd.

Elasticity of biomembranes studied by dynamic light scattering

NASA Astrophysics Data System (ADS)

Fujime, Satoru; Miyamoto, Shigeaki

1991-05-01

Combination of osmotic swelling and dynamic light scattering makes it possible to measure the elastic modulus of biomembranes. By this technique we have observed a drastic increase in membrane flexibility on activation of Na/glucose cotransporters in membrane vesicles prepared from brush-borders of rat small intestine and on activation by micromolar [Ca2] of exocytosis in secretory granules isolated from rat pancreatic acinar cells and bovine adrenal chromaffin cells. 1 .

Matrix Rigidity Activates Wnt Signaling through Down-regulation of Dickkopf-1 Protein*

PubMed Central

Barbolina, Maria V.; Liu, Yiuying; Gurler, Hilal; Kim, Mijung; Kajdacsy-Balla, Andre A.; Rooper, Lisa; Shepard, Jaclyn; Weiss, Michael; Shea, Lonnie D.; Penzes, Peter; Ravosa, Matthew J.; Stack, M. Sharon

2013-01-01

Cells respond to changes in the physical properties of the extracellular matrix with altered behavior and gene expression, highlighting the important role of the microenvironment in the regulation of cell function. In the current study, culture of epithelial ovarian cancer cells on three-dimensional collagen I gels led to a dramatic down-regulation of the Wnt signaling inhibitor dickkopf-1 with a concomitant increase in nuclear β-catenin and enhanced β-catenin/Tcf/Lef transcriptional activity. Increased three-dimensional collagen gel invasion was accompanied by transcriptional up-regulation of the membrane-tethered collagenase membrane type 1 matrix metalloproteinase, and an inverse relationship between dickkopf-1 and membrane type 1 matrix metalloproteinase was observed in human epithelial ovarian cancer specimens. Similar results were obtained in other tissue-invasive cells such as vascular endothelial cells, suggesting a novel mechanism for functional coupling of matrix adhesion with Wnt signaling. PMID:23152495

Matrix rigidity activates Wnt signaling through down-regulation of Dickkopf-1 protein.

PubMed

Barbolina, Maria V; Liu, Yiuying; Gurler, Hilal; Kim, Mijung; Kajdacsy-Balla, Andre A; Rooper, Lisa; Shepard, Jaclyn; Weiss, Michael; Shea, Lonnie D; Penzes, Peter; Ravosa, Matthew J; Stack, M Sharon

2013-01-04

Cells respond to changes in the physical properties of the extracellular matrix with altered behavior and gene expression, highlighting the important role of the microenvironment in the regulation of cell function. In the current study, culture of epithelial ovarian cancer cells on three-dimensional collagen I gels led to a dramatic down-regulation of the Wnt signaling inhibitor dickkopf-1 with a concomitant increase in nuclear β-catenin and enhanced β-catenin/Tcf/Lef transcriptional activity. Increased three-dimensional collagen gel invasion was accompanied by transcriptional up-regulation of the membrane-tethered collagenase membrane type 1 matrix metalloproteinase, and an inverse relationship between dickkopf-1 and membrane type 1 matrix metalloproteinase was observed in human epithelial ovarian cancer specimens. Similar results were obtained in other tissue-invasive cells such as vascular endothelial cells, suggesting a novel mechanism for functional coupling of matrix adhesion with Wnt signaling.

Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

NASA Astrophysics Data System (ADS)

Kamiya, Koki; Kawano, Ryuji; Osaki, Toshihisa; Akiyoshi, Kazunari; Takeuchi, Shoji

2016-09-01

Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

Functional and molecular characterization of multiple K-Cl cotransporter isoforms in corneal epithelial cells

PubMed Central

Capó-Aponte, José E.; Wang, Zheng; Bildin, Victor N.; Iserovich, Pavel; Pan, Zan; Zhang, Fan; Pokorny, Kathryn S.; Reinach, Peter S.

2009-01-01

The dependence of regulatory volume decrease (RVD) activity on potassium–chloride cotransporter (KCC) isoform expression was characterized in corneal epithelial cells (CEC). During exposure to a 50% hypotonic challenge, the RVD response was larger in SV40-immortalized human CEC (HCEC) than in SV40-immortalized rabbit CEC (RCEC). A KCC inhibitor—[(dihydroindenyl)oxy] alkanoic acid (DIOA)—blocked RVD more in HCEC than RCEC. Under isotonic conditions, N-ethylmaleimide (NEM) produced KCC activation and transient cell shrinkage. Both of these changes were greater in HCEC than in RCEC. Immunoblot analysis of HCEC, RCEC, primary human CEC (pHCEC), and primary bovine CEC (BCEC) plasma membrane enriched fractions revealed KCC1, KCC3, and KCC4 isoform expression, whereas KCC2 was undetectable. During a hypotonic challenge, KCC1 membrane content increased more rapidly in HCEC than in RCEC. Such a challenge induced a larger increase and more transient p44/42MAPK activation in HCEC than RCEC. On the other hand, HCEC and RCEC p38MAPK phosphorylation reached peak activations at 2.5 and 15 min, respectively. Only in HCEC, pharmacological manipulation of KCC activity modified the hypotonicity-induced activation of p44/42MAPK, whereas p38MAPK phosphorylation was insensitive to such procedures in both cell lines. Larger increases in HCEC KCC1 membrane protein content correlate with their ability to undergo faster and more complete RVD. Furthermore, pharmacological activation of KCC increased p44/42MAPK phosphorylation in HCEC but not in RCEC, presumably a reflection of low KCC membrane expression in RCEC. These findings suggest that KCC1 plays a role in (i) maintaining isotonic steady-state cell volume homeostasis, (ii) recovery of isotonic cell volume after a hypotonic challenge through RVD, and (iii) regulating hypotonicity-induced activation of the p44/42MAPK signaling pathway required for cell proliferation. PMID:17418819

Chemoelectrical energy conversion of adenosine triphosphate

NASA Astrophysics Data System (ADS)

Sundaresan, Vishnu Baba; Sarles, Stephen Andrew; Leo, Donald J.

2007-04-01

Plant and animal cell membranes transport charged species, neutral molecules and water through ion pumps and channels. The energy required for moving species against established concentration and charge gradients is provided by the biological fuel - adenosine triphosphate (ATP) -synthesized within the cell. The adenosine triphosphatase (ATPases) in a plant cell membrane hydrolyze ATP in the cell cytoplasm to pump protons across the cell membrane. This establishes a proton gradient across the membrane from the cell exterior into the cell cytoplasm. This proton motive force stimulates ion channels that transport nutrients and other species into the cell. This article discusses a device that converts the chemical energy stored in adenosine triphosphate into electrical power using a transporter protein, ATPase. The V-type ATPase proteins used in our prototype are extracted from red beet(Beta vulgaris) tonoplast membranes and reconstituted in a bilayer lipid membrane or BLM formed from POPC and POPS lipids. A pH7 medium that can support ATP hydrolysis is provided on both sides of the membrane and ATP is dissolved in the pH7 buffer on one side of the membrane. Hydrolysis of ATP results in the formation of a phosphate ion and adenosine diphosphate. The energy from the reaction activates ATPase in the BLM and moves a proton across the membrane. The charge gradient established across the BLM due to the reaction and ion transport is converted into electrical current by half-cell reference electrodes. The prototype ATPase cell with an effective BLM area of 4.15 mm2 carrying 15 μl of ATPase proteins was observed to develop a steady state peak power output of 70 nW, which corresponds to a specific power of 1.69 μW/cm2 and a current density of 43.4 μA/cm2 of membrane area.

Overview of online two-dimensional liquid chromatography based on cell membrane chromatography for screening target components from traditional Chinese medicines.

PubMed

Muhammad, Saqib; Han, Shengli; Xie, Xiaoyu; Wang, Sicen; Aziz, Muhammad Majid

2017-01-01

Cell membrane chromatography is a simple, specific, and time-saving technique for studying drug-receptor interactions, screening of active components from complex mixtures, and quality control of traditional Chinese medicines. However, the short column life, low sensitivity, low column efficiency (so cannot resolve satisfactorily mixture of compounds), low peak capacity, and inefficient in structure identification were bottleneck in its application. Combinations of cell membrane chromatography with multidimensional chromatography such as two-dimensional liquid chromatography and high sensitivity detectors like mass have significantly reduced many of the above-mentioned shortcomings. This paper provides an overview of the current advances in online two-dimensional-based cell membrane chromatography for screening target components from traditional Chinese medicines with particular emphasis on the instrumentation, preparation of cell membrane stationary phase, advantages, and disadvantages compared to alternative approaches. The last section of the review summarizes the applications of the online two-dimensional high-performance liquid chromatography based cell membrane chromatography reported since its emergence to date (2010-June 2016). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of counterions in the membrane-disruptive properties of pH-sensitive lysine-based surfactants.

PubMed

Nogueira, D R; Mitjans, M; Infante, M R; Vinardell, M P

2011-07-01

Surfactants are among the most versatile and widely used excipients in pharmaceuticals. This versatility, together with their pH-responsive membrane-disruptive activity and low toxicity, could also enable their potential application in drug delivery systems. Five anionic lysine-based surfactants which differ in the nature of their counterion were studied. Their capacity to disrupt the cell membrane was examined under a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model for endosomal membranes. The surfactants showed pH-sensitive hemolytic activity and improved kinetics at the endosomal pH range. Low concentrations resulted in negligible hemolysis at physiological pH and high membrane lytic activity at pH 5.4, which is in the range characteristic of late endosomes. With increasing concentration, the surfactants showed an enhanced capacity to lyse cell membranes, and also caused significant membrane disruption at physiological pH. This observation indicates that, at high concentrations, surfactant behavior is independent of pH. The mechanism of surfactant-mediated membrane destabilization was addressed, and scanning electron microscopy studies were also performed to evaluate the effects of the compounds on erythrocyte morphology as a function of pH. The in vitro cytotoxicity of the surfactants was assessed by MTT and NRU assays with the 3T3 cell line. The influence of different types of counterion on hemolytic activity and the potential applications of these surfactants in drug delivery are discussed. The possibility of using pH-sensitive surfactants for endosome disruption could hold great promise for intracellular drug delivery systems in future therapeutic applications. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

[Bioactive glass 45S5-silk fibroin membrane supports proliferation and differentiation of human dental pulp stem cells].

PubMed

Lyu, Xiaoshuai; Li, Zhengmao; Wang, Haiyan; Yang, Xuechao

2015-12-01

To investigate the effect of bioactivity glass 45S5- silk fibroin(BG45S5- SF) membrane on growth, proliferation and differentiation of human dental pulp stem cells(hDPSC), and to provide new ideas and method for the regeneration of pulp-dentine complex. hDPSC seed on pure silk fibroin membrane (protein membrane group) and BG45S5-SF membrane with different concentrations(1 000, 5 000 mg/L, composite membrane group A and B, respectively) were prepared, and the materials were incubated in cell culture fluid for 24 h. No material membrane orifice plate was used as blank control group. Contact angle meter was used to measure surface contact angle of protein membrane and composite membrane group(each group had three repeated holes). Cell proliferation was assessed by cell counting kit- 8 on the 4, 7, 14, and 21 days. The state of adhesion and growth of hDPSC on the materials surface was evaluated by scanning electron microscopy and cytoskeleton staining; and alkaline phosphatase (ALP) activity was measured to evaluate the cell differentiation potential. The expression of odontoblastic differentiation-related genes was measured by real-time PCR. Surface contact angle of the protein membrane group and composite membrane group A and group B were 89.51° ± 0.12°, 70.32° ± 0.07° and 71.31° ± 0.09° respectively. hDPSC adhered well on each materials surface on the 7, 14, 21 days, ALP activity and differentiation genes of composite membrane group A and B rised more significantly than the blank control group and protein membrane group did (P<0.05). Dentin matrix protein1(DMP- 1), dentin sialoprotein(DSP), ALP, osteocalcin(OC) mRNA expression reached peak on the 14 days in group A, and in group B on the 21 days. Bone sialoprotein(BSP) mRNA expression in both group A and B reached peak on the 21 days. BG45S5- SF membrane is able to support the proliferation and showed the potential of odontoblastic differentiation for hDPSC. This finding suggests that BG45S5-SF membrane was a kind of tissue engineering film material with the regeneration potential for pulp-dentine complex.

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. Copyright © 2015 The Authors. Artificial Organs published by Wiley Periodicals, Inc. on behalf of International Center for Artificial Organs and Transplantation (ICAOT).

Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax Requires CADM1/TSLC1 for Inactivation of the NF-κB Inhibitor A20 and Constitutive NF-κB Signaling

PubMed Central

Thomas, Remy; van der Weyden, Louise; Rauch, Dan; Ratner, Lee; Nyborg, Jennifer K.; Ramos, Juan Carlos; Takai, Yoshimi; Shembade, Noula

2015-01-01

Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells. PMID:25774694

Active Coupled Oscillators in the Inner Ear

NASA Astrophysics Data System (ADS)

Strimbu, Clark Elliott

Auditory and vestibular systems are endowed with an active process that enables them to detect signals as small as a few Angstroms; they also exhibit frequency selectivity; show strong nonlinearities; and can exhibit as spontaneous activity. Much of this active process comes from the sensory hair cells at the periphery of the auditory and vestibular systems. Each hair cell is capped by an eponymous hair bundle, a specialized structure that transduces mechanical forces into electrical signals. Experiments on mechanically decoupled cells from the frog sacculus have shown that individual hair bundles behave in an active manner analogous to an intact organ suggesting a common cellular basis for the active processes seen in many species. In particular, mechanically decoupled hair bundles show rapid active movements in response to transient stimuli and exhibit spontaneous oscillations. However, a single mechanosensitive hair cell is unable to match the performance of an entire organ. In vivo, hair bundles are often coupled to overlying membranes, gelatinous extracellular matrices. We used an in vitro preparation of the frog sacculus in which the otolithic membrane has been left intact. Under natural coupling conditions, there is a strong degree of correlation across the saccular epithelium, suggesting that the collective response of many cells contributes to the extreme sensitivity of this organ. When the membrane is left intact, the hair bundles do not oscillate spontaneously, showing that the natural coupling and loading tunes them into a quiescent regime. However, when stimulated by a pulse, the bundles show a rapid biphasic response that is abolished when the transduction channels are blocked. The active forces generated by the bundles are sufficient to move the overlying membrane.

Membrane microparticles and diseases.

PubMed

Wu, Z-H; Ji, C-L; Li, H; Qiu, G-X; Gao, C-J; Weng, X-S

2013-09-01

Membrane microparticles (MPs) are plasma membrane-derived vesicles shed by various types of activated or apoptotic cells including platelets, monocytes, endothelial cells, red blood cells, and granulocytes. MPs are being increasingly recognized as important regulators of cell-to-cell interactions. Recent evidences suggest they may play important functions not only in homeostasis but also in the pathogenesis of a number of diseases such as vascular diseases, cancer, infectious diseases and diabetes mellitus. Accordingly, inhibiting the production of MPs may serve as a novel therapeutic strategy for these diseases. Here we review recent advances on the mechanism underlying the generation of MPs and the role of MPs in vascular diseases, cancer, diabetes, inflammation, and pathogen infection.

Fungicidal mechanisms of cathelicidins LL-37 and CATH-2 revealed by live-cell imaging.

PubMed

Ordonez, Soledad R; Amarullah, Ilham H; Wubbolts, Richard W; Veldhuizen, Edwin J A; Haagsman, Henk P

2014-01-01

Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides kill C. albicans rapidly. CATH-2 is the most active peptide and kills C. albicans within 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to the C. albicans cell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreases C. albicans cell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37.

Effect of aniracetam on phosphatidylinositol transfer protein alpha in cytosolic and plasma membrane fractions of astrocytes subjected to simulated ischemia in vitro.

PubMed

Gabryel, Bozena; Chalimoniuk, Małgorzata; Małecki, Andrzej; Strosznajder, Joanna B

2005-01-01

Brain ischemia affects phosphoinositide metabolism and the level of lipid-derived second messengers. Phosphatidylinositol transfer proteins (PI-PTs) are responsible for the transport of phosphatidylinositol (PI) and other phospholipids through membranes. Isoform of PI-TPs (PI-TPalpha) is an essential component in ensuring substrate supply for phospholipase C (PLC). The current study was conducted to examine potential effect of aniracetam on PI-TPalpha expression and to characterize the PI-TPalpha isoform distribution between membrane and cytosol fractions of astrocytes exposed to simulated ischemia in vitro. After 8 h period of ischemia, the level of PI-TPalpha was significantly higher in cytosol (by about 28%) as well as in membrane fraction (by about 80%) in comparison with control. We have found that aniracetam treatment of astrocytes in normoxia significantly increased the level of PI-TPalpha in membrane fraction with a maximal effect at 0.1 microM concentration of aniracetam (by about 195% of control). In membrane fractions of ischemic cells, aniracetam increased PI-TPalpha expression in a concentration-dependent manner. In ischemic cells, aniracetam (10 microM) has elevated PI-TPalpha expression up to 155% and 428% in cytosolic and membrane fractions in comparison with ischemic untreated cells, respectively. The study has shown that aniracetam significantly activates PI-TPalpha in cell membrane fraction and this effect might be connected with previously described activation of MAP kinase cascade.

Interactions between peroxiredoxin 2, hemichrome and the erythrocyte membrane.

PubMed

Bayer, Simone B; Low, Felicia M; Hampton, Mark B; Winterbourn, Christine C

2016-12-01

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H 2 O 2 , but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H 2 O 2 or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.

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.

Multiplex lithography for multilevel multiscale architectures and its application to polymer electrolyte membrane fuel cell

NASA Astrophysics Data System (ADS)

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

2015-09-01

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.

Molecular recognition and colorimetric detection of cholera toxin by poly(diacetylene) liposomes incorporating G{sub m1} ganglioside

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, J.J.; Charych, D.

1997-03-19

Molecular recognition sites on cell membranes serve as the main communication channels between the inside of a cell and its surroundings. Upon receptor binding, cellular messages such as ion channel opening or activation of enzymes are triggered. In this report, we demonstrate that artificial cell membranes made from conjugated lipid polymers (poly(diacetylene)) can, on a simple level, mimic membrane processes of molecular recognition and signal transduction. The ganglioside GM1 was incorporated into poly(diacetylene) liposomes. Molecular recognition of cholera toxin at the interface of the liposome resulted in a change of the membrane color due to conformational charges in the conjugatedmore » (ene-yne) polymer backbone. The `colored liposomes` might be used as simple colorimetric sensors for drug screening or as new tools to study membrane-membrane or membrane-receptor interactions. 21 refs., 3 figs.« less

Timing is everything

PubMed Central

Carr, Heather S; Frost, Jeffrey A

2013-01-01

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion. PMID:23792411

Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery.

PubMed

Yamada, Asako; Mitsueda, Asako; Hasan, Mahadi; Ueda, Miho; Hama, Susumu; Warashina, Shota; Nakamura, Takashi; Harashima, Hideyoshi; Kogure, Kentaro

2016-03-01

Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.

Calcium signaling and cell proliferation.

PubMed

Pinto, Mauro Cunha Xavier; Kihara, Alexandre Hiroaki; Goulart, Vânia A M; Tonelli, Fernanda M P; Gomes, Katia N; Ulrich, Henning; Resende, Rodrigo R

2015-11-01

Cell proliferation is orchestrated through diverse proteins related to calcium (Ca(2+)) signaling inside the cell. Cellular Ca(2+) influx that occurs first by various mechanisms at the plasma membrane, is then followed by absorption of Ca(2+) ions by mitochondria and endoplasmic reticulum, and, finally, there is a connection of calcium stores to the nucleus. Experimental evidence indicates that the fluctuation of Ca(2+) from the endoplasmic reticulum provides a pivotal and physiological role for cell proliferation. Ca(2+) depletion in the endoplasmatic reticulum triggers Ca(2+) influx across the plasma membrane in an phenomenon called store-operated calcium entries (SOCEs). SOCE is activated through a complex interplay between a Ca(2+) sensor, denominated STIM, localized in the endoplasmic reticulum and a Ca(2+) channel at the cell membrane, denominated Orai. The interplay between STIM and Orai proteins with cell membrane receptors and their role in cell proliferation is discussed in this review. Copyright © 2015 Elsevier Inc. All rights reserved.

Site-specific regulation of the GEF Cdc24p by the scaffold protein Far1p during yeast mating

PubMed Central

Wiget, Philippe; Shimada, Yukiko; Butty, Anne-Christine; Bi, Efrei; Peter, Matthias

2004-01-01

Receptor-mediated cell polarization via heterotrimeric G-proteins induces cytoskeletal rearrangements in a variety of organisms. In yeast, Far1p is required for orienting cell growth towards the mating partner by linking activated Gβγ to the guanine-nucleotide exchange factor (GEF) Cdc24p, which activates the Rho-type GTPase Cdc42p. Here we investigated the role of Far1p in the regulation of Cdc24p in vivo. Using time-lapse microscopy of mating cells and artificial membrane targeting of Far1p, we show that Far1p is necessary and sufficient to recruit Cdc24p to the plasma membrane. Wild-type Far1p contains a PH-like domain, which is required for its membrane localization in vivo. Interestingly, expression of membrane-targeted Far1p causes toxicity, most likely by activating Cdc42p uniformly at the cell cortex. The ability of full-length Far1p to function as an activator of Cdc24p in vivo requires its interaction with Cdc24p and Gβγ. Our results imply that Gβγ not only targets Far1p to the correct site but may also trigger a conformational change in Far1p that is required for its ability to activate Cdc24p in vivo. PMID:14988725

Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

PubMed

Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

2015-12-01

The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. Copyright © 2015 by the Genetics Society of America.

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

PubMed

Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

2015-11-01

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. Copyright © 2015. Published by Elsevier B.V.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells*

PubMed Central

Arredouani, Abdelilah; Ruas, Margarida; Collins, Stephan C.; Parkesh, Raman; Clough, Frederick; Pillinger, Toby; Coltart, George; Rietdorf, Katja; Royle, Andrew; Johnson, Paul; Braun, Matthias; Zhang, Quan; Sones, William; Shimomura, Kenju; Morgan, Anthony J.; Lewis, Alexander M.; Chuang, Kai-Ting; Tunn, Ruth; Gadea, Joaquin; Teboul, Lydia; Heister, Paula M.; Tynan, Patricia W.; Bellomo, Elisa A.; Rutter, Guy A.; Rorsman, Patrik; Churchill, Grant C.; Parrington, John; Galione, Antony

2015-01-01

Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca2+ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca2+ from the endolysosomal system, resulting in localized Ca2+ signals. We show here that NAADP-mediated Ca2+ release from endolysosomal Ca2+ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca2+ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca2+ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca2+ release from acidic Ca2+ storage organelles in stimulus-secretion coupling in β cells. PMID:26152717

Apoptosis-inducing activity of HPLC fraction from Voacanga globosa (Blanco) Merr. on the human colon carcinoma cell.

PubMed

Acebedo, Alvin Resultay; Amor, Evangeline Cancio; Jacinto, Sonia Donaldo

2014-01-01

Voacanga globosa (Blanco), a plant endemic to the Philippines, is traditionally used especially by indigenous people of Bataan in the treatment of ulcers, wounds and tumorous growths. This study aimed to provide scientific evidence to therapeutic properties by determining cytotoxic and pro-apoptotic activity of HPLC fractions from leaves on HCT116 human colon carcinoma and A549 human lung carcinoma cell lines. Ethanolic extraction was performed on V globosa leaves followed by hexane and ethyl acetate partitioning. Silica gel column chromatography and high performance liquid chromatography (HPLC) produced MP1, MP2 and MP3 fractions. Cytotoxic activity of the fractions was determined through MTT assay against the cancer cell lines HCT116 and A549 and the non-cancer AA8 Chinese hamster ovarian cell line. Pro-apoptotic activities of the most active fractions were further assessed through DAPI staining, TUNEL assay and JC-1 mitochondrial membrane potential assay with HCT116 cells. While the MP1 fraction exerted no significant activity against all cell lines tested, MP2 and MP3 fractions demonstrated high toxicity against HCT116 and A549 cells. The MP3 fraction induced formation of apoptotic bodies, condensed DNA and other morphological changes consistent with apoptosis of HCT116 cells and TUNEL assay showed significant increase in DNA fragmentation over time. In these cells, the MP3 fraction also induced mitochondrial membrane destabilization, which is generally associated with the beginning of apoptosis. Phytochemical analysis demonstrated the presence only of saponins and terpenoids in the MP3 fraction. The results indicate that the MP3 fraction exerts cytotoxic activity on HCT116 cells via induction of apoptosis triggered by loss of mitochondrial membrane potential crucial for cell survival.

Mechanisms of bicarbonate secretion: lessons from the airways.

PubMed

Bridges, Robert J

2012-08-01

Early studies showed that airway cells secrete HCO(3)(-) in response to cAMP-mediated agonists and HCO(3)(-) secretion was impaired in cystic fibrosis (CF). Studies with Calu-3 cells, an airway serous model with high expression of CFTR, also show the secretion of HCO(3)(-) when cells are stimulated with cAMP-mediated agonists. Activation of basolateral membrane hIK-1 K(+) channels inhibits HCO(3)(-) secretion and stimulates Cl(-) secretion. CFTR mediates the exit of both HCO(3)(-) and Cl(-) across the apical membrane. Entry of HCO(3)(-) on a basolateral membrane NBC or Cl(-) on the NKCC determines which anion is secreted. Switching between these two secreted anions is determined by the activity of hIK-1 K(+) channels.

Phosphatidic Acid and Lipid Sensing by mTOR

PubMed Central

Foster, David A.

2013-01-01

Mammalian target of rapamycin (mTOR) has been implicated as a sensor of nutrient sufficiency for dividing cells and is activated by essential amino acids and glucose. However, cells also require lipids for membrane biosynthesis. A central metabolite in the synthesis of membrane phospholipids is phosphatidic acid (PA), which is required for the stability and activity of mTOR complexes. While PA is commonly generated by the phospholipase D-catalyzed hydrolysis of phosphatidylcholine, PA is also generated by diacylglycerol kinases and lysophosphatidic acid acyltransferases, which are at the center of phospholipid biosynthesis. It is proposed that the responsiveness of mTOR/TOR to PA evolved as a means for sensing lipid precursors for membrane biosynthesis prior to doubling the mass of a cell and dividing. PMID:23507202

A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

PubMed

York, Joanne; Nunberg, Jack H

2018-01-01

For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

Modelling three-dimensional cochlear micromechanics within the guinea pig organ of Corti

NASA Astrophysics Data System (ADS)

Ni, Guangjian; Elliott, Stephen J.

2018-05-01

The active amplification process in the mammalian cochlea depends on a complex interaction between cells within the organ of Corti. A three-dimensional (3D) model was developed using the finite element method based on anatomy for the apical end in the guinea pig cochlea, which is comprised of 3D discrete hair cells, 3D continuous membranes and fluid. The basilar membrane, tectorial membrane and the reticular lamina are modelled with orthotropic materials. The Y-shape structures formed by the outer hair cell (OHC), the Deiters' cell and Deiters' cell phalangeal process are also included to account for the structural longitudinal coupling. The motion within the organ of Corti was first simulated in response to a pressure difference loading on the basilar membrane, in order to calculate the passive vibration pattern. Then, the outer hair cells somatic electromotility was implemented by applying a voltage across the OHC walls to investigate its contribution to membranes motion.

Ultraviolet radiation-induced interleukin 6 release in HeLa cells is mediated via membrane events in a DNA damage-independent way.

PubMed

Kulms, D; Pöppelmann, B; Schwarz, T

2000-05-19

Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damage and activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 degrees C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominant over nuclear effects. UV-induced IL-6 release is mediated via NFkappaB since the NFkappaB inhibitor MG132 or transfection of cells with a super-repressor form of the NFkappaB inhibitor IkappaB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage.

SLP-65 signal transduction requires Src homology 2 domain-mediated membrane anchoring and a kinase-independent adaptor function of Syk.

PubMed

Abudula, Abulizi; Grabbe, Annika; Brechmann, Markus; Polaschegg, Christian; Herrmann, Nadine; Goldbeck, Ingo; Dittmann, Kai; Wienands, Jürgen

2007-09-28

The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca(2+)-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca(2+) mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca(2+) mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca(2+) initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.

Involvement of phosphoinositide 3-kinase and PTEN protein in mechanism of activation of TRPC6 protein in vascular smooth muscle cells.

PubMed

Monet, Michaël; Francoeur, Nancy; Boulay, Guylain

2012-05-18

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry after the stimulation of a G(q)-protein-coupled or tyrosine-kinase receptor. TRPC6 translocates to the plasma membrane upon stimulation and remains there as long as the stimulus is present. However, the mechanism that regulates the trafficking and activation of TRPC6 are unclear. In this study we showed phosphoinositide 3-kinase and its antagonistic phosphatase, PTEN, are involved in the activation of TRPC6. The inhibition of PI3K by PIK-93, LY294002, or wortmannin decreased carbachol-induced translocation of TRPC6 to the plasma membrane and carbachol-induced net Ca(2+) entry into T6.11 cells. Conversely, a reduction of PTEN expression did not affect carbachol-induced externalization of TRPC6 but increased Ca(2+) entry through TRPC6 in T6.11 cells. We also showed that the PI3K/PTEN pathway regulates vasopressin-induced translocation of TRPC6 to the plasma membrane and vasopressin-induced Ca(2+) entry into A7r5 cells, which endogenously express TRPC6. In summary, we provided evidence that the PI3K/PTEN pathway plays an important role in the translocation of TRPC6 to the plasma membrane and may thus have a significant impact on Ca(2+) signaling in cells that endogenously express TRPC6.

Involvement of Phosphoinositide 3-Kinase and PTEN Protein in Mechanism of Activation of TRPC6 Protein in Vascular Smooth Muscle Cells*

PubMed Central

Monet, Michaël; Francoeur, Nancy; Boulay, Guylain

2012-01-01

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca2+ entry after the stimulation of a Gq-protein-coupled or tyrosine-kinase receptor. TRPC6 translocates to the plasma membrane upon stimulation and remains there as long as the stimulus is present. However, the mechanism that regulates the trafficking and activation of TRPC6 are unclear. In this study we showed phosphoinositide 3-kinase and its antagonistic phosphatase, PTEN, are involved in the activation of TRPC6. The inhibition of PI3K by PIK-93, LY294002, or wortmannin decreased carbachol-induced translocation of TRPC6 to the plasma membrane and carbachol-induced net Ca2+ entry into T6.11 cells. Conversely, a reduction of PTEN expression did not affect carbachol-induced externalization of TRPC6 but increased Ca2+ entry through TRPC6 in T6.11 cells. We also showed that the PI3K/PTEN pathway regulates vasopressin-induced translocation of TRPC6 to the plasma membrane and vasopressin-induced Ca2+ entry into A7r5 cells, which endogenously express TRPC6. In summary, we provided evidence that the PI3K/PTEN pathway plays an important role in the translocation of TRPC6 to the plasma membrane and may thus have a significant impact on Ca2+ signaling in cells that endogenously express TRPC6. PMID:22493444

Constant change: dynamic regulation of membrane transport by calcium signalling networks keeps plants in tune with their environment.

PubMed

Kleist, Thomas J; Luan, Sheng

2016-03-01

Despite substantial variation and irregularities in their environment, plants must conform to spatiotemporal demands on the molecular composition of their cytosol. Cell membranes are the major interface between organisms and their environment and the basis for controlling the contents and intracellular organization of the cell. Membrane transport proteins (MTPs) govern the flow of molecules across membranes, and their activities are closely monitored and regulated by cell signalling networks. By continuously adjusting MTP activities, plants can mitigate the effects of environmental perturbations, but effective implementation of this strategy is reliant on precise coordination among transport systems that reside in distinct cell types and membranes. Here, we examine the role of calcium signalling in the coordination of membrane transport, with an emphasis on potassium transport. Potassium is an exceptionally abundant and mobile ion in plants, and plant potassium transport has been intensively studied for decades. Classic and recent studies have underscored the importance of calcium in plant environmental responses and membrane transport regulation. In reviewing recent advances in our understanding of the coding and decoding of calcium signals, we highlight established and emerging roles of calcium signalling in coordinating membrane transport among multiple subcellular locations and distinct transport systems in plants, drawing examples from the CBL-CIPK signalling network. By synthesizing classical studies and recent findings, we aim to provide timely insights on the role of calcium signalling networks in the modulation of membrane transport and its importance in plant environmental responses. © 2015 John Wiley & Sons Ltd.

Oxidative Stress and Maxi Calcium-Activated Potassium (BK) Channels

PubMed Central

Hermann, Anton; Sitdikova, Guzel F.; Weiger, Thomas M.

2015-01-01

All cells contain ion channels in their outer (plasma) and inner (organelle) membranes. Ion channels, similar to other proteins, are targets of oxidative impact, which modulates ion fluxes across membranes. Subsequently, these ion currents affect electrical excitability, such as action potential discharge (in neurons, muscle, and receptor cells), alteration of the membrane resting potential, synaptic transmission, hormone secretion, muscle contraction or coordination of the cell cycle. In this chapter we summarize effects of oxidative stress and redox mechanisms on some ion channels, in particular on maxi calcium-activated potassium (BK) channels which play an outstanding role in a plethora of physiological and pathophysiological functions in almost all cells and tissues. We first elaborate on some general features of ion channel structure and function and then summarize effects of oxidative alterations of ion channels and their functional consequences. PMID:26287261

Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide

PubMed Central

Vellani, Vittorio; Mapplebeck, Sarah; Moriondo, Andrea; Davis, John B; McNaughton, Peter A

2001-01-01

The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. Removing external Ca2+ enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca2+ produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium also potentiates activation, and PKC activation then has no further effect. The results are consistent with a model in which phosphorylation of VR1 by PKC increases the probability of channel gating by agonists, and in which dephosphorylation occurs by a calcium-dependent process. PMID:11483711

Timing is everything: Rac1 controls Net1A localization to regulate cell adhesion.

PubMed

Carr, Heather S; Frost, Jeffrey A

2013-01-01

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion.

Rga6 is a fission yeast Rho GAP involved in Cdc42 regulation of polarized growth

PubMed Central

Revilla-Guarinos, M. T.; Martín-García, Rebeca; Villar-Tajadura, M. Antonia; Estravís, Miguel; Coll, Pedro M.; Pérez, Pilar

2016-01-01

Active Cdc42 is essential for the establishment of polarized growth. This GTPase is negatively regulated by the GTPase-activating proteins (GAPs), which are important for the spatial specificity of Cdc42 function. Rga4 is the only GAP described as negative regulator of fission yeast Cdc42. We report here that Rga6, another fission yeast Cdc42 GAP, shares some functions with Rga4. Cells lacking Rga6 are viable but slightly shorter and broader than wild type, and cells lacking Rga6 and Rga4 simultaneously are rounded. In these cells, active Cdc42 is observed all around the membrane. These additive effects indicate that both GAPs collaborate in the spatial regulation of active Cdc42. Rga6 localizes to the plasma membrane, forming clusters different from those formed by Rga4. A polybasic region at the Rga6 C-terminus is responsible for its membrane localization. Rga6-GFP fluorescence decreases considerably at the growing tips, and this decrease is dependent on the actin cables. Of note, in the absence of Rga6, the amplitude of active Cdc42 oscillations at the tips decreases, and less GTP-Cdc42 accumulates at the new end of the cells. We propose that Rga6 collaborates with Rga4 to spatially restrict active Cdc42 at the cell tips and maintain cell dimensions. PMID:26960792

Litsea cubeba leaf essential oil from Vietnam: chemical diversity and its impacts on antibacterial activity.

PubMed

Nguyen, H V; Meile, J-C; Lebrun, M; Caruso, D; Chu-Ky, S; Sarter, S

2018-03-01

The threat of bacterial resistance to antibiotics has created an urgent need to develop new antimicrobials. The aim of this study was to characterize the chemical diversity of Litsea cubeba leaf essential oil (EO) and its impacts on the antibacterial activity against pathogenic bacteria. Essential oils collected from seven provinces in North Vietnam (n = 25) were characterized by their high content in either 1,8-cineole or linalool. Linalool-type EOs were more effective against the eight bacterial strains tested than 1,8-cineole-type. Oil samples, LC19 (50% 1,8-cineole) and BV27 (94% linalool), were selected to investigate their antibacterial mechanisms against Escherichia coli. A strong bactericidal effect was observed after 4 and 2 h of exposure respectively. Microscopic analysis of treated E. coli cultures clearly showed that EOs caused changes in cell morphology, loss of integrity and permeability of the cell membrane, as well as DNA loss. However, the effects of both EOs were distinct. LC19 mostly affected cell membrane, led to a significant cell filamentation rate and altered cell width, whereas BV27 damaged cell membrane integrity leading to cell permeabilization and altered nucleoid morphology with the appearance of spot and visibly altered compaction. This study aimed to characterize the chemical diversity of Litsea cubeba leaf essential oil (EO) and its impacts on its antibacterial activity. Two major chemotypes (1,8-cineole or linalool rich) were identified in North Vietnam and both were bactericidal against several pathogenic bacteria. A distinct inhibitory effect of EO samples on Escherichia coli was observed. 1,8-cineole-rich sample (LC19) affected cell membrane, led to cell filamentation and perturbation of cell width, while the linalool-rich one (BV27) induced damages in the cell membrane and changes in the nucleoid morphology. The study demonstrates the importance of considering chemotype variations in terms of chemical composition as well as the mode of action. © 2017 The Society for Applied Microbiology.

Regulating the Membrane Transport Activity and Death of Cells via Electroosmotic Manipulation.

PubMed

Hui, Tsz Hin; Kwan, Kin Wah; Chun Yip, Timothy Tak; Fong, Hong Wai; Ngan, Kai Cheong; Yu, Miao; Yao, Shuhuai; Wan Ngan, Alfonso Hin; Lin, Yuan

2016-06-21

Although the volume of living cells has been known to heavily influence their behavior and fate, a method allowing us to control the cell size in a programmable manner is still lacking. Here, we develop a technique in which precise changes in the cellular volume can be conveniently introduced by varying the voltage applied across a Nafion membrane that separates the culture medium from a reservoir. It is found that, unlike sudden osmotic shocks, active ion transport across the membrane of leukemia K562 cells will not be triggered by a gradual change in the extracellular osmolarity. Furthermore, when subjected to the same applied voltage, different lung and nasopharyngeal epithelial cancer cells will undergo larger volumetric changes and have a 5-10% higher death rate compared to their normal counterparts. We show that such distinct response is largely caused by the overexpression of aquaporin-4 in tumor cells, with knockout of this water channel protein resulting in a markedly reduced change in the cellular volume. Finally, by taking into account the exchange of water/ion molecules across the Nafion film and the cell membrane, a theoretical model is also proposed to describe the voltage-induced size changes of cells, which explain our experimental observations very well. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Quantitative and Mechanistic Assessment of Model Lipophilic Drugs in Micellar Solutions in the Transport Kinetics Across MDR1-MDCK Cell Monolayers.

PubMed

Ho, Norman F H; Nielsen, James; Peterson, Michelle; Burton, Philip S

2016-02-01

An approach to characterizing P-glycoprotein (Pgp) interaction potential for sparingly water-soluble compounds was developed using bidirectional transport kinetics in MDR1-MDCK cell monolayers. Paclitaxel, solubilized in a dilute polysorbate 80 (PS80) micellar solution, was used as a practical example. Although the passage of paclitaxel across the cell monolayer was initially governed by the thermodynamic activity of the micelle-solubilized drug solution, Pgp inhibition was sustained by the thermodynamic activity (i.e., critical micelle concentration) of the PS80 micellar solution bathing the apical (ap) membrane. The mechanistic understanding of the experimental strategies and treatment of data was supported by a biophysical model expressed in the form of transport events occurring at the ap and basolateral (bl) membranes in series whereas the vectorial directions of the transcellular kinetics were accommodated. The derived equations permitted the stepwise quantitative delineation of the Pgp efflux activity (inhibited and uninhibited by PS80) and the passive permeability coefficient of the ap membrane, the passive permeability at the bl membrane and, finally, the distinct coupling of these with efflux pump activity to identify the rate-determining steps and mechanisms. The Jmax/KM(∗) for paclitaxel was in the order of 10(-4) cm/s and the ap- and bl-membrane passive permeability coefficients were asymmetric, with bl-membrane permeability significantly greater than ap. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Challenges in the Development of Functional Assays of Membrane Proteins

PubMed Central

Tiefenauer, Louis; Demarche, Sophie

2012-01-01

Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

Color-Coded Organelles.

ERIC Educational Resources Information Center

McLaughlin, Esther; And Others

1994-01-01

Describes how red beets can be used to demonstrate a variety of membrane phenomena. Some of the activities include observation of vacuoles; vacuoles in intact cells; isolation of vacuoles in physiological studies; demonstration of membrane integrity; and demonstration of ion diffusion and active transport with purified vacuoles. (ZWH)

Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells

PubMed Central

1975-01-01

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane. PMID:163833

Endocytosis of Nanoscale Systems for Cancer Treatments.

PubMed

Chen, Kai; Li, Xue; Zhu, Hongyan; Gong, Qiyong; Luo, Kui

2017-04-28

Advances of nanoscale systems for cancer treatment have been involved in enabling highly regulated site-specific localization to sub cellular organelles hidden beneath cell membranes. Thus far, the cellular entry of these nanoscale systems has been not fully understood. Endocytosisis a form of active transport in which cell transports elected extracellular molecules (such as proteins, viruses, micro-organisms and nanoscale systems) are allowed into cell interiors by engulfing them in an energy-dependent process. This process appears at the plasma membrane surface and contains internalization of the cell membrane as well as the membrane proteins and lipids of cell. There are multiform pathways of endocytosis for nanoscale systems. Further comprehension for the mechanisms of endocytosis is achieved with a combination of efficient genetic manipulations, cell dynamic imaging, and chemical endocytosis inhibitors. This review provides an account of various endocytic pathways, itemizes current methods to study endocytosis of nanoscale systems, discusses some factors associated with cellular uptake for nanoscale systems and introduces the trafficking behavior for nanoscale systems with active targeting. An insight into the endocytosis mechanism is urgent and significant for developing safe and efficient nanoscale systems for cancer diagnosis and therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Role-Playing Mitosis.

ERIC Educational Resources Information Center

Wyn, Mark A.; Stegink, Steven J.

2000-01-01

Introduces a role playing activity that actively engages students in the learning process of mitosis. Students play either chromosomes carrying information, or cells in the cell membrane. (Contains 11 references.) (Author/YDS)

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome.

PubMed

Balajthy, András; Somodi, Sándor; Pethő, Zoltán; Péter, Mária; Varga, Zoltán; Szabó, Gabriella P; Paragh, György; Vígh, László; Panyi, György; Hajdu, Péter

2016-08-01

In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.

UVA Irradiation of Dysplastic Keratinocytes: Oxidative Damage versus Antioxidant Defense

PubMed Central

Nechifor, Marina T.; Niculiţe, Cristina M.; Urs, Andreea O.; Regalia, Teodor; Mocanu, Mihaela; Popescu, Alexandra; Manda, Gina; Dinu, Diana; Leabu, Mircea

2012-01-01

UVA affects epidermal cell physiology in a complex manner, but the harmful effects have been studied mainly in terms of DNA damage, mutagenesis and carcinogenesis. We investigated UVA effects on membrane integrity and antioxidant defense of dysplastic keratinocytes after one and two hours of irradiation, both immediately after exposure, and 24 h post-irradiation. To determine the UVA oxidative stress on cell membrane, lipid peroxidation was correlated with changes in fatty acid levels. Membrane permeability and integrity were assessed by propidium iodide staining and lactate dehydrogenase release. The effects on keratinocyte antioxidant protection were investigated in terms of catalase activity and expression. Lipid peroxidation increased in an exposure time-dependent manner. UVA exposure decreased the level of polyunsaturated fatty acids, which gradually returned to its initial value. Lactate dehydrogenase release showed a dramatic loss in membrane integrity after 2 h minimum of exposure. The cell ability to restore membrane permeability was noted at 24 h post-irradiation (for one hour exposure). Catalase activity decreased in an exposure time-dependent manner. UVA-irradiated dysplastic keratinocytes developed mechanisms leading to cell protection and survival, following a non-lethal exposure. The surviving cells gained an increased resistance to apoptosis, suggesting that their pre-malignant status harbors an abnormal ability to control their fate. PMID:23222638

Accelerating bioelectric functional development of neural stem cells by graphene coupling: Implications for neural interfacing with conductive materials.

PubMed

Guo, Rongrong; Zhang, Shasha; Xiao, Miao; Qian, Fuping; He, Zuhong; Li, Dan; Zhang, Xiaoli; Li, Huawei; Yang, Xiaowei; Wang, Ming; Chai, Renjie; Tang, Mingliang

2016-11-01

In order to govern cell-specific behaviors in tissue engineering for neural repair and regeneration, a better understanding of material-cell interactions, especially the bioelectric functions, is extremely important. Graphene has been reported to be a potential candidate for use as a scaffold and neural interfacing material. However, the bioelectric evolvement of cell membranes on these conductive graphene substrates remains largely uninvestigated. In this study, we used a neural stem cell (NSC) model to explore the possible changes in membrane bioelectric properties - including resting membrane potentials and action potentials - and cell behaviors on graphene films under both proliferation and differentiation conditions. We used a combination of single-cell electrophysiological recordings and traditional cell biology techniques. Graphene did not affect the basic membrane electrical parameters (capacitance and input resistance), but resting membrane potentials of cells on graphene substrates were more strongly negative under both proliferation and differentiation conditions. Also, NSCs and their progeny on graphene substrates exhibited increased firing of action potentials during development compared to controls. However, graphene only slightly affected the electric characterizations of mature NSC progeny. The modulation of passive and active bioelectric properties on the graphene substrate was accompanied by enhanced NSC differentiation. Furthermore, spine density, synapse proteins expressions and synaptic activity were all increased in graphene group. Modeling of the electric field on conductive graphene substrates suggests that the electric field produced by the electronegative cell membrane is much higher on graphene substrates than that on control, and this might explain the observed changes of bioelectric development by graphene coupling. Our results indicate that graphene is able to accelerate NSC maturation during development, especially with regard to bioelectric evolvement. Our findings provide a fundamental understanding of the role of conductive materials in tuning the membrane bioelectric properties in a graphene model and pave the way for future studies on the development of methods and materials for manipulating membrane properties in a controllable way for NSC-based therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

ELMOD1 Stimulates ARF6-GTP Hydrolysis to Stabilize Apical Structures in Developing Vestibular Hair Cells.

PubMed

Krey, Jocelyn F; Dumont, Rachel A; Wilmarth, Philip A; David, Larry L; Johnson, Kenneth R; Barr-Gillespie, Peter G

2018-01-24

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we analyzed mice of both sexes from a strain lacking functional ELMOD1 [roundabout ( rda )]. In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5; large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cell's apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles compared with controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle. SIGNIFICANCE STATEMENT Assembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia. Copyright © 2018 the authors 0270-6474/18/380843-15$15.00/0.

The cytotoxic effects of titanium oxide and zinc oxide nanoparticles oh Human Cervical Adenocarcinoma cell membranes

NASA Astrophysics Data System (ADS)

Mironava, Tatsiana; Applebaum, Ariella; Applebaum, Eliana; Guterman, Shoshana; Applebaum, Kayla; Grossman, Daniel; Gordon, Chris; Brink, Peter; Wang, H. Z.; Rafailovich, Miriam

2013-03-01

The importance of titanium dioxide (TiO2) and zinc oxide (ZnO), inorganic metal oxides nanoparticles (NPs) stems from their ubiquitous applications in personal care products, solar cells and food whitening agents. Hence, these NPs come in direct contact with the skin, digestive tracts and are absorbed into human tissues. Currently, TiO2 and ZnO are considered safe commercial ingredients by the material safety data sheets with no reported evidence of carcinogenicity or ecotoxicity, and do not classify either NP as a toxic substance. This study examined the direct effects of TiO2 and ZnO on HeLa cells, a human cervical adenocarcinonma cell line, and their membrane mechanics. The whole cell patch-clamp technique was used in addition to immunohistochemistry staining, TEM and atomic force microscopy (AFM). Additionally, we examined the effects of dexamethasone (DXM), a glucocorticoid steroid known to have an effect on cell membrane mechanics. Overall, TiO2 and ZnO seemed to have an adverse effect on cell membrane mechanics by effecting cell proliferation, altering cellular structure, decreasing cell-cell adhesion, activating existing ion channels, increasing membrane permeability, and possibly disrupting cell signaling.

An amino acid composition criterion for membrane active antimicrobials

NASA Astrophysics Data System (ADS)

Schmidt, Nathan; Lai, Ghee Hwee; Mishra, Abhijit; Bong, Dennis; McCray, Paul, Jr.; Selsted, Michael; Ouellette, Andre; Wong, Gerard

2011-03-01

Membrane active antimicrobials (AMPs) are short amphipathic peptides with broad spectrum anti microbial activity. While it is believed that their hydrophobic and cationic moieties are responsible for membrane-based mechanisms of action, membrane disruption by AMPs is manifested in a diversity of outcomes, such as pore formation, blebbing, and budding. This complication, along with others, have made a detailed, molecular understanding of AMPs difficult. We use synchrotron small angle xray scattering to investigate the interaction of model bacterial and eukaryotic cell membranes with archetypes from beta-sheet AMPs (e.g. defensins) and alpha-helical AMPs (e.g. magainins). The relationship between membrane composition and peptide induced changes in membrane curvature and topology is examined. By comparing the membrane rearrangement and phase behavior induced by these different peptides we will discuss the importance of amino acid composition on AMP design.

Biochemical and pharmacological characterization of the thyrotropin releasing hormone (TRH) receptor from clonal GH sub 4 C sub 1 pituitary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phillips, W.J.

1987-01-01

The effect of drugs with anesthetic properties on the activity of the pituitary thyrotropin-releasing hormone (TRH) receptor was determined in the clonal GH{sub 4}C{sub 1} somatomammotropic cell line. Classic local anesthetics and other drugs with anesthetic activity inhibited binding of ({sup 3}H)methyl-TRH to cell receptors at concentrations known to produce anesthetic effects on the membrane. The inhibition of TRH receptor binding by tetracaine was competitive and temperature and pH dependent. Verapamil and tetracaine inhibited TRH-stimulated prolactin secretion at concentrations that inhibited peptide binding. TRH-stimulated prolactin secretion was equivalent with or without Ca{sup 2+} channel activity. Verapamil and tetracaine also inhibitedmore » basal prolactin and secretion stimulated by drugs that bypass membrane receptors, db-cAMP and TPA. These results indicate that inhibition of TRH binding and responses by diverse drugs results from an anesthetic effect on the cell membrane.« less

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways*

PubMed Central

Lecat, Sandra; Matthes, Hans W.D.; Pepperkok, Rainer; Simpson, Jeremy C.; Galzi, Jean-Luc

2015-01-01

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. PMID:25759509

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

PubMed

Lecat, Sandra; Matthes, Hans W D; Pepperkok, Rainer; Simpson, Jeremy C; Galzi, Jean-Luc

2015-05-01

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

An adaptor role for cytoplasmic Sam68 in modulating Src activity during cell polarization.

PubMed

Huot, Marc-Etienne; Brown, Claire M; Lamarche-Vane, Nathalie; Richard, Stéphane

2009-04-01

The Src-associated substrate during mitosis with a molecular mass of 68 kDa (Sam68) is predominantly nuclear and is known to associate with proteins containing the Src homology 3 (SH3) and SH2 domains. Although Sam68 is a Src substrate, little is known about the signaling pathway that link them. Src is known to be activated transiently after cell spreading, where it modulates the activity of small Rho GTPases. Herein we report that Sam68-deficient cells exhibit loss of cell polarity and cell migration. Interestingly, Sam68-deficient cells exhibited sustained Src activity after cell attachment, resulting in the constitutive tyrosine phosphorylation and activation of p190RhoGAP and its association with p120rasGAP. Consistently, we observed that Sam68-deficient cells exhibited deregulated RhoA and Rac1 activity. By using total internal reflection fluorescence microscopy, we observed Sam68 near the plasma membrane after cell attachment coinciding with phosphorylation of its C-terminal tyrosines and association with Csk. These findings show that Sam68 localizes near the plasma membrane during cell attachment and serves as an adaptor protein to modulate Src activity for proper signaling to small Rho GTPases.

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

PubMed

Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

2018-01-23

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Poloxamer 188 (p188) as a membrane resealing reagent in biomedical applications.

PubMed

Moloughney, Joseph G; Weisleder, Noah

2012-12-01

Maintenance of the integrity of the plasma membrane is essential for maintenance of cellular function and prevention of cell death. Since the plasma membrane is frequently exposed to a variety of mechanical and chemical insults the cell has evolved active processes to defend against these injuries by resealing disruptions in the plasma membrane. Cell membrane repair is a conserved process observed in nearly every cell type where intracellular vesicles are recruited to sites of membrane disruption where they can fuse with themselves or the plasma membrane to create a repair patch. When disruptions are extensive or there is an underlying pathology that reduces the membrane repair capacity of a cell this defense mechanism may prove insufficient and the cell could die due to breakdown of the plasma membrane. Extensive loss of cells can compromise the integrity and function of tissues and leading to disease. Thus, methods to increase membrane resealing capacity could have broad utility in a number of disease states. Efforts to find reagents that can modulate plasma membrane reseal found that specific tri-block copolymers, such as poloxamer 188 (P188, or Pluronic F68), can increase the structural stability and resealing of the plasma membrane. Here we review several current patents and patent applications that present inventions making use of P188 and other copolymers to treat specific disease states such as muscular dystrophy, heart failure, neurodegenerative disorders and electrical injuries, or to facilitate biomedical applications such as transplantation. There appears to be promise for the application of poloxamers in the treatment of various diseases, however there are potential concerns with toxicity with long term application and bioavailability in some cases.

Using 1H2O MR to measure and map sodium pump activity in vivo.

PubMed

Springer, Charles S

2018-06-01

The cell plasma membrane Na + ,K + -ATPase [NKA] is one of biology's most [if not the most] significant enzymes. By actively transporting Na + out [and K + in], it maintains the vital trans-membrane ion concentration gradients and the membrane potential. The forward NKA reaction is shown in the Graphical Abstract [which is elaborated in the text]. Crucially, NKA does not operate in isolation. There are other transporters that conduct K + back out of [II, Graphical Abstract] and Na + back into [III, Graphical Abstract] the cell. Thus, NKA must function continually. Principal routes for ATP replenishment include mitochondrial oxidative phosphorylation, glycolysis, and creatine kinase [CrK] activity. However, it has never been possible to measure, let alone map, this integrated, cellular homeostatic NKA activity in vivo. Active trans-membrane water cycling [AWC] promises a way to do this with 1 H 2 O MR. Inthe Graphical Abstract, the AWC system is characterized by active contributions totheunidirectional rate constants for steady-state water efflux and influx, respectively, k io (a) and k oi (a). The discovery, validation, and initial exploration of active water cycling are reviewed here. Promising applications in cancer, cardiological, and neurological MRI are covered. This initial work employed paramagnetic Gd(III)chelate contrast agents [CAs]. However, the significant problems associated with in vivo CA use are also reviewed. A new analysis of water diffusion-weighted MRI [DWI] is presented. Preliminary results suggest a non-invasive way to measure the cell number density [ρ (cells/μL)], the mean cell volume [V (pL)], and the cellular NKA metabolic rate [ c MR NKA (fmol(ATP)/s/cell)] with high spatial resolution. These crucial cell biology properties have not before been accessible invivo. Furthermore, initial findings indicate their absolute values can be determined. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

Modulation of hyaluronan synthase activity in cellular membrane fractions.

PubMed

Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

2009-10-30

Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers.

PubMed

Lee-Chang, Catalina; Bodogai, Monica; Moritoh, Kanako; Chen, Xin; Wersto, Robert; Sen, Ranjan; Young, Howard A; Croft, Michael; Ferrucci, Luigi; Biragyn, Arya

2016-04-15

B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL(+)MHC class-I(Hi)CD86(Hi)B cells of unknown origin. In this article, we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result, activated B1a/4BL cells induce expression of granzyme B in CD8(+)T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus, for the first time, to our knowledge, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8(+)T cells. Copyright © 2016 by The American Association of Immunologists, Inc.

Prostate cell membrane chromatography-liquid chromatography-mass spectrometry for screening of active constituents from Uncaria rhynchophylla.

PubMed

He, Jianyu; Han, Shengli; Yang, Fangfang; Zhou, Nan; Wang, Sicen

2013-01-01

Uncaria rhynchophylla is a traditional Chinese medicinal herb used to treat hypertension and convulsive disorders such as epilepsy. Rat prostate cell membrane chromatography combined with liquid chromatography-mass spectrometry (LC-MS) was used to identify active constituents from U. rhynchophylla extracts. Four compounds (corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline) were discovered. Competitive binding assay results indicated that the four compounds were in direct competition at a single common binding site and interacted with α1A adrenergic receptors (α1A-AR) in a manner similar to tamsulosin. Affinity constant values of the four compounds binding with α1A-AR were also measured using rat prostate cell membrane chromatography (CMC). Finally, their pharmacodynamic effects were tested on rat caudal arteries. This CMC combined LC-MS system offers a means of drug discovery by screening natural medicinal herbs for new pharmacologically active molecules targeting specific receptors.

Dynamic regulation of mechanosensitive channels: capacitance used to monitor patch tension in real time

NASA Astrophysics Data System (ADS)

Suchyna, Thomas M.; Besch, Steven R.; Sachs, Frederick

2004-03-01

All cells, from bacteria to human, are mechanically sensitive. The most rapid of these membrane protein transducers are mechanosensitive ion channels, ionic pores in the membrane that open and close in response to membrane tension. In specific sensory organs, these channels serve the senses of touch and hearing, and inform the central nervous system about the filling of hollow organs such as the bladder. Non-specialized cells use these channels to report on changes in cell volume and local strain. To preserve dynamic sensitivity, sensory receptors adapt to steady-state stimuli. Here we show that in rat astrocytes, the most abundant cells in the brain, this apparent adaptation to the stimulus is actually an inactivation. We have been able to track the time course of local strain by measuring attofarad changes in membrane capacitance and show that it is not correlated with loss of channel activity. The reduction in current with time is caused by an increased occupancy of low conductance states, and a reduction in the probability of opening, not a relaxation of local stress. The occupancy of these substates depends on the integrity of the cell's cytoplasm. However, while disruption of the cytoskeleton leads to a loss of inactivation, it leaves activation unaffected. The activation process is voltage-insensitive, closely correlated with changes in capacitance, and seems to arise solely from stress in the bilayer. The inactivation rate decreases with depolarization, and kinetic analysis suggests that the process involves multiple cytoplasmic ligands. Surprisingly, multivalent ions such as Gd+3 and Ca+2 that bind to the lipids and affect channel gating, do not affect the strain-induced increase in membrane capacitance; contrary to expectations, membrane elasticity is unchanged.

Progesterone receptor membrane component 1 as the mediator of the inhibitory effect of progestins on cytokine-induced matrix metalloproteinase 9 activity in vitro.

PubMed

Allen, Terrence K; Feng, Liping; Grotegut, Chad A; Murtha, Amy P

2014-02-01

Progesterone (P4) and the progestin, 17α-hydroxyprogesterone caproate, are clinically used to prevent preterm births (PTBs); however, their mechanism of action remains unclear. Cytokine-induced matrix metalloproteinase 9 (MMP-9) activity plays a key role in preterm premature rupture of the membranes and PTB. We demonstrated that the primary chorion cells and the HTR8/SVneo cells (cytotrophoblast cell line) do not express the classical progesterone receptor (PGR) but instead a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1), whose role remains unclear. Using HTR8/SVneo cells in culture, we further demonstrated that 6 hours pretreatment with medroxyprogesterone acetate (MPA) and dexamethasone (Dex) but not P4 or 17α-hydroxyprogesterone hexanoate significantly attenuated tumor necrosis factor α-induced MMP-9 activity after a 24-hour incubation period. The inhibitory effect of MPA, but not Dex, was attenuated when PGRMC1 expression was successfully reduced by PGRMC1 small interfering RNA. Our findings highlight a possible novel role of PGRMC1 in mediating the effects of MPA and in modulating cytokine-induced MMP-9 activity in cytotrophoblast cells in vitro.

Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate.

PubMed

Morono, Yuki; Takano, Suguru; Miyanaga, Kazuhiko; Tanji, Yasunori; Unno, Hajime; Hori, Katsutoshi

2004-03-01

Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.

A laser microsurgical method of cell wall removal allows detection of large-conductance ion channels in the guard cell plasma membrane

NASA Technical Reports Server (NTRS)

Miedema, H.; Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1999-01-01

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

Activity ranking of synthetic analogs targeting vascular endothelial growth factor receptor 2 by an integrated cell membrane chromatography system.

PubMed

Wang, Dongyao; Lv, Diya; Chen, Xiaofei; Liu, Yue; Ding, Xuan; Jia, Dan; Chen, Langdong; Zhu, Zhenyu; Cao, Yan; Chai, Yifeng

2015-12-01

Evaluating the biological activities of small molecules represents an important part of the drug discovery process. Cell membrane chromatography (CMC) is a well-developed biological chromatographic technique. In this study, we have developed combined SMMC-7721/CMC and HepG2/CMC with high-performance liquid chromatography and time-of-flight mass spectrometry to establish an integrated screening platform. These systems was subsequently validated and used for evaluating the activity of quinazoline compounds, which were designed and synthesized to target vascular endothelial growth factor receptor 2. The inhibitory activities of these compounds towards this receptor were also tested using a classical caliper mobility shift assay. The results revealed a significant correlation between these two methods (R(2) = 0.9565 or 0.9420) for evaluating the activities of these compounds. Compared with traditional methods of evaluating the activities analogous compounds, this integrated cell membrane chromatography screening system took less time and was more cost effective, indicating that it could be used as a practical method in drug discovery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biosynthesis of edeine: II. Localization of edeine synthetase within Bacillus brevis Vm4.

PubMed

Kurylo-Borowska, Z

1975-07-14

Edeine-synthesizing polyenzymes, associated with a complex of sytoplasmic membrane and DNA, were obtained from gently lysed cells of Bacillus brevis Vm4. The polyenzymes-membrane-DNA complex, isolated from dells intensively synthesizing edeines (18--20 h culture) contained edeine B. Edeine B was found to be bound covalently t o the edeine synthetase. The amount of edeine bound to polyenzymes was 0.1--0.3 mumol/mg protein, depending on the age of cells. Detachment of deeine synthetase with a covalently bound edeine B from the membrane-DNA complex was accomplished by a treatment with (NH4)2-SO4 at 45--55% saturation or by DEAE-cellulose column fractionation. In contrast to other components of the complex, the edeine-polyenzymes fragment was not adsorbed to the DEAE-cellulose. Sephadex G-200 column chromatography separated the edeine-polyenzymes complex into 3 fractions. Edeine-polyenzymes complex, obtained from lysozyme-Brij-58-DNAase treated cells, contained edeine B bound to two protein fractions of mol. wt 210 000 and 160 000. Edeine-polyenzymes complex detached from the complex with the membrane and DNA contained edeine B, bound only to protein fraction of mol. wt 210 000. Edeine A was not found in the edeine-polyenzymes complex. No accumulation of free antibiotics within 16--22 h old cells of B. brevis Vm4 was detected. The edeine-polyenzymes complex associated with the DNA-membrane complex has shown no antimicrobial activity. By treating of above with alkali, edeine B of specific activity: 80 units/mjmol was released. The complex of DNA-membrane associated with edeine-polyenzymes complex was able to synthesize DNA, under the conditions described for synthesis, directed by a DNA-membrane complex. Edeine when associated with this complex did not effect the DNA-synthesizing activity.

Toxicity of bovicin HC5 against mammalian cell lines and the role of cholesterol in bacteriocin activity.

PubMed

Paiva, Aline Dias; de Oliveira, Michelle Dias; de Paula, Sérgio Oliveira; Baracat-Pereira, Maria Cristina; Breukink, Eefjan; Mantovani, Hilário Cuquetto

2012-11-01

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by Bacteria and some Archaea. The assessment of the toxic potential of antimicrobial peptides is important in order to apply these peptides on an industrial scale. The aim of the present study was to investigate the in vitro cytotoxic and haemolytic potential of bovicin HC5, as well as to determine whether cholesterol influences bacteriocin activity on model membranes. Nisin, for which the mechanism of action is well described, was used as a reference peptide in our assays. The viability of three distinct eukaryotic cell lines treated with bovicin HC5 or nisin was analysed by using the MTT assay and cellular morphological changes were determined by light microscopy. The haemolytic potential was evaluated by using the haemoglobin liberation assay and the role of cholesterol on bacteriocin activity was examined by using model membranes composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DPoPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The IC(50) of bovicin HC5 and nisin against Vero cells was 65.42 and 13.48 µM, respectively. When the MTT assay was performed with MCF-7 and HepG2 cells, the IC(50) obtained for bovicin HC5 was 279.39 and 289.30 µM, respectively, while for nisin these values were 105.46 and 112.25 µM. The haemolytic activity of bovicin HC5 against eukaryotic cells was always lower than that determined for nisin. The presence of cholesterol did not influence the activity of either bacteriocin on DOPC model membranes, but nisin showed reduced carboxyfluorescein leakage in DPoPC membranes containing cholesterol. In conclusion, bovicin HC5 only exerted cytotoxic effects at concentrations that were greater than the concentration needed for its biological activity, and the presence of cholesterol did not affect its interaction with model membranes.

[Cell entry mechanisms of coronaviruses].

PubMed

Taguchi, Fumihiro; Matsuyama, Shutoku

2009-12-01

Enveloped viruses enter into cells via fusion of their envelope and cellular membrane. Spike (S) protein of coronavirus (CoV) is responsible for entry events. We studied the cell entry mechanisms of two different CoVs, murine coronavirus mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV). MHV-JHM that induces syncytia in infected cells entered directly from cell surface, i.e., fusion of envelope and plasma membrane, whereas SARS-CoV and MHV-2 that fail to induce syncytia entered via endosome in a protease-dependent fashion, i.e., fusion of envelope and endosomal membrane. The latter viruses entered directly from cell surface, when receptor-bound viruses were treated with proteases that activate fusion activity of their S proteins. The entry pathway of SARS-CoV could influence the severity of the disease. It was also reveled that a highly neurovirulent JHM spread in a receptor-independent fashion, which could result in a high neuropathogenicity of the virus.

Buwchitin: a ruminal peptide with antimicrobial potential against Enterococcus faecalis

NASA Astrophysics Data System (ADS)

Oyama, Linda B.; Crochet, Jean-Adrien; Edwards, Joan E.; Girdwood, Susan E.; Cookson, Alan R.; Fernandez-Fuentes, Narcis; Hilpert, Kai; Golyshin, Peter N.; Golyshina, Olga V.; Privé, Florence; Hess, Matthias; Mantovani, Hilario C.; Creevey, Christopher J.; Huws, Sharon A.

2017-07-01

Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

Physiological stimuli evoke two forms of endocytosis in bovine chromaffin cells.

PubMed

Chan, S A; Smith, C

2001-12-15

1. Exocytosis and endocytosis were measured following single, or trains of, simulated action potentials (sAP) in bovine adrenal chromaffin cells. Catecholamine secretion was measured by oxidative amperometry and cell membrane turnover was measured by voltage clamp cell capacitance measurements. 2. The sAPs evoked inward Na(+) and Ca(2+) currents that were statistically identical to those evoked by native action potential waveforms. On average, a single secretory granule underwent fusion following sAP stimulation. An equivalent amount of membrane was then quickly internalised (tau = 560 ms). 3. Stimulation with sAP trains revealed a biphasic relationship between cell firing rate and endocytic activity. At basal stimulus frequencies (single to 0.5 Hz) cells exhibited a robust membrane internalisation that then diminished as firing increased to intermediate levels (1.9 and 6 Hz). However at the higher stimulation rates (10 and 16 Hz) endocytic activity rebounded and was again able to effectively maintain cell surface near pre-stimulus levels. 4. Treatment with cyclosporin A and FK506, inhibitors of the phosphatase calcineurin, left endocytosis characteristics unaltered at the lower basal stimulus levels, but blocked the resurgence in endocytosis seen in control cells at higher sAP frequencies. 5. Based on these findings we propose that, under physiological electrical stimulation, chromaffin cells internalise membrane via two distinct pathways that are separable. One is prevalent at basal stimulus frequencies, is lessened with increased firing, and is insensitive to cyclosporin A and FK506. A second endocytic form is activated by increased firing frequencies, and is selectively blocked by cyclosporin A and FK506.

Physiological stimuli evoke two forms of endocytosis in bovine chromaffin cells

PubMed Central

Chan, Shyue-An; Smith, Corey

2001-01-01

Exocytosis and endocytosis were measured following single, or trains of, simulated action potentials (sAP) in bovine adrenal chromaffin cells. Catecholamine secretion was measured by oxidative amperometry and cell membrane turnover was measured by voltage clamp cell capacitance measurements. The sAPs evoked inward Na+ and Ca2+ currents that were statistically identical to those evoked by native action potential waveforms. On average, a single secretory granule underwent fusion following sAP stimulation. An equivalent amount of membrane was then quickly internalised (τ = 560 ms). Stimulation with sAP trains revealed a biphasic relationship between cell firing rate and endocytic activity. At basal stimulus frequencies (single to 0.5 Hz) cells exhibited a robust membrane internalisation that then diminished as firing increased to intermediate levels (1.9 and 6 Hz). However at the higher stimulation rates (10 and 16 Hz) endocytic activity rebounded and was again able to effectively maintain cell surface near pre-stimulus levels. Treatment with cyclosporin A and FK506, inhibitors of the phosphatase calcineurin, left endocytosis characteristics unaltered at the lower basal stimulus levels, but blocked the resurgence in endocytosis seen in control cells at higher sAP frequencies. Based on these findings we propose that, under physiological electrical stimulation, chromaffin cells internalise membrane via two distinct pathways that are separable. One is prevalent at basal stimulus frequencies, is lessened with increased firing, and is insensitive to cyclosporin A and FK506. A second endocytic form is activated by increased firing frequencies, and is selectively blocked by cyclosporin A and FK506. PMID:11744761

Tumor cell cholesterol depletion and V-ATPase inhibition as an inhibitory mechanism to prevent cell migration and invasiveness in melanoma.

PubMed

Costa, Gildeíde Aparecida; de Souza, Sávio Bastos; da Silva Teixeira, Layz Ribeiro; Okorokov, Lev A; Arnholdt, Andrea Cristina Vetö; Okorokova-Façanha, Anna L; Façanha, Arnoldo Rocha

2018-03-01

V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H + pump affects cholesterol mobilization to the plasma membrane. Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H + transport in membrane vesicles, and intact cell H + fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET). Cholesterol depletion by 5mM MβCD was found to be inhibitory to the hydrolytic and H + pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H + efflux in live cells at the same extent of MβCD. We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin. The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma. Copyright © 2017 Elsevier B.V. All rights reserved.

Membrane lipid-protein interactions modify the regulatory role of adenosine-deaminase complexing protein: a phase fluorometry study of a malignancy marker

NASA Astrophysics Data System (ADS)

Parola, Abraham H.; Porat, Nurith; Caiolfa, Valeria R.; Gill, David; Kiesow, Lutz A.; Weisman, Mathew; Nemschitz, S.; Yaron, Dahlia; Singer, Karen; Solomon, Ethel

1990-05-01

The role of membrane lipid-protein interactions in malignant cell transformation was examined with adenosine deaminase (ADA) as a representative membrane protein. ADA's activity changes dramatically in transformed cells and accordingly it is a malignancy marker. Yet, the mechanisms controlling its variable activity are unknown. We undertook the spectroscopic deciphering of its interactions with its lipidic environment in normal and malignant cells. ADA exists in two interconvertible forms, small (45 KD) and large (21OKD). The large form consists of two small catalytic subunits (55-ADA) and a dimeric complexing protein ADCP. The physiological role of ADCP was not known either. Our studies were carried out at three levels.: 1. Solution enzyme kinetics, 2. The interaction of 55-ADA with ADCP reconstituted in liposomes: Effect of cholesterol and 3. Multifrequency phase modulation spectrofluorometry of pyrene-labeled 55-ADA bound to ADCP on the membranes of normal and RSV or RSV Ts68 transformed chick embryo fibroblasts. We found: 1. ADCP has an allosteric regulatory role on 55-ADA, which may be of physiological relevance: It inhibits 55-ADA activity at low physiological adenosine concentrations but accelerates deamination at high substrate concentration. 2. When reconstituted in DMPC liposomes, it retains 55-ADA activity (in its absence the activity is lost) and upon rigidification with cholesterol, a three fold increase in 55-ADA activity is attained, contrary to ADCP's regulatory activity when free of lipids. 3. The reduced ADA activity in transformed chick embryo fibroblasts is associated with increased membrane lipid fluidity (reduced order parameter), reduced accessibility of ADCP and increase rotational dynamics of the complex. We thus obtained spectroscopic deciphering of the vertical motion of ADCP, controlled by lipid-protein interaction, resulting in variable activity of this malignancy marker.

Ultrastructural Complexity of Nuclear Components During Early Apoptotic Phases in Breast Cancer Cells

PubMed Central

Castelli, Christian; Losa, Gabriele A.

2001-01-01

Fractal morphometry was used to investigate the ultrastructural features of the plasma membrane, perinuclear membrane and nuclear chromatin in SK‐BR‐3 human breast cancer cells undergoing apoptosis. Cells were incubated with 1 μM calcimycin (A23187) for 24 h. Cells in the early stage of apoptosis had fractal dimension (FD) values indicating that their plasma membranes were less rough (lower FD) than those of control cells, while their perinuclear membranes were unaffected. Changes of the chromatin texture within the entire nucleus and in selected nuclear domains were more pronounced in treated cells. This confirms that the morphological reorganization imputable to a loss of structural complexity (reduced FD) occurs in the early stage of apoptosis, is accompanied by the inhibition of distinct enzymatic events and precedes the onset of conventional cellular markers, which can only be detected during the active phases of the apoptotic process. PMID:11790854

TRPV2.

PubMed

Kojima, Itaru; Nagasawa, Masahiro

2014-01-01

Transient receptor potential vanilloid type 2, TRPV2, is a calcium-permeable cation channel belonging to the TRPV channel family. This channel is activated by heat (>52 °C), various ligands, and mechanical stresses. In most of the cells, a large portion of TRPV2 is located in the endoplasmic reticulum under unstimulated conditions. Upon stimulation of the cells with phosphatidylinositol 3-kinase-activating ligands, TRPV2 is translocated to the plasma membrane and functions as a cation channel. Mechanical stress may also induce translocation of TRPV2 to the plasma membrane. The expression of TRPV2 is high in some types of cells including neurons, neuroendocrine cells, immune cells involved in innate immunity, and certain types of cancer cells. TRPV2 may modulate various cellular functions in these cells.

Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

DOE PAGES

Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; ...

2015-06-16

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas G12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas G12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

PubMed Central

Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li-Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

2015-01-01

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. PMID:26080442

The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices

PubMed Central

Miller, Evan W.; Slak Rupnik, Marjan

2013-01-01

Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca2+]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network. PMID:24324777

Charge-Triggered Membrane Insertion of Matrix Metalloproteinase-7, Supporter of Innate Immunity and Tumors.

PubMed

Prior, Stephen H; Fulcher, Yan G; Koppisetti, Rama K; Jurkevich, Alexander; Van Doren, Steven R

2015-11-03

Matrix metalloproteinase-7 (MMP-7) sheds signaling proteins from cell surfaces to activate bacterial killing, wound healing, and tumorigenesis. The mechanism targeting soluble MMP-7 to membranes has been investigated. Nuclear magnetic resonance structures of the zymogen, free and bound to membrane mimics without and with anionic lipid, reveal peripheral binding to bilayers through paramagnetic relaxation enhancements. Addition of cholesterol sulfate partially embeds the protease in the bilayer, restricts its diffusion, and tips the active site away from the bilayer. Its insertion of hydrophobic residues organizes the lipids, pushing the head groups and sterol sulfate outward toward the enzyme's positive charge on the periphery of the enlarged interface. Fluorescence probing demonstrates a similar mode of binding to plasma membranes and internalized vesicles of colon cancer cells. Binding of bilayered micelles induces allosteric activation and conformational change in the auto-inhibitory peptide and the adjacent scissile site, illustrating a potential intermediate in the activation of the zymogen. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chitosan derivatives targeting lipid bilayers: Synthesis, biological activity and interaction with model membranes.

PubMed

Martins, Danubia Batista; Nasário, Fábio Domingues; Silva-Gonçalves, Laiz Costa; de Oliveira Tiera, Vera Aparecida; Arcisio-Miranda, Manoel; Tiera, Marcio José; Dos Santos Cabrera, Marcia Perez

2018-02-01

The antimicrobial activity of chitosan and derivatives to human and plant pathogens represents a high-valued prospective market. Presently, two low molecular weight derivatives, endowed with hydrophobic and cationic character at different ratios were synthesized and characterized. They exhibit antimicrobial activity and increased performance in relation to the intermediate and starting compounds. However, just the derivative with higher cationic character showed cytotoxicity towards human cervical carcinoma cells. Considering cell membranes as targets, the mode of action was investigated through the interaction with model lipid vesicles mimicking bacterial, tumoral and erythrocyte membranes. Intense lytic activity and binding are demonstrated for both derivatives in anionic bilayers. The less charged compound exhibits slightly improved selectivity towards bacterial model membranes, suggesting that balancing its hydrophobic/hydrophilic character may improve efficiency. Observing the aggregation of vesicles, we hypothesize that the "charge cluster mechanism", ascribed to some antimicrobial peptides, could be applied to these chitosan derivatives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rappa, Germana; College of Pharmacy, Roseman University of Health Sciences, Henderson, NV 89104; Mercapide, Javier

2013-04-01

Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that threemore » distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1–positive structures appeared in three sizes (small, ≤40 nm; intermediates ∼40–80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1–containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma. - Highlights: ► First report of release of prominin-1–containing microvesicles from cancer cells. ► Pro-metastatic role of prominin-1–containing microvesicles in FEMX-I melanoma. ► Down-regulation of prominin-1 results in decreased nuclear localization of β-catenin. ► Wnt signaling as mediator of the pro-metastatic activity of prominin-1.« less

Engineering antimicrobial peptides with improved antimicrobial and hemolytic activities.

PubMed

Zhao, Jun; Zhao, Chao; Liang, Guizhao; Zhang, Mingzhen; Zheng, Jie

2013-12-23

The rapid rise of antibiotic resistance in pathogens becomes a serious and growing threat to medicine and public health. Naturally occurring antimicrobial peptides (AMPs) are an important line of defense in the immune system against invading bacteria and microbial infection. In this work, we present a combined computational and experimental study of the biological activity and membrane interaction of the computationally designed Bac2A-based peptide library. We used the MARTINI coarse-grained molecular dynamics with adaptive biasing force method and the umbrella sampling technique to investigate the translocation of a total of 91 peptides with different amino acid substitutions through a mixed anionic POPE/POPG (3:1) bilayer and a neutral POPC bilayer, which mimic the bacterial inner membrane and the human red blood cell (hRBC) membrane, respectively. Potential of mean force (PMF, free energy profile) was obtained to measure the free energy barrier required to transfer the peptides from the bulk water phase to the water-membrane interface and to the bilayer interior. Different PMF profiles can indeed identify different membrane insertion scenarios by mapping out peptide-lipid energy landscapes, which are correlated with antimicrobial activity and hemolytic activity. Computationally designed peptides were further tested experimentally for their antimicrobial and hemolytic activities using bacteria growth inhibition assay and hemolysis assay. Comparison of PMF data with cell assay results reveals a good correlation of the peptides between predictive transmembrane activity and antimicrobial/hemolytic activity. Moreover, the most active mutants with the balanced substitutions of positively charged Arg and hydrophobic Trp residues at specific positions were discovered to achieve the improved antimicrobial activity while minimizing red blood cell lysis. Such substitutions provide more effective and cooperative interactions to distinguish the peptide interaction with different lipid bilayers. This work provides a useful computational tool to better understand the mechanism and energetics of membrane insertion of AMPs and to rationally design more effective AMPs.

Membrane-proximal TRAIL species are incapable of inducing short circuit apoptosis signaling: Implications for drug development and basic cytokine biology.

PubMed

Tatzel, Katharina; Kuroki, Lindsay; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T; Goedegebuure, S Peter; Powell, Matthew A; Mutch, David G; Hawkins, William G; Spitzer, Dirk

2016-03-03

TRAIL continues to garner substantial interest as a recombinant cancer therapeutic while the native cytokine itself serves important tumor surveillance functions when expressed in membrane-anchored form on activated immune effector cells. We have recently developed the genetically stabilized TRAIL platform TR3 in efforts to improve the limitations associated with currently available drug variants. While in the process of characterizing mesothelin-targeted TR3 variants using a single chain antibody (scFv) delivery format (SS-TR3), we discovered that the membrane-tethered cytokine had a substantially increased activity profile compared to non-targeted TR3. However, cell death proceeded exclusively via a bystander mechanism and protected the mesothelin-positive targets from apoptosis rather than leading to their elimination. Incorporation of a spacer-into the mesothelin surface antigen or the cancer drug itself-converted SS-TR3 into a cis-acting phenotype. Further experiments with membrane-anchored TR3 variants and the native cytokine confirmed our hypothesis that membrane-proximal TRAIL species lack the capacity to physically engage their cognate receptors coexpressed on the same cell membrane. Our findings not only provide an explanation for the "peaceful" coexistence of ligand and receptor of a representative member of the TNF superfamily but give us vital clues for the design of activity-enhanced TR3-based cancer therapeutics.

Effects of unsaturated fatty acids on the kinetics of voltage‐gated proton channels heterologously expressed in cultured cells

PubMed Central

Kawanabe, Akira

2016-01-01

Key points Arachidonic acid (AA) greatly enhances the activity of the voltage‐gated proton (Hv) channel, although its mechanism of action and physiological function remain unclear.In the present study, we analysed the effects of AA on proton currents through Hv channels heterologously expressed in HEK293T cells.The dramatic increase in proton current amplitude elicited by AA was accompanied by accelerated activation kinetics and a leftward shift in the voltage‐dependence of activation.Mutagenesis studies suggest the two aforementioned effects of AA reflect two distinct structural mechanisms.Application of phospholipase A2, which liberates AA from phospholipids in the membrane, also enhances Hv channel activity, supporting the idea that AA modulates Hv channel activity within physiological contexts. Abstract Unsaturated fatty acids are key components of the biological membranes of all cells, and precursors of mediators for cell signalling. Arachidonic acid (AA) is an unsaturated fatty acid known to modulate the activities of various ion channels, including the voltage‐gated proton (Hv) channel, which supports the rapid production of reactive oxygen species (ROS) in phagocytes through regulation of pH and membrane potential. However, the molecular mechanisms and physiological functions of the effects of AA on Hv channels remain unclear. In the present study, we report an electrophysiological analysis of the effects of AA on the mouse Hv channel (mHv1) heterologously expressed in HEK293T cells. Application of AA to excised inside‐out patch membranes rapidly induced a robust increase in the amplitude of the proton current through mHv1. The current increase was accompanied by accelerated activation kinetics and a small leftward shift of the current–voltage relationship. In monomeric channels lacking the coiled‐coil region of the channel protein, the shift in the current–voltage relationship was diminished but activation and deactivation remained accelerated. Studies with several AA derivatives showed that double bonds and hydrophilic head groups are essential for the effect of AA, although charge was not important. The application of phospholipase A2 (PLA2), which generates AA from cell membrane phospholipids, stimulated mHv1 activity to a similar extent as direct application of ∼20 μm AA, suggesting that endogenous AA may regulate Hv channel activity. PMID:26563684

D120 and K152 within the PH Domain of T Cell Adapter SKAP55 Regulate Plasma Membrane Targeting of SKAP55 and LFA-1 Affinity Modulation in Human T Lymphocytes

PubMed Central

Witte, Amelie; Meineke, Bernhard; Sticht, Jana; Philipsen, Lars; Kuropka, Benno; Müller, Andreas J.; Freund, Christian

2017-01-01

ABSTRACT The β2-integrin lymphocyte function-associated antigen 1 (LFA-1) is needed for the T cell receptor (TCR)-induced activation of LFA-1 to promote T cell adhesion and interaction with antigen-presenting cells (APCs). LFA-1-mediated cell-cell interactions are critical for proper T cell differentiation and proliferation. The Src kinase-associated phosphoprotein of 55 kDa (SKAP55) is a key regulator of TCR-mediated LFA-1 signaling (inside-out/outside-in signaling). To gain an understanding of how SKAP55 controls TCR-mediated LFA-1 activation, we assessed the functional role of its pleckstrin homology (PH) domain. We identified two critical amino acid residues within the PH domain of SKAP55, aspartic acid 120 (D120) and lysine 152 (K152). D120 facilitates the retention of SKAP55 in the cytoplasm of nonstimulated T cells, while K152 promotes SKAP55 membrane recruitment via actin binding upon TCR triggering. Importantly, the K152-dependent interaction of the PH domain with actin promotes the binding of talin to LFA-1, thus facilitating LFA-1 activation. These data suggest that K152 and D120 within the PH domain of SKAP55 regulate plasma membrane targeting and TCR-mediated activation of LFA-1. PMID:28052935

Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.

PubMed

Haupt, Sara; Malik, Zvi; Ehrenberg, Benjamin

2014-01-01

Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

Enhancement of death-receptor induced caspase-8-activation in the death-inducing signalling complex by uncoupling of oxidative phosphorylation.

PubMed

Vier, Juliane; Gerhard, Monika; Wagner, Hermann; Häcker, Georg

2004-01-01

Signalling through the death receptor CD95 induces apoptosis by formation of a signalling complex at the cell membrane and subsequent caspase-8 and caspase-3-activation. Treatment of Jurkat T cells with protonophores across the mitochondrial membrane such as 2,4-dinitrophenol (DNP) enhances the death-inducing capacity of CD95. In this study, we show that this enhancement is due to the specific acceleration of caspase-8-processing and activation at the CD95-receptor. DNP-treatment did not affect NF-kappaB-induction by CD95. Immunoprecipitation experiments showed that the amounts of the adapter FADD/MORT1 and pro-caspase-8 at the CD95-receptor were not altered by DNP. Subcellular fractionation studies revealed that the amount of mature caspase-8 but not pro-caspase at the membrane was increased following CD95-stimulation in the presence of DNP. As a consequence of caspase-activation, c-FLIP-levels in the cytosol decreased. In Jurkat cells overexpressing c-FLIPS, DNP was still able to enhance caspase-activation. The enhancing capacity of DNP was seen in some cell lines (Jurkat, CEM and HeLa) but not in SKW6 cells and was also found in mitogen-stimulated human T cells. Furthermore, the enhancement extended to TRAIL-induced caspase-activation. Thus, a mechanism exists by which caspase-8-activation can be accelerated at death receptors and this mechanism can be triggered by targeting mitochondrial oxidative phosphorylation.

Role of two adaptor molecules SLP-76 and LAT in the PI3K signaling pathway in activated T cells.

PubMed

Shim, Eun Kyung; Jung, Seung Hee; Lee, Jong Ran

2011-03-01

Previously, we identified p85, a subunit of PI3K, as one of the molecules that interacts with the N-terminal region of Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76). We also demonstrated that tyrosine phosphorylation either at the 113 and/or 128 position is sufficient for the association of SLP-76 with the Src homology 2 domain near the N terminus of p85. The present study further examines the role of the association of these two molecules on the activation of PI3K signaling cascade. Experiments were done to determine the role of SLP-76, either wild-type, tyrosine mutants, or membrane-targeted forms of various SLP-76 constructs, on the membrane localization and phosphorylation of Akt, which is an event downstream of PI3K activation. Reconstitution studies with these various SLP-76 constructs in a Jurkat variant cell line that lacks SLP-76 or linker for activation of T cells (LAT) show that the activation of PI3K pathway following TCR ligation requires both SLP-76 and LAT adaptor proteins. The results suggest that SLP-76 associates with p85 after T cell activation and that LAT recruits this complex to the membrane, leading to Akt activation.

INHIBITION OF MYCOLIC ACID TRANSPORT ACROSS THE MYCOBACTERIUM TUBERCULOSIS PLASMA MEMBRANE

PubMed Central

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

2011-01-01

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

Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk Is Essential for Toll-like Receptor Activation and Cellular Signaling*

PubMed Central

Abdulkhalek, Samar; Amith, Schammim Ray; Franchuk, Susan L.; Jayanth, Preethi; Guo, Merry; Finlay, Trisha; Gilmour, Alanna; Guzzo, Christina; Gee, Katrina; Beyaert, Rudi; Szewczuk, Myron R.

2011-01-01

The signaling pathways of mammalian Toll-like receptors (TLRs) are well characterized, but the precise mechanism(s) by which TLRs are activated upon ligand binding remains poorly defined. Recently, we reported a novel membrane sialidase-controlling mechanism that depends on ligand binding to its TLR to induce mammalian neuraminidase-1 (Neu1) activity, to influence receptor desialylation, and subsequently to induce TLR receptor activation and the production of nitric oxide and proinflammatory cytokines in dendritic and macrophage cells. The α-2,3-sialyl residue of TLR was identified as the specific target for hydrolysis by Neu1. Here, we report a membrane signaling paradigm initiated by endotoxin lipopolysaccharide (LPS) binding to TLR4 to potentiate G protein-coupled receptor (GPCR) signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP9) activation to induce Neu1. Central to this process is that a Neu1-MMP9 complex is bound to TLR4 on the cell surface of naive macrophage cells. Specific inhibition of MMP9 and GPCR Gαi-signaling proteins blocks LPS-induced Neu1 activity and NFκB activation. Silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction or siRNA transfection of macrophage cells and MMP9 knock-out primary macrophage cells significantly reduced Neu1 activity and NFκB activation associated with LPS-treated cells. These findings uncover a molecular organizational signaling platform of a novel Neu1 and MMP9 cross-talk in alliance with TLR4 on the cell surface that is essential for ligand activation of TLRs and subsequent cellular signaling. PMID:21873432

The Potassium Channel, Kir3.4 Participates in Angiotensin II-Stimulated Aldosterone Production by a Human Adrenocortical Cell Line

PubMed Central

Oki, Kenji; Plonczynski, Maria W.; Lam, Milay Luis; Gomez-Sanchez, Elise P.

2012-01-01

Angiotensin II (A-II) regulation of aldosterone secretion is initiated by inducing cell membrane depolarization, thereby increasing intracellular calcium and activating the calcium calmodulin/calmodulin kinase cascade. Mutations in the selectivity filter of the KCNJ5 gene coding for inward rectifying potassium channel (Kir)3.4 has been found in about one third of aldosterone-producing adenomas. These mutations result in loss of selectivity of the inward rectifying current for potassium, which causes membrane depolarization and opening of calcium channels and activation of the calcium calmodulin/calmodulin kinase cascade and results in an increase in aldosterone secretion. In this study we show that A-II and a calcium ionophore down-regulate the expression of KCNJ5 mRNA and protein. Activation of Kir3.4 by naringin inhibits A-II-stimulated membrane voltage and aldosterone secretion. Overexpression of KCNJ5 in the HAC15 cells using a lentivirus resulted in a decrease in membrane voltage, intracellular calcium, expression of steroidogenic acute regulatory protein, 3-β-hydroxysteroid dehydrogenase 3B2, cytochrome P450 11B1 and cytochrome P450 11B2 mRNA, and aldosterone synthesis. In conclusion, A-II appears to stimulate aldosterone secretion by depolarizing the membrane acting in part through the regulation of the expression and activity of Kir3.4. PMID:22798349

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

DOE PAGES

Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; ...

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. Here, we present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity.more » 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. Moreover, we observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Finally, 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.« less

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.

[The influence of the actoprotectors on lipid peroxidation and erythrocyte membranes in rats poisoned with malathion ].

PubMed

Myshkin, V A; Guliaeva, I A; Ibatullina, R B; Savlukov, A I; Enikeev, D A; Sergeeva, S A

2004-01-01

Actoprotecting properties ofbemitil, tietasol in combination with atropin were studied in red cell membranes and lipid peroxidation of rats poisoned with MI in a dose 320 mg/kg (0.9 LD50). Atropin treatment showed a low effect. The addition of bemitil and tietasol normalized electric charge and osmotic resistance in red cell membranes, activity of superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase and content of lipid peroxidation products--ketodienes and TBA-reacting products. Efficacy of the combined treatment is due primarily to noncholinergic mechanism of action of bemitil and tietasol--stimulation of endogenic antioxidant systems of erythron and antiradical activity (bemitil).

Annexin A2 in Proliferative Vitreoretinopathy

DTIC Science & Technology

2017-10-01

cells , leading to formation of an epiretinal membrane, retinal detachment, and loss of vision. At present, there are no reliable means of...type versus annexin A2- deficient mice, [2] define the role of A2 in the function of activated macrophages and RPE cells in PVR, and [3] examine the...expression is needed in both macrophages and RPE cells , and that A2 is extensively expressed within cells of epiretinal membranes in human PVR. Our

[Extracellular matrix--regulation of cancer invasion and metastasis].

PubMed

Watanabe, Hideto

2010-11-01

Cancer cell invasion comprises steps in the destruction of the basement membrane and migration of cells into the connective tissue. These cells further migrate into lymph ducts and small vessels to reach metastasis. The extracellular matrix (ECM) provides a microenvironment for cells, and its destruction is associated with cancer cell invasion. Among matrix metalloproteinases (MMPs), both MMP-2 and 9 digest type IV collagen, a major component of the basement membrane, and MMP-14/MT1-MMP, a membrane-type MMP, activates MMP-2. Thus, these MMPs play a central role in cancer cell invasion. MMPs also cleave latent forms of growth factors and signaling molecules, releasing and activating them, which influence neo-vascularization and cancer apoptosis. Like proteins, carbohydrates are known to be involved in cancer invasion. Hyaluronan is known to both stimulate and inhibit cancer invasion, depending on its molecular size. Heparanase, which digests heparan sulfate, is known to facilitate cancer invasion and metastasis. In summary, ECM provides a microenvironment that regulates cell behavior and its structure altered by MMPs affects cancer cell invasion.

In vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate.

PubMed

Beloti, Márcio M; de Oliveira, Paulo T; Gimenes, Rossano; Zaghete, Maria A; Bertolini, Márcio J; Rosa, Adalberto L

2006-11-01

This study was aimed at investigating the in vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT). Osteoblastic cells were obtained from human alveolar bone fragments and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured on P(VDF-TrFE)/BT and expanded polytetrafluoroethylene (e-PTFE--control) membranes in 24-well plates. Cell adhesion and spreading were evaluated at 30 min, and 4 and 24 h. For proliferation assay, cells were cultured for 1, 7, and 10 days. Cell viability was detected by trypan blue at 7 and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 7, 14, and 21 days. Cultures were stained with Alizarin red at 21 days, for detection of mineralized matrix. Data were compared by ANOVA and Student t test. Cell attachment (p = 0.001), cell number (p = 0.001), and ALP activity (p = 0.0001) were greater on P(VDF-TrFE)/BT. Additionally, doubling time was greater on P(VDF-TrFE)/BT (p = 0.03), indicating a decreased proliferation rate. Bone-like nodule formation took place only on P(VDF-TrFE)/BT. The present results showed that both membranes are biocompatible. However, P(VDF-TrFE)/BT presented a better in vitro biocompatibility and allowed bone-like nodule formation. Therefore, P(VDF-TrFE)/BT could be an alternative membrane to be used in guided tissue regeneration.

Interaction of capsaicinoids with cell membrane models does not correlate with pungency of peppers

NASA Astrophysics Data System (ADS)

Geraldo, Vananélia P. N.; Ziglio, Analine C.; Gonçalves, Débora; Oliveira, Osvaldo N.

2017-04-01

Mixed monolayers were prepared using phospholipids in order to mimic cell membranes and fractions of capsaicinoids (extracted from Malagueta, Caps-M, and Bhut Jolokia, Caps-B, peppers). According to their surface-pressure isotherms and polarization-modulated infrared reflection absorption spectra (PM-IRRAS), weak molecular-level interactions were observed between Caps and phospholipids. Both Caps-M and Caps-B penetrated into the alkyl tail region of the monolayer, interacted with the phosphate group of the phospholipids and affected hydration of their Cdbnd O groups. Since the physiological activity of Caps is not governed solely by interaction with cell membranes, it should require participation of a neuronal membrane receptor, e.g. vanilloid receptor (TRPV1).

Diorcinol D Exerts Fungicidal Action against Candida albicans through Cytoplasm Membrane Destruction and ROS Accumulation

PubMed Central

Li, Ying; Chang, Wenqiang; Zhang, Ming; Li, Xiaobin; Jiao, Yang; Lou, Hongxiang

2015-01-01

Candida albicans, which is the most common human fungal pathogen, causes high mortality among immunocompromised patients. Antifungal drug resistance becomes a major challenge for the management of Candida infection. Diorcinol D (DD), a diphenyl ether derivative isolated from an endolichenic fungus, exerted fungicidal action against Candida species. In this study, we investigated the possible mechanism of its antifungal activity. The change of membrane dynamics and permeability suggested that the cell membrane was disrupted by the treatment of DD. This was further supported by the evidences of intracellular glycerol accumulation, alteration of cell ultrastructure, and down-regulation of genes involved in cell membrane synthesis. In addition, the treatment of C. albicans with DD resulted in the elevation of reactive oxygen species (ROS), which caused the dysfunction of mitochondria. These altogether suggested that DD exerted its antifungal activity through cytoplasmic membrane destruction and ROS accumulation. This finding is helpful to uncover the underlying mechanisms for the diphenyl ether derivatives and provides a potential application in fighting clinical fungal infections. PMID:26047493

Necroptosis Execution Is Mediated by Plasma Membrane Nanopores Independent of Calcium.

PubMed

Ros, Uris; Peña-Blanco, Aida; Hänggi, Kay; Kunzendorf, Ulrich; Krautwald, Stefan; Wong, W Wei-Lynn; García-Sáez, Ana J

2017-04-04

Necroptosis is a form of regulated necrosis that results in cell death and content release after plasma membrane permeabilization. However, little is known about the molecular events responsible for the disruption of the plasma membrane. Here, we find that early increase in cytosolic calcium in TNF-induced necroptosis is mediated by treatment with a Smac mimetic via the TNF/RIP1/TAK1 survival pathway. This does not require the activation of the necrosome and is dispensable for necroptosis. Necroptosis induced by the activation of TLR3/4 pathways does not trigger early calcium flux. We also demonstrate that necroptotic plasma membrane rupture is mediated by osmotic forces and membrane pores around 4 nm in diameter. This late permeabilization step represents a hallmark in necroptosis execution that is cell and treatment independent and requires the RIP1/RIP3/MLKL core. In support of this, treatment with osmoprotectants reduces cell damage in an in vivo necroptosis model of ischemia-reperfusion injury. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity.

PubMed

Adu-Gyamfi, Emmanuel; Kim, Lori S; Jardetzky, Theodore S; Lamb, Robert A

2016-09-01

Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

PubMed

O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

2012-12-18

Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

Expression of DNAJB12 or DNAJB14 Causes Coordinate Invasion of the Nucleus by Membranes Associated with a Novel Nuclear Pore Structure

PubMed Central

Goodwin, Edward C.; Motamedi, Nasim; Lipovsky, Alex; Fernández-Busnadiego, Rubén; DiMaio, Daniel

2014-01-01

DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity. PMID:24732912

Membrane Transport Phenomena (MTP)

NASA Technical Reports Server (NTRS)

Mason, Larry W.

1996-01-01

The development of the seal between the membrane and the Fluid Optical Cells (FOC) has been a high priority activity. This seal occurs at an interface in the instrument where three key functions must be realized: (1) physical membrane support, (2) fluid sealing, and (3) unobscured optical transmission.

Size-dependent protein segregation at membrane interfaces

PubMed Central

Schmid, Eva M; Bakalar, Matthew H; Choudhuri, Kaushik; Weichsel, Julian; Ann, HyoungSook; Geissler, Phillip L; Dustin, Michael L; Fletcher, Daniel A

2016-01-01

Membrane interfaces formed at cell-cell junctions are associated with characteristic patterns of membrane protein organization, such as E-cadherin enrichment in epithelial junctional complexes and CD45 exclusion from the signaling foci of immunological synapses. To isolate the role of protein size in these processes, we reconstituted membrane interfaces in vitro using giant unilamellar vesicles decorated with synthetic binding and non-binding proteins. We show that size differences between binding and non-binding proteins can dramatically alter their organization at membrane interfaces in the absence of active contributions from the cytoskeleton, with as little as a ~5 nm increase in non-binding protein size driving its exclusion from the interface. Combining in vitro measurements with Monte Carlo simulations, we find that non-binding protein exclusion is also influenced by lateral crowding, binding protein affinity, and thermally-driven membrane height fluctuations that transiently limit access to the interface. This simple, sensitive, and highly effective means of passively segregating proteins has implications for signaling at cell-cell junctions and protein sorting at intracellular contact points between membrane-bound organelles. PMID:27980602

Plasma membrane signaling in HIV-1 infection.

PubMed

Abbas, Wasim; Herbein, Georges

2014-04-01

Plasma membrane is a multifunctional structure that acts as the initial barrier against infection by intracellular pathogens. The productive HIV-1 infection depends upon the initial interaction of virus and host plasma membrane. Immune cells such as CD4+ T cells and macrophages contain essential cell surface receptors and molecules such as CD4, CXCR4, CCR5 and lipid raft components that facilitate HIV-1 entry. From plasma membrane HIV-1 activates signaling pathways that prepare the grounds for viral replication. Through viral proteins HIV-1 hijacks host plasma membrane receptors such as Fas, TNFRs and DR4/DR5, which results in immune evasion and apoptosis both in infected and uninfected bystander cells. These events are hallmark in HIV-1 pathogenesis that leads towards AIDS. The interplay between HIV-1 and plasma membrane signaling has much to offer in terms of viral fitness and pathogenicity, and a better understanding of this interplay may lead to development of new therapeutic approaches. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking. Copyright © 2013 Elsevier B.V. All rights reserved.

Important factors for cell-membrane permeabilization by gold nanoparticles activated by nanosecond-laser irradiation

PubMed Central

Yao, Cuiping; Rudnitzki, Florian; Hüttmann, Gereon; Zhang, Zhenxi; Rahmanzadeh, Ramtin

2017-01-01

Purpose Pulsed-laser irradiation of light-absorbing gold nanoparticles (AuNPs) attached to cells transiently increases cell membrane permeability for targeted molecule delivery. Here, we targeted EGFR on the ovarian carcinoma cell line OVCAR-3 with AuNPs. In order to optimize membrane permeability and to demonstrate molecule delivery into adherent OVCAR-3 cells, we systematically investigated different experimental conditions. Materials and methods AuNPs (30 nm) were functionalized by conjugation of the antibody cetuximab against EGFR. Selective binding of the particles was demonstrated by silver staining, multiphoton imaging, and fluorescence-lifetime imaging. After laser irradiation, membrane permeability of OVCAR-3 cells was studied under different conditions of AuNP concentration, cell-incubation medium, and cell–AuNP incubation time. Membrane permeability and cell viability were evaluated by flow cytometry, measuring propidium iodide and fluorescein isothiocyanate–dextran uptake. Results Adherently growing OVCAR-3 cells can be effectively targeted with EGFR-AuNP. Laser irradiation led to successful permeabilization, and 150 kDa dextran was successfully delivered into cells with about 70% efficiency. Conclusion Antibody-targeted and laser-irradiated AuNPs can be used to deliver molecules into adherent cells. Efficacy depends not only on laser parameters but also on AuNP:cell ratio, cell-incubation medium, and cell–AuNP incubation time. PMID:28848345

Salvianolic acid B protects hepatocytes from H2O2 injury by stabilizing the lysosomal membrane.

PubMed

Yan, Xiao-Feng; Zhao, Pei; Ma, Dong-Yan; Jiang, Yi-Lu; Luo, Jiao-Jiao; Liu, Liu; Wang, Xiao-Ling

2017-08-07

To investigate the capability of salvianolic acid B (Sal B) to protect hepatocytes from hydrogen peroxide (H 2 O 2 )/carbon tetrachloride (CCl 4 )-induced lysosomal membrane permeabilization. Cell Counting Kit-8 assay was used to measure cell viability. Apoptosis and death were assayed through flow cytometry. BrdU incorporation was used to detect cell proliferation. Serum alanine aminotransferase activity and liver malondialdehyde (MDA) content were measured. Liver histopathological changes were evaluated using hematoxylin-eosin staining. Lysosomal membrane permeability was detected with LysoTracker Green-labeled probes and acridine orange staining. The levels of protein carbonyl content (PCC), cathepsins (Cat)B/D, and lysosome-associated membrane protein 1 (LAMP1) were evaluated through western blotting. Cytosol CatB activity analysis was performed with chemiluminescence detection. The mRNA level of LAMP1 was evaluated through quantitative real-time polymerase chain reaction. Results indicated that H 2 O 2 induced cell injury/death. Sal B attenuated H 2 O 2 -induced cell apoptosis and death, restored the inhibition of proliferation, decreased the amount of PCC, and stabilized the lysosome membrane by increasing the LAMP1 protein level and antagonizing CatB/D leakage into the cytosol. CCl 4 also triggered hepatocyte death. Furthermore, Sal B effectively rescued hepatocytes by increasing LAMP1 expression and by reducing lysosomal enzyme translocation to the cytosol. Sal B protected mouse embryonic hepatocytes from H 2 O 2 /CCl 4 -induced injury/death by stabilizing the lysosomal membrane.

siRNA-based Analysis of the Abrogation of the Protective Function of Membrane-associated Catalase of Tumor Cells.

PubMed

Bauer, Georg

2017-02-01

Tumor cells, in contrast to non-malignant cells, show sustained expression of membrane-associated NADPH oxidase-1 and therefore generate extracellular superoxide anions and their dismutation product H 2 O 2 In order to prevent intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling, tumor cells need to express membrane-associated catalase that interferes with HOCl and nitric oxide/peroxynitrite signaling. Catalase is attached to tumor cells through the activity of transglutaminase-2 and is prevented from superoxide anion-dependent inhibition through coexpression of membrane-associated superoxide dismutase. Therefore, specific inhibition of membrane-associated catalase should reactivate intercellular ROS/RNS-dependent apoptosis-inducing signaling. These processes are analyzed here through small interfering RNA-mediated knockdown of essential signaling compounds. This allows to establish a rather comprehensive picture of intercellular ROS/RNS signaling that may be instrumental for future therapeutic approaches. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Oleyl group-functionalized insulating gate transistors for measuring extracellular pH of floating cells

NASA Astrophysics Data System (ADS)

Imaizumi, Yuki; Goda, Tatsuro; Toya, Yutaro; Matsumoto, Akira; Miyahara, Yuji

2016-01-01

The extracellular ionic microenvironment has a close relationship to biological activities such as by cellular respiration, cancer development, and immune response. A system composed of ion-sensitive field-effect transistors (ISFET), cells, and program-controlled fluidics has enabled the acquisition of real-time information about the integrity of the cell membrane via pH measurement. Here we aimed to extend this system toward floating cells such as T lymphocytes for investigating complement activation and pharmacokinetics through alternations in the plasma membrane integrity. We functionalized the surface of tantalum oxide gate insulator of ISFET with oleyl-tethered phosphonic acid for interacting with the plasma membranes of floating cells without affecting the cell signaling. The surface modification was characterized by X-ray photoelectron spectroscopy and water contact angle measurements. The Nernst response of -37.8 mV/pH was obtained for the surface-modified ISFET at 37 °C. The oleyl group-functionalized gate insulator successfully captured Jurkat T cells in a fluidic condition without acute cytotoxicity. The system was able to record the time course of pH changes at the cells/ISFET interface during the process of instant addition and withdrawal of ammonium chloride. Further, the plasma membrane injury of floating cells after exposure by detergent Triton™ X-100 was successfully determined using the modified ISFET with enhanced sensitivity as compared with conventional hemolysis assays.

Oleyl group-functionalized insulating gate transistors for measuring extracellular pH of floating cells

PubMed Central

Imaizumi, Yuki; Goda, Tatsuro; Toya, Yutaro; Matsumoto, Akira; Miyahara, Yuji

2016-01-01

Abstract The extracellular ionic microenvironment has a close relationship to biological activities such as by cellular respiration, cancer development, and immune response. A system composed of ion-sensitive field-effect transistors (ISFET), cells, and program-controlled fluidics has enabled the acquisition of real-time information about the integrity of the cell membrane via pH measurement. Here we aimed to extend this system toward floating cells such as T lymphocytes for investigating complement activation and pharmacokinetics through alternations in the plasma membrane integrity. We functionalized the surface of tantalum oxide gate insulator of ISFET with oleyl-tethered phosphonic acid for interacting with the plasma membranes of floating cells without affecting the cell signaling. The surface modification was characterized by X-ray photoelectron spectroscopy and water contact angle measurements. The Nernst response of −37.8 mV/pH was obtained for the surface-modified ISFET at 37 °C. The oleyl group-functionalized gate insulator successfully captured Jurkat T cells in a fluidic condition without acute cytotoxicity. The system was able to record the time course of pH changes at the cells/ISFET interface during the process of instant addition and withdrawal of ammonium chloride. Further, the plasma membrane injury of floating cells after exposure by detergent Triton™ X-100 was successfully determined using the modified ISFET with enhanced sensitivity as compared with conventional hemolysis assays. PMID:27877886

Electrical filtering in gerbil isolated type I semicircular canal hair cells

NASA Technical Reports Server (NTRS)

Rennie, K. J.; Ricci, A. J.; Correia, M. J.

1996-01-01

1. Membrane potential responses of dissociated gerbil type I semicircular canal hair cells to current injections in whole cell current-clamp have been measured. The input resistance of type I cells was 21.4 +/- 14.3 (SD) M omega, (n = 25). Around the zero-current potential (Vz = -66.6 +/- 9.3 mV, n = 25), pulsed current injections (from approximately -200 to 750 pA) produced only small-amplitude, pulse-like changes in membrane potential. 2. Injecting constant current to hyperpolarize the membrane to around -100 mV resulted in a approximately 10-fold increase in membrane resistance. Current pulses superimposed on this constant hyperpolarization produced larger and more complex membrane potential changes. Depolarizing currents > or = 200 pA caused a rapid transient peak voltage before a plateau. 3. Membrane voltage was able to faithfully follow sine-wave current injections around Vz over the range 1-1,000 Hz with < 25% attenuation at 1 kHz. A previously described K conductance, IKI, which is active at Vz, produces the low input resistance and frequency response. This was confirmed by pharmacologically blocking IKI. This conductance, present in type I cells but not type II hair cells, would appear to confer on type I cells a lower gain, but a much broader bandwidth at Vz, than seen in type II cells.

Effects of steroid hormones on nuclear membrane and membrane-bound heterochromatin from breast cancer cells evaluated by fractal morphometry.

PubMed

Losa, G A; Graber, R; Baumann, G; Nonnenmacher, T F

1999-10-01

To evaluate the effect of steroid hormones on the ultrastructure of nuclear heterochromatin and perinuclear membranes in human MCF-7 breast cancer cells. MCF-7 cells were cultured briefly (five minutes) in the presence of 10(-9) M estrogen 17 beta-estradiol, a stimulator of cell proliferation and/or 10(-9) M glucocorticoid dexamethasone. Changes in the morphologic complexity of nuclear membrane-bound heterochromatin (NMBHC) and nuclear membranes (ENM) were assessed by means of the fractal capacity dimension, D, a noneuclidean geometric descriptor of complex, irregular bodies. 17 beta-estradiol (10(-9) M) enhanced the ultrastructural irregularity of NMBHC, as documented by the increased value of D, whereas dexamethasone (10(-9) M) reduced it when compared to NMBHC from untreated MCF-7 control cells. In contrast, neither steroid modified ENM ultrastructure. Changes in the nuclear heterochromatin complexity induced by estrogen 17 beta-estradiol occurred concomitantly with functional changes at the cell periphery, such as activation of the phospholipase C, a cell membrane-associated enzyme involved in signal transduction. Dexamethasone reduced the ultrastructural complexity of NMBHC without affecting functional processes. Fractal morphometry proved its usefulness in quantifying early ultrastructural changes in nuclear components induced in MCF-7 cells by steroid hormones, 17 beta-estradiol and dexamethasone.

Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis

PubMed Central

Basso, Pauline; Ragno, Michel; Elsen, Sylvie; Reboud, Emeline; Golovkine, Guillaume; Bouillot, Stephanie; Huber, Philippe; Lory, Stephen; Faudry, Eric

2017-01-01

ABSTRACT   Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa. In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication. PMID:28119472

Cross talk between ceramide and redox signaling: implications for endothelial dysfunction and renal disease.

PubMed

Li, Pin-Lan; Zhang, Yang

2013-01-01

Recent studies have demonstrated that cross talk between ceramide and redox signaling modulates various cell activities and functions and contributes to the development of cardiovascular diseases and renal dysfunctions. Ceramide triggers the generation of reactive oxygen species (ROS) and increases oxidative stress in many mammalian cells and animal models. On the other hand, inhibition of ROS-generating enzymes or treatment of antioxidants impairs sphingomyelinase activation and ceramide production. As a mechanism, ceramide-enriched signaling platforms, special cell membrane rafts (MR) (formerly lipid rafts), provide an important microenvironment to mediate the cross talk of ceramide and redox signaling to exert a corresponding regulatory role on cell and organ functions. In this regard, activation of acid sphingomyelinase and generation of ceramide mediate the formation of ceramide-enriched membrane platforms, where transmembrane signals are transmitted or amplified through recruitment, clustering, assembling, or integration of various signaling molecules. A typical such signaling platform is MR redox signaling platform that is centered on ceramide production and aggregation leading to recruitment and assembling of NADPH oxidase to form an active complex in the cell plasma membrane. This redox signaling platform not only conducts redox signaling or regulation but also facilitates a feedforward amplification of both ceramide and redox signaling. In addition to this membrane MR redox signaling platform, the cross talk between ceramide and redox signaling may occur in other cell compartments. This book chapter focuses on the molecular mechanisms, spatial-temporal regulations, and implications of this cross talk between ceramide and redox signaling, which may provide novel insights into the understanding of both ceramide and redox signaling pathways.